Early Emergence and Long-Term Persistence of HIV-Infected T-Cell Clones in Children.

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.IMPORTANCE 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. Pediatric immune systems differ significantly from adults, consisting primarily of naive T cells, which have low expression of the HIV-1 coreceptor CCR5. This difference may result in variances in the number or size of infected cell clones that persist in children on ART. Here, we provide the most extensive analysis of the integration landscape of HIV-1 in children. We found that, despite the largely naive cell populations in neonatal immune systems, patterns of HIV-1 integration and the size of infected cell clones are as large and widespread as those in adults. Furthermore, selection for integration events in proto-oncogenes were observed in children despite early ART. If such cell clones persist for the life span of these individuals, there may be long-term consequences that have yet to be realized.

Combined HIV-1 sequence and integration site analysis informs viral dynamics and allows reconstruction of replicating viral ancestors.

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 Infected T Cells Can Proliferate in vivo Without Inducing Expression of the Integrated Provirus.

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.

HIV-1 in lymph nodes is maintained by cellular proliferation during antiretroviral therapy.

To investigate the possibility that HIV-1 replication in lymph nodes sustains the reservoir during 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 present 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.

Clones of infected cells arise early in HIV-infected individuals.

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 × 105 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.

Lower pre-ART intra-participant HIV-1 pol diversity may not be associated with virologic failure in adults.

BACKGROUND: Identifying pre-ART factors associated with the emergence of HIV-1 drug resistance is critical for optimizing strategies to prevent virologic failure. A previous study reported that lower pre-ART HIV-1 pol diversity was associated with higher risk of virologic failure in HIV-1-infected children. To investigate this association in adults, we measured HIV-1 diversity with deep sequencing in pre-ART samples from adults with well-characterized virologic outcomes in a study (A5142) of initial ART conducted by the AIDS Clinical Trials Group (ACTG). METHODS: We identified 22 cases in ACTG A5142 who experienced virologic failure with drug resistance mutations in RT and 44 matched controls who did not experience virologic failure. cDNA was synthesized from plasma HIV-1 RNA. Each cDNA molecule was tagged with a unique primer ID and RT codons 41-103 were amplified and deep sequenced. Sequences with the same tag were aligned and a consensus was generated to reduce PCR and sequencing errors. Diversity was calculated by measuring average pairwise distance (APD) of the consensus sequences. An exact conditional logistic regression model with percent APD as the risk factor estimated the odds ratio for VF and the corresponding 95% confidence interval. RESULTS: Consensus single-genome sequences and diversity estimates of pol were obtained for pre-ART samples from 21 cases and 42 controls. The median (IQR) pre-ART percent APD was 0.71 (0.31-1.13) in cases and 0.58 (0.32-0.94) in controls. A possible trend was found for higher diversity being associated with greater risk of virologic failure in adults (OR = 2.2 per one percent APD increase, 95% CI = [0.8, 7.2]; p = 0.15). CONCLUSIONS: This study in adults suggests there is a positive association between higher pre-ART pol diversity and the risk of virologic failure in adults rather than an inverse relationship reported in children.

HIV-1 persistence following extremely early initiation of antiretroviral therapy (ART) during acute HIV-1 infection: An observational study.

