Human Endogenous Retrovirus Type K (HERV-K) Particles Package and Transmit HERV-K-Related Sequences. - PDF Download Free (2024)

JVI Accepted Manuscript Posted Online 29 April 2015 J. Virol. doi:10.1128/JVI.00544-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Human Endogenous Retroviruses Type-K (HERV-K) Virus

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Particles Package and Transmit HERV-K-Related

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Sequences

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Rafael Contreras-Galindo1, Mark H. Kaplan1, Derek Dube1, Marta J. Gonzalez-Hernandez1,2,

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Susana Chan1, Fan Meng3,4, Manhong Dai3, Gilbert S Omenn1,5,6,7, Scott D. Gitlin1,7,10, and

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David M. Markovitz1,2,8,9

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Authors’ affiliations:

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Department of Internal Medicine1, Programs in Immunology2, Molecular and Behavioral

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Neuroscience Institute3, Department of Psychiatry4, Department of Computational Medicine and

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Bioinformatics5, Department of Human Genetics6, Comprehensive Cancer Center7, Cancer

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Biology8, and Cellular and Molecular Biology9, University of Michigan, Ann Arbor, MI 48109,

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USA.10 Veteran Affairs Health System, Ann Arbor, MI 48105, USA

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Running title: Packaging and transmission of endogenous retroviruses

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Corresponding author:

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David M. Markovitz 1

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Department of Internal Medicine

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Division of Infectious Diseases

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University of Michigan, Ann Arbor, MI

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48109-5640, USA

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Tel# (734) 647-1786

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Fax# (734) 764-0101

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E-mail: [emailprotected]

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Word count for the abstract: 250

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Word count for the text: 13481

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Keywords: HERV-K packaging, HERV-K transmission, Reverse Transcription, chromosomal

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integration, episome formation, HIV-1

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Abstract

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Human Endogenous Retroviruses make up 8% of the human genome. While the youngest

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of these retroviruses, HERV-K (HML-2), termed HK2, is able to code for all viral proteins and

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produce virus-like particles, it is not known if these virus particles package and transmit HK2-

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related sequences. Here we analyzed the capacity of HK2 for packaging and transmitting HK2

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sequences. We created a HK2 probe, termed Bogota, which can be packaged into HK2 viruses,

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and transfected it into cells that make HK2 particles. Supernatants of the transfected cells, which

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contained HK2 viral particles, were then added to target cells and the transmissibility of the HK2

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Bogota reporter was tracked by G418 resistance. Our studies revealed that contemporary HK2

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virions produced by some teratocarcinoma and breast cancer cell lines, as well as by peripheral

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blood lymphocytes from lymphoma patients, can package HK2 Bogota probes, and these viruses

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transmitted these probes to other cells. After transmission, HK2 Bogota transcripts undergo

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reverse transcription, a step impaired by antiretroviral agents or by introduction of mutations into

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the probe sequences required for reverse transcription. HK2 viruses were more efficiently

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transmitted in the presence of HK2 Rec or HIV-1 Tat and Vif. Transmitted Bogota probes

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formed episomes, but did not integrate into the cellular genome. Resistance to integration might

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explain the relatively low number of HK2 insertions that were acquired during the last 25 million

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years of evolution. Whether transient transmission of modern HK2 sequences, which encode two

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putative oncoproteins, can lead to disease remains to be studied.

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Importance

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Retroviruses have invaded the genome of human ancestors over millions of years, yet

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these viruses have been generally inactivated during evolution, with only remnants of these

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infectious sequences remaining in the human genome. One of these viruses, termed HK2, is still

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capable of producing virus particles, although these particles have been regarded as being non-

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infectious. Using a genetic probe derived from HK2, we have discovered that HK2 viruses

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produced in modern humans can package HK2 sequences and transmit them to various other

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cells. Furthermore, the genetic sequences packaged in HK2 undergo reverse transcription. The

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transmitted probe circularized in the cell and failed to integrate into the cellular genome. These

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findings suggest that modern HK2 viruses can package viral RNA and transmit it to other cells.

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Contrary to previous views, we provide evidence of an extracellular viral phase of modern HK2

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viruses. We have no evidence of sustained, spreading infection.

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Introduction

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Actively replicating retroviruses infected the germline multiple times over the millions of

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years of human evolution. These sequences were transmitted vertically in a Mendelian fashion to

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the next generations and therefore became a stable part of the inherited genetic material. Relics

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of these retroviral infections presently make up approximately 8% of the modern human genome.

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These retroviral sequences have acquired multiple mutations, and sometimes deletions, leading

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to the widely-held assumption that they are no longer competent to replicate [1-3]. The most

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recent of those infections were attributed to the endogenous retroviruses HERV-K (HML-2),

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termed HK2 in this manuscript [4,5]. After infection, HK2 integrated into the germline DNA to

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form proviruses consisting of four retroviral genes (gag, pro, pol and env) and two regulatory

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genes, np9 and rec. Np9 and Rec proteins are alternate splice products that are expressed

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depending upon the presence or absence of a 292 bp segment in the env gene, respectively. The

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coding region of HK2 is flanked by two Long Terminal Repeats (LTRs). Approximately 3000

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proviral remnants of the HK2 group remain in the modern human genome [6,7]. About 85% of

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these HK2 elements exist as solitary LTRs (Solo LTR), which originated by recombination

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between the 5’ and 3’ LTRs of full-length proviruses, removing the internal gag, pro, pol, and

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env genes [8]. To date, approximately two hundred HK2 elements have been found in the DNA

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of the modern human as full-length proviruses [3,9,10]. A few HK2 loci have been copied by

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segmental duplication. About one hundred of these elements copied by recombination into the

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centromeres of several human chromosomes. Upon integration, HK2 created identical 5-6 bp

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sequences, known as target site duplications, flanking each side of the provirus. hom*ologous

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recombination between some HK2, however, created hybrid proviruses with different flanking

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target site sequences [11]. 5

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Certain HK2 elements are present only in the genome of humans but not in other

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primates, and therefore represent the youngest retroviruses to enter the human genome [4].

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Further, the youngest eleven HK2 are polymorphic in the human population [3,9,12]. As HK2 is

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the most recent retrovirus to enter the genome, it is not surprising that HK2 is still

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transcriptionally active [13-19]. Expression of HK2 viral RNA, proteins, and virus-like particles

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(VLPs) are detectable in breast cancer, leukemia, melanoma, and teratocarcinoma cell lines.

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These viruses, however, are widely considered to be non-infectious [15,20-23]. In addition to the

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findings of HERV-K expression in cancer cell lines, we have found HK2 RNA, proteins, and

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VLPs in the blood of patients with HIV-1 infection, lymphoma, and breast cancer [9-10,24-27];

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the capacity of these viruses to package HK2 sequences and transmit them to other cells remains

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to be tested.

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While no single human endogenous retrovirus sequence thus far identified in the genome

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encodes a complete infectious virus, the diverse HK2 loci in the human genome retain coding

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capacity for every functional retroviral protein [4,10,28-30]: the Gag protein, necessary for

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particle formation and release [31-35]; the Pro protein, necessary for cleavage and maturation of

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viral proteins [35-37]; the Pol protein, necessary for RNA-dependent DNA replication and

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integration [38-42]; the accessory protein Rec, which exports unspliced viral RNA from the

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nucleus to the cytoplasm [43,44]; and functional Env proteins [45]. Recombination or trans-

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complementation between these proviruses could lead to the formation of viruses that would be

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able to package and transmit HK2 sequences [9, 46-48], as seen with endogenous retroviruses in

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mice [49-51].

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Recently, several groups have begun to suspect that modern HK2 may still retain the

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capacity to replicate. By analyzing HK2 sequences, Belshaw et al. revealed that during millions 6

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of years of HK2 evolution, the env gene, which is only necessary for virus infectivity, has been

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under purifying selection, meaning that this gene has accumulated synonymous mutations to

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preserve infectious capacity and suggesting that a pool of replication-competent HK2 might still

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be present in modern humans [6]. Subsequently, two groups resurrected infectious viruses using

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existing HK2 fragments of the human genome [52,53]; molecular recombination among present-

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day HK2 proviruses produced viruses competent to replicate, providing proof of principle of the

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possibility of HK2 replication in ancient and perhaps modern humans (53). Our laboratory has

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recently demonstrated that HK2 viral genomes found in HK2 particles are able to proceed

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through reverse transcription, and have utilized a life cycle with a degree of genomic flexibility

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in which both RNA- and DNA-containing viruses are capable of mediating infection (54).

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Finally, we looked for evidence of HK2 transmission in modern humans by analyzing the viral

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genome found in viruses produced by living patients [9]. Sequence analysis of the viral env gene

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in HIV-1-infected, but not breast cancer patients, revealed evidence consistent with HK2

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transmission: i) active recombination, suggesting the viral RNA was packaged into the particles

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and went through reverse transcription; and ii) accumulation of synonymous mutations, a sign of

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purifying selection that could result from copying of loci within the individual. These

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observations suggest, but do not prove, that HK2 sequences could still be packaged into HK2

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particles and transmitted to other cells under certain conditions [9,25].

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Proof that HK2 sequences can be packaged and transmitted to other cells in modern

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humans is challenging to obtain using standard virology techniques, as all human cells contain

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HK2. It is difficult to differentiate between viral DNA, RNA, and protein products generated by

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de novo transmission from the already existent endogenous HK2. We have devised an innovative

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strategy to assess packaging and transmission of HK2 viruses, based on experiments previously 7

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employed to demonstrate that L1 elements are mobile [55-58]. We constructed a molecular

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probe, termed Bogota, which has a presumed packaging sequence and, the minimal HK2 viral

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sequences necessary to undergo reverse transcription, as well as a neomycin resistance reporter

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cassette. The genome of Bogota however, does not encode any retroviral protein. The Bogota

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probe was designed so that it could be packaged into HK2 virus, and then used to track cell-to-

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cell transmission and reverse transcription of genetic material packaged within HK2 viruses

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produced in human cells under varying conditions.

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In the present manuscript, we report that HK2 viruses produced under certain conditions

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can indeed package HK2-related sequences and transmit these sequences to other cells.

