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The integration of human immunodeficiency virus type 1 (HIV-1) into chromosomal DNA establishes the long-term persistence of HIV-1 as proviruses despite effective antiretroviral therapy (ART). Characterizing proviruses is difficult because of their rarity in individuals on long-term suppressive ART, their highly polymorphic sequences and genetic structures, and the need for efficient amplification and sequencing of the provirus and its integration site.

The prevalence of HIV-1 drug resistance is increasing worldwide and monitoring its emergence is important for the successful management of populations receiving combination antiretroviral therapy. It is likely that pre-existing drug resistance mutations linked on the same viral genomes are predictive of treatment failure. Because of the large number of sequences generated by ultrasensitive single-genome sequencing (uSGS) and other similar next-generation sequencing methods, it is difficult to assess each sequence individually for linked drug resistance mutations. Several software/programs exist to report the frequencies of individual mutations in large data sets, but they provide no information on linkage of resistance mutations. In this study, we report the HIV-DRLink program, a research tool that provides resistance mutation frequencies as well as their genetic linkage by parsing and summarizing the Sierra output from the Stanford HIV Database. The HIV-DRLink program should only be used on data sets generated by methods that eliminate artifacts due to polymerase chain reaction recombination, for example, standard single-genome sequencing or uSGS. HIV-DRLink is exclusively a research tool and is not intended to inform clinical decisions.

BACKGROUNDHIV-1 viremia that is not suppressed by combination antiretroviral therapy (ART) is generally attributed to incomplete medication adherence and/or drug resistance. We evaluated individuals referred by clinicians for nonsuppressible viremia (plasma HIV-1 RNA above 40 copies/mL) despite reported adherence to ART and the absence of drug resistance to the current ART regimen.METHODSSamples were collected from at least 2 time points from 8 donors who had nonsuppressible viremia for more than 6 months. Single templates of HIV-1 RNA obtained from plasma and viral outgrowth of cultured cells and from proviral DNA were amplified by PCR and sequenced for evidence of clones of cells that produced infectious viruses. Clones were confirmed by host-proviral integration site analysis.RESULTSHIV-1 genomic RNA with identical sequences were identified in plasma samples from all 8 donors. The identical viral RNA sequences did not change over time and did not evolve resistance to the ART regimen. In 4 of the donors, viral RNA sequences obtained from plasma matched those sequences from viral outgrowth cultures, indicating that the viruses were replication competent. Integration sites for infectious proviruses from those 4 donors were mapped to the introns of the MATR3, ZNF268, ZNF721/ABCA11P, and ABCA11P genes. The sizes of the clones were estimated to be from 50 million to 350 million cells.CONCLUSIONThese findings show that clones of HIV-1-infected cells producing virus can cause failure of ART to suppress viremia. The mechanisms involved in clonal expansion and persistence need to be defined to effectively target viremia and the HIV-1 reservoir.FUNDINGNational Cancer Institute, NIH; Howard Hughes Medical Research Fellows Program, Howard Hughes Medical Institute; Bill and Melinda Gates Foundation; Office of AIDS Research; American Cancer Society; National Cancer Institute through a Leidos subcontract; National Institute for Allergy and Infectious Diseases, NIH, to the I4C Martin Delaney Collaboratory; University of Rochester Center for AIDS Research and University of Rochester HIV/AIDS Clinical Trials Unit.

COVID-19 vaccines were first administered on 15 December 2020, marking an important transition point for the spread of SARS-CoV-2 in the United States (U.S.). Prior to this point in time, the virus spread to an almost completely immunologically naïve population, whereas subsequently, vaccine-induced immune pressure and prior infections might be expected to influence viral evolution. Accordingly, we conducted a study to characterize the spread of SARS-CoV-2 in the U.S. pre-vaccination, investigate the depth and uniformity of genetic surveillance during this period, and measure and otherwise characterize changing viral genetic diversity, including by comparison with more recently emergent variants of concern (VOCs). In 2020, SARS-CoV-2 spread across the U.S. in three phases distinguishable by peaks in the numbers of infections and shifting geographical distributions. Virus was genetically sampled during this period at an overall rate of ~1.2%, though there was a substantial mismatch between case rates and genetic sampling nationwide. Viral genetic diversity tripled over this period but remained low in comparison to other widespread RNA virus pathogens, and although 54 amino acid changes were detected at frequencies exceeding 5%, linkage among them was not observed. Based on our collective observations, our analysis supports a targeted strategy for worldwide genetic surveillance as perhaps the most sensitive and efficient means of detecting new VOCs.

HIV infection is not curable with current antiretroviral therapy (ART) because a small fraction of CD4+ T cells infected prior to ART initiation persists. Understanding the nature of this latent reservoir and how it is created is essential to development of potentially curative strategies. The discovery that a large fraction of the persistently infected cells in individuals on suppressive ART are members of large clones greatly changed our view of the reservoir and how it arises. Rather than being the products of infection of resting cells, as was once thought, HIV persistence is largely or entirely a consequence of infection of cells that are either expanding or are destined to expand, primarily due to antigen-driven activation. Although most of the clones carry defective proviruses, some carry intact infectious proviruses; these clones comprise the majority of the reservoir. A large majority of both the defective and the intact infectious proviruses in clones of infected cells are transcriptionally silent; however, a small fraction expresses a few copies of unspliced HIV RNA. A much smaller fraction is responsible for production of low levels of infectious virus, which can rekindle infection when ART is stopped. Further understanding of the reservoir will be needed to clarify the mechanism(s) by which provirus expression is controlled in the clones of cells that constitute the reservoir.

In vivo clonal expansion of HIV-infected T cells is an important mechanism of viral persistence. In some cases, clonal expansion is driven by HIV proviral DNA integrated into one of a handful of genes. To investigate this phenomenon in vitro, we infected primary CD4+ T cells with an HIV construct expressing GFP and, after nearly 2 mo of culture and multiple rounds of activation, analyzed the resulting integration site distribution. In each of three replicates from each of two donors, we detected large clusters of integration sites with multiple breakpoints, implying clonal selection. These clusters all mapped to a narrow region within the STAT3 gene. The presence of hybrid transcripts splicing HIV to STAT3 sequences supports a model of LTR-driven STAT3 overexpression as a driver of preferential growth. Thus, HIV integration patterns linked to selective T cell outgrowth can be reproduced in cell culture. The single report of an HIV provirus in a case of AIDS-associated B-cell lymphoma with an HIV provirus in the same part of STAT3 also has implications for HIV-induced malignancy.

