Proviral Genome Research Articles

Presence of a Shared 5'-Leader Sequence in Ancestral Human and Mammalian Retroviruses and Its Transduction into Feline Leukemia Virus.

Recombination events induce significant genetic changes, and this process can result in virus genetic diversity or in the generation of novel pathogenicity. We discovered a new recombinant feline leukemia virus (FeLV) gag gene harboring an unrelated insertion, termed the X region, which was derived from Felis catus endogenous gammaretrovirus 4 (FcERV-gamma4). The identified FcERV-gamma4 proviruses have lost their coding capabilities, but some can express their viral RNA in feline tissues. Although the X-region-carrying recombinant FeLVs appeared to be replication-defective viruses, they were detected in 6.4% of tested FeLV-infected cats. All isolated recombinant FeLV clones commonly incorporated a middle part of the FcERV-gamma4 5'-leader region as an X region. Surprisingly, a sequence corresponding to the portion contained in all X regions is also present in at least 13 endogenous retroviruses (ERVs) observed in the cat, human, primate, and pig genomes. We termed this shared genetic feature the commonly shared (CS) sequence. Despite our phylogenetic analysis indicating that all CS-sequence-carrying ERVs are classified as gammaretroviruses, no obvious closeness was revealed among these ERVs. However, the Shannon entropy in the CS sequence was lower than that in other parts of the provirus genome. Notably, the CS sequence of human endogenous retrovirus T had 73.8% similarity with that of FcERV-gamma4, and specific signals were detected in the human genome by Southern blot analysis using a probe for the FcERV-gamma4 CS sequence. Our results provide an interesting evolutionary history for CS-sequence circulation among several distinct ancestral viruses and a novel recombined virus over a prolonged period.IMPORTANCE Recombination among ERVs or modern viral genomes causes a rapid evolution of retroviruses, and this phenomenon can result in the serious situation of viral disease reemergence. We identified a novel recombinant FeLV gag gene that contains an unrelated sequence, termed the X region. This region originated from the 5' leader of FcERV-gamma4, a replication-incompetent feline ERV. Surprisingly, a sequence corresponding to the X region is also present in the 5' portion of other ERVs, including human endogenous retroviruses. Scattered copies of the ERVs carrying the unique genetic feature, here named the commonly shared (CS) sequence, were found in each host genome, suggesting that ancestral viruses may have captured and maintained the CS sequence. More recently, a novel recombinant FeLV hijacked the CS sequence from inactivated FcERV-gamma4 as the X region. Therefore, tracing the CS sequences can provide unique models for not only the modern reservoir of new recombinant viruses but also the genetic features shared among ancient retroviruses.

Differentiating Immune Cell Targets in Gut-Associated Lymphoid Tissue for HIV Cure.

The single greatest challenge to an HIV cure is the persistence of latently infected cells containing inducible, replication-competent proviral genomes, which constitute only a small fraction of total or infected cells in the body. Although resting CD4+ T cells in the blood are a well-known source of viral rebound, more than 90% of the body's lymphocytes reside elsewhere. Many are in gut tissue, where HIV DNA levels per million CD4+ T cells are considerably higher than in the blood. Despite the significant contribution of gut tissue to viral replication and persistence, little is known about the cell types that support persistence of HIV in the gut; importantly, T cells in the gut have phenotypic, functional, and survival properties that are distinct from T cells in other tissues. The mechanisms by which latency is established and maintained will likely depend on the location and cytokine milieu surrounding the latently infected cells in each compartment. Therefore, successful HIV cure strategies require identification and characterization of the exact cell types that support viral persistence, particularly in the gut. In this review, we describe the seeding of the latent HIV reservoir in the gut mucosa; highlight the evidence for compartmentalization and depletion of T cells; summarize the immunologic consequences of HIV infection within the gut milieu; propose how the damaged gut environment may promote the latent HIV reservoir; and explore several immune cell targets in the gut and their place on the path toward HIV cure.

CD4 is expressed on a heterogeneous subset of hematopoietic progenitors, which persistently harbor CXCR4 and CCR5-tropic HIV proviral genomes in vivo.