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Furthermore, these sequences undergo reverse transcription. Efficient transmission of the HK2

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sequences requires conserved sequences necessary for reverse transcription and is inhibited by

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reverse transcriptase inhibitors. Production of transmission-competent HK2 viruses occurs in

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certain cancer cell lines and peripheral blood lymphocytes of lymphoma patients. Expression of

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transmissible HK2 virus increases with the over-expression of the HK2 accessory protein Rec,

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and is further enhanced by the HIV-1 accessory proteins Tat and Vif. We therefore have

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developed a strategy to assess packaging and transmissibility of endogenous retroviruses

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particles and have shown that modern HK2 viruses are competent to package HK2 sequences

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and transmit them to other cells. Upon passaging, we have demonstrated that the packaged

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genetic material undergoes reverse transcription. The viral sequences, however, do not undergo

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genomic integration into chromosomal DNA.

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Materials and Methods

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Ethics Statement

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Blood samples were collected from lymphoma patients after receiving informed consent

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from a protocol approved by the Institutional Review Board of the University of Michigan

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Medical School (IRBMED).

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Construction of HERV-K Bogota plasmids and mutants

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Bogota constructs were derived from the HERV-K113 provirus (9472 bp, Acc. No

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AY037928.1) present in the bacterial artificial chromosome (BAC) clone RP11-398B1 obtained

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from BacPac Resources (https://bacpac.chori.org). Bogota was engineered to contain sequences

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necessary for reverse transcription including the LTRs, the primer binding site (PBS), and the

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polypurine tract (PPT), whereas all essential and accessory genes gag, pol, env, and rec were

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mutated or deleted. The Bogota construct contains only a portion of the gag gene and lacks pol,

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most of the env, and the rec gene. Therefore, Bogota constructs retain K113 sequence from the

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5’LTR to the middle portion of gag (bases 366 to 2665 of K113 genome), and the 3’LTR with a

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final portion of the env sequence (bases 8265-9472 of K113 genome). Either a neo cassette

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(HERV-KBogota

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replaced the internal K113 genes [55]. Bogota constructs were cloned into the pCEP4 vector

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(Invitrogen, http://www.lifetechnologies.com). The expression of Bogota is driven by the CMV

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promoter, which replaced the U3 portion of the 5’LTR sequence (bases 1-366). Bogota mutants

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were constructed by deleting either the PBS (TGG TGC CCA ACG TGG AGG, 971-988 bp) the

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PPT site (AAA AGA AAA GGG GGA, 8488-8502 bp), or the last 220 bp of the 3’LTR. Further

neo)

or a neo cassette in reverse orientation with an intron (HERV-KBogota

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oenR)

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description of the cloning strategies is available upon request.

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Other plasmid constructs

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Dr. Paul Bieniasz kindly provided the plasmid CHKCP coding for the infectious HERV-

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KCON and the plasmid pCR3.1/K-Rec. HERV-KCON contains a puromycin reporter cassette

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inserted into the env gene and its expression is driven by the CMV promoter [52]. The plasmid

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pCR3.1/K-Rec codes for the HERV-K108 Rec protein. The VSVG plasmid codes for the

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Vesicular Stomatitis Virus (VSV) Envelope. The HIV-1 Tat coding plasmid pcDNA3.1-Tat86

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was derived from the parent vector pcDNA3.1+/Tat101-flag (Eric Verdin, NIH AIDS Research

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and Reference Reagent Program, http://www.aidsreagent.org). The plasmids pcDNA-HVif, and

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pcDNA-Vphu encode the HIV-1 accessory proteins Vif, and Vpu, respectively. The sequences of

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Vif and Vpu are codon optimized for expression in human cells and are further described

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elsewhere (Stephan Bour and Klaus Strebel, NIH AIDS Research and Reference Reagent

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Program, http://www.aidsreagent.org). The pcDNA3.1 empty vector control was made by

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excising the Tat insert with BamHI, followed by religation of the backbone.

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Culture of cell lines, peripheral blood lymphocytes, and transfection

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The human cell lines 293T, NCCIT, PA-1, MCF-7, and the hamster CHO, rat C6, and

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buffalo green monkey BGMK cell lines were maintained in DMEM supplemented with 10%

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fetal bovine serum (FBS) and penicillin/streptomycin. 293T is derived from embryonic kidney

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cells. The HK2 particle-producing NCCIT cell line is derived from male teratocarcinoma cells.

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PA-1 is derived from female ovarian teratocarcinoma cells. MCF-7 is derived from breast

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adenocarcinoma cells. The feline G355 cells were cultured in modified McCoy's 5a medium with

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the same additives. The quail QT6 cells were cultured in Ham's F-12 medium supplemented with

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10% tryptose phosphate broth and 5% bovine calf serum. Blood samples were collected from

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lymphoma patients after receiving informed consent from a protocol approved by the University

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of Michigan Institutional Review Board. Peripheral blood lymphocytes (PBLs) were isolated

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from whole blood by Ficoll-Hypaque gradient and separated from macrophages by plate

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adherence. PBLs were maintained in RPMI medium supplemented with 10% FBS,

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penicillin/streptomycin, and stimulated with 5 μg of phytohemagglutinin (PHA-P; Sigma-

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Aldrich, http://www.sigmaaldrich.com)/ml and 10 U of interleukin-2 (Sigma-Aldrich,

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http://www.sigmaaldrich.com)/ml. Cells were transiently transfected in 10 cm plates at 5 X 106

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cells/plate or in six-well plates at 1 X 105 cells/well using FuGENE®HD (Roche,

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http://www.roche-applied-science.com), according to the manufacturers protocol. After 24 h,

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cells were washed 5 times with phosphate buffered saline (PBS), and medium was replaced.

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Virus-particle-containing supernatants were collected after an additional 24 to 48 hours. Control

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experiments included mock transfections.

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Immunoprecipitation of HK2 particles.

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Virus-containing supernatant of NCCIT cells transfected with HERV-KBogota constructs

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was cleared of cellular debris with two centrifugation steps at 1000 X g at 4 oC, followed by

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filtration through a 0.45 μm Puradisc Filter (Whatman, http://www.whatman.com). Viral

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particles were concentrated by ultracentrifugation through a 20% sucrose cushion, and

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resuspended in 600 μl of NH Buffer (0.8% NaCl, 10 mM Hepes pH 7.4), and DNase treated

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using the Turbo DNA-free kit (Ambion, http://www.lifetechnologies.com). Viral particles were

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pre-cleared with 50 μl of Protein A/G Plus Agarose beads (Santa Cruz Biotech, 11

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http://www.scbt.com) for 30 min with rocking. The beads were then removed by centrifugation

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for 3 min at 2000 X g, and the supernatant split into two equal volumes for immunoprecipitation.

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To one tube, 6 μg of control mouse IgG2a was added. To the second, 6 μg of mouse anti-HERV-

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K Env (HERM-1811-5, Austral Biologicals, http://www.australbiologicals.com) was added.

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Both samples were incubated for 90 min with rocking, after which 50 μl of Protein A/G Plus

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Agarose beads were added to each tube. The samples were then incubated for an additional 2 h.

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The samples were centrifuged for 3 min at 2000 X g to pellet the beads, the supernatant

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removed, and washed three times with 300 μl NH Buffer with centrifugation. The samples were

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then once again split into two tubes, one for western blot analysis and one for qRT-PCR analysis.

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Western Blot Analysis following Immunoprecipitation

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To prepare western blot samples, the bound Protein A/G beads were resuspended in 40 μl

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of NH Buffer and 10 μl of 5 X Laemmli sample buffer with dithiothreitol. Samples were boiled

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for 10 min to lyse and denature viral proteins and loaded onto SDS-PAGE. HK2 capsid

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expression was detected using a rabbit anti-HERV-K Gag antibody developed by the group of

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Norbert Bannert [59], and a mouse anti-rabbit IgG antibody conjugated to horseradish

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peroxidase. SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific,

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http://www.piercenet.com) was added and protein bands detected with X-ray Film.

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Viral and cellular RNA extraction

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Viral RNA was extracted from purified and DNAse-treated viral particles, from cell-free

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supernatants, or HK2 Env-immunoprecipitated samples using the QIAamp Viral RNA Mini Kit

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(Qiagen,

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immunoprecipitated samples, the Protein A/G beads were removed by centrifugation after viral

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lysis. Viral RNA was eluted from the QIAamp columns in equal volumes and stored at -80 oC.

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Cellular RNA was extracted with the QIAmp RNA blood mini-kit following the manufacturer’s

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instructions (Qiagen, http://www.qiagen.com) and treated with RNAse-free DNAse as

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recommend by the manufacturer. The elimination of DNA was confirmed by PCR without RT

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using primers that bind HERV-K gag and Bogota neo. No PCR amplification product was

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detected in the cellular or viral RNA samples, confirming the absence of genomic or plasmid

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DNA.

http://www.qiagen.com)

following

the

manufacturer’s

protocol.

For

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Quantitative RT-PCR

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Viral and cellular RNA was analyzed for gene expression of the endogenous retroviruses

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HK2 np9, rec, gag, and env; HERV-W gag, and env; HERV-H env, and HERV-KBogota neo as

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well as the housekeeping genes (gapdh, cox-2, β-actin) by Real time RT-PCR using the primers

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described in Table 1. Quantitative RT-PCR was done using the Brilliant III Ultrafast SYBR

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Green qRT-PCR Kit (Agilent technologies, http://www.genomics.agilent.com) in the StepOne

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Plus

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Quantitation analyses were performed using the ΔΔcT method.

Real-Time

PCR

System (Applied Biosystems,

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http://www.lifetechnologies.com).

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Virus Transmission

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Cells that may or may not produce transmissible HERV-K endogenous virus particles

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were transfected with HERV-K Bogota probe constructs and co-transfected with plasmids

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expressing HERV-K Rec or HIV-1 regulatory proteins where indicated. After 24 hours, the

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transfection media was removed, and cells washed at least five times with PBS in order to

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eliminate any residual plasmid. Transfected cells were incubated in serum-free media for 24-48

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additional hours. Production of infectious HERV-KCON viruses was performed as described

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previously [52]. Briefly, plasmid CHKCP encoding HERV-KCON was transfected into 293T cells

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with or without plasmids expressing HERV-K Rec and VSV-G. Virus-containing supernatant

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was treated with DNAse and cleared of cellular debris with two centrifugations at 1000 X g at 4

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o

C, followed by filtration through a 0.45 μM Whatman Puradisc Filter (Whatman,

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http://www.whatman.com). Infection was performed by exposing the filtered virus-containing

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supernatant onto 60-70% confluent target cells seeded in 6-well or 10 cm plates along with fresh

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media in the presence of 5 μg/ml of polybrene. In some experiments, target cells were incubated

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in the presence or absence of nucleoside and non-nucleoside reverse transcriptase inhibitors

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(RTIs). Cells transduced with HERV-K particles containing Bogota probes were selected in

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medium containing 250-500 μg/ml of neomycin for 2-4 weeks. Cells transduced with HERV-

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KCON were selected in 2.5 μg/ml puromycin for approximately 10 days. Then colonies were

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stained with Coomassie blue for visualization. The number of resistant colonies appearing as

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plaques was scored, and when indicated, the frequency of transmission was determined by

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calculating the percent of resistant colonies formed by either RTI-treated cells or Bogota mutants

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in relation to the number of colonies observed in the untreated, Bogota transmitted cells.