The characterisation of the HIV-1 reservoir, which consists of replication-competent integrated proviruses that persist on antiretroviral therapy (ART), is made difficult by the rarity of intact proviruses relative to those that are defective. While the only conclusive test for the replication-competence of HIV-1 proviruses is carried out in cell culture, genetic characterization of genomes by near full-length (NFL) PCR and sequencing can be used to determine whether particular proviruses have insertions, deletions, or substitutions that render them defective. Proviruses that are not excluded by having such defects can be classified as genetically intact and, possibly, replication competent. Identifying and quantifying proviruses that are potentially replication-competent is important for the development of strategies towards a functional cure. However, to date, there are no programs that can be incorporated into deep-sequencing pipelines for the automated characterization and annotation of HIV genomes. Existing programs that perform this work require manual intervention, cannot be widely installed, and do not have easily adjustable settings. Here, we present HIVIntact, a python-based software tool that characterises genomic defects in NFL HIV-1 sequences, allowing putative intact genomes to be identified in-silico. Unlike other applications that assess the genetic intactness of HIV genomes, this tool can be incorporated into existing sequence-analysis pipelines and applied to large next-generation sequencing datasets.

Understanding HIV-1 persistence despite antiretroviral therapy (ART) is of paramount importance. Both single-genome sequencing (SGS) and integration site analysis (ISA) provide useful information regarding the structure of persistent HIV DNA populations; however, until recently, there was no way to link integration sites to their cognate proviral sequences. Here, we used multiple-displacement amplification (MDA) of cellular DNA diluted to a proviral endpoint to obtain full-length proviral sequences and their corresponding sites of integration. We applied this method to lymph node and peripheral blood mononuclear cells from 5 ART-treated donors to determine whether groups of identical subgenomic sequences in the 2 compartments are the result of clonal expansion of infected cells or a viral genetic bottleneck. We found that identical proviral sequences can result from both cellular expansion and viral genetic bottlenecks occurring prior to ART initiation and following ART failure. We identified an expanded T cell clone carrying an intact provirus that matched a variant previously detected by viral outgrowth assays and expanded clones with wild-type and drug-resistant defective proviruses. We also found 2 clones from 1 donor that carried identical proviruses except for nonoverlapping deletions, from which we could infer the sequence of the intact parental virus. Thus, MDA-SGS can be used for “viral reconstruction” to better understand intrapatient HIV-1 evolution and to determine the clonality and structure of proviruses within expanded clones, including those with drug-resistant mutations. Importantly, we demonstrate that identical sequences observed by standard SGS are not always sufficient to establish proviral clonality.

HIV-1 integrates its genome into the DNA of host cells. Consequently, HIV-1 genomes are copied with the host cell DNA during cellular division., Little is known about the emergence and persistence of human immunodeficiency virus (HIV)-infected T-cell clones in perinatally infected children. We analyzed peripheral blood mononuclear cells (PBMCs) for clonal expansion in 11 children who initiated antiretroviral therapy (ART) between 1.8 and 17.4 months of age and with viremia suppressed for 6 to 9 years. We obtained 8,662 HIV type 1 (HIV-1) integration sites from pre-ART samples and 1,861 sites from on-ART samples. Expanded clones of infected cells were detected pre-ART in 10/11 children. In 8 children, infected cell clones detected pre-ART persisted for 6 to 9 years on ART. A comparison of integration sites in the samples obtained on ART with healthy donor PBMCs infected ex vivo showed selection for cells with proviruses integrated in BACH2 and STAT5B. Our analyses indicate that, despite marked differences in T-cell composition and dynamics between children and adults, HIV-infected cell clones are established early in children, persist for up to 9 years on ART, and can be driven by proviral integration in proto-oncogenes.

The latent HIV-1 reservoir is comprised of stably integrated and intact proviruses with limited to no viral transcription. It has been proposed that latent infection may be maintained by methylation of pro-viral DNA. Here, for the first time, we investigate the cytosine methylation of a replication competent provirus (AMBI-1) found in a T cell clone in a donor on antiretroviral therapy (ART). Methylation profiles of the AMBI-1 provirus were compared to other proviruses in the same donor and in samples from three other individuals on ART, including proviruses isolated from lymph node mononuclear cells (LNMCs) and peripheral blood mononuclear cells (PBMCs). We also evaluated the apparent methylation of cytosines outside of CpG (i.e., CpH) motifs. We found no evidence for methylation in AMBI-1 or any other provirus tested within the 5′ LTR promoter. In contrast, CpG methylation was observed in the env-tat-rev overlapping reading frame. In addition, we found evidence for differential provirus methylation in cells isolated from LNMCs vs. PBMCs in some individuals, possibly from the expansion of infected cell clones. Finally, we determined that apparent low-level methylation of CpH cytosines is consistent with occasional bisulfite reaction failures. In conclusion, our data do not support the proposition that latent HIV infection is associated with methylation of the HIV 5′ LTR promoter.

HIV persists during antiretroviral therapy (ART) as integrated proviruses in cells descended from a small fraction of the CD4+ T cells infected prior to the initiation of ART. To better understand what controls HIV persistence and the distribution of integration sites (IS), we compared about 15,000 and 54,000 IS from individuals pre-ART and on ART, respectively, with approximately 395,000 IS from PBMC infected in vitro. The distribution of IS in vivo is quite similar to the distribution in PBMC, but modified by selection against proviruses in expressed genes, by selection for proviruses integrated into one of 7 specific genes, and by clonal expansion. Clones in which a provirus integrated in an oncogene contributed to cell survival comprised only a small fraction of the clones persisting in on ART. Mechanisms that do not involve the provirus, or its location in the host genome, are more important in determining which clones expand and persist., Author summary In HIV-infected individuals, a small fraction of the infected cells persist and divide. This reservoir persists during fully suppressive ART and can rekindle the infection if ART is discontinued. Because the number of possible sites of HIV DNA integration is very large, each infected cell, and all of its descendants, can be identified by the site where the provirus is integrated (IS). To understand the selective forces that determine the fates of infected cells in vivo, we compared the distribution of HIV IS in freshly-infected cells to cells from HIV-infected donors sampled both before and during ART. We found that, as previously reported, integration favors highly-expressed genes. However, over time, the fraction of cells with proviruses integrated in highly-expressed genes decreases, implying that they grow less well. There are exceptions to this broad negative selection. When a provirus is integrated in a specific region in one of seven genes, the proviruses affect the expression of the target gene, promoting growth and/or survival of the cell. Although this effect is striking, it is only a minor component of the forces that promote the growth and survival of the population of infected cells during ART.