BACKGROUND: It is unknown if extremely early initiation of antiretroviral therapy (ART) may lead to long-term ART-free HIV remission or cure. As a result, we studied 2 individuals recruited from a pre-exposure prophylaxis (PrEP) program who started prophylactic ART an estimated 10 days (Participant A; 54-year-old male) and 12 days (Participant B; 31-year-old male) after infection with peak plasma HIV RNA of 220 copies/mL and 3,343 copies/mL, respectively. Extensive testing of blood and tissue for HIV persistence was performed, and PrEP Participant A underwent analytical treatment interruption (ATI) following 32 weeks of continuous ART. METHODS AND FINDINGS: Colorectal and lymph node tissues, bone marrow, cerebral spinal fluid (CSF), plasma, and very large numbers of peripheral blood mononuclear cells (PBMCs) were obtained longitudinally from both participants and were studied for HIV persistence in several laboratories using molecular and culture-based detection methods, including a murine viral outgrowth assay (mVOA). Both participants initiated PrEP with tenofovir/emtricitabine during very early Fiebig stage I (detectable plasma HIV-1 RNA, antibody negative) followed by 4-drug ART intensification. Following peak viral loads, both participants experienced full suppression of HIV-1 plasma viremia. Over the following 2 years, no further HIV could be detected in blood or tissue from PrEP Participant A despite extensive sampling from ileum, rectum, lymph nodes, bone marrow, CSF, circulating CD4+ T cell subsets, and plasma. No HIV was detected from tissues obtained from PrEP Participant B, but low-level HIV RNA or DNA was intermittently detected from various CD4+ T cell subsets. Over 500 million CD4+ T cells were assayed from both participants in a humanized mouse outgrowth assay. Three of 8 mice infused with CD4+ T cells from PrEP Participant B developed viremia (50 million input cells/surviving mouse), but only 1 of 10 mice infused with CD4+ T cells from PrEP Participant A (53 million input cells/mouse) experienced very low level viremia (201 copies/mL); sequence confirmation was unsuccessful. PrEP Participant A stopped ART and remained aviremic for 7.4 months, rebounding with HIV RNA of 36 copies/mL that rose to 59,805 copies/mL 6 days later. ART was restarted promptly. Rebound plasma HIV sequences were identical to those obtained during acute infection by single-genome sequencing. Mathematical modeling predicted that the latent reservoir size was approximately 200 cells prior to ATI and that only around 1% of individuals with a similar HIV burden may achieve lifelong ART-free remission. Furthermore, we observed that lymphocytes expressing the tumor marker CD30 increased in frequency weeks to months prior to detectable HIV-1 RNA in plasma. This study was limited by the small sample size, which was a result of the rarity of individuals presenting during hyperacute infection. CONCLUSIONS: We report HIV relapse despite initiation of ART at one of the earliest stages of acute HIV infection possible. Near complete or complete loss of detectable HIV in blood and tissues did not lead to indefinite ART-free HIV remission. However, the small numbers of latently infected cells in individuals treated during hyperacute infection may be associated with prolonged ART-free remission.

No evidence of HIV replication in children on antiretroviral therapy.

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.

Single-cell analysis of HIV-1 transcriptional activity reveals expression of proviruses in expanded clones during ART.

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.

Proviruses with identical sequences comprise a large fraction of the replication-competent HIV reservoir.

The major obstacle to curing HIV infection is the persistence of cells with intact proviruses that can produce replication-competent virus. This HIV reservoir is believed to exist primarily in CD4+ T-cells and is stable despite years of suppressive antiretroviral therapy. A potential mechanism for HIV persistence is clonal expansion of infected cells, but how often such clones carry replication-competent proviruses has been controversial. Here, we used single-genome sequencing to probe for identical HIV sequence matches among viruses recovered in different viral outgrowth cultures and between the sequences of outgrowth viruses and proviral or intracellular HIV RNA sequences in uncultured blood mononuclear cells from eight donors on suppressive ART with diverse proviral populations. All eight donors had viral outgrowth virus that was fully susceptible to their current ART drug regimen. Six of eight donors studied had identical near full-length HIV RNA sequences recovered from different viral outgrowth cultures, and one of the two remaining donors had identical partial viral sequence matches between outgrowth virus and intracellular HIV RNA. These findings provide evidence that clonal expansion of HIV-infected cells is an important mechanism of reservoir persistence that should be targeted to cure HIV infection.

Clonally expanded CD4+ T cells can produce infectious HIV-1 in vivo.

Reservoirs of infectious HIV-1 persist despite years of combination antiretroviral therapy and make curing HIV-1 infections a major challenge. Most of the proviral DNA resides in CD4(+)T cells. Some of these CD4(+)T cells are clonally expanded; most of the proviruses are defective. It is not known if any of the clonally expanded cells carry replication-competent proviruses. We report that a highly expanded CD4(+) T-cell clone contains an intact provirus. The highly expanded clone produced infectious virus that was detected as persistent plasma viremia during cART in an HIV-1-infected patient who had squamous cell cancer. Cells containing the intact provirus were widely distributed and significantly enriched in cancer metastases. These results show that clonally expanded CD4(+)T cells can be a reservoir of infectious HIV-1.

Therapeutic Vaccination With Dendritic Cells Loaded With Autologous HIV Type 1-Infected Apoptotic Cells.