301 14

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Detection of Bogota Integration

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Identification of Bogota integration into the target cell genome was performed on

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genomic DNA isolated from G418® cells. The existence of Bogota DNA, either in episomal form

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or integrated into chromosomes, was assessed by several methodologies. i) PCR: DNA was

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screened for Bogota integration by PCR using primers that bind to the neo gene, NEO437F and

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NEO1808R (see Table 1). PCR reactions were performed using Fast-start Taq DNA polymerase

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(Roche Applied Science, http://www.roche-applied-science.com) using the following conditions:

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One cycle at 95 °C for 2 min, followed by 35 cycles at 94 °C for 15 sec, 55 °C for 20 sec, and 72

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°C for 30 sec. PCR products were purified using the QIAquick PCR purification kit (Qiagen,

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http://www.qiagen.com) and sequenced. ii) Deep-sequencing analyses: Paired-end libraries were

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prepared from the genomic DNA samples, and Bogota elements were enriched for by

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hybridization with a biotinylated probe set spanning a consensus sequence of the HK2 LTR. The

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hybridized DNA was then captured with streptavidin beads, washed, and re-amplified prior to

315

deep sequencing using the Illumina HiSeq 2000 system [60]. Potential integration sites were

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determined by PCR with primers that bind to the HK2 LTR and their potential flanking genomic

317

sequences. iii) Inverse PCR: Five μg genomic DNA was digested with SpeI, or HindIII (New

318

England Biolabs, http://www.neb.com). The digested DNA was ligated using T4 DNA ligase

319

(3200 U; New England Biolabs, http://www.neb.com) in a volume of 600 μL at 16 °C for 16 h.

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The ligated DNA was ethanol-precipitated and dissolved in 50 μL of distilled water. The inverse

321

PCR was performed using the Expand Long Range dNTPack PCR kit (Roche Applied Science,

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http://www.roche-applied-science.com). Two μL of DNA were used in a primary PCR reaction

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in a 50 μL volume containing 2.5 mM MgCl2, 500 μM dNTPs, 300 nM of each primer

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(NEO1720F and NEO210R, see Table 1), and 3.5 units of Expand Long Range Enzyme Mix.

15

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PCR was performed in one cycle of 95 °C for 2 min, followed by 30 cycles of 94 °C for 10 sec,

326

63 °C for 30 sec, and 68 °C for 15 min, followed by a final extension step at 68 °C for 30 min.

327

One μL of the resultant product was subjected to a secondary PCR reaction using the same

328

conditions, except that we used primers NEO1808F and NEO 173R (see Table 1). PCR products

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from the second amplification were purified with QIAquick PCR purification kit (Qiagen,

330

http://www.qiagen.com) and sequenced [61]. iv) Alu and B2 RAN-PCR: We performed Alu PCR

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and B2 Ran PCR to account for post-transmission Bogota insertion into areas of highly abundant

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species-specific repetitive elements. PCR was performed using primers that bind to human Alu

333

[62] or B2 rodent [63] sequences and complementary primers that bind the HK2 LTR of Bogota

334

using the conditions described in the citations [62,63]. v) ATLAS suppression PCR. We adapted

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a protocol for the amplification typing of L1 active subfamilies (ATLAS), that enables the

336

selective amplification of DNA fragments containing the ends of human-specific L1s and

337

flanking sequence as described [64], using primers specific for the Bogota virus instead of L1

338

primers.

339 340

In silico sequence analysis

341

The env sequences obtained in supernatants from HK2 particle-producing cell lines were

342

BLASTed to the HERVd and NCBI databases. The sequences were aligned in BioEdit and

343

exported to the MEGA 6 matrix. Viral RNA sequences were aligned to known HK2 proviruses

344

[3,9]. Phylogenetic trees were constructed and corroborated by the neighbor-joining method,

345

using the statistical bootstrap test (10000 replicates) of inferred phylogeny and the Kimura-2

346

parameter model [65,66]. ORFs were calculated using translated-BLAST in the NCBI database.

16

347

Highlighter plots were generated using the highlighter tool of the Los Alamos HIV sequence

348

database (http://www.hiv.lanl.gov).

349 350

Test for recombination

351

We evaluated sequences for potential recombination events using several methods. First,

352

we inspected the neighbor-joining tree for each data set. Recombination of large portions of

353

different elements may lead to branches with unresolved topology, resulting in taxonomic units

354

(TUs) that either protrude far beyond the other taxa or fall far short in comparison. The potential

355

recombinant sequences were verified and the parent sequences identified using RIP 3.0

356

(http://www.hiv.lanl.gov). This program used a sliding window (200 bp in this study) that moves

357

over an alignment containing the query sequence and all the possible parental proviruses. Best

358

matches are marked if they are significant by using an internal statistical test. Recombination

359

analyses were corroborated using the recombination analysis tool (RAT) program [67]. We

360

verified the sequence similarity between the putative parent and query sequence on each side of

361

the recombination spot. On several occasions, recombinants were more than 99% similar to each

362

parental sequence.

363 364 365 366 367 368 17

369

Results

370

Design of a strategy to test HK2 packaging and transmission: the Bogota system

371

To examine whether present-day HK2 viruses can package and transmit HK2-related

372

transcripts, we engineered a molecular HK2 probe, termed Bogota, which uses the transmission

373

of resistance to the aminoglycoside neomycin (G418®) to detect transmission into target cells.

374

Similar approaches that use neomycin resistance as a surrogate marker have been created to

375

successfully demonstrate the retrotransposition of L1 elements in human cells [55-58]. We

376

therefore cloned a neomycin (neo) resistance reporter cassette consisting of the simian virus 40

377

(SV-40) promoter, the neomycin resistance gene, and a 3’ polyadenylation signal into a defective

378

HERV-K clone derived from provirus K113 [12]. This clone HERV-KBogota neo also contains the

379

sequences necessary to fully undergo reverse transcription: the primer binding site (PBS), the

380

polypurine tract (PPT), and the R, and U5 segments of the LTRs. This construct only contains

381

partial segments of the gag and env TM genes, and therefore Bogota is incompetent to make any

382

retroviral protein itself (Fig. 1). The Bogota construct, HERV-KBogota neo, was designed to contain

383

a sense neo reporter cassette (Fig. 1). Expression of the Bogota construct is driven by the CMV

384

promoter, which replaced the U3 portion of the 5’ LTR, in order to achieve high level of

385

expression and avoid the variable expression of HERV-K seen in different cell types [14,68].

386

Transfection of Bogota into HK2 particle-producing cells results in production of Bogota

387

retroviral transcripts that were predicted to be co-packaged into HK2 VLPs, as Bogota contains

388

the putative HK2 packaging signal. When these VLPs are used for transmission, they can confer

389

G418® resistance onto the target cell only when the Bogota genome undergoes reverse

390

transcription and forms episomal forms (1LTR and 2LTR) or integrates into chromosomal DNA

391

(Fig. 1). However, if small traces of contamination of the HERV-KBogota 18

neo

construct are

392

inadvertently transfected to a detector cell, it can create G418® resistance without the need for

393

reverse transcription. Therefore, a second construct, HERV-KBogota oenR, was designed to contain

394

an antisense neo resistance indicator gene (oenR) disrupted by the intron IVS 2 of the λ-globin

395

gene in the opposite transcriptional orientation (Fig. 1). The arrangement of HERV-KBogota oenR

396

ensures that G418® resistant target cells arise only when transmitted HERV-KBogota

397

transcripts are spliced, packaged into VLPs, reverse transcribed, and expressed by their own

398

promoter [55-58]. Cells will remain sensitive to neomycin (G418®) if Bogota transcripts cannot

399

be spliced and reverse-transcribed, and the neo resistance product therefore not synthetized.

oenR

400 401

Evaluation of cell lines that might produce transmissible HK2 viruses

402

We first evaluated several possible cancer cell lines that might produce HK2 VLPs that can

403

package and transmit HK2 sequences. While some breast cancer, teratocarcinoma, leukemia, and

404

melanoma cell lines produce HK2 VLPs, these viruses appear to be non- infectious [15,20-23].

405

Bieda et al. characterized the phenotype of HK2 VLPs produced by the teratocarcinoma cell

406

lines GH, Tera-1, NCCIT, 2102Ep, and Tera-2; one additional cell line, PA-1, served as a virus-

407

negative control [20]. Some VLPs released by NCCIT cells appeared to have morphological and

408

biochemical properties of transmissible viruses: i) by electron microscopy, free virus particles

409

are seen with processed cores, suggesting mature viruses; ii) western blot analysis detected

410

processed Gag protein in those particles. However, Env protein was not detected by western

411

blotting in those particles with the anti-HK2 sera used in this study, suggesting that the particles

412

cannot be infectious [20]. In experiments conducted in our laboratory, however, by electron

413

microscopy we saw that the VLPs derived from NCCIT cells can be immunostained with anti-

19

414

HERV-K Env antibody. This antibody further detected processed Env protein in VLP

415

preparations by western blotting (Fig. 2), suggesting that VLPs produced from NCCIT cells are

416

promising candidates for transmissible HK2 viruses. The transmembrane (TM) band observed in

417

the western blot is very weak when compared to the full Env bands suggesting that the HERV-K

418

Env protein is hardly cleaved and not fusogenic. However, the small portion of HERV-K viruses

419

with processed or cleaved TM protein could be infectious.

420

In a further step, we analyzed the viral env genome sequence contained in HK2 VLPs

421

produced by breast cancer, melanoma, and teratocarcinoma cell lines, looking for evidence of

422

viral RNA replication by reverse transcription as demonstrated by sequence diversification and

423

recombination. Analysis of HK2 env sequences found in the supernatants of the NCCIT cell

424

line, but not other cell lines, revealed evidence of virus diversity and recombination, suggesting

425

that the HK2 genome replicated through reverse transcription (Fig. S1). It is possible that ex vivo

426

RT-PCR recombination artificially created these recombinant HK2 env sequences detected in our

427

study [69]. However, the detection of recombinant env sequences (45 % of the sequences) only

428

in the supernatants of NCCIT cells, but not other cell lines, argues that these recombinant env

429

sequences arose by HK2 replication through reverse transcription in the NCCIT cells and not

430

artifactually. It is important to also clarify that many more HK2 proviruses are expressed in

431

NCCIT cells as compared to other cell lines studied (Fig. S1), and therefore the occurrence of

432

recombination might be related to the diversity of HK2 proviruses that are expressed.