HIV persists during antiretroviral therapy (ART) as integrated proviruses in cells descended from a small fraction of the CD4+ T cells infected prior to the initiation of ART. To better understand what controls HIV persistence and the distribution of integration sites (IS), we compared about 16,000 and 54,000 IS from individuals pre-ART and on ART, respectively, with approximately 385,000 IS from PBMC infected in vitro. The distribution of IS in vivo is quite similar to the distribution in PBMC, modified by selection against proviruses in expressed genes, by selection for proviruses integrated into one of 6 specific genes, and by clonal expansion. The clones in which a provirus integrated in an oncogene contributed to the survival of the clone comprise only a small fraction of the clones that persist in HIV-infected individuals on ART. Mechanisms that do not involve the provirus, or its location in the host genome, are more important in determining which clones expand and persist.Author SummaryIn HIV-infected individuals, a small fraction of the infected cells persist and divide. This reservoir persists on ART and can rekindle the infection if ART is discontinued. Because the number of possible sites of HIV DNA integration is very large, each infected cell, and all of its descendants, can be identified by the site where the provirus is integrated (IS). To understand the selective forces that determine the fates of infected cells in vivo, we compared the distribution of HIV IS in freshly-infected cells to cells from HIV-infected donors sampled both before and during ART. We found that, as has been previously reported, integration favors highly-expressed genes. However, over time the fraction of cells with proviruses integrated in highly-expressed genes decreases, implying that they grow less well. There are exceptions to this broad negative selection. When a provirus is integrated in a specific region in one of six genes, the proviruses affect the expression of the target gene, promoting growth and/or survival of the cell. Although this effect is striking, it is only a minor component of the forces that promote the growth and survival of the population of infected cells during ART.

Little is known about the emergence and persistence of HIV-infected T cell clones in perinatally-infected children. We analyzed peripheral blood mononuclear cells for clonal expansion in 11 children who initiated antiretroviral therapy (ART) between 1.8-17.4 months of age and with viremia suppressed for 6-9 years. We obtained 8,662 HIV-1 integration sites from pre-ART and 1,861 sites on ART. Expanded clones of infected cells were detected pre-ART in 10/11 children. In 8 children, infected cell clones detected pre-ART persisted for 6-9 years on ART. A comparison of integration sites in the samples obtained on ART with healthy donor PBMC infected ex-vivo showed selection for cells with proviruses integrated in BACH2 and STAT5B. Our analyses indicate that, despite marked differences in T cell composition and dynamics between children and adults, HIV-infected cell clones are established early in children, persist for up to 9 years on ART, and can be driven by proviral integration in proto-oncogenes.

BACKGROUNDHIV-1 viremia that is not suppressed by combination antiretroviral therapy (ART) is generally attributed to incomplete medication adherence and/or drug resistance. We evaluated individuals referred for non-suppressible viremia (plasma HIV-1 RNA above 40 copies/ml) who reported adherence to ART and did not show drug resistance to their current regimen.METHODSSamples were collected from at least two time points from eight donors who had non-suppressible viremia for more than six months on ART. Single templates of HIV-1 RNA obtained from plasma and viral outgrowth of cultured cells and from proviral DNA were PCR-amplified and sequenced for evidence of clones of cells that produced infectious viruses. Clones were identified by host-proviral integration site analysis.RESULTSHIV-1 genomic RNAs with identical sequences were identified in plasma samples from all eight donors. The identical viral RNA sequences did not change over time and lacked resistance to the ART regimen. In four of the donors, viral RNA sequences obtained from plasma matched those sequences from viral outgrowth cultures, indicating that the viruses were replication-competent. Integration sites for infectious proviruses from those four donors were mapped to introns of theMATR3,ZNF268,ZNF721/ABCA11P, andABCA11Pgenes. The sizes of the clones were from 50 million to 350 million cells.CONCLUSIONClones of HIV-1-infected cells producing virus can cause failure of ART to suppress viremia despite medication adherence and absence of drug resistance. The mechanisms involved in clonal expansion and persistence need to be defined to eliminate viremia and the HIV-1 reservoir.

Combination antiretroviral therapy (cART) controls but does not eradicate HIV infection, HIV persistence is the principal obstacle to curing infections. The proportion of defective proviruses increases during cART, but the dynamics of this process are not well understood, and a quantitative analysis of how the proviral landscape is reshaped after cART is initiated is critical to understanding how HIV persists. Here, we studied longitudinal samples from HIV infected individuals undergoing long term cART using multiplexed Droplet Digital PCR (ddPCR) approaches to quantify the proportion of deleted proviruses in lymphocytes. In most individuals undergoing cART, HIV proviruses that contain gag are lost more quickly than those that lack gag. Increases in the fraction of gag-deleted proviruses occurred only after 1&ndash, 2 years of therapy, suggesting that the immune system, and/or toxicity of viral re-activation helps to gradually shape the proviral landscape. After 10&ndash, 15 years on therapy, there were as many as 3.5&ndash, 5 times more proviruses in which gag was deleted or highly defective than those containing intact gag. We developed a provirus-specific ddPCR approach to quantify individual clones. Investigation of a clone of cells containing a deleted HIV provirus integrated in the HORMAD2 gene revealed that the cells underwent a massive expansion shortly after cART was initiated until the clone, which was primarily in effector memory cells, dominated the population of proviruses for over 6 years. The expansion of this HIV-infected clone had substantial effects on the overall proviral population.

Background Increased transcription of the human endogenous retrovirus group HERV-K (HML-2) is often seen during disease. Although the mechanism of its tissue-specific activation is unclear, research shows that LTR CpG hypomethylation alone is not sufficient to induce its promoter activity and that the transcriptional milieu of a malignant cell contributes, at least partly, to differential HML-2 expression. Results We analyzed the relationship between LTR sequence variation and promoter expression patterns in human breast cancer cell lines, finding them to be positively correlated. In particular, two proviruses (3q12.3 and 11p15.4) displayed increased activity in almost all tumorigenic cell lines sampled. Using a transcription factor binding site prediction algorithm, we identified two unique binding sites in each 5′ LTR that appeared to be associated with inducing promoter activity during neoplasia. Genomic analysis of the homologous proviruses in several non-human primates indicated post-integration genetic drift in two transcription factor binding sites, away from the ancestral sequence and towards the active form. Based on the sequences of 2504 individuals from the 1000 Genomes Project, the active form of the 11p15.4 site was found to be polymorphic within the human population, with an allele frequency of 51%, whereas the activating mutation in the 3q12.3 provirus was fixed in humans but not present in the orthologous provirus in chimpanzees or gorillas. Conclusions These data suggest that stage-specific transcription factors at least partly contribute to LTR promoter activity during transformation and that, in some cases, transcription factor binding site polymorphisms may be responsible for the differential HML-2 expression often seen between individuals. Electronic supplementary material The online version of this article (10.1186/s12977-018-0441-2) contains supplementary material, which is available to authorized users.