Latent HIV infection of long-lived cells is a barrier to viral clearance. Hematopoietic stem and progenitor cells are a heterogeneous population of cells, some of which are long-lived. CXCR4-tropic HIVs infect a broad range of HSPC subtypes, including hematopoietic stem cells, which are multi-potent and long-lived. However, CCR5-tropic HIV infection is limited to more differentiated progenitor cells with life spans that are less well understood. Consistent with emerging data that restricted progenitor cells can be long-lived, we detected persistent HIV in restricted HSPC populations from optimally treated people. Further, genotypic and phenotypic analysis of amplified env alleles from donor samples indicated that both CXCR4- and CCR5-tropic viruses persisted in HSPCs. RNA profiling confirmed expression of HIV receptor RNA in a pattern that was consistent with in vitro and in vivo results. In addition, we characterized a CD4high HSPC sub-population that was preferentially targeted by a variety of CXCR4- and CCR5-tropic HIVs in vitro. Finally, we present strong evidence that HIV proviral genomes of both tropisms can be transmitted to CD4-negative daughter cells of multiple lineages in vivo. In some cases, the transmitted proviral genomes contained signature deletions that inactivated the virus, eliminating the possibility that coincidental infection explains the results. These data support a model in which both stem and non-stem cell progenitors serve as persistent reservoirs for CXCR4- and CCR5-tropic HIV proviral genomes that can be passed to daughter cells.

Complete genome sequencing and characterization revealed a recombinant subgroup B isolate of avian leukosis virus with a subgroup J-like U3 region

One natural recombinant subgroup B avian leukosis virus (ALV) with a subgroup J-like U3 region was isolated from commercial native chickens that experienced disease in 2014 and named GX14FF03. GX14FF03 was isolated by DF-1 cell culture and then identified with ELISA detection of avian leukosis virus p27 group-specific antigen, the detection of subtype specific PCR, and indirect immunofluorescence assay with ALV-B-specific monoclonal antibody. Its complete proviral genome was sequenced and compared with the reference strains of ALVs and found that the gag and pol were relatively conservative. The gp85 of GX14FF03 showed 91.3-96.2% amino acid identity to the other ALV-B reference strains and 36.0-37.1% identity to the ALV-J reference strains, and its U3 region showed 49.4-89.3% nucleotide identity to ALV-A, B, C, D, E, K reference strains and 91.6-95.3% identity to ALV-J reference strains. Phylogenetic analysis of U3 region showed that GX14FF03 and ALV-J reference strains were in the same cluster. Moreover, an additional AIB REP1 retroviral transcription regulatory element was found in GX14FF04 U3 region which was only presenting in ALV-J strains. These results suggested that isolate GX14FF03 may be a recombinant ALV-B with the ALV-J-like U3 region.

Proviral Latency, Persistent Human Immunodeficiency Virus Infection, and the Development of Latency Reversing Agents.

Quiescent proviral genomes that persist during human immunodeficiency virus type 1 (HIV-1) infection despite effective antiretroviral therapy (ART) can fuel rebound viremia after ART interruption and is a central obstacle to the cure of HIV infection. The induction of quiescent provirus is the goal of a new class of potential therapeutics, latency reversing agents (LRAs). The discovery, development, and testing of HIV LRAs is a key part of current efforts to develop latency reversal and viral clearance strategies to eradicate established HIV infection. The development of LRAs is burdened by many uncertainties that make drug discovery difficult. The biology of HIV latency is complex and incompletely understood. Potential targets for LRAs are host factors, and the potential toxicities of host-directed therapies in individuals that are otherwise clinically stable may be unacceptable. Assays to measure latency reversal and assess the effectiveness of potential therapeutics are complex and incompletely validated. Despite these obstacles, novel LRAs are under development and beginning to enter combination testing with viral clearance strategies. It is hoped that the steady advances in the development of LRAs now being paired with emerging immunotherapeutics to clear persistently infected cells will soon allow measurable clinical advances toward an HIV cure.

Paucity of Intact Non-Induced Provirus with Early, Long-Term Antiretroviral Therapy of Perinatal HIV Infection.