433 434 435 20

436

Packaging of Bogota into HK2 viruses

437

Having determined that the HK2 VLPs produced by the NCCIT cell line have the

438

potential to be transmissible, we next addressed whether the Bogota probe can be packaged into

439

these VLPs by transfecting the Bogota construct into NCCIT cells and analyzing the released

440

HK2 VLPs for co-packaging of Bogota transcripts. The packaging of Bogota transcripts in HK2

441

VLPs was evaluated using two different methodologies. In the first approach, the enveloped

442

HK2 VLPs were immunoprecipitated with anti-HK-2 Env antibody, and in the second approach,

443

total VLPs were pelleted by ultracentrifugation; the amount of Bogota transcripts packaged into

444

those VLPs was measured by real time RT-PCR. For the immunoprecipitation studies, VLPs

445

produced

446

immunoprecipitated using anti-HERV-K Env antibody or control non-specific IgG. VLPs

447

immunoprecipitated with anti-HERV-K Env antibody reacted with anti-HERV-K Gag antibody

448

in western blot analysis, confirming the precipitation of HK2 VLPs using this methodology (Fig.

449

3A). Immunoprecipitation of HK2 VLPs with non-specific IgG was detected only at a low level.

450

We next measured the abundance of the RNA transcripts HK2 gag and Bogota neo contained in

451

immunoprecipitated HK2 VLPs, using glyceraldehyde-3-phosphate dehydrogenase (gapdh) as a

452

control. Bogota neo and HK2 gag RNA transcripts were enriched in VLPs immunoprecipitated

453

with HERV-K Env antibody as compared to the VLPs precipitated with non-specific IgG (Fig.

454

3B). The levels of gapdh transcripts decreased in IP samples compared to the raw supernatant.

455

The residual low levels of gapdh in IP samples is expected, as this has been reported in studies of

456

other retroviruses [70-78], which can package different non-viral RNA sequences at low levels.

457

Further, enrichment of HK2 gag and Bogota neo transcripts found in HK2 VLPs was 5 to 20

were

concentrated

from

NCCIT

21

supernatants

by

ultracentrifugation

and

458

times greater when compared to that of gapdh transcripts (Fig 3B), suggesting that Bogota

459

transcripts are efficiently packaged into HK2 viral particles.

460

We next pelleted retroviral VLPs by ultracentrifugation to examine Bogota packaging in

461

total, as opposed to immunoprecipitated, HK2 VLPs. We quantitated the abundance of Bogota

462

neo, HK2 np9, rec, gag, and env type 2 transcripts. We then compared the amounts of these

463

transcripts in relation to the levels of expression of the cellular housekeeping genes gapdh and β-

464

actin, the mitochondrial gene cytochrome c oxidase II (cox-2), as well as transcripts from the

465

endogenous retrovirus families HERV-W and HERV-H (Fig. 3C). In order to determine the

466

packaging efficiency of these transcripts into the pelleted retroviral VLPs, we compared the

467

levels of transcripts found in the pelleted retroviral VLPs to the ones found in the cellular RNA

468

using a previously validated methodology [78-80]. Of these transcripts, HK2 genes and Bogota

469

HK2 appear to be the most abundant retroviral transcripts in NCCIT cellular RNA. A small

470

degree of expression of the endogenous retroviruses HERV-W and HERV-H was detected in the

471

cellular RNA as well, as opposed to the high levels of expression of the housekeeping genes

472

gapdh and β-actin. In the pelleted retroviral VLPs, however, we detected higher amounts of HK2

473

and Bogota transcripts as compared to the levels of expression of the housekeeping genes.

474

Transcripts of the HERV-H family were also detected in the pelleted retroviral VLPs, but at a

475

low level, and transcripts of the HERV-W family were not detected (Fig. 3C).

476

We calculated the relative packaging efficiency of each RNA transcript by dividing the

477

expression levels of each transcript found in pelleted retroviral VLPs by its expression level

478

found in cellular RNA and normalizing these numbers to the packaging efficiency level of

479

HERV-H env, which was arbitrarily set to 1. The relative packaging efficiencies of HK2 and

480

Bogota transcripts were 2-5 logs higher than the packaging efficiency of HERV-H env. The 22

481

relative packaging efficiency could not be calculated for HERV-W transcripts, as they were not

482

detectable in pelleted retroviral VLPs from NCCIT cells (Fig. 3D). Calculation of the packaging

483

efficiencies of cellular and mitochondrial housekeeping genes produced negative values,

484

suggesting they are not selectively packaged into pelleted retroviral VLPs. These results strongly

485

suggest that HK2 and Bogota transcripts are selectively packaged into pelleted retroviral VLPs

486

from NCCIT cells transfected with the Bogota construct. In contrast, transcripts from

487

housekeeping genes, as well as from other endogenous retroviral families, seem not to be

488

selectively packaged into pelleted retroviral VLPs, arguing that these HK2 RNAs are packaged

489

into structures other than HK2 VLPs, such as exosomes that would co-exist in these pellets”.

490 491

Infectivity of HK2 viruses containing Bogota transcripts

492

Having determined that Bogota transcripts are efficiently packaged into HK2 VLPs, we

493

next determined whether these particles could transmit Bogota from one cell to another. In order

494

to accomplish this, we tested whether HK2 VLPs from NCCIT containing the Bogota genome,

495

which uses neomycin resistance as a surrogate marker for transmissibility, can transmit

496

neomycin (G418®) resistance to target cells (Fig. 4A). As a negative control, we transfected

497

293T cells, which do not produce detectable HK2 VLPs and therefore should not package

498

Bogota and transmit neomycin (G418®) resistance to target cells. As a positive control for the

499

transmissibility assay, we artificially produced infectious HK2 VLPs in 293T cells by

500

transfecting the cells with the molecular clone CHKCP, a plasmid that produces the infectious

501

HK2 virus HERV-KCON. This virus uses the puromycin resistance marker for infectivity [52],

502

and HERV-KCON infected cells were selected by their resistance to puromycin. As infectivity of

23

503

HERV-KCON is improved by expression in trans of HK2 Rec and the Vesicular Stomatitis Virus

504

envelope protein (VSV-G), we also co-transfected CHKCP with Rec and VSV-G (Fig. 4B)

505

constructs. As expected, HERV-KCON expression in 293T cells in the presence of Rec and VSV-

506

G produced VLPs that infected target cells grown in puromycin as previously reported (52, Fig.

507

4B). Expression of Bogota in 293T cells, however, did not produce transmissible VLPs, even in

508

the presence of Rec and VSV-G (Fig. 4B). These results demonstrate that cell lines that do not

509

express transmissible HK2 particles are not able to transmit Bogota into target cells.

510

We then addressed whether Bogota transcripts produced in HK2 VLP-producing cell

511

lines can be transmitted to target cells. We transfected HERV-KBogota

512

absence of a Rec construct into the HK2 particle-producing NCCIT cells and the non-virus

513

producing cell lines 293T and PA-1. Co-transfection with Rec has been shown to increase the

514

production of transmissible particles [52]. VLPs were collected and used to transmit Bogota onto

515

target cells, and these cells were selected with G418® resistance. The Bogota probe packaged

516

into VLPs produced by the NCCIT cells was transmitted into target cells (Fig. 4C). Notably,

517

expression in trans of the Rec protein increased the production of transmissible HK2 VLPs by ~

518

10 fold. Expression of the Bogota probe in non-virus producing 293T and PA-1 cells, either in

519

the presence or absence of Rec, did not lead to production of transmissible HK2 VLPs. G418®

520

resistant cells were never obtained when target cells were exposed to VLPs of cells transfected

521

with the backbone vector pCEP4, the expression vector used to clone the Bogota probe (data not

522

shown).

523 524

24

neo

in the presence or

525

Reverse transcription of transmitted Bogota HK2 sequences

526

We next determined whether transmission of the Bogota genome into target cells

527

followed a reverse transcription step. We transfected NCCIT cells with the HERV-KBogota oenR,

528

which contains an antisense copy of the neo gene disrupted by an intron, and assessed the

529

transmissibility of this genome into target cells. We observed that HK2 VLPs produced by the

530

NCCIT cell line transmit the HERV-KBogota

531

HERV-KBogota

532

resistance in target cells (Fig. 4D). Production of transmissible HK2 VLPs containing the

533

HERV-KBogota oenR probe was also enhanced in the presence of Rec. G418® resistant cells could

534

have arisen only if a spliced HERV-KBogota oenR genome is packaged into VLPs, transmitted into

535

the target cell, replicated through reverse transcription to a sense strand encoding the neo

536

resistance gene, integrated into chromosomal DNA or formed episomal forms, and was then

537

transcribed to produce G418® resistance.

oenR

oenR

probe into target cells, suggesting that the

genome was spliced and reverse transcribed in order to generate G418®

538

Transmission of HK2 Bogota into target cells thus appears to require a reverse

539

transcription step. As Bogota itself does not encode a reverse transcriptase, the RT activity is

540

hypothesized to come from the HK2 VLP in which it is packaged. Thus, we next studied in

541

further detail whether the reverse transcriptase enzyme indeed modulates reverse transcription of

542

the Bogota sequence. We tested the capacity of the HK2 Bogota genome to undergo reverse

543

transcription in the presence of reverse transcriptase inhibitors, molecules known to terminate the

544

reverse transcription of a wide variety of retroviruses [81,82]. Transmission of Bogota into target

545

cells was reduced by ~ 80% when target cells were subjected to treatment with the nucleoside

546

analogue reverse transcriptase inhibitors Zidovudine (AZT) and Lamivudine (3TC).

547

Transmission of the Bogota genome was not inhibited when the experiments were conducted in 25

548

the presence of the HIV-1-specific reverse transcriptase inhibitor Efavirenz (Fig. 5A), suggesting

549

that the reverse transcription of the Bogota genome is modulated by an HK2 specific reverse

550

transcriptase. We also determined whether reverse transcription of the HK2 Bogota genome

551

depends on substrate sequences necessary for reverse transcription, notably the primer-binding

552

site (PBS), the polypurine tract (PPT), and the U5 LTR [82]. We deleted these sequences in the

553

HK2 Bogota genome (Fig. 5B), and tested the reverse transcription efficiency of Bogota.