AGS-004 consists of matured autologous dendritic cells co-electroporated with in vitro transcribed RNA encoding autologous HIV antigens. In an open-label, single arm sub-study of AGS-004-003, AGS-004 was administered monthly to suppressed participants who started antiretroviral therapy (ART) during acute HIV infection. HIV-1 specific T cell responses were measured by multicolor flow cytometry after 3–4 doses. The frequency of resting CD4+ T-cell infection (RCI) was measured by quantitative viral outgrowth assay. Participants demonstrating increased immune response postvaccination were eligible for analytic treatment interruption (ATI). AGS-004 induced a positive immune response defined as ≥2-fold increase from baseline in the number of multifunctional HIV-1 specific CD28+/CD45RA− CD8+ effector/memory cytoxic T-lymphocytes (CTLs) in all six participants. All participants underwent ATI with rebound viremia at a median of 29 days. Immune correlates between time to viral rebound and the induction of effector CTLs were determined. Baseline RCI was low in most participants (0.043–0.767 IUPM). One participant had a >2-fold decrease (0.179–0.067 infectious units per million [IUPM]) in RCI at week 10. One participant with the lowest RCI had the longest ATI. AGS-004 dendritic cell administration increased multifunctional HIV-specific CD28+/CD45RA− CD8+ memory T cell responses in all participants, but did not permit sustained ART interruption. However, greater expansion of CD28−/CCR7−/CD45RA− CD8+ effector T cell responses correlated with a longer time to viral rebound. AGS-004 may be a useful tool to augment immune responses in the setting of latency reversal and eradication strategies.

Efforts to cure HIV-1 infection require better quantification of the HIV-1 reservoir, particularly the clones of cells harboring replication-competent (intact) proviruses, termed repliclones. The digital droplet PCR assays commonly used to quantify intact proviruses do not differentiate among specific repliclones, thus the dynamics of repliclones are not well defined. The major challenge in tracking repliclones is the relative rarity of the cells carrying specific intact proviruses. To date, detection and accurate quantification of repliclones requires in-depth integration site sequencing. Here, we describe a simplified workflow using integration site-specific qPCR (IS-qPCR) to determine the frequencies of the proviruses integrated in individual repliclones. We designed IS-qPCR to determine the frequencies of repliclones and clones of cells that carry defective proviruses in samples from three donors. Comparing the results of IS-qPCR with deep integration site sequencing data showed that the two methods yielded concordant estimates of clone frequencies (r = 0.838). IS-qPCR is a potentially valuable tool that can be applied to multiple samples and cell types over time to measure the dynamics of individual repliclones and the efficacy of treatments designed to eliminate them.

We hypothesized that HIV-1 with dual-class but not single-class drug resistance mutations linked on the same viral genome, present in the virus population before initiation of antiretroviral therapy (ART), would be associated with failure of ART to suppress viremia. To test this hypothesis, we utilized an ultrasensitive single-genome sequencing assay that detects rare HIV-1 variants with linked drug resistance mutations (DRMs). A case (ART failure) control (nonfailure) study was designed to assess whether linkage of DRMs in pre-ART plasma samples was associated with treatment outcome in the nevirapine/tenofovir/emtricitabine arm of the AIDS Clinical Trials Group A5208/Optimal Combined Therapy After Nevirapine Exposure (OCTANE) Trial 1 among women who had received prior single-dose nevirapine. Ultrasensitive single-genome sequencing revealed a significant association between pre-ART HIV variants with DRMs to 2 drug classes linked on the same genome (dual class) and failure of combination ART with 3 drugs to suppress viremia. In contrast, linked, single-class DRMs were not associated with ART failure. We conclude that linked dual-class DRMs present before the initiation of ART are associated with ART failure, whereas linked single-class DRMs are not.

Background HIV-1 proviruses can persist during ART in clonally-expanded populations of CD4+ T cells. To date, few examples of an expanded clones containing replication-competent proviruses exist, although it is suspected to be common. One such clone, denoted AMBI-1 (Maldarelli et al., 2014), was also a source of persistent viremia on ART, begging the question of how the AMBI-1 clone can survive despite infection with a replication-competent, actively-expressing provirus. We hypothesized that only a small fraction of cells within the AMBI-1 clone are activated to produce virus particles during cell division while the majority remain latent despite division, ensuring their survival. To address this question, we determined the fraction of HIV-1 proviruses within the AMBI-1 clone that expresses unspliced cell-associated RNA during ART and compared this fraction to 33 other infected T cell clones within the same individual. Results In total, 34 different clones carrying either intact or defective proviruses in "Patient 1" from Maldarelli et al. (2014) were assessed. We found that 2.3% of cells within the AMBI-1 clone contained unspliced HIV-1 RNA. Highest levels of HIV-1 RNA were found in the effector memory (EM) T cell subset. The fraction of cells within clones that contained HIV-1 RNA was not different in clones with intact (median 2.3%) versus defective (median 3.5%) proviruses (p = 0.2). However, higher fractions and levels of RNA were found in cells with proviruses containing multiple drug resistance mutations, including those contributing to rebound viremia. Conclusion These findings show that the vast majority of HIV-1 proviruses within expanded T cell clones, including intact proviruses, may be transcriptionally silent at any given time, implying that infected T cells may be able to be activated to proliferate without inducing the expression of the integrated provirus or, alternatelively, may be able to proliferate without cellular activation. The results of this study suggest that the long, presumed correlation between the level of cellular and proviral activation may not be accurate and, therefore, requires further investigation.

To investigate the possibility that HIV-1 replication in lymph nodes sustains the reservoir during antiretroviral therapy (ART), we looked for evidence of viral replication in 5 donors after up to 13 years of viral suppression. We characterized proviral populations in lymph nodes and peripheral blood before and during ART, evaluated the levels of viral RNA expression in single lymph node and blood cells, and characterized the proviral integration sites in paired lymph node and blood samples. Proviruses with identical sequences, identical integration sites, and similar levels of RNA expression were found in lymph nodes and blood samples collected during ART, and no single sequence with significant divergence from the pretherapy population was detected in either blood or lymph nodes. These findings show that all detectable persistent HIV-1 infection is consistent with maintenance in lymph nodes by clonal proliferation of cells infected before ART and not by ongoing viral replication during ART.