The latent reservoir is a major barrier to HIV eradication. Reservoir size is emerging as an important biomarker to assess the likelihood of HIV remission in the absence of antiretroviral therapy (ART) and may be reduced by earlier initiation of ART that restricts HIV spread into CD4+ T cells. Reservoir size is traditionally measured with a quantitative viral outgrowth assay (QVOA) that induces replication-competent HIV production through in vitro stimulation of resting CD4+ T cells. However, the recent identification of replication-intact, non-induced proviral genomes (NIPG) suggests the QVOA significantly underestimates (by 62-fold) latent reservoir size in chronically-infected adults. Whether formation and persistence of Intact, NIPG is thwarted by early ART initiation and long-term virologic suppression in perinatal infection is unclear. Here, we show that the latent reservoir in 11 early treated, long-term suppressed perinatally infected children and adolescents was not inducible by QVOA and dominated by defective, NIPG. Single genome analysis of 164 NIPG from 232 million cultured resting CD4+ T cells revealed no replication-intact, near-full length sequences. Forty-three (26%) NIPG contained APOBEC3G-mediated hypermutation, 115 (70%) NIPG contained large internal deletions, one NIPG contained nonsense mutations and indels, and 5 (3%) NIPG were assigned as “Not Evaluable” due to multiple failed sequencing attempts that precluded further classification. The lack of replication competent inducible provirus and intact NIPG in this cohort indicate early, long-term ART of perinatal infection leads to marked diminution of replication-competent HIV-1 reservoirs, creating a favorable state towards interventions aimed at virologic remission.

A Novel Human T-lymphotropic Virus Type 1c Molecular Variant in an Indigenous Individual from New Caledonia, Melanesia.

BackgroundHuman T-Lymphotropic Virus type 1 (HTLV-1) is endemic among people of Melanesian descent in Papua New Guinea, Solomon Islands and Vanuatu, and in Indigenous populations from Central Australia. Molecular studies revealed that these Australo-Melanesian strains constitute the highly divergent HTLV-1c subtype. New Caledonia is a French overseas territory located in the Southwest Pacific Ocean. HTLV-1 situation is poorly documented in New Caledonia and the molecular epidemiology of HTLV-1 infection remains unknown.ObjectivesStudying 500 older adults Melanesian natives from New Caledonia, we aim to evaluate the HTLV-1 seroprevalence and to molecularly characterize HTLV-1 proviral strains.Study designPlasma from 262 men and 238 females (age range: 60–96 years old, mean age: 70.5) were screened for anti-HTLV-1 antibodies by particle agglutination (PA) and indirect immunofluorescence assay (IFA). Serological confirmation was obtained using Western blot assay. DNAs were extracted from peripheral blood buffy coat of HTLV-1 seropositive individuals, and subjected to four series of PCR (LTR-gag; pro-pol; pol-env and tax-LTR). Primers were designed from highly common conserved regions of the major HTLV-1 subtypes to characterize the entire HTLV-1 proviral genome.ResultsAmong 500 samples, 3 were PA and IFA positive. The overall seroprevalence was 0.6%. The DNA sample from 1 New Caledonian woman (NCP201) was found positive by PCR and the complete HTLV-1 proviral genome (9,033-bp) was obtained. The full-length HTLV-1 genomic sequence from a native woman from Vanuatu (EM5), obtained in the frame of our previous studies, was also characterized. Both sequences belonged to the HTLV-1c Australo-Melanesian subtype. The NCP201 strain exhibited 0.3% nucleotide divergence with the EM5 strain from Vanuatu. Furthermore, divergence reached 1.1% to 2.9% with the Solomon and Australian sequences respectively. Phylogenetic analyses on a 522-bp-long fragment of the gp21-env gene showed the existence of two major clades. The first is composed of strains from Papua New Guinea; the second includes strains from all neighboring archipelagos (Solomon, Vanuatu, New Caledonia), and Australia. Interestingly, this second clade itself is divided into two sub-clades: strains from Australia on one hand, and strains from Solomon Islands, Vanuatu and New Caledonia on the other hand.ConclusionsThe HTLV-1 seroprevalence (0.6%) in the studied adult population from New Caledonia appears to be low. This seroprevalence is quite similar to the situation observed in Vanuatu and Solomon Islands. However it is very different to the one encountered in Central Australia. Taken together, these results demonstrated that Australo-Melanesia is endemic for HTLV-1 infection with a high diversity of HTLV-1c strains and a clear geographic clustering according to the island of origin of HTLV-1 infected persons.