554

Deletion of PBS, PPT, and U5 LTR sequences reduced the transmission of Bogota into target

555

cells by more than 80%, suggesting that reverse transcription of the Bogota genome required

556

these substrate sequences (Fig. 5C). Thus, deletions introduced into key elements necessary for

557

reverse transcription of the HK2 Bogota probe significantly reduced transmissibility.

558

Transmission was however not abolished, suggesting that remaining nucleotide sequences/leaky

559

mutations allow for residual reverse transcription.

560 561

Lack of integration of transmitted HK2 Bogota reporters

562

Having determined that Bogota genomes contained in HK2 VLPs produced by NCCIT

563

cells were transmitted and reverse transcribed in target cells, we tested whether Bogota could

564

integrate into chromosomal DNA or form episomes (1LTR or 2LTR episomes, Fig. 6A). We

565

further verified whether or not HK2 Bogota DNA is detected in transmitted G418® resistant cell

566

clones by amplifying the Bogota surrogate antibiotic marker, the neo resistance gene. PCR

567

detected neo DNA in Bogota-G418® resistant cells (human NCCIT or hamster CHO cells), but

568

not in G418-susceptible cells (Fig. 6A and 6B).

26

569

Having detected Bogota DNA in G418® resistant cells, we aimed to detect the possible

570

Bogota integration forms. After completion of reverse transcription, retroviral cDNA could be

571

integrated into the host chromosomal DNA or be circularized into episomal forms; the 1LTR or

572

2LTR circles. 2LTRs can be formed upon autointegration, sometimes called suicidal integration

573

or circularization of the unintegrated provirus, by the viral Integrase. hom*ologous recombination

574

among the LTRs of linear unintegrated proviruses can further generate 1LTR episomes.

575

Episomal integration forms have been detected for infectious HIV-1 and resurrected HK2 viruses

576

[60, 83-85]. We assessed the existence of Bogota episomal forms in DNA isolated from G418®

577

resistant cells by inverse PCR using primers that bind to the neo resistance gene in the outwards

578

orientation (Fig. 6A). We detected 1LTR forms of the inferred Bogota construct in all G418®

579

resistant cells exposed to HK2 viruses containing Bogota but not in untreated cells (Fig. 6B); the

580

DNA sequence of these episomal forms was confirmed by sequencing to be Bogota. Less

581

frequently, the inverse PCR reaction amplified the 2LTR circles, suggesting preferential

582

formation or amplification of 1LTR over 2LTR episomes (Fig. 6B). As 1LTR forms arise by

583

recombination between the 5’ and 3’ LTRs of unintegrated viral cDNA [83-85], we analyzed the

584

1LTR sequence for signs of recombination. Sequence analysis revealed that the LTR produced in

585

the 1LTR episome arose by recombination between the 5’ and 3’ LTR of Bogota (data not

586

shown). These data suggest that Bogota DNA recircularized into episomal forms. Attempts to

587

detect chromosomal integration of Bogota into the genome of the target G418® resistant cells

588

was unsuccessful in spite of the use of multiple methodologies: i) deep-sequencing analysis [60];

589

ii) inverse PCR in DNA digested with restriction enzymes and re-circularized with DNA ligase

590

[61]; iii) Alu and B2 RAN-PCR [62,63]; and iv) ATLAS suppression PCR [64] (see Materials

591

and Methods).

27

592

Production of transmissible HK2 viruses by cell lines other than NCCIT

593

As the Bogota probe packaged into HK2 particles proved to be a useful indicator of HK2

594

transmission, we used it next to assess whether or not cell lines other than NCCIT produce

595

transmissible HK2 VLPs. We tested the capacity of transmission of putative HK2 VLPs

596

produced by the breast cancer cell line MCF-7 and the peripheral blood lymphocytes (PBLs)

597

from patients with lymphoma, in which some recombinant HK2 viral genomes are detected,

598

suggesting the possibility of transmissible HK2 viruses [25,86]. When compared to the standard

599

particle-producing NCCIT cells, transfection of HK2 Bogota into MCF-7 and PBLs from

600

patients with lymphoma results in the production of a low number of transmissible HK2 particles

601

that conferred G418® resistance onto target 293T cells (Fig. 7A). Transmission of these HK2

602

VLPs was again reduced by ~ 75% when sequences necessary for reverse transcription, the PBS,

603

the PPT, and the U5 LTR were deleted from the Bogota genome (Fig. 7A). No transmission was

604

seen with the empty vector. While it is possible that the small number of transmissible units

605

detected in our studies reflects differences in the efficiency of transfection of MCF-7 and PBLs,

606

as compared to NCCIT, the more limited production may be due to the lower titer of HK2

607

produced by the MCF-7 cell line and PBLs of patients with lymphoma.

608 609

Tropism of HK2 viruses

610

We determined the tropism of HK2 VLPs produced by NCCIT cells using the Bogota

611

system. The capacity of transmission of HK2 VLPs was evaluated in target human (NCCIT and

612

293T), hamster CHO, rat C6, buffalo green monkey BGMK, feline G355, and quail QT6 cells

613

(Fig. 7B). Cells were selected by G418® resistance. No transmission was seen with the empty 28

614

vector. We observed that HK2 VLPs were efficiently transmitted to human and hamster cells.

615

Rat and feline cells were less susceptible to transmission. HK2 could not be transmitted to

616

buffalo green monkey and quail cells (Fig. 7B). These results suggest that human and hamster

617

cells are permissive to transmission of HK2 VLPs. Similar results were reported when

618

investigators assessed the infectious capacity of resurrected HK2 in human, hamster, and feline

619

cell lines [52,53], further suggesting that Bogota is being transmitted in an HK2 virion.

620 621

Expression of HIV-1 Tat and Vif increase HK2 transmission

622

We next asked whether expression of HIV-1 viral proteins increases the production of

623

transmissible HK2 VLPs. The rationale behind this approach is that HIV infection, and

624

specifically the expression of the HIV-1 Tat and Vif proteins, has been found in previous studies

625

to enhance expression of HK2 [9,10,24,26-27,88,89]. Expression of HIV-1 Tat and Vif, but not

626

Vpu, in NCCIT cells increased the production of transmissible HK2 VLPs containing the Bogota

627

probe, as determined by measuring the number of G418® resistant clones (Fig. 8). A synergistic

628

effect on the production of transmissible HK2 VLPs was observed when HIV-1 Tat and Vif were

629

expressed together. Furthermore, production of transmissible HK2 VLPs increased further when

630

expression of HIV-1 Tat or Vif took place in the presence of the HK2 Rec protein, and increased

631

transmission of HK2 VLP resulted when the cells were stimulated with the combination of all

632

these proteins (Fig. 8). These results suggest that the HIV-1 accessory proteins Tat and Vif

633

enhance the production, and hence transmission, of HK2 VLPs produced in NCCIT cells.

29

634

Discussion

635

The prevailing wisdom for many years has been that no endogenous retrovirus is capable

636

of infection in modern humans due to mutations in their viral sequences. Beyond this

637

assumption, experimental approaches to examine the transmission capacity of endogenous

638

retroviruses in humans have been hampered by the inability to distinguish de novo infection from

639

the proviruses already present in the genome. Recently, two groups reconstructed what appeared

640

to be the most recent HK2 infectious retrovirus to have endogenized in the human species by

641

correcting the mutations found in present-day HK2 sequences to make these viruses infectious

642

[52,53]. To assess the transmissibility of these ancient HK2 viruses, these investigators

643

introduced surrogate markers (antibiotic resistance or fluorescent tags) to trace their mobility in

644

modern human cells. Although these viruses were transmitted to other human cells, and are a

645

very helpful tool to examine endogenous retrovirus biology, it has remained unclear whether

646

contemporary endogenous retroviruses, those that are encoded by the modern human genome,

647

can be transmitted. We have devised a molecular system that traces the transmissibility of

648

modern HK2 viruses using the molecular probe Bogota, which uses a surrogate marker to

649

indicate transmission of HK2 viruses. Moreover, transmission of the Bogota probe is dependent

650

on sequences necessary to undergo reverse transcription. This probe therefore can assess not only

651

transmissibility, but also reverse transcription of modern HK2 viral genomes.

652

In the present study we have shown that HK2 viruses from cell lines that produce HK2

653

viruses with mature cores and processed Gag and Env proteins were capable of transmitting the

654

Bogota marker to other cells [20]. Bieda et al reported that, in contrast to other teratocarcinoma

655

cell lines producing HK2 viruses, the cell line NCCIT produced free mature viral particles with

656

processed Gag protein, despite Env protein being undetectable in these studies [20]. However, 30

657

we have detected the Env protein in HK2 particles released by NCCIT cells and have found

658

evidence of transmissibility of those particles using the Bogota probe. Transfer of Bogota cannot

659

take place if this probe is expressed in non-HK2 particle producing cells. Our studies indicate

660

that HK2 viruses produced by NCCIT cells and MCF-7 cells and the peripheral blood

661

lymphocytes from lymphoma patients produce transmission competent HK2 viral particles, and

662

that the Bogota sequences contained in these particles further underwent reverse transcription.

663

Furthermore, HK2 viruses could be transmitted, to some extent, to certain non-human cells,

664

suggesting that the tropism of HK2 viruses is not limited to human cells, an observation

665

compatible with those of other groups [52,53]. We cannot rule out the possibility that the broad

666

tropism of HK2 particles depends on other human retroviral envelopes able to pseudotype HK2

667

particles. At least an Env protein would be required for virus transmission, since other studies

668

have failed to transmit unenveloped HK2 particles, such as unenveloped HERV-KCON [52]. Even

669

though our studies indicate HK2 RNA is packaged selectively into HK2 enveloped VLPs,

670

particles that allow for the transmission RNA to target cells, the possibility exists that

671

transmission of some RNA was mediated by exosomes [87].

672

The transmission of Bogota packaged into HK2 particles was enhanced in the presence of

673

retroviral regulatory proteins. Expression in trans of HK2 Rec, a protein that facilitates the

674

export of incompletely spliced RNA from the nucleus to the cytoplasm [43,44], increased the

675

transmission of Bogota by ~10 fold. Similar results were obtained when the resurrected

676

infectious HERV-KCON virus was expressed in the presence of HK2 Rec [53]. In addition,

677

expression in trans of the HIV-1 accessory proteins Tat and Vif increased the production of

678

transmissible HK2 Bogota. The HIV-1 Tat protein likely activates expression of HK2 viruses

679

through transactivation of the HK2 viral promoter; we previously reported that HIV-1 Tat 31

680

upregulated HK2 expression by modulating the binding of the transcription factors NF-κB and

681

NFAT in the HK2 LTR [88,89]. Expression of the HIV-1 protein Vif could facilitate the

682

transmissibility of HK2 virus by interfering with cytidine deaminases of the APOBEC family;

683

these proteins introduce mutations into the viral genome by deaminating minus strand cytidines

684

during reverse transcription [90-92]. APOBEC deaminases have been shown to inhibit infection

685

with HK2 viruses [52,93], and therefore expression of HIV-1 Vif may counteract this effect.

