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Defective proviruses rapidly accumulate during acute HIV-1 infection
 
 
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"To cure HIV, you want to get rid of the proviruses without defects," says senior study author Robert Siliciano, M.D., Ph.D., an infectious disease physician and molecular biologist at the Johns Hopkins University School of Medicine. "But our work shows that the standard assays used to do that are measuring forms of the virus that are not really relevant to these cure strategies."
 
"Although early ART initiation limits the size of the latent reservoir, it does not have a profound effect on the composition of proviral populations, with the vast majority of proviruses in both CP- and AP-treated adults containing defects.....these results have implications for assessing HIV-1 cure strategies. Because there is no correlation between the QVOA or DNA PCR and the number of intact proviruses, these assays cannot be used to accurately predict the true reservoir size, even for individuals who are treated early in infection.....Notably, because the nature of the defects described here indicates that many defective proviruses may not be eliminated by eradication strategies, defective proviruses could obscure the measurement of real changes in the rarer population of intact proviruses that must be eliminated to achieve a cure."
 
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Nature Medicine August 8 2016
 
Katherine M Bruner1, Alexandra J Murray1, Ross A Pollack1, Mary G Soliman1, Sarah B Laskey1, Adam A Capoferri1,2, Jun Lai1, Matthew C Strain3, Steven M Lada4, Rebecca Hoh5, Ya-Chi Ho1, Douglas D Richman3,4,6, Steven G Deeks5, Janet D Siliciano1 & Robert F Siliciano1,2
 
Although antiretroviral therapy (ART) suppresses viral replication to clinically undetectable levels, human immunodeficiency virus type 1 (HIV-1) persists in CD4+ T cells in a latent form that is not targeted by the immune system or by ART1, 2, 3, 4, 5. This latent reservoir is a major barrier to curing individuals of HIV-1 infection. Many individuals initiate ART during chronic infection, and in this setting, most proviruses are defective6. However, the dynamics of the accumulation and the persistence of defective proviruses during acute HIV-1 infection are largely unknown. Here we show that defective proviruses accumulate rapidly within the first few weeks of infection to make up over 93% of all proviruses, regardless of how early ART is initiated. By using an unbiased method to amplify near-full-length proviral genomes from HIV-1-infected adults treated at different stages of infection, we demonstrate that early initiation of ART limits the size of the reservoir but does not profoundly affect the proviral landscape. This analysis allows us to revise our understanding of the composition of proviral populations and estimate the true reservoir size in individuals who were treated early versus late in infection. Additionally, we demonstrate that common assays for measuring the reservoir do not correlate with reservoir size, as determined by the number of genetically intact proviruses. These findings reveal hurdles that must be overcome to successfully analyze future HIV-1 cure strategies.
 
HIV-1 establishes infection in CD4+ T cells1, 2, 7, generating a latent reservoir with an extremely long half-life (44 months) that necessitates life-long treatment4, 8. Efforts to eradicate this reservoir include the 'shock-and-kill' approach in which latent proviruses are induced so that infected cells can be eliminated9, 10, 11. The latent reservoir was initially defined by using a quantitative viral outgrowth assay (QVOA)1, 4, 8, 12, 13. PCR can also detect proviral DNA14, 15, 16; however, the QVOA and PCR assays correlate poorly17. We recently examined discrepancies between the assays by sequencing proviruses containing the gag gene from QVOA wells that were negative for viral outgrowth6. Most proviruses were defective, but 12% were intact, and some of these could be reactivated following a second round of T cell activation6. However, these studies were limited to individuals who initiated ART late in the course of infection and only analyzed gag+ proviruses in cells that had been expanded for weeks in culture6, which may have altered the proportions of different proviral populations. Because immediate ART is now recommended for all infected individuals18 and the size (and perhaps other characteristics) of the reservoir differs in individuals who are treated early versus late in infection19, 20, 21, we sought to define the composition of proviral populations in individuals who were treated during acute infection. As cure efforts advance, it is essential to understand how defective proviruses accumulate, persist in individuals and affect reservoir assay measurements. Therefore, we conducted a proviral analysis on unmanipulated samples from HIV-1-infected adults who were treated at different stages of infection.
 