First Complete Genome Sequence of a Simian Foamy Virus Isolate from a Cynomolgus Macaque.

We report here the first complete proviral genome sequence (DDBJ/ENA/GenBank accession no. LC094267) of a simian foamy virus, SFVmfa/Cy5061, isolated from a cynomolgus macaque (Macaca fascicularis). This proviral genome consists of 12,965 nucleotides and has five open reading frames, gag, pol, env, tas, and bet, as with other foamy viruses.

Concomitant emergence of the antisense protein gene of HIV-1 and of the pandemic

Recent experiments provide sound arguments in favor of the in vivo expression of the AntiSense Protein (ASP) of HIV-1. This putative protein is encoded on the antisense strand of the provirus genome and entirely overlapped by the env gene with reading frame -2. The existence of ASP was suggested in 1988, but is still controversial, and its function has yet to be determined. We used a large dataset of ∼23,000 HIV-1 and SIV sequences to study the origin, evolution, and conservation of the asp gene. We found that the ASP ORF is specific to group M of HIV-1, which is responsible for the human pandemic. Moreover, the correlation between the presence of asp and the prevalence of HIV-1 groups and M subtypes appeared to be statistically significant. We then looked for evidence of selection pressure acting on asp Using computer simulations, we showed that the conservation of the ASP ORF in the group M could not be due to chance. Standard methods were ineffective in disentangling the two selection pressures imposed by both the Env and ASP proteins-an expected outcome with overlaps in frame -2. We thus developed a method based on careful evolutionary analysis of the presence/absence of stop codons, revealing that ASP does impose significant selection pressure. All of these results support the idea that asp is the 10th gene of HIV-1 group M and indicate a correlation with the spread of the pandemic.

Sources of Error in Mammalian Genetic Screens.

Genetic screens are invaluable tools for dissection of biological phenomena. Optimization of such screens to enhance discovery of candidate genes and minimize false positives is thus a critical aim. Here, we report several sources of error common to pooled genetic screening techniques used in mammalian cell culture systems, and demonstrate methods to eliminate these errors. We find that reverse transcriptase-mediated recombination during retroviral replication can lead to uncoupling of molecular tags, such as DNA barcodes (BCs), from their associated library elements, leading to chimeric proviral genomes in which BCs are paired to incorrect ORFs, shRNAs, etc. This effect depends on the length of homologous sequence between unique elements, and can be minimized with careful vector design. Furthermore, we report that residual plasmid DNA from viral packaging procedures can contaminate transduced cells. These plasmids serve as additional copies of the PCR template during library amplification, resulting in substantial inaccuracies in measurement of initial reference populations for screen normalization. The overabundance of template in some samples causes an imbalance between PCR cycles of contaminated and uncontaminated samples, which results in a systematic artifactual depletion of GC-rich library elements. Elimination of contaminating plasmid DNA using the bacterial endonuclease Benzonase can restore faithful measurements of template abundance and minimize GC bias.

Defective proviruses rapidly accumulate during acute HIV-1 infection.

Although antiretroviral therapy (ART) suppresses viral replication to clinically undetectable levels, HIV-1 persists in CD4+ T cells in a latent form not targeted by the immune system or ART1–5. This latent reservoir is a major barrier to cure. Many individuals initiate ART during chronic infection, and in this setting, most proviruses are defective6. However, the dynamics of the accumulation and 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. 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 ART initiation limits the size of the reservoir but does not profoundly impact 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 treated early vs. late in infection. Additionally, we demonstrate that common assays for measuring the reservoir do not correlate with reservoir size. These findings reveal hurdles that must be overcome to successfully analyze future HIV-1 cure strategies.

APOBEC3 proteins can copackage and comutate HIV-1 genomes.