686

These findings are consistent with those from our group and others, who have found increased

687

expression of HK2 viruses in the setting of HIV-1 infection [9-10,24-27, 94]. The consequence

688

of this increased expression of transmissible HK2 viruses, for both HIV-1 infected cells and the

689

HIV-1-infected individual, remains to be elucidated. However, it is important to note that

690

expression of HK2 epitopes, and perhaps transmissible HK2 viruses, on HIV-1 infected cells

691

render these cells susceptible to lysis by immune T-cells and antibody responses directed against

692

HK2, creating a tool that could conceivably be used to eradicate HIV-1 infection [95-97].

693

Our studies indicate that modern HK2 viruses may indeed remain transmissible and that

694

genetic material packaged in these viral particles can undergo reverse transcription. Several lines

695

of evidence suggest that expression and transmission of Bogota required reverse transcription: i)

696

transmission of Bogota oenR, a probe that uses a neo resistance gene disrupted by an intron and in

697

an antisense orientation, could have generated G418® resistance in target cells only by removing

698

the intron through splicing and positioning the neo gene in the sense orientation after reverse

699

transcription (Fig. 4D); ii) transmission of Bogota was impaired by introducing genetic

700

mutations in sequences required for reverse transcription: the PBS, the PPT, and the U5LTR; iii)

701

transfer of Bogota was reduced substantially in the presence of nucleoside analogue reverse

702

transcriptase inhibitors; and iv) detection of Bogota episomal integration (1LTR and 2LTR). 32

703

We also examined the fate of Bogota cDNA in target cells, and whether it integrated into

704

chromosomal DNA and/or formed 1LTR and 2LTR episomes [60,83-85]. While we detected

705

abundant Bogota integration in episomal forms, we were unable to detect Bogota integration into

706

chromosomal DNA using multiple methodological approaches. The inability of Bogota to

707

integrate into cellular DNA could be attributed to properties inherent to the Bogota system.

708

Bogota may have lacked unique sequences necessary for integration, or perhaps the viral cDNA

709

product was so short as to favor autointegration and not chromosomal integration. Replication of

710

the Bogota episomes might be attributed to the presence of an SV-40 origin of replication

711

upstream of the neo resistance gene. The SV-40 large T antigen, an antigen expressed in the cell

712

line 293T used as the target cell in certain of our transmission experiments, is known to promote

713

episomal DNA replication in the nucleus of plasmids with the SV-40 origin of replication [98].

714

Interestingly, however, it has also been shown that plasmids containing the SV-40 origin of

715

replication can replicate in hamster CHO cells (cells we have also used as the target cells in the

716

transmission studies), even though they lack the expression of the SV-40 large T antigen,

717

suggesting that Bogota episomes containing the SV-40 origin of replication might similarly be

718

able to replicate autonomously in CHO cells [99]. It appears, however, that the replication of

719

these SV-40 ORI containing episomes required the expression of the scaffold/matrix attachment

720

region (S/MAR) protein from the human beta-interferon gene cluster as well as transcription of

721

sequences upstream of the S/MAR gene [100]. In any case, we have very consistently detected

722

Bogota episomes in several target cell lines that apparently do not express the SV-40 large T

723

antigen (the human NCCIT, the hamster CHO, the rat C6, and the feline G355), suggesting that

724

the replication of the Bogota episomes is not dependent on the SV40 large T antigen. How these

725

cells maintain the replication of the Bogota episome remains unclear, but it is possible that trans-

33

726

acting factors other than the large T antigen modulate the SV-40 origin of replication in the

727

Bogota plasmid.

728

Although the Bogota probe seems to favor episomal replication when transmitted in an

729

autonomous form, infection and retroviral integration of HK2 into the genome of an infected cell

730

has been demonstrated using resurrected HK2 viruses, HERV-KCON and Phoenix [53-54], as well

731

as the autointegration of HERV-KCON to form episomes [60]. Thus, HK2 is in principle able to

732

integrate into the genome of human cells. So, it is surprising that the majority of HK2 viruses led

733

to transmissible viral genomes that resulted in circular forms rather than genomic integrations.

734

Perhaps, unlike the Integrase enzyme encoded by the consensus HERV-KCON and Phoenix

735

viruses, which are functional and allow integration, none of the HK2 elements in the cells tested

736

in our studies contribute a functional Integrase protein to the VLPs. It is likely that HK2

737

episomes can be transcribed and translated into proteins able to be packaged into new particles,

738

as the Bogota episomes were able produce a protein that confers G418® resistance. It is still

739

unclear to us whether the recombinant viral genomes found in the VLPs produced by NCCIT

740

come from these episomal forms or whether they are derived from a recombinant integrated

741

provirus. Our data suggest that cellular or viral factors may prevent transmission of HK2 virus

742

from integrating in the human genome; this could explain why only about 3000 HK2 sequences

743

integrated in the last 25 million years of human evolution [3,9]. This may be due to the

744

previously described cell restriction factors or to even more potent restriction mechanisms in the

745

germ cells of primates. Thus, our inability to detect chromosomal integration may well reflect a

746

biological reality, in that passage of truly infectious HK2 is a rare event in modern humans. It is

747

important to note that the transmission of HK2 particles was seen most impressively with

748

particles produced by the NCCIT cell line, in which a great portion of the repertoire of HK2 34

749

particle-associated sequences are recombinant and the expression profile of HK2 proviruses was

750

more diverse. As HIV-1 viral proteins further increased the transmission of HK2 viruses

751

produced by NCCIT, it is likely that expression of more HK2 proviruses enhances the production

752

of transmissible HK2 viruses. Whether transmissible HK2 viruses are produced in HIV-1

753

patients remains to be further explored. However, the findings in this study suggest that the

754

increased expression, occurrence of recombination, and evidence of purifying selection of HK2

755

found in the HIV-1 setting [9,10, 24, 26, 27, 88, 93] may be indicative of production of

756

transmissible HK2 viruses in HIV-1 infected individuals.

757

What pathogenic effect a modern transmissible HK2 virus may have needs to be

758

investigated. HK2 expression, and production of HK2 particles, has been linked to cancers, in

759

particular teratocarcinoma, breast cancer, melanoma, and lymphoma [15,20-23,25]. Our data

760

have revealed that HK2 virus released from certain cell lines can be transmitted from one cell to

761

the next, and therefore transmissible HK2 viruses might contribute to the pathogenesis of cancer.

762

It is conceivable that with viral recombination and sequence diversification, more transmissible

763

particles can be created. As we could not find passaged HK2 integrated into chromosomes, a

764

mechanism of insertional mutagenesis mediated by HK2 seems unlikely to contribute to the

765

pathogenesis of cancer. Nonetheless, transmission of HK2 viral sequences could enhance the

766

burden of HK2 proteins, such as Rec, Np9, and Env, which have been associated with cancer

767

development [reviewed in 101]. The data presented here suggest that, contrary to previous views,

768

certain modern HERVs retain the ability to be reverse transcribed and transmitted to new cells.

769

Furthermore, the transmitted HK2 sequences circularize preferentially in the target cells,

770

generating episomes in abundance. However, as no chromosomal integration was seen, the

35

771

potential for true replication of modern HERVs with ongoing, continuous viral spread has not yet

772

been demonstrated.

773

36

774

Acknowledgments

775

We thank Joseph Zahn and Anjan K. Saha for their thoughtful insights into the manuscript and

776

Paul Bieniasz (Rockefeller University) for providing us with the plasmids HERV-KCON CHKCP

777

and pCR3.1/K-Rec. The plasmids pcDNA3.1-Tat86, pcDNA-HVif, and pcDNA-Vphu were

778

obtained through the AIDS Research and Reference Reagent Program, Division of AIDS. We

779

thank Dr. Norbert Bannert for supplying the rabbit anti-HERV-K Gag antibody. We thank José

780

L. García-Pérez and John M. Moran for providing the pCEP4, pBSKS, and pINT plasmids. This

781

work was supported by grant RO1 CA144043 from the National Institutes of Health to D.M.M.,

782

grant K22 CA177824 from the National Cancer Institute, and a Research Supplement to Promote

783

Diversity in Health-Related Research 3R01CA144043-03S1 from the National Institutes of

784

Health to R.C-G., and grant 05-5089 from the Concerned Parents for AIDS Research (CPFA) to

785

M.H.K., D.D., and M.J.G-H. were supported by the Molecular Mechanisms of Microbial

786

Pathogenesis Training Grant from the University of Michigan (5T32AI007528-13). D.D. was

787

also supported by an NIH Ruth L. Kirschstein NRSA Individual Postdoctoral Fellowship (1 F32

788

AI106189-01). M.J.G-H. was also supported by a Rackham Merit Fellowship and by the NIH

789

Ruth L. Kirschstein NRSA Individual Predoctoral Fellowship to Promote Diversity in Health-

790

Related Research grant 1F31CA150523-01. G.S.O. acknowledges support from NIH grants RM-

791

08-029, P30U54ES017885, U54DA021519, and UL1RR24986.

792 793

37

794 795 796

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88. Gonzalez-Hernandez MJ, Swanson MD, Contreras-Galindo R, Cookinham S, King

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SR, Noel RJ Jr, Kaplan MH, Markovitz DM. 2012. Expression of human endogenous

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retrovirus type K (HML-2) is activated by the Tat protein of HIV-1. J. Virol. 86: 7790-

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89. Gonzalez-Hernandez MJ, Cavalcoli JD, Sartor MA, Contreras-Galindo R, Meng F,

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Dai M, Dube D, Saha AK, Gitlin SD, Omenn GS, Kaplan MH, Markovitz DM. 2014.

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Regulation of the human endogenous retrovirus K (HML-2) transcriptome by the HIV-1

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90. Mangeat B, Turelli P, Caron G, Friedli M, Perrin L, Trono D. 2003. Broad

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antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse

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transcripts. Nature. 424: 99-103.