We developed a novel, unbiased single-genome-amplification method that captures intact and defective proviruses. Because deletions probably occur during minus strand synthesis before the second strand transfer event of reverse transcription, primers were designed to capture deletions arising during this process (Fig. 1a)22, 23, 24. PCRs were performed at limiting dilution to prevent in vitro recombination and competition between short and long templates. Using a nested PCR approach, we performed a near-full-length outer PCR followed by inner PCRs for the gag and env genes to confirm clonality. Next, six overlapping inner PCRs were performed on all of the wells at limiting dilution (Fig. 1b). The PCR products were directly sequenced to minimize PCR-induced error.
 
We examined proviral DNA in purified resting CD4+ T cells from ten subjects who initiated ART during the chronic phase of infection (CP; ART started >180 d after infection) (Supplementary Table 1). Analysis was performed on freshly isolated cells to prevent bias from in vitro expansion. Notably, 98% of proviruses were defective (Fig. 1c,d and Supplementary Fig. 1). The most common defects were internal deletions (80%), which varied in size (15 bp to ~8 kb) and in location in the genome. Some proviruses had 3′ deletions affecting the env, tat, rev and nef genes. We also identified proviruses with 5′ deletions affecting the gag and pol genes, as well as others with very large deletions (>6 kb) that encompassed most of the HIV-1 genes (Fig. 1c,d). The 5′ deletions and the very large deletions, which represent 40% of all sequences, were not identified in a previous screen because they contain deletions in the gag gene6. Proviruses with small (15- to 97-bp) deletions at the packaging signal and the major splice donor site represented 5% of sequences (Fig. 1d). These proviruses are probably replication defective owing to a failure to correctly make spliced HIV-1 RNAs or to package genomes into virions6. Some clones contained multiple deletions, suggesting multiple template-switching events during reverse transcription (Fig. 1c), and others contained sequence inversions or insertions (Fig. 1d). For each type of deletion, proviruses with sequence homology at the deletion junctions were found (Fig. 1e), consistent with the idea of template switching during reverse transcription25. In control experiments, plasmids containing reference (NL4-3) or patient-derived proviruses were diluted into human genomic DNA and amplified by the same procedure with no defects observed (data not shown). Additionally, analysis of the deletions showed nonrandom distribution within the HIV-1 genome, with distinct hotspots (Supplementary Fig. 2), and we observed identical deletions in independent amplifications from the same subject, reflecting clonal expansion of infected cells (see below). Taken together, these results indicate that the deletions we observed occurred in vivo and did not result from PCR recombination. We also identified 7% of proviruses with guanine-to-adenine (G-to-A) hypermutations that were induced by the cytidine deaminases APOBEC3F and APOBEC3G, which are HIV-1 restriction factors. An additional 8% of proviruses contained both deletions and hypermutation, indicating that these processes can occur together during reverse transcription (Fig. 1d). Almost all of the hypermutated proviruses contained mutated start codons in the gag, gag–pol and nef open-reading frames (ORFs) due to the presence of an obligatory position 2 glycine codon which creates the consensus recognition site for APOBEC3G, ATGGGT26, 27. Additionally, all of the hypermutated sequences had multiple internal stop codons in the larger ORFs (gag, gag–pol, env and nef), and only a small fraction of hypermutated sequences could make functional gene products (Supplementary Fig. 3). These defects and common deletions affecting key viral ORFs (Fig. 1c,d) probably prevent many defective proviruses from being eliminated by eradication strategies that depend on viral protein expression. All of the subjects had undetectable viral loads (<50 copies/ml) for >8 months before sampling, and all but one had low frequencies of 2–long terminal repeat (2-LTR) circles in cells (<25 copies per 106 resting CD4+ T cells), which represented less than 8% of the total number of proviruses (Supplementary Fig. 4a,b). The labile nature of linear unintegrated HIV-1 DNA and the low level of 2-LTR circles suggest that the majority of sequences examined were integrated proviruses28. Of 152 near-full-length sequences examined, only three sequences (2%) were intact. These data indicate that defective proviruses are much more common than previously shown (Fig. 1d).
 