Although APOBEC3 cytidine deaminases A3G, A3F, A3D and A3H are packaged into virions and inhibit viral replication by inducing G-to-A hypermutation, it is not known whether they are copackaged and whether they can act additively or synergistically to inhibit HIV-1 replication. Here, we showed that APOBEC3 proteins can be copackaged by visualization of fluorescently-tagged APOBEC3 proteins using single-virion fluorescence microscopy. We further determined that viruses produced in the presence of A3G + A3F and A3G + A3H, exhibited extensive comutation of viral cDNA, as determined by the frequency of G-to-A mutations in the proviral genomes in the contexts of A3G (GG-to-AG) and A3D, A3F or A3H (GA-to-AA) edited sites. The copackaging of A3G + A3F and A3G + A3H resulted in an additive increase and a modest synergistic increase (1.8-fold) in the frequency of GA-to-AA mutations, respectively. We also identified distinct editing site trinucleotide sequence contexts for each APOBEC3 protein and used them to show that hypermutation of proviral DNAs from seven patients was induced by A3G, A3F (or A3H), A3D and A3G + A3F (or A3H). These results indicate that APOBEC3 proteins can be copackaged and can comutate the same genomes, and can cooperate to inhibit HIV replication.

Isolation, identification and evolution analysis of a novel subgroup of avian leukosis virus isolated from a local Chinese yellow broiler in South China.

Avian leukosis virus (ALV) causes high mortality associated with tumor formation and decreased fertility, and results in major economic losses in the poultry industry worldwide. Recently, a putative novel ALV subgroup virus named ALV-K was observed in Chinese local chickens. In this study, a novel ALV strain named GD14LZ was isolated from a Chinese local yellow broiler in 2014. The proviral genome was sequenced and phylogenetically analyzed. The replication ability and pathogenicity of this virus were also evaluated. The complete proviral genome sequence of GD14LZ was 7482 nt in length, with a genetic organization typical of replication-competent type C retroviruses lacking viral oncogenes. Sequence analysis showed that the gag, pol and gp37 genes of GD14LZ have high sequence similarity to those of other ALV strains (A-E subgroups), especially to those of ALV-E. The gp85 gene of the GD14LZ isolate showed a low sequence similarity to those other ALV strains (A-E subgroups) but showed high similarity to strains previously described as ALV-K. Phylogenetic analysis of gp85 also suggested that the GD14LZ isolate was related to ALV-K strains. Further study showed that this isolate replicated more slowly and was less pathogenic than other ALV strains. These results indicate that the GD14LZ isolate belongs to the novel subgroup ALV-K and probably arose by recombination of ALV-K with endogenous viruses with low replication and pathogenicity. This virus might have existed in local Chinese chickens for a long time.

Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems.

CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs), act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5’ long terminal repeat (LTR), for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination.

P6 - Ultra-deep sequencing of HIV-1 near full-length and partial proviral genomes reveals high genetic diversity among Brazilian blood donors

P6 - Ultra-deep sequencing of HIV-1 near full-length and partial proviral genomes reveals high genetic diversity among Brazilian blood donors

7. High-Throughput Sequencing of AAV Proviral Libraries from the Human Population Reveals Novel Variants with Unprecedented Intra-and Inter-Tissue Diversity