BACKGROUND: We report the results of a phase I/II, open-label, single-arm clinical trial to evaluate the safety and anti-human immunodeficiency virus type 1 (HIV-1) efficacy of an autologous dendritic cell (DC)-based HIV-1 vaccine loaded with autologous HIV-1-infected apoptotic cells. METHODS: Antiretroviral therapy (ART)-naive individuals were enrolled, and viremia was suppressed by ART prior to delivery of 4 doses of DC-based vaccine. Participants underwent treatment interruption 6 weeks after the third vaccine dose. The plasma HIV-1 RNA level 12 weeks after treatment interruption was compared to the pre-ART (ie, baseline) level. RESULTS: The vaccine was safe and well tolerated but did not prevent viral rebound during treatment interruption. Vaccination resulted in a modest but significant decrease in plasma viremia from the baseline level (from 4.53 log10 copies/mL to 4.27 log10 copies/mL;P= .05). Four of 10 participants had a >0.70 log10 increase in the HIV-1 RNA load in plasma following vaccination, despite continuous ART. Single-molecule sequencing of HIV-1 RNA in plasma before and after vaccination revealed increases in G>A hypermutants in gag and pol after vaccination, which suggests cytolysis of infected cells. CONCLUSIONS: A therapeutic HIV-1 vaccine based on DCs loaded with apoptotic bodies was safe and induced T-cell activation and cytolysis, including HIV-1-infected cells, in a subset of study participants. CLINICAL TRIALS REGISTRATION: NCT00510497.

Well-mixed plasma and tissue viral populations in RT-SHIV-infected macaques implies a lack of viral replication in the tissues during antiretroviral therapy.

BACKGROUND: Determining the anatomic compartments that contribute to plasma HIV-1 is critical to understanding the sources of residual viremia during combination antiretroviral therapy (ART). We analyzed viral DNA and RNA populations in the plasma and tissues from macaques infected with SIV containing HIV-1 RT (RT-SHIV) to identify possible sources of persistent viremia and to investigate the effect of ART on viral replication in tissues. Tissues were collected at necropsy from four pigtailed macaques infected for 30 weeks with a diverse population of RT-SHIV. Two animals (6760 and 8232) were untreated and two animals (8030 and 8272) were treated with efavirenz, tenofovir, and emtricitabine for 20 weeks. RESULTS: A total of 1800 single-genome RT-SHIV pol and env DNA and RNA sequences were analyzed from the plasma, PBMCs, axillary and mesenteric lymph nodes, spleen, thymus, small intestine, bone marrow, lung, and brain. Analyses of intracellular DNA and RNA populations revealed that the majority of proviruses in tissues from untreated animal 8232 were not expressed, whereas a greater proportion of proviruses in tissues were expressed from 6760. Few intracellular RNA sequences were detected in treated animals and most contained inactivating mutations, such as frame shifts or large deletions. Phylogenetics showed that RT-SHIV DNA populations in tissues were not different from virus in contemporary plasma samples in the treated or untreated animals, demonstrating a lack of anatomic compartmentalization and suggesting that plasma viremia is derived from multiple tissue sources. No sequence divergence was detected in the plasma or between tissues in the treated animals after 20 weeks of ART indicating a lack of ongoing replication in tissues during treatment. CONCLUSIONS: Virus populations in plasma and tissues did not differ significantly in either treated or untreated macaques, suggesting frequent exchange of virus or infected cells between tissues and plasma, consistent with non-compartmentalized and widely disseminated infection. There was no genetic evidence of ongoing replication in tissues during suppressive ART.

PAPNC, a novel method to calculate nucleotide diversity from large scale next generation sequencing data.

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 R(2) 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.

Histone deacetylase inhibitor romidepsin induces HIV expression in CD4 T cells from patients on suppressive antiretroviral therapy at concentrations achieved by clinical dosing.