Accurate definition of the HIV-1 reservoir on antiretroviral therapy (ART) is of paramount importance to the development of curative strategies. Much of this reservoir is derived from clonal expansion of latently infected CD4+ T cells. Methods used to characterize the reservoir include near full-length single-genome sequencing (NFL-SGS) and integration site analysis (ISA). However, current technologies do not link the intact proviruses detected by NFL-SGS to their sites of integration. Therefore, we developed a method to obtain both near full-length single-proviral sequences and their sites of integration. We call our method full-length integrated proviral single-genome sequencing (FLIP-SGS). Genomic DNA from ACH2 and CEM cells mixed at 1:1,000, or patient samples were diluted to a single proviral endpoint. An in-house, optimized whole genome amplification (WGA) method was performed on wells at the endpoint, generating multiple copies of all DNA molecules within each well. The number of proviral copies after WGA was determined by droplet digital PCR targeting the long terminal region (LTR). Forty per cent of each WGA reaction was used to obtain the provirus–host integration sites with ISA (linker ligation, nested PCR, and Illumina sequencing). The remaining fraction was used to amplify the full-length proviruses in four overlapping fragments (LTR-pol, gag-int, int-env, and env-LTR) for Sanger sequencing. WGA performed on the endpoint-diluted ACH2:CEM DNA amplified single-copy HIV-1 proviral templates greater than 500-fold, making it possible to obtain unique integration sites from single proviruses in ACH2 cells, including one that was previously reported (in the NT5C3A gene on chromosome 7) and two that were not previously reported (in the EIF4ENIF1 gene of chromosome 22 and an unknown region of chromosome 6). Near full-length PCR amplification and Sanger sequencing was performed on proviruses integrated in the NT5C3A gene. FLIP-SGS was applied to peripheral blood mononuclear cells from one HIV-1 infected donor with viremia suppressed on ART and yielded integration sites of four genomes that appear to contain large internal deletions. We report a method for near full-length HIV-1 single-genome sequencing combined with host integration site detection that we call FLIP-SGS. This assay will further define clonal expansion of infected CD4+ T cells as a mechanism that maintains the HIV-1 reservoir and as the source of identical sequences observed during therapy and rebound, rather than from ongoing replication.

There are conflicting reports regarding the presence of low-level HIV replication during suppressive antiretroviral therapy (ART). We simulated varying levels of replication and estimated the number of generations needed to obtain linked, drug resistance mutations to explore the effects of replication during ART. HIV replication was simulated with varying population sizes (10 to 3,000,000). Each population size was modeled ten times. Each genome was given a Poisson-distributed number of mutations according to its length and the average replication error rate (3.4 × 10−5 sub/nt/cycle). Simulations were run a maximum of 20,000 generations with endpoints defined as detection of a variant with resistance mutations to at least two ARVs. In all simulations, variants that were resistant to all three ARVs emerged in less than 20,000 generations. The time to emergence ranged from 148–16,156 generations in the various simulations, depending on the replicating population size (4.8 months to 44.3 years if the generation time is 1 day). Clinically detectable virologic failure can result from linkage of two mutations conferring resistance to two ARVs in a regimen. In our simulations, two linked mutations emerged in from 9 to 6,429 generations (9 days to 17.6 years). Our simulations suggest that in patients continually suppressed on ART for at least 10 years, the replicating population size would have to be less than ten, or virologic failure would have occurred from emergence of two ARV-resistant variants. Because most patients on ART do not experience virologic failure, our simulations suggest that any residual replicating population on ART is very small and thus not likely to either sustain or significantly contribute to the HIV reservoir.

Clonal expansion of HIV infected cells plays an important role in the formation and persistence of the reservoir that allows the virus to persist, in DNA form, despite effective antiretroviral therapy. We used integration site analysis to ask if there is a similar clonal expansion of SIV infected cells in macaques. We show that the distribution of HIV and SIV integration sites in vitro is similar and that both viruses preferentially integrate in many of the same genes. We obtained approximately 8000 integration sites from blood samples taken from SIV-infected macaques prior to the initiation of ART, and from blood, spleen, and lymph node samples taken at necropsy. Seven clones were identified in the pre-ART samples; one persisted for a year on ART. An additional 100 clones were found only in on-ART samples; a number of these clones were found in more than one tissue. The timing and extent of clonal expansion of SIV-infected cells in macaques and HIV-infected cells in humans is quite similar. This suggests that SIV-infected macaques represent a useful model of the clonal expansion of HIV infected cells in humans that can be used to evaluate strategies intended to control or eradicate the viral reservoir., Author summary Although antiretroviral therapy (ART) effectively blocks HIV replication, infected people are not cured. As a part of its normal replication cycle, HIV inserts (integrates) a DNA copy of its genome into the genome of infected host cells, which allows the virus to persist as long as the infected cells survive. Not only can these infected cells survive, they can grow and divide, increasing the numbers of infected cells without viral replication. The ability of the infected cells to proliferate plays an important role in maintaining the numbers of infected cells (and the infection) in people on successful therapy. However, there are some important experiments that cannot easily be done with samples that can be obtained from HIV infected people. SIV infected macaques are often used as a model to do experiments that cannot be done in HIV infected people. We show here that the distribution of HIV and SIV integration sites is similar, and that, in infected macaques, the timing and extent of the proliferation of SIV infected cells is also quite similar to HIV infected cells in humans. This shows that the SIV/macaque system can be used to model the clonal expansion of HIV infected cells.

In HIV-infected individuals on long-term antiretroviral therapy (ART), more than 40% of the infected cells are in clones. Although most HIV proviruses present in individuals on long-term ART are defective, including those in clonally expanded cells, there is increasing evidence that clones carrying replication-competent proviruses are common in patients on long-term ART and form part of the HIV reservoir that makes it impossible to cure HIV infection with current ART alone. Given the importance of clonal expansion in HIV persistence, we determined how soon after HIV acquisition infected clones can grow large enough to be detected (clones larger than ca. 1 × 10(5) cells). We studied 12 individuals sampled in early HIV infection (Fiebig stage III–V/VI) and 5 who were chronically infected. The recently infected individuals were started on ART at or near the time of diagnosis. We isolated more than 6,500 independent integration sites from peripheral blood mononuclear cells before ART was initiated and after 0.5–18 years of suppressive ART. Some infected clones could be detected approximately 4 weeks after HIV infection and some of these clones persisted for years. The results help to explain how the reservoir is established early and persists for years.