Recombinant adeno-associated viruses (rAAVs) remain the most promising gene delivery vehicle for human gene therapy. The development of novel rAAVs with high transduction efficiency and specific tropisms primarily relies on PCR cloning of natural variants from primate tissues, or the engineering of capsids via directed evolution and rational design. Faithful clinical translation of leading therapeutic AAV serotypes derived from non-human tissues remains a hot-button issue. Here, we employed high-throughput variant profiling of natural AAV proviral libraries from the human population to identify novel AAV variants with unique tissue tropisms. We screened 844 surgical specimens, encompassing a wide range of tissue types and disease states from 455 patients by robust signature PCR. We found that >80% of human tissues are AAV positive with notable serotype frequencies (AAV2/3 chimera > AAV8 > AAV2 > AAV6) distributed among different tissues. Notably, we also used qPCR primers against the conserved AAV rep sequence to quantify the abundance of pAAV proviral genomes in human tissues. Next, to ensure the highest return on identifying novel variants, we employed single-molecule, real-time (SMRT) sequencing to characterize full-length rep and cap sequences. Our preliminary findings from a single patient tissue sample revealed more than 800 unique DNA sequence variants (originating from single nucleotide polymorphisms, genetic recombination, and de novo mutations) that account for more than 50 unique amino acid sequence variants. We also detected significant inter-patient variation, as well as high inter-tissue variability within individual patients. These findings suggest that AAV exhibits the capacity for high genomic diversity and accelerated intra-host evolution. These characteristics, which are shared among many other viruses, are presumed to promote survival against the host immune response, and fitness in tissue-specific cellular environments.We also validated a selection of AAV8 variants by assessing their capacity for vector packaging and in vivo gene transfer. We found that a subset of variants has a stronger competency for transcytosis and tropism to liver and muscle than the conventional AAV8 serotype. Remarkably, one AAV8 variant can cross the blood-brain barrier and target the CNS more efficiently than AAV9 after intravenous injection of adult mice. Another noteworthy discovery is the sequence variability found in the assembly-activating protein (AAP) gene, which is encoded by an alternative open reading frame of the cap gene. The diversity within AAP may define the efficacy of AAV packaging among variants, and implicate its role in AAV evolution and fitness.By exploiting AAV-host tissues as a natural incubator for viral evolution to potentiate new AAV capsid sequences, and employing a strategy to profile intact capsid sequences on a high-throughput scale, this study highlights the unprecedented diversity and evolutionary capacity of AAV. Importantly, these new findings reshape the versatility of rAAV as the ideal biotherapy for targeting a range of normal and/or diseased tissues.

Inhibition of Heat Shock Protein 90 Prevents HIV Rebound

HIV evades eradication because transcriptionally dormant proviral genomes persist in long-lived reservoirs of resting CD4+ T cells and myeloid cells, which are the source of viral rebound after cessation of antiretroviral therapy. Dormant HIV genomes readily produce infectious virus upon cellular activation because host transcription factors activated specifically by cell stress and heat shock mediate full-length HIV transcription. The molecular chaperone heat shock protein 90 (Hsp90) is overexpressed during heat shock and activates inducible cellular transcription factors. Here we show that heat shock accelerates HIV transcription through induction of Hsp90 activity, which activates essential HIV-specific cellular transcription factors (NF-κB, NFAT, and STAT5), and that inhibition of Hsp90 greatly reduces gene expression mediated by these factors. More importantly, we show that Hsp90 controls virus transcription in vivo by specific Hsp90 inhibitors in clinical development, tanespimycin (17-(allylamino)-17-demethoxygeldanamycin) and AUY922, which durably prevented viral rebound in HIV-infected humanized NOD scid IL-2Rγ−/− bone marrow-liver-thymus mice up to 11 weeks after treatment cessation. Despite the absence of rebound viremia, we were able to recover infectious HIV from PBMC with heat shock. Replication-competent virus was detected in spleen cells from these nonviremic Hsp90 inhibitor-treated mice, indicating the presence of a tissue reservoir of persistent infection. Our novel findings provide in vivo evidence that inhibition of Hsp90 activity prevents HIV gene expression in replication-competent cellular reservoirs that would typically cause rebound in plasma viremia after antiretroviral therapy cessation. Alternating or supplementing Hsp90 inhibitors with current antiretroviral therapy regimens could conceivably suppress rebound viremia from persistent HIV reservoirs.

Ultra-Deep Sequencing of HIV-1 near Full-Length and Partial Proviral Genomes Reveals High Genetic Diversity among Brazilian Blood Donors.