Persistent latent reservoir of replication-competent proviruses in memory CD4 T cells is a major obstacle to curing HIV infection. Pharmacological activation of HIV expression in latently infected cells is being explored as one of the strategies to deplete the latent HIV reservoir. In this study, we characterized the ability of romidepsin (RMD), a histone deacetylase inhibitor approved for the treatment of T-cell lymphomas, to activate the expression of latent HIV. In an in vitro T-cell model of HIV latency, RMD was the most potent inducer of HIV (EC50 = 4.5 nM) compared with vorinostat (VOR; EC50 = 3,950 nM) and other histone deacetylase (HDAC) inhibitors in clinical development including panobinostat (PNB; EC50 = 10 nM). The HIV induction potencies of RMD, VOR, and PNB paralleled their inhibitory activities against multiple human HDAC isoenzymes. In both resting and memory CD4 T cells isolated from HIV-infected patients on suppressive combination antiretroviral therapy (cART), a 4-hour exposure to 40 nM RMD induced a mean 6-fold increase in intracellular HIV RNA levels, whereas a 24-hour treatment with 1 µM VOR resulted in 2- to 3-fold increases. RMD-induced intracellular HIV RNA expression persisted for 48 hours and correlated with sustained inhibition of cell-associated HDAC activity. By comparison, the induction of HIV RNA by VOR and PNB was transient and diminished after 24 hours. RMD also increased levels of extracellular HIV RNA and virions from both memory and resting CD4 T-cell cultures. The activation of HIV expression was observed at RMD concentrations below the drug plasma levels achieved by doses used in patients treated for T-cell lymphomas. In conclusion, RMD induces HIV expression ex vivo at concentrations that can be achieved clinically, indicating that the drug may reactivate latent HIV in patients on suppressive cART.

Lack of detectable HIV-1 molecular evolution during suppressive antiretroviral therapy.

A better understanding of changes in HIV-1 population genetics with combination antiretroviral therapy (cART) is critical for designing eradication strategies. We therefore analyzed HIV-1 genetic variation and divergence in patients' plasma before cART, during suppression on cART, and after viral rebound. Single-genome sequences of plasma HIV-1 RNA were obtained from HIV-1 infected patients prior to cART (N = 14), during suppression on cART (N = 14) and/or after viral rebound following interruption of cART (N = 5). Intra-patient population diversity was measured by average pairwise difference (APD). Population structure was assessed by phylogenetic analyses and a test for panmixia. Measurements of intra-population diversity revealed no significant loss of overall genetic variation in patients treated for up to 15 years with cART. A test for panmixia, however, showed significant changes in population structure in 2/10 patients after short-term cART (<1 year) and in 7/10 patients after long-term cART (1-15 years). The changes consisted of diverse sets of viral variants prior to cART shifting to populations containing one or more genetically uniform subpopulations during cART. Despite these significant changes in population structure, rebound virus after long-term cART had little divergence from pretherapy virus, implicating long-lived cells infected before cART as the source for rebound virus. The appearance of genetically uniform virus populations and the lack of divergence after prolonged cART and cART interruption provide strong evidence that HIV-1 persists in long-lived cells infected before cART was initiated, that some of these infected cells may be capable of proliferation, and that on-going cycles of viral replication are not evident.

Analysis of 454 sequencing error rate, error sources, and artifact recombination for detection of Low-frequency drug resistance mutations in HIV-1 DNA.

BACKGROUND: 454 sequencing technology is a promising approach for characterizing HIV-1 populations and for identifying low frequency mutations. The utility of 454 technology for determining allele frequencies and linkage associations in HIV infected individuals has not been extensively investigated. We evaluated the performance of 454 sequencing for characterizing HIV populations with defined allele frequencies. RESULTS: We constructed two HIV-1 RT clones. Clone A was a wild type sequence. Clone B was identical to clone A except it contained 13 introduced drug resistant mutations. The clones were mixed at ratios ranging from 1% to 50% and were amplified by standard PCR conditions and by PCR conditions aimed at reducing PCR-based recombination. The products were sequenced using 454 pyrosequencing. Sequence analysis from standard PCR amplification revealed that 14% of all sequencing reads from a sample with a 50:50 mixture of wild type and mutant DNA were recombinants. The majority of the recombinants were the result of a single crossover event which can happen during PCR when the DNA polymerase terminates synthesis prematurely. The incompletely extended template then competes for primer sites in subsequent rounds of PCR. Although less often, a spectrum of other distinct crossover patterns was also detected. In addition, we observed point mutation errors ranging from 0.01% to 1.0% per base as well as indel (insertion and deletion) errors ranging from 0.02% to nearly 50%. The point errors (single nucleotide substitution errors) were mainly introduced during PCR while indels were the result of pyrosequencing. We then used new PCR conditions designed to reduce PCR-based recombination. Using these new conditions, the frequency of recombination was reduced 27-fold. The new conditions had no effect on point mutation errors. We found that 454 pyrosequencing was capable of identifying minority HIV-1 mutations at frequencies down to 0.1% at some nucleotide positions. CONCLUSION: Standard PCR amplification results in a high frequency of PCR-introduced recombination precluding its use for linkage analysis of HIV populations using 454 pyrosequencing. We designed a new PCR protocol that resulted in a much lower recombination frequency and provided a powerful technique for linkage analysis and haplotype determination in HIV-1 populations. Our analyses of 454 sequencing results also demonstrated that at some specific HIV-1 drug resistant sites, mutations can reliably be detected at frequencies down to 0.1%.