Because the dynamics of proviral accumulation remain unclear, we sought to determine how rapidly defective proviruses accumulate. We examined the proviral populations in subjects who were treated during the acute or early phase of infection (AP; ART started within 100 d in all subjects and within 60 d in eight of nine subjects) (Supplementary Table 1). Unexpectedly, only 7% of proviruses were intact, even in subjects 2453, 2454, 3693 and 2443, who initiated ART within the first few weeks of infection (Fig. 2 and Supplementary Fig. 5). Although the number of individuals subjected to this extensive analysis was small, the results were very consistent between subjects. The remaining proviral sequences contained defects similar to those in CP-treated subjects (Fig. 2a,d), and there was no significant difference in the fraction of intact proviruses as compared to that in CP-treated subjects (Supplementary Fig. 6a). AP-treated subjects had a higher fraction of hypermutated sequences (35% versus 14%; P = 0.0036; Supplementary Fig. 6b) and a significantly lower fraction of deleted sequences (57% versus 82%; P = 0.0028; Supplementary Fig. 6c), suggesting that hypermutation is particularly important during acute infection, perhaps due to upregulation of APOBEC3G and APOBEC3F by type I interferons29, 30, 31. Notably, defects were even detectable in a single round of in vitro infection (41% of sequences; Fig. 2b,d). Consistent with this result, we readily detected defective proviruses in unfractionated CD4+ T cells from subjects with viremia who are in the chronic phase of infection, representing 65% of sequences (Fig. 2c,d). In these subjects, the provirus populations include both proviruses in newly infected cells and archived proviruses in the reservoir. These results indicate that defective proviruses are probably generated during the initial rounds of replication after transmission, as defective proviruses are generated at a high frequency from the process of reverse transcription (Fig. 2b). Because many of the proviruses identified have defects that would preclude high-level expression of viral genes, cells carrying these proviruses would presumably be less susceptible to elimination through viral cytopathic effects or through lysis by cytotoxic T lymphocytes. Thus, even individuals treated very early after infection have large numbers of defective proviruses. Taken together, these results demonstrate that defective proviruses arise commonly, accumulate rapidly within 2–3 weeks after infection and persist in vivo.
 
Proliferation of cells carrying replication-competent proviruses could be a major barrier to eradication of the virus32, 33, 34. Integration-site analysis, pioneered by Schröder et al.35, has provided critical evidence for the proliferation of infected cells in vivo but does not reveal whether the proviruses are replication competent33, 34, 36. Our unbiased analysis allowed us to detect expanded cellular clones in vivo and evaluate the presence of defects. Among 312 sequences from nine AP-treated (Fig. 3a) and ten CP-treated subjects (Fig. 3b), 38 were from expanded cellular clones that were identified to be identical sequences arising from independent amplifications. Notably, all of these expanded clones contained gross defects that preclude replication. In subjects 2529, 2609, CP03, CP05 and CP10, expanded clones represented over 25% of all sequences (Fig. 3c,d). Subject 2521 contained an expanded clone that represented 11% of all proviral sequences (Fig. 3a,c) after just 17 months of infection and 8 months on suppressive ART, indicating that expanded clones do not require years to accumulate to large proportions. Our results indicate that the majority of expanded clones are defective. However, considering that <7% of proviruses are intact, rare expanded clones may carry replication-competent virus and contribute to HIV-1 persistence, as was seen in a recent clinical case study37. Although cells harboring defective proviruses can undergo clonal expansion with minimal consequences to the host cells, cells harboring replication-competent viruses could presumably expand by either activation of the cell but not the provirus38 or by proliferation and survival of the cells despite viral gene expression and virion production.
 