In 1970 the independent and simultaneous discovery of reverse transcriptase in retroviruses (then RNA tumor viruses) by David Baltimore and Howard Temin revolutionized molecular biology and laid the foundations for retrovirology and cancer biology. In this historical review we describe the formulation of the controversial provirus hypothesis by Temin, which ultimately was proven by his discovery of reverse transcriptase in Rous sarcoma virus virions. Baltimore arrived at the same discovery through his studies on replication of RNA-containing viruses, starting with poliovirus and then moving to vesicular stomatitis virus, where he discovered a virion RNA polymerase. Subsequent studies of reverse transcriptase led to the elucidation of the mechanism of retrovirus replication, the discovery of oncogenes, the advent of molecular cloning, the search for human cancer viruses, and the discovery and treatment of HIV/AIDS.

Significance Reservoirs of HIV-infected cells persist during antiretroviral therapy, and understanding persistence is essential to develop HIV curative strategies. During replication, HIV integrates into the host genome; most proviruses are not infectious, but some with replication-competent HIV persist. Cells with integrated HIV can proliferate, potentially expanding the reservoir, but whether cells with replication-competent HIV actually undergo expansion is unknown. HIV reactivation is often lethal to infected cells, and others have reported finding no replication-competent HIV in expanded populations. We describe a highly expanded clone containing infectious HIV that was the source of viremia for years in a patient. Clonally expanded populations can represent a long-lived reservoir of HIV. Curative strategies will require targeting this persistence mechanism.

Understanding the origin of HIV variants during viral rebound may provide insight into the composition of the HIV reservoir and has implications for the design of curative interventions. HIV single-genome sequences were obtained from 10 AIDS Clinical Trials Group participants who underwent analytic antiretroviral therapy (ART) interruption (ATI). Rebounding variants were compared with those in pre-ART plasma in all 10 participants and with on-ART peripheral blood mononuclear cell (PBMC)-associated DNA and RNA (CA-RNA) in 7/10 participants. The highest viral diversities were found in the DNA and CA-RNA populations. In 3 of 7 participants, we detected multiple, identical DNA and CA-RNA sequences during suppression on ART that exactly matched plasma HIV sequences. Hypermutated DNA and CA-RNA were detected in four participants, contributing to diversities in these compartments that were higher than in the pre-ART and post-ATI plasma. Shifts in the viral rebound populations could be detected in some participants over the 2- to 3-month observation period. These findings suggest that a source of initial rebound viremia could be populations of infected cells that clonally expanded prior to and/or during ART, some of which were already expressing HIV RNA before treatment was interrupted. These clonally expanding populations of HIV-infected cells may represent an important target for strategies aimed at achieving reservoir reduction and sustained virologic remission. IMPORTANCE Antiretroviral therapy alone cannot eradicate the HIV reservoir, and viral rebound is generally rapid after treatment interruption. It has been suggested that clonal expansion of HIV-infected cells is an important mechanism of HIV reservoir persistence, but the contribution of these clonally proliferating cells to the rebounding virus is unknown. We report a study of AIDS Clinical Trials Group participants who underwent treatment interruption and compared rebounding plasma virus with that found within cells prior to treatment interruption. We found several incidences in which plasma HIV variants exactly matched that of multiple proviral DNA copies from infected blood cells sampled before treatment interruption. In addition, we found that these cells were not dormant but were generating unspliced RNA transcripts before treatment was interrupted. Identification of the HIV reservoir and determining its mechanisms for persistence may aid in the development of strategies toward a cure for HIV. (This study was presented in part at the Conference on Retroviruses and Opportunistic Infections, Seattle, WA, February 23 to 26 2015.)

International audience; Reverse-transcribing viruses, which synthesize a copy of genomic DNA from an RNA template, are widespread in animals, plants, algae, and fungi (1, 2). This broad distribution suggests the ancient origin(s) of these viruses, possibly [...]

Human endogenous retrovirus (HERV-K (HML-2)) proviruses are among the few endogenous retroviral elements in the human genome that retain coding sequence. HML-2 expression has been widely associated with human disease states, including different types of cancers as well as with HIV-1 infection. Understanding of the potential impact of this expression requires that it be annotated at the proviral level. Here, we utilized the high throughput capabilities of next-generation sequencing to profile HML-2 expression at the level of individual proviruses and secreted virions in the teratocarcinoma cell line Tera-1. We identified well-defined expression patterns, with transcripts emanating primarily from two proviruses located on chromosome 22, only one of which was efficiently packaged. Interestingly, there was a preference for transcripts of recently integrated proviruses, over those from other highly expressed but older elements, to be packaged into virions. We also assessed the promoter competence of the 5’ long terminal repeats (LTRs) of expressed proviruses via a luciferase assay following transfection of Tera-1 cells. Consistent with the RNASeq results, we found that the activity of most LTRs corresponded to their transcript levels.

Lorenzo-Redondo et al. recently analyzed HIV RNA sequences in plasma virus and proviral DNA sequences in lymph nodes (LN) and peripheral blood mononuclear cells (PBMC) from samples collected over a 6-month period from 3 individuals following initiation of antiretroviral therapy (ART) and concluded that ongoing HIV replication occurred in LN despite ART and that this replication maintained the HIV reservoir. We analyzed the same sequences and found that the dataset was very limited (median of 5 unique RNA or DNA sequences per sample) after accounting for polymerase chain reaction resampling and hypermutation and that the few remaining DNA sequences after 3 and 6 months on ART were not more diverse or divergent from those in pre-ART in any of the individuals studied. These findings, and others, lead us to conclude that the claims of ongoing replication on ART made by Lorenzo-Redondo et al. are not justified from the dataset analyzed in their publication.

Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2-18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression.

It remains controversial whether current antiretroviral therapy (ART) fully suppresses the cycles of HIV replication and viral evolution in vivo. If replication persists in sanctuary sites such as the lymph nodes, a high priority should be placed on improving ART regimes to target these sites. To investigate the question of ongoing viral replication on current ART regimens, we analyzed HIV populations in longitudinal samples from 10 HIV-1-infected children who initiated ART when viral diversity was low. Eight children started ART at less than ten months of age and showed suppression of plasma viremia for seven to nine years. Two children had uncontrolled viremia for fifteen and thirty months, respectively, before viremia suppression, and served as positive controls for HIV replication and evolution. These latter 2 children showed clear evidence of virus evolution, whereas multiple methods of analysis bore no evidence of virus evolution in any of the 8 children with viremia suppression on ART. Phylogenetic trees simulated with the recently reported evolutionary rate of HIV-1 on ART of 6 × 10-4 substitutions/site/month bore no resemblance to the observed data. Taken together, these data refute the concept that ongoing HIV replication is common with ART and is the major barrier to curing HIV-1 infection.