BackgroundHere, we aimed to gain a comprehensive picture of the HIV-1 diversity in the northeast and southeast part of Brazil. To this end, a high-throughput sequencing-by-synthesis protocol and instrument were used to characterize the near full length (NFLG) and partial HIV-1 proviral genome in 259 HIV-1 infected blood donors at four major blood centers in Brazil: Pro-Sangue foundation (São Paulo state (SP), n 51), Hemominas foundation (Minas Gerais state (MG), n 41), Hemope foundation (Recife state (PE), n 96) and Hemorio blood bank (Rio de Janeiro (RJ), n 70).Materials and MethodsA total of 259 blood samples were obtained from 195 donors with long-standing infections and 64 donors with a lack of stage information. DNA was extracted from the peripheral blood mononuclear cells (PBMCs) to amplify the HIV-1 NFLGs from five overlapping fragments. The amplicons were molecularly bar-coded, pooled, and sequenced by Illumina paired-end protocol.ResultsOf the 259 samples studied, 208 (80%) NFLGs and 49 (18.8%) partial fragments were de novo assembled into contiguous sequences and successfully subtyped. Of these 257 samples, 183 (71.2%) were pure subtypes consisting of clade B (n = 167, 65%), C (n = 10, 3.9%), F1 (n = 4, 1.5%), and D (n = 2, 0.7%). Recombinant viruses were detected in 74 (28.8%) samples and consist of unique BF1 (n = 41, 15.9%), BC (n = 7, 2.7%), BCF1 (n = 4, 1.5%), CF1 and CDK (n = 1, 0.4%, each), CRF70_BF1 (n = 4, 1.5%), CRF71_BF1 (n = 12, 4.7%), and CRF72_BF1 (n = 4, 1.5%). Evidence of dual infection was detected in four patients coinfected with the same subtype (n = 3) and distinct subtype (n = 1).ConclusionBased on this work, subtype B appears to be the prevalent subtype followed by a high proportion of intersubtype recombinants that appeared to be arising continually in this country. Our study represents the largest analysis of the viral NFLG ever undertaken worldwide and provides insights into the understanding the genesis of the HIV-1 epidemic in this particular area of South America and informs vaccine design and clinical trials.

Functional interactions of archaea, bacteria and viruses in a hypersaline endolithic community.

Halite endoliths in the Atacama Desert represent one of the most extreme ecosystems on Earth. Cultivation-independent methods were used to examine the functional adaptations of the microbial consortia inhabiting halite nodules. The community was dominated by haloarchaea and functional analysis attributed most of the autotrophic CO2 fixation to one unique cyanobacterium. The assembled 1.1 Mbp genome of a novel nanohaloarchaeon, Candidatus Nanopetramus SG9, revealed a photoheterotrophic life style and a low median isoelectric point (pI) for all predicted proteins, suggesting a 'salt-in' strategy for osmotic balance. Predicted proteins of the algae identified in the community also had pI distributions similar to 'salt-in' strategists. The Nanopetramus genome contained a unique CRISPR/Cas system with a spacer that matched a partial viral genome from the metagenome. A combination of reference-independent methods identified over 30 complete or near complete viral or proviral genomes with diverse genome structure, genome size, gene content and hosts. Putative hosts included Halobacteriaceae, Nanohaloarchaea and Cyanobacteria. Despite the dependence of the halite community on deliquescence for liquid water availability, this study exposed an ecosystem spanning three phylogenetic domains, containing a large diversity of viruses and predominance of a 'salt-in' strategy to balance the high osmotic pressure of the environment.

The diversity of the structure and genomic integration sites of HTLV-1 provirus in MT-2 cell lines.

A human T-lymphotropic virus Type 1 (HTLV-1) positive cell line, MT-2, derived from human cord leukocytes co-culturing with adult T cell leukemia/lymphoma (ATL) cells is commonly used in HTLV-1 research; however, the details of provirus integrated in MT-2 genome have not yet been characterized. In this study, five types of HTLV-1 proviral sequences were detected in 11 different sites of the genome in a reference MT-2 cell line. The five types of HTLV-1 proviral sequences were one complete proviral genome, two types of proviruses with deletion of large internal viral sequences (5.3 and 3.9kB), one provirus with a large deletion (6.2kB) from 5'LTR to position 6257, and one provirus of LTR only. The provirus with identical deletion of large internal viral sequence (5.3kB) was found to be integrated into six different sites (chromosomes). A complete provirus and three of four types of defective provirus were consistently detected in two other MT-2 cell lines cultured in different laboratories. Not only Tax/Rex RNA and HBZ RNA, but also the transcriptional product for a specific defective provirus, were detectable in all three MT-2 cell lines. Because it has been reported that defective provirus is frequently detected in ATL cells, these results may be important in understanding the mechanism of HTLV-1 proviral polymorphism, which may be related to leukemogenesis. In addition, the large variation in integrated HTLV-1 proviruses makes it important for researchers to exercise caution in their assessment and interpretation of results using MT-2 cell lines.