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91. Harris RS, Bishop KN, Sheehy AM, Craig HM, Petersen-Mahrt SK, Watt IN,

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Neuberger MS, Malim MH. 2003. DNA deamination mediates innate immunity to

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Kramer B, McMichael AJ, Rambaut A, Iversen AK. 2008. Conserved footprints of

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endogenous retrovirus HERV-K(HML2) sequences. J. Virol. 82: 8743-61.

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94. Bhardwaj N, Maldarelli F, Mellors J, Coffin JM. 2014. HIV-1 infection leads to

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increased transcription of human endogenous retrovirus HERV-K (HML-2) proviruses in

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vivo but not to increased virion production. J Virol. 88: 11108-20.

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95. Garrison KE, Jones RB, Meiklejohn DA, Anwar N, Ndhlovu LC, Chapman JM,

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Ostrowski MA, Nixon DF. 2007. T cell responses to human endogenous retroviruses in

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HIV-1 infection. PLoS Pathog. 3: e165.

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96. Tandon R, SenGupta D, Ndhlovu LC, Vieira RG, Jones RB, York VA, Vieira VA,

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Sharp ER, Wiznia AA, Ostrowski MA, Rosenberg MG, Nixon DF. 2011.

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Identification of human endogenous retrovirus (HERV)-specific T cell responses in

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vertically HIV-1-infected subjects. J. Virol. 85: 11526–11531.

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specific T cells eliminate diverse HIV-1/2 and SIV primary isolates. J. Clin. Invest. 122:

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98. Chittenden T, Frey A, Levine AJ. 1991. Regulated replication of an episomal simian virus 40 origin plasmid in COS7 cells. J Virol. 65:5944-5951.

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99. Piechaczek C, Fetzer C, Baiker A, Bode J, Lipps HJ. 1999. A vector based on the

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Glover DJ, Lipps HJ, Jans DA. 2005. Towards safe, non-viral therapeutic gene

expression in humans. Nat Rev Genet. 6:299-310. 101.

Hohn O, Hanke K, Bannert N. 2013. HERV-K(HML-2), the Best Preserved

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Family of HERVs: Endogenization, Expression, and Implications in Health and Disease.

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Brinzevich D, Young GR, Sebra R, Ayllon J, Maio SM, Deikus G, Chen BK,

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endogenous retrovirus K (HML-2) envelopes derived from human primary lymphocytes.

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1097 1098 1099 1100 1101 1102 1103 51

1104

Figure legends

1105

Figure 1. HK2 Packaging and Transmission Assay. (A) Organization of HK2 proviruses. ORFs

1106

coded by the gag, prt, pol, and env genes, and the accessory rec gene, are indicated by closed

1107

rectangles. The 5’ and 3’ LTRs (light grey boxes) at each side of the provirus, and the Surface

1108

(SU) and Transmembrane (TM) domains of the encoded Env protein are shown. (B) An

1109

overview of the packaging and transmission assay of HERV-K Bogota reporters. The constructs

1110

HERV-KBogota neo and HERV-KBogota oenR were derived from the K113 provirus by replacing the

1111

retroviral genes with a neomycin (neo) resistance indicator gene to tag the retroviral transcripts.

1112

An SV-40 promoter and a 3’ polyadenylation signal (A’) regulate the expression of neo.

1113

Although Bogota transcripts do not code for retroviral proteins, the transcripts retain sequences

1114

necessary for reverse transcription (the primer binding site (PBS), the polypurine tract (PPT),

1115

and the U3, R, and U5 parts of the LTRs). As the LTR of HK2 is not functional in every cell

1116

line, a CMV promoter replaced the U3 portion of the 5’ LTR. Bogota retroviral transcripts

1117

contain the packaging signal of HK2 and so can be packaged into HK2 viral particles. Upon cell

1118

transmission, these transcripts confer G418® resistance only when they are reverse transcribed

1119

and then integrated into chromosomal DNA or form episomes (1LTR and 2LTR). HERV-KBogota

1120

oenR

1121

in the sense direction. The splice donor (SD) and splice acceptor (SA) sites of the intron are

1122

indicated. Transcripts originating from the HERV-K Bogota oenR promoter can splice out the intron,

1123

but contain only an antisense copy of the neo gene. G418® resistant colonies should arise only

1124

when, upon packaging and transmission, these transcripts are reverse transcribed, producing a

1125

sense copy of the neo resistance gene, which could be integrated into the genome or into

1126

episomal forms, and expressed from their own promoter.

(right) contains an antisense copy of the neo gene disrupted by intron 2 of the γ-globin gene

52

1127

Figure 2. Western blot analysis of HK2 Env protein found in the cell-associated lysate and cell-

1128

free VLPs released from NCCIT cells. Cell lysates and VLPs contained in the supernatants of

1129

human NCCIT and PA-1 teratocarcinoma cells were assessed for the presence of the HK2 Env

1130

protein using the anti-HK2 Env antibody HERM1811-5 (Austral Biologicals, San Ramon, CA)

1131

as previously described [9]. Two bands of 90 and 80 kb were detected in the cell lysate of

1132

NCCIT but not PA-1 cells. These bands are consistent with the unprocessed precursor Env

1133

protein, with or without the signal peptide. These bands were also detected in cell-free VLPs of

1134

NCCIT cells together with a band of ~ 37 Kb that corresponds to the TM subunit cleaved after

1135

Env processing [102].

1136 1137

Figure 3. Packaging of Bogota transcripts in HK2 viral particles. (A-B) Immunoprecipitation of

1138

HERV-K virions containing Bogota transcripts. NCCIT cells were transfected with HERV-

1139

KBogota

1140

ultracentrifugation prior to immunoprecipitation with either an anti-HERV-K Env antibody or a

1141

control anti-mouse IgG2a. (A) Cleared supernatant and the immunoprecipitated samples were

1142

analyzed for HERV-K capsid enrichment by western blotting with an anti-HERV-K Gag

1143

antibody. The untreated supernatant is about five-fold diluted as compared to the volumes

1144

obtained in the concentrated and immunoprecipitated samples, and therefore equal amounts of

1145

the supernatants could not be added in the gel due to well-volume constraints. The low

1146

background level detected with the anti-IgG2a immunoprecipitation may be non-specific. (B)

1147

RNA from immunoprecipitated particles was quantitated for HERV-K gag, Bogota neo, or non-

1148

viral gapdh by qRT-PCR. The average fold enrichment for each target in particles precipitated

1149

with anti-HERV-K Env as compared to those precipitated by control anti-mouse IgG2a is shown.

neo

and viral particles were collected 48 h after. Viral particles pelleted by

53

1150

(C) RNA expression levels of HERV-K (np9, rec, gag, and env type 2), Bogota neo, HERV-W

1151

(gag, env), HERV-H env, gapdh, cox-2, and b-actin were analyzed by qRT-PCR in Bogota-

1152

transfected NCCIT cells (white bars) and pelleted retroviral-like particles (pRVLPs, black bars).

1153

The log10 RNA titers show the average quantitation ± SD of at least three independent

1154

experiments expressed in arbitrary units. (D) Packaging efficiency of Bogota transcripts in

1155

NCCIT-produced viral particles. The relative fold-change of packaging efficiency was calculated

1156

by dividing expression levels of target RNA copies in NCCIT pRVLPs by their expression levels

1157

in total cellular RNA. Values are presented as the mean ± SD of at least three independent

1158

experiments. A value of 0 is given for HERV-W gag and env due to the absence of these

1159

transcripts in NCCIT pRVLPs.

1160 1161

Figure 4. Transmission of HK2 viral particles. (A) Methodology of the transmissibility assay.

1162

Cells were transfected with Bogota constructs or indicated plasmid mixtures. Supernatants were

1163

collected 48 h post transfection, and the particles incubated with target 293T. Cells exposed to

1164

HK2 VLPs were selected for G418® resistance and the apparent transmissibility units calculated

1165

by colony forming units (CFU) per plate. (B) Positive control: infectivity of the resurrected

1166

HERV-KCON, produced by the CHKCP plasmid, following transfection into 293T cells. HERV-

1167

KCON (VSV-G)-pseudotyped virions were produced in 293T cells in the presence or absence of

1168

Rec. HERV-KCON-infected cells were selected with puromycin. A similar transmissibility assay

1169

using HERV-KBogota neo did not reveal production of transmissible HK2 particles in 293T cells,

1170

which was predicted, as 293T cell do not make HK2 particles. (C) Transmissible units of HK2

1171

particles containing Bogota transcripts produced by 293T, and the germ-cell tumor cell lines PA-

1172

1 and NCCIT, in the presence or absence of Rec. The target cells were 293T cells. (D) 54

1173

Transmissible units of NCCIT-producing HK2 particles containing Bogota transcripts with neo

1174

markers [the neo gene displayed in sense orientation or antisense orientation and disrupted with

1175

an intron (see text and Materials and Methods for further explanation)] in the presence or

1176

absence of Rec. The target cells were 293T cells. Values represent the mean ± SD of at least

1177

three independent experiments.

1178 1179

Figure 5. Transmission of HK2 particles into 293T target cells requires reverse transcription. (A)

1180

Inhibition of transmission of HK2 particles containing Bogota transcripts by treatment of the

1181

target cells with nucleoside reverse transcriptase inhibitors (NRTIs). Frequency of transmission

1182

of HK2 particles containing Bogota transcripts in the presence or absence of 50 μM AZT, 3TC

1183

or Efavirenz. (B) Genomic position of sequences required for reverse transcription modified in

1184

Bogota mutant constructs. The primer binding site (PBS), necessary to initiate reverse

1185

transcription, the polypurine tract (PPT), which resists digestion by the RNAseH activity of the

1186

reverse transcriptase (RT) enzyme and primes plus-strand DNA synthesis, and the last 220 bp of

1187

the 3’LTR containing a portion of R and U5, required for template switching during reverse

1188

transcription, were deleted by site-directed mutagenesis to test the effect of these sequences

1189

during reverse transcription of Bogota transcripts. (C) Transmission of HK2 particles containing

1190

Bogota transcripts and reverse transcription-associated mutant transcripts. Frequency of infection

1191

of HK2 particles containing Bogota or mutant δPBS, δPPT, or δ3’RU5 transcripts. Values

1192

represent the mean ± SD of at least three independent experiments.