Our analyses show that the fraction of intact proviruses is much lower than originally thought, even in individuals who were treated early. We have previously shown that intact proviruses replicate well in vitro when reconstructed6. Furthermore, when wells that were negative for viral outgrowth in the QVOA were reactivated, additional replication-competent virus was isolated6. Taken together these data suggest that some, if not all, of these intact proviruses are competent for viral replication and that the number of intact proviruses is probably the closest estimate to the true size of the latent reservoir. Given these results, we sought to more accurately estimate the true reservoir size in AP- and CP-treated individuals, as defined by the number of intact proviruses, and compare that result to current assays, such as the QVOA and DNA PCR. We used a validated droplet digital method to measure proviral DNA with gag primers14, 39 (gag+ proviruses) and corrected for proviruses that were deleted in gag (total number of infected cells). The DNA PCR assay gave frequencies of infected cells that were dramatically higher than the frequency of cells with intact proviruses (P < 0.0001 for CP- or AP-treated subjects). The QVOA gave frequencies of infected cells that were dramatically lower than the frequency of cells with intact proviruses (P < 0.001 for CP-treated subjects; P < 0.01 for AP-treated subjects) (Fig. 4a,b). We found that the QVOA potentially underestimates the latent reservoir by a median of 27-fold in CP-treated subjects and 25-fold in AP-treated subjects (Fig. 4c), whereas DNA PCR overestimated the reservoir size by a median of 188-fold in CP-treated subjects and 13-fold in AP-treated subjects (Fig. 4c). Notably, there was no correlation between the number of intact proviruses and either the QVOA or the DNA PCR results (Supplementary Fig. 7a–c). Additionally, the relationships between the QVOA, the number of intact proviruses and proviral DNA varied greatly from person to person in both CP-treated subjects (Fig. 4d and Supplementary Fig. 7d) and AP-treated subjects (Fig. 4e and Supplementary Fig. 7e). This precludes the use of either the QVOA or DNA PCR as a surrogate marker for the number of intact proviruses, which is probably the closest estimate of the true size of the latent reservoir.
 
This unbiased screen demonstrates that the fraction of intact proviruses is considerably smaller than was previously shown and that defects accumulate as early as 2–3 weeks after infection. Although unexpected, the rapid accumulation is consistent with the observation that defects can be observed in 40% of proviruses generated in a single-round in vitro infection (Fig. 2d) and the fact that cells harboring defective proviruses may be less susceptible to immune clearance or cytopathic effects. With these new data, we were able to revise our estimates of the reservoir size in both CP- and AP-treated individuals. If all of the intact proviruses could be induced in vivo, then the true size of the latent reservoir would be, on average, 12 infectious proviruses per million resting CD4+ T cells (median of AP-treated) in individuals who were treated in the acute or early phase of infection and 37 infectious proviruses per million resting CD4+ T cells (median of CP-treated) in individuals who were treated in the chronic phase of infection, with substantial person-to-person variation. Furthermore, our results change our understanding of the effect of early ART on the proviral landscape. Although early ART initiation limits the size of the latent reservoir, it does not have a profound effect on the composition of proviral populations, with the vast majority of proviruses in both CP- and AP-treated adults containing defects. Finally, these results have implications for assessing HIV-1 cure strategies. Because there is no correlation between the QVOA or DNA PCR and the number of intact proviruses, these assays cannot be used to accurately predict the true reservoir size, even for individuals who are treated early in infection. Indeed, any analysis of subgenomic regions of proviruses to evaluate viral evolution or reservoir reduction must be reconsidered in this context, as the majority of sequences studied will be from defective proviruses40. Notably, because the nature of the defects described here indicates that many defective proviruses may not be eliminated by eradication strategies, defective proviruses could obscure the measurement of real changes in the rarer population of intact proviruses that must be eliminated to achieve a cure.

 
 
 
 
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