Endogenous retroviral sequences provide a molecular fossil record of ancient infections whose analysis might illuminate mechanisms of viral extinction. A close relative of gammaretroviruses, HERV-T, circulated in primates for ~25 million years (MY) before apparent extinction within the past ~8 MY. Construction of a near-complete catalog of HERV-T fossils in primate genomes allowed us to estimate a ~32 MY old ancestral sequence and reconstruct a functional envelope protein (ancHTenv) that could support infection of a pseudotyped modern gammaretrovirus. Using ancHTenv, we identify monocarboxylate transporter-1 (MCT-1) as a receptor used by HERV-T for attachment and infection. A single HERV-T provirus in hominid genomes includes an env gene (hsaHTenv) that has been uniquely preserved. This apparently exapted HERV-T env could not support virion infection but could block ancHTenv mediated infection, by causing MCT-1 depletion from cell surfaces. Thus, hsaHTenv may have contributed to HERV-T extinction, and could also potentially regulate cellular metabolism. DOI: http://dx.doi.org/10.7554/eLife.22519.001, eLife digest Over millions of years, viruses and the animals that they infect have been locked in a battle for survival, where each has needed to evolve ways to counteract the effects of the other. While the evolution of ancient animals can be studied by looking at the fossilized remains of their extinct relatives, studying how ancient viruses have evolved is more difficult as they usually do not leave behind physical traces of their existence. However, a family of viruses called retroviruses is a notable exception to this rule. Retroviruses have a step in their life cycle in which their genetic material is integrated into the genome (the name for an organism’s complete set of genetic material) of the cell that they have infected. In rare cases, when that cell is a precursor of a sperm or egg cell, then the viral genes may then be passed on to the animal’s offspring, ultimately leaving genetic traces that can be studied in modern animals. This acts as a genetic ‘fossil record’ of extinct viruses. HERV-T was a retrovirus that spread among our primate ancestors for about 25 million years before its extinction roughly 11 million years ago. Blanco-Melo et al. have now analyzed the genetic remains left by HERV-T in the genomes of humans and related primates, and were able to use this information to recreate a protein that made up the outer envelope that surrounded the virus. Further experiments showed that this viral protein helped HERV-T to infect human cells by interacting with a protein called MCT1 on the cell surface. Blanco-Melo et al. also found a particular HERV-T gene that was unexpectedly well preserved in the human genome. The gene retained its ability to produce an envelope protein for about 13 to 19 million years. It is likely that ancient primates ‘hijacked’ the viral gene and used the protein it produced to remove the MCT1 protein from the surface of their own cells. Without MCT1 on the surface, HERV-T was unable to infect the cells. Thus, these findings present an example of how viruses themselves can provide the genetic material that animals use to combat them, potentially leading to their extinction. DOI: http://dx.doi.org/10.7554/eLife.22519.002

A quantitative real-time PCR (qRT-PCR) assay with single-copy sensitivity targeting HIV-1 gag RNA (the gag single-copy assay [gSCA]) has been used widely to quantify plasma viremia below the limit of detection of clinical assays in patients on effective antiretroviral therapy (ART), but viral RNA in 15 to 30% of samples amplifies inefficiently because of primer/probe mismatches. We sought to develop improved single-copy assays with increased sensitivity by improving nucleic acid recovery, designing qRT-PCR primers and a probe for a highly conserved region of integrase in the HIV-1 pol gene (the integrase single-copy assay [iSCA]), and increasing the plasma volume tested (Mega-iSCA). We evaluated gSCA versus iSCA in paired plasma samples from 10 consecutive patients with viremia of >1,000 copies/ml and 25 consecutive patients on suppressive ART. Three of 10 viremic samples amplified inefficiently with gSCA compared to the Roche Cobas Ampliprep/TaqMan 2.0, whereas all 10 samples amplified efficiently with iSCA. Among 25 samples from patients on suppressive ART, 8 of 12 samples that were negative for HIV-1 RNA by gSCA had detectable HIV-1 RNA by iSCA, and iSCA detected 3-fold or higher HIV-1 RNA levels compared to gSCA in 10 of 25 samples. Large-volume plasma samples (>20 ml) from 7 patients were assayed using Mega-iSCA, and HIV-1 RNA was quantifiable in 6, including 4 of 5 that were negative by standard-volume iSCA. These improved assays with superior sensitivity will be useful for evaluating whether in vivo interventions can reduce plasma viremia and for assessing relationships between residual viremia and other virologic parameters, including the inducible proviral reservoir.

Estimating viral diversity in infected patients can provide insight into pathogen evolution and emergence of drug resistance. With the widespread adoption of deep sequencing, it is important to develop tools to accurately calculate population diversity from very large datasets. Current methods for estimating diversity that are based on multiple alignments are not practical to apply to such data. In this study, the authors report a novel method (Pairwise Alignment Positional Nucleotide Counting, PAPNC) for estimating population diversity from 454 sequence data. The diversity measurements determined using this method were comparable to those calculated by average pairwise difference (APD) of multiply aligned sequences using MEGA5. Diversities were estimated for 9 patient plasma HIV samples sequenced with Titanium 454 technology and by single-genome sequencing (SGS). Diversities calculated from deep sequencing using PAPNC ranged from 0.002 to 0.021 while APD measurements calculated from SGS data ranged proximately from 0.001 to 0.018, with the difference being attributable to PCR error (contributing background diversity of 0.0016 in a control sample). Comparison of APDs estimated from 100 sets of sequences drawn at random from 454 generated data and from corresponding SGS data showed very close correlation between the two methods with R2 of 0.96, and differing on average by about 1% (after correction for PCR error). The authors have developed a novel method that is good for calculating genetic diversities for large scale datasets from next generation sequencing. It can be implemented easily as a function in available variation calling programs like SAMtools or haplotype reconstruction software for nucleotide genetic diversity calculation. A Perl script implementing this method is available upon request.

Xenotropic murine leukemia virus-related virus (XMRV) infection was incorrectly associated with prostate cancer and chronic fatigue syndrome (CFS) in recent years. In this forum, we discuss the story of XMRV and how we can apply lessons learned here to inform the debate surrounding cancers associated with human endogenous retroviruses (HERVs).