Multiple sources of contamination in samples from patients reported to have XMRV infection.

Xenotropic murine leukemia virus (MLV)-related retrovirus (XMRV) was reported to be associated with prostate cancer by Urisman, et al. in 2006 and chronic fatigue syndrome (CFS) by Lombardi, et al. in 2009. To investigate this association, we independently evaluated plasma samples from 4 patients with CFS reported by Lombardi, et al. to have XMRV infection and from 5 healthy controls reported to be XMRV uninfected. We also analyzed viral sequences obtained from supernatants of cell cultures found to contain XMRV after coculture with 9 clinical samples from 8 patients. A qPCR assay capable of distinguishing XMRV from endogenous MLVs showed that the viral sequences detected in the CFS patient plasma behaved like endogenous MLVs and not XMRV. Single-genome sequences (N = 89) from CFS patient plasma were indistinguishable from endogenous MLVs found in the mouse genome that are distinct from XMRV. By contrast, XMRV sequences were detected by qPCR in 2 of the 5 plasma samples from healthy controls (sequencing of the qPCR product confirmed XMRV not MLV). Single-genome sequences (N = 234) from the 9 culture supernatants reportedly positive for XMRV were indistinguishable from XMRV sequences obtained from 22Rv1 and XMRV-contaminated 293T cell-lines. These results indicate that MLV DNA detected in the plasma samples from CFS patients evaluated in this study was from contaminating mouse genomic DNA and that XMRV detected in plasma samples from healthy controls and in cultures of patient samples was due to cross-contamination with XMRV (virus or nucleic acid).

Amplifying and quantifying HIV-1 RNA in HIV infected individuals with viral loads below the limit of detection by standard clinical assays.

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) (13, 19) and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) (12, 20). The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate. The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive (3, 7, 12, 14, 15),is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination (19). However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

The human papillomavirus type 8 E2 tethering protein targets the ribosomal DNA loci of host mitotic chromosomes.

For many papillomaviruses, the viral protein E2 tethers the viral genome to the host mitotic chromosomes to ensure persistent, long-term maintenance of the genome during cell division. Our previous studies of E2 proteins from different genera of papillomaviruses have shown that they bind to different regions of the host chromosomes during mitosis. For example, bovine papillomavirus type 1 (BPV-1) E2 binds to all chromosomes as small speckles in complex with the cellular protein Brd4. In contrast, the human papillomavirus type 8 (HPV-8) E2 protein binds as large speckles at the pericentromeric regions of chromosomes. Here we show that these speckles do not contain Brd4, and unlike that of BPV-1, the N-terminal Brd4-interacting domain of HPV-8 E2 is not required for chromosome binding. In contrast to BPV-1 E2, the HPV-8 E2 protein targets the short arms of acrocentric mitotic chromosomes. Furthermore, the E2 protein interacts with the repeated ribosomal DNA genes found in this location and colocalizes with UBF, the RNA polymerase I transcription factor. Therefore, HPV-8 E2 genome tethering occurs by a Brd4-independent mechanism through a novel interaction with specific regions of mitotic chromosomes. Thus, a wide range of viruses have adopted the strategy of linking their genomes to host chromosomes, but individual viruses use different chromosomal targets. Characterization of these targets will enable the development of antiviral therapies to eliminate the viral genomes from infected cells.