1193

55

1194

Figure 6. Bogota reverse-transcribed DNA sequences are found in episomal forms in target

1195

cells. (A) Schematic representation of the possible integration outcomes of Bogota reverse-

1196

transcribed cDNAs. After reverse transcription, Bogota cDNA may integrate into chromosomal

1197

DNA or form episomes (1LTR or 2LTR forms). Bogota integration forms can be detected with

1198

primers E1 and E2 by inverse PCR (see Table 1). Primers E3 and E4 can detect Bogota neo

1199

DNA, which can be present in any of the integration forms. (B) Detection of Bogota DNA and

1200

Bogota episomal forms in recipient cells. DNA was extracted from target cells with Bogota-

1201

transmitted neomycin resistance. DNA of the neo resistance gene, as well as Bogota 1LTRs and

1202

2LTRs, were detected by PCR and inverse PCR, respectively, using the primers described above.

1203

Target cells included the human NCCIT cell line and the hamster CHO cell line. DNA from

1204

Bogota neo was detected in all the neomycin resistant clones, but not in untreated, neomycin

1205

susceptible cells. Episomal 1LTR Bogota form was detected in all target cells, yet the 2 LTR

1206

form was detected only in NCCIT clones 1 and 2, and the CHO clones 4 and 5. Sequencing

1207

confirmed the presence of episomal forms. The HERV-KBogota neo plasmid served as a positive

1208

control for neo DNA, and a clone containing a 1LTR sequence obtained in one of the

1209

experiments performed in this investigation served as positive control for 1LTR amplification.

1210 1211

Figure 7. Transmissibility of HK2 particles produced from cancer cells and virus tropism. (A)

1212

Transmission of HK2 particles produced from cancer cell lines and lymphocytes from lymphoma

1213

patients to target 293T cells. Frequency of transmissibility of HK2 particles containing Bogota

1214

and reverse transcription-associated δPBS, δPPT, or δ3’RU5 mutant transcripts produced in

1215

NCCIT cells, the breast cancer MCF-7 cell line, and the peripheral blood lymphocytes (PBLs) of

1216

lymphoma patients (PBLs1 corresponds to a patient with large B-cell lymphoma and PBLs2 56

1217

corresponds to a patient with follicular lymphoma). (B) Tropism of HK2 particles produced in

1218

NCCIT cells containing Bogota transcripts. HK2 particles containing Bogota transcripts were

1219

incubated with hamster CHO, rat C6, buffalo green monkey BGMK, feline G355, quail QT6,

1220

and human NCCIT and 293T cell lines and selected for G418®. Transmissible units per plate

1221

represent the mean ± SD of at least three independent experiments.

1222 1223

Figure 8. Expression of HIV-1 accessory proteins increases the production of transmissible HK2

1224

particles. NCCIT cells were transfected with the indicator Bogota construct and plasmids

1225

expressing the HIV-1 Vpu, Tat, and Vif proteins. HK2 particles produced in treated cells were

1226

collected and assessed for transmissibility to 293T target cells by using the Bogota neomycin

1227

selection assay. The graph shows the transmissible units of HK2 particles containing Bogota

1228

transcripts stimulated by overexpression of HIV-1 proteins in the presence or absence of Rec.

1229

Expression of HIV-1 Tat or Vif, but not Vpu, increases the production of transmissible HK2

1230

particles. Furthermore, an additive effect in the transmissible units of HK2 particles is seen when

1231

HIV-1 Tat and Vif, together with HK2 Rec, are expressed in NCCIT cells. Values represent the

1232

mean ± SD of at least three independent experiments.

1233 1234 1235 1236 1237 57

1238

Table 1. List of primers Knp9F Knp9R KrecF KrecR KgagRTF KgagRTR Kenvtype2F Kenvtype2R BogotaneoF BogotaneoR HERV-WenvF

5’-CCA ACG TGG AGG CTT TTC TCT AG-3’ 5’-GTA CAC CTG CAG TCT CCG TCT CC -3’ 5’-GAG GCT GGC GGG ATC CTC-3’ 5’-ACA AAG CTT CCT ACG TCA TCA TGG CCC G -3’ 5’-AGC AGG TCA GGT GCC TGT AAC ATT-3’ 5’-TGG TGC CGT AGG ATT AAG TCT CCT-3’ 5’-AGA CAC CGC AAT CGA GCA CCG TTG A-3’ 5’-ATC AAG GCT GCA AGC AGC ATA CTC-3’ 5’-ATG TTT CGC TTG GTG GTC GAA TGG-3’ 5’-ACC TTG CTC CTG CCG AGA AAG TAT-3’ 5’-TCA TAT CTA AGC CCC GCA AC-3’

HERV-WenvR HERV-WgagF HERV-WgagR HERV-HenvF HERV-HenvR GAPDHF GAPDHR Cox-2F Cox-2R B-actinF B-actinR E1 NEO1720F E2 NEO210R E3 NEO437F E4 NEO1808R

5’-CGT TCC ATG TCC CCA TTT AG-3’ 5’-TCA GGT CAA CAA TAG GAT GAC AAC A-3’ 5’-CAA TGA GGG TCT ACA CTG GGA ACT-3’ 5’-GTC GGT TTA GGA CTT TCT GC-3’ 5’-TGT GGG AAC CTA GAG CGG GA-3’ 5’-TGC ACC ACC AAC TGC TTA GCA CCC-3’ 5’-CTT GAT GAC ATC ATA TTT GGC AGG-3’ 5’-TGA CTA TGG CTA CAA AAG CTG GG-3’ 5’-GCA AAC ATC ATG TTT GAG CCC T-3’ 5’-GTG GGG CGC CCC AGG CAC CA-3’ 5’-CTC CTT AAT GTC ACG CAC GAT TTC-3’ 5’-TGC GCT GAC AGC CGG AAC ACG-3’ 5’-GAC CGC TTC CTC GTG CTT TAC G-3’ 5’-GAG CCC CTG ATG CTC TTC GTC C-3’ 5’-CAT TGA ACA AGA TGG ATT GCA CGC-3’

1239 1240 1241 1242 1243 1244 1245 58

Human Endogenous Retrovirus Type K (HERV-K) Particles Package and Transmit HERV-K-Related Sequences. - PDF Download Free (2024)

FAQs

What is an endogenous retrovirus in humans? ›

Endogenous retroviruses (ERVs) have long been thought as 'junk DNA' or 'fossil records' of ancestral retrovirus invasions. ERVs can be transcriptionally active and critical for human development and health. ERVs are involved in pathological processes such as virus infection, immune response, and aging.

How is an endogenous retrovirus transmitted to offspring? ›

In mammals, modern retroviruses usually infect white blood cells. If, however, a retrovirus happens to infect a sperm cell or egg cell and if that sperm or egg cell happens to participate in fertilization, the resulting child will have a copy of the virus DNA in ever single one of her cells!

What are human endogenous retroviruses schizophrenia? ›

Schizophrenia is a complex disorder, characterized by the interplay between genetic and environmental factors. Human endogenous retroviruses (HERVs), genetic elements that originated from infections by exogenous retroviruses millions of years ago, comprise ~8% of the human genome.

Do endogenous retroviruses make aging go viral? ›

In summary, endogenous retroviruses can promote cell senescence and tissue ageing, and are potential drug targets to increase human healthspan. The study also raises the possibility that viral contagion has a role in ageing.

Are retroviruses harmful? ›

Retroviruses are a family of viruses that are grouped together based on how they are structured and how they replicate within a host. Besides human immunodeficiency virus (HIV), the virus that causes AIDS, there a two other retroviruses that can cause human illness.

What are the human diseases caused by retroviruses? ›

Retroviruses are associated with a wide variety of diseases including an array of malignancies, immunodeficiencies, and neurologic disorders. Syndromes as seemingly diverse as arthritis, osteopetrosis, and anemia can all result from retroviral infection.

What are the symptoms of retrovirus? ›

Symptoms of the HTLV Type I Retrovirus
  • Feeling tired.
  • Swollen lymph nodes.
  • Excess thirst.
  • Fever.
  • Frequent infections and illnesses.
  • Nausea and vomiting.
  • Skin problems.
  • Bone problems.
Jul 17, 2023

How does a retrovirus infection begin? ›

The retroviruses that infect humans are spread in bodily fluids. You may get them through sexual contact or exposure to infected blood or tissue. They can also be passed to a fetus during pregnancy or childbirth.

Are retroviruses curable? ›

Currently, there's no cure for retroviral infections. But a variety of treatments can help to keep them managed.

Are retroviruses permanent? ›

Reverse transcription and integration make retroviral infection permanent, as integrated proviruses are only rarely lost from the cellular genome: “A retrovirus is forever.” Integration of viral DNA into the host genome is ipso facto mutagenic.

Do human endogenous retroviruses contribute to multiple sclerosis and if so how? ›

Two human endogenous retroviruses of the HERV-W family can act as cofactors triggering multiple sclerosis (MS): MS-associated retrovirus (MSRV) and ERVWE1. Endogenous retroviral elements are believed to have integrated in our ancestors' DNA millions of years ago.

Do I have schizophrenia gene? ›

Schizophrenia tends to run in families, but no single gene is thought to be responsible. It's more likely that different combinations of genes make people more vulnerable to the condition.

Does retrovirus cause lifelong infection? ›

Unlike most RNA viruses, retroviruses establish lifelong infections. In most cases, the “natural” host-virus interaction appears to be benign, with the virus having little, if any, pathogenic effect during the natural lifespan of the host.

Do endogenous retroviruses cause disease? ›

ERVs preserve functions of exogenous retroviruses to various extents. ERVs are both parasites and symbionts. Although the most pathogenic elements are eliminated by selection, some pathogenicity may remain. Some recently endogenized elements of mice and cats are known to cause disease.

Can retroviruses be passed down? ›

Usually this kind of viral genetic material isn't passed down from generation to generation. But some ancient retroviruses gained the ability to infect germ cells, such as egg or sperm, that do pass their DNA down to future generations.

What is an example of an endogenous disease? ›

Endogenous infections result from the procedure itself, whereby a patient is exposed to their own microbial flora. For example, a sedated patient during flexible bronchoscopy may breathe in oral secretions during the procedure and contract pneumonia.

What are endogenous viral elements in humans? ›

An endogenous viral element (EVE) is a DNA sequence derived from a virus, and present within the germline of a non-viral organism. EVEs may be entire viral genomes (proviruses), or fragments of viral genomes.

Can retroviruses infect human cells? ›

A retrovirus is a virus that uses RNA as its genomic material. Upon infection with a retrovirus, a cell converts the retroviral RNA into DNA, which in turn is inserted into the DNA of the host cell. The cell then produces more retroviruses, which infect other cells.

Do humans have endogenous reverse transcriptase? ›

Human endogenous retrovirus-K (HERV-K) reverse transcriptase (RT) structure and biochemistry reveals remarkable similarities to HIV-1 RT and opportunities for HERV-K–specific inhibition.

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