Endogenous retroviruses (ERVs) represent ancestral sequences of modern retroviruses or their extinct relatives. The majority of ERVs cluster alongside exogenous retroviruses into two main groups based on phylogenetic analyses of the reverse transcriptase (RT) enzyme. Class I includes gammaretroviruses, and class II includes lentiviruses and alpha-, beta-, and deltaretroviruses. However, analyses of the transmembrane subunit (TM) of the envelope glycoprotein ( env ) gene result in a different topology for some retroviruses, suggesting recombination events in which heterologous env sequences have been acquired. We previously demonstrated that the TM sequences of five of the six genera of orthoretroviruses can be divided into three types, each of which infects a distinct set of vertebrate classes. Moreover, these classes do not always overlap the host range of the associated RT classes. Thus, recombination resulting in acquisition of a heterologous env gene could in theory facilitate cross-species transmissions across vertebrate classes, for example, from mammals to reptiles. Here we characterized a family of class II avian ERVs, “TgERV-F,” that acquired a mammalian gammaretroviral env sequence. Although TgERV-F clusters near a sister clade to alpharetroviruses, its genome also has some features of betaretroviruses. We offer evidence that this unusual recombinant has circulated among several avian orders and may still have infectious members. In addition to documenting the infection of a nongalliform avian species by a mammalian retrovirus, TgERV-F also underscores the importance of env sequences in reconstructing phylogenies and supports a possible role for env swapping in allowing cross-species transmissions across wide taxonomic distances. IMPORTANCE Retroviruses can sometimes acquire an envelope gene ( env ) from a distantly related retrovirus. Since env is a key determinant of host range, such an event affects the host range of the recombinant virus and can lead to the creation of novel retroviral lineages. Retroviruses insert viral DNA into the host DNA during infection, and therefore vertebrate genomes contain a “fossil record” of endogenous retroviral sequences thought to represent past infections of germ cells. We examined endogenous retroviral sequences in avian genomes for evidence of recombination events involving env . Although cross-species transmissions of retroviruses between vertebrate classes (from mammals to birds, for example) are thought to be rare, we here characterized a group of avian retroviruses that acquired an env sequence from a mammalian retrovirus. We offer evidence that this unusual recombinant circulated among songbirds 2 to 4 million years ago and has remained active into the recent past.

Mutations in the human immunodeficiency virus type 1 (HIV-1) genome have been hypothesized to occur at all nucleotide positions, including those that confer drug resistance, as the result of high rates of HIV-1 replication, mutation, and recombination [1–3]. Consequently, after exposure to one antiretroviral drug such as nevirapine (NVP), given as a single dose to prevent mother-to-child HIV-1 transmission, drug-resistant variants can rapidly emerge [4–7]. It is well established that such drug-resistant variants, when detected by standard genotype, can compromise virologic responses to combination antiretroviral therapy (cART) [8–10]. For example, in the OCTANE/A5208 trial 1, conducted in African women with prior exposure to single-dose NVP (sdNVP) and who subsequently initiated NVP-based cART, NVP resistance detected by standard genotype at study entry was associated with virologic failure (VF) or death, and lopinavir/ritonavir (LPV/r) was superior to NVP-based cART in sdNVP-exposed women [11]. The impact of minor populations of drug-resistant variants on the response to initial cART has been more controversial, with some studies reporting their association with VF and others finding no association [12–19]. We reported that the risk of VF with NVP-based cART was significantly associated with low frequency (>1%), NVP-resistant variants in African women with prior exposure to sdNVP (OCTANE/A5208 trial 1) [20]. In women who had never been exposed to sdNVP (OCTANE/A5208 trial 2), however, NVP- or LPV/r-based cART showed equivalent virologic efficacy [21]. To investigate the different outcomes of the NVP-based cART arms of trial 1 and trial 2, we performed allele-specific polymerase chain reaction (PCR) on pre-cART samples from women without prior sdNVP exposure (trial 2), to quantify frequencies of the 3 most common NVP resistance mutations (K103N, Y181C, and G190A) and their association with VF or death.

We previously identified two novel endogenous murine leukemia virus proviruses, PreXMRV-1 and PreXMRV-2, and showed that they most likely recombined during xenograft passaging of a human prostate tumor in mice to generate xenotropic murine leukemia virus-related virus (XMRV). To determine the recombination potential of PreXMRV-1 and PreXMRV-2, we examined the generation of replication-competent retroviruses (RCRs) over time in a cell culture system. We observed that either virus alone was noninfectious and the RNA transcripts of the viruses were undetectable in the blood and spleen of nude mice that carry them. To determine their potential to generate RCRs through recombination, we transfected PreXMRV-1 and PreXMRV-2 into 293T cells and used the virus produced to infect fresh cells; the presence of reverse transcriptase activity at 10 days postinfection indicated the presence of RCRs. Population sequencing of proviral DNA indicated that all RCRs contained the gag and 5′ half of pol from PreXMRV-2 and the long terminal repeat, 3′ half of pol (including integrase), and env from PreXMRV-1. All crossovers were within sequences of at least 9 identical nucleotides, and crossovers within each of two selected recombination zones of 415 nucleotides (nt) in the 5′ untranslated region and 982 nt in pol were required to generate RCRs. A recombinant with the same genotype as XMRV was not detected, and our analysis indicates that the probability of generating an identical RCR is vanishingly small. In addition, the studies indicate that the process of RCR formation is primarily driven by selection for viable cis and trans elements from the parental proviruses.

It has been proposed that most drug-resistant mutants, resulting from a single-nucleotide change, exist at low frequency in human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) populations in vivo prior to the initiation of antiretroviral therapy (ART). To test this hypothesis and to investigate the emergence of resistant mutants with drug selection, we developed a new ultrasensitive allele-specific PCR (UsASP) assay, which can detect drug resistance mutations at a frequency of ≥0.001% of the virus population. We applied this assay to plasma samples obtained from macaques infected with an SIV variant containing HIV-1 reverse transcriptase (RT) (RT-simian-human immunodeficiency [SHIV] mne ), before and after they were exposed to a short course of efavirenz (EFV) monotherapy. We detected RT inhibitor (RTI) resistance mutations K65R and M184I but not K103N in 2 of 2 RT-SHIV-infected macaques prior to EFV exposure. After three doses over 4 days of EFV monotherapy, 103N mutations (AAC and AAT) rapidly emerged and increased in the population to levels of ∼20%, indicating that they were present prior to EFV exposure. The rapid increase of 103N mutations from 6 or more infected cells per replication cycle.