HIV-1 Genome Research Articles

HIV and AIDS

In the quarter-century since the first report of AIDS in the United States, HIV infection has spread throughout the population, disproportionately affecting black women, Hispanic women, and men who have sex with men. The prognosis for persons infected with HIV has improved dramatically with the introduction and evolution of highly active antiretroviral therapy (HAART). The underlying principle of HAART is that a combination of potent antiretrovirals, each of which requires different mutations in the HIV genome for resistance to develop, can suppress replication sufficiently to prevent mutation and the emergence of resistance. The prospect that currently available antiretroviral therapy (ART) regimens may suppress HIV replication indefinitely provides the hope that infected patients will have life expectancies similar to those of age-matched uninfected individuals. For these patients, HIV care has shifted from an emphasis on treatment and prevention of the complications of HIV disease itself to a focus on suppression of HIV replication and management of short- and long-term complications of HIV, ART toxicities, and aging. This chapter describes the epidemiology, pathophysiology and pathogenesis, prevention, diagnosis, and management of acute and chronic HIV infection and AIDS, with figures and tables illustrating each chapter section. This review contains 9 figures, 32 tables, and 249 references

HIV and AIDS

In the quarter-century since the first report of AIDS in the United States, HIV infection has spread throughout the population, disproportionately affecting black women, Hispanic women, and men who have sex with men. The prognosis for persons infected with HIV has improved dramatically with the introduction and evolution of highly active antiretroviral therapy (HAART). The underlying principle of HAART is that a combination of potent antiretrovirals, each of which requires different mutations in the HIV genome for resistance to develop, can suppress replication sufficiently to prevent mutation and the emergence of resistance. The prospect that currently available antiretroviral therapy (ART) regimens may suppress HIV replication indefinitely provides the hope that infected patients will have life expectancies similar to those of age-matched uninfected individuals. For these patients, HIV care has shifted from an emphasis on treatment and prevention of the complications of HIV disease itself to a focus on suppression of HIV replication and management of short- and long-term complications of HIV, ART toxicities, and aging. This chapter describes the epidemiology, pathophysiology and pathogenesis, prevention, diagnosis, and management of acute and chronic HIV infection and AIDS, with figures and tables illustrating each chapter section. This review contains 9 figures, 32 tables, and 249 references

Nuclear Import of HIV-1.

The delivery of the HIV-1 genome into the nucleus is an indispensable step in retroviral infection of non-dividing cells, but the mechanism of HIV-1 nuclear import has been a longstanding debate due to controversial experimental evidence. It was commonly believed that the HIV-1 capsid would need to disassemble (uncoat) in the cytosol before nuclear import because the capsid is larger than the central channel of nuclear pore complexes (NPCs); however, increasing evidence demonstrates that intact, or nearly intact, HIV-1 capsid passes through the NPC to enter the nucleus. With the protection of the capsid, the HIV-1 core completes reverse transcription in the nucleus and is translocated to the integration site. Uncoating occurs while, or after, the viral genome is released near the integration site. These independent discoveries reveal a compelling new paradigm of this important step of the HIV-1 life cycle. In this review, we summarize the recent studies related to HIV-1 nuclear import, highlighting the spatial–temporal relationship between the nuclear entry of the virus core, reverse transcription, and capsid uncoating.

Deep sequencing of HIV-1 reveals extensive subtype variation and drug resistance after failure of first-line antiretroviral regimens in Nigeria.

BackgroundDeep sequencing could improve understanding of HIV treatment failure and viral population dynamics. However, this tool is often inaccessible in low- and middle-income countries.ObjectivesTo determine the genetic patterns of resistance emerging in West African HIV-1 subtypes during first-line virological failure, and the implications for future antiretroviral options.Patients and methodsParticipants were selected from a Nigerian cohort of people living with HIV who had failed first-line ART and subsequently switched to second-line therapy. Whole HIV-1 genome sequences were generated from first-line virological failure samples with Illumina MiSeq. Mutations detected at ≥2% frequency were analysed and compared by subtype.ResultsHIV-1 sequences were obtained from 101 participants (65% female, median age 30 years, median 32.9 months of nevirapine- or efavirenz-based ART). Thymidine analogue mutations (TAMs) were detected in 61%, other core NRTI mutations in 92% and NNRTI mutations in 99%. Minority variants (<20% frequency) comprised 18% of all mutations. K65R was more prevalent in CRF02_AG than G subtypes (33% versus 7%; P = 0.002), and ≥3 TAMs were more common in G than CRF02_AG (52% versus 24%; P = 0.004). Subtype G viruses also contained more RT cleavage site mutations. Cross-resistance to at least one of the newer NNRTIs, doravirine, etravirine or rilpivirine, was predicted in 81% of participants.ConclusionsExtensive drug resistance had accumulated in people with West African HIV-1 subtypes, prior to second-line ART. Deep sequencing significantly increased the detection of resistance-associated mutations. Caution should be used if considering newer-generation NNRTI agents in this setting.

English

Rilpivirine (RPV) and Etravirine (ETR) are second-generation non-nucleoside reverse transcriptase inhibitors (NNRTIs) that are not used for HIV-1 treatment in Kenya. In this cross-sectional study, we sequenced and analyzed the reverse transcriptase and pol regions of HIV-1 genome from 140 HIV infected individuals from Busia County Referral Hospital, Western Kenya, who were on anti-HIV treatment with confirmed virologic failure. All the participants were on first-generation NRTI&rsquo;s and NNRTI&rsquo;s for more than 12 months at the time of the study. Briefly, HIV RNA was extracted from plasma samples and sequenced to analyze for the presence of HIV-1 drug resistance mutations. The study findings showed that approximately 46% of the population had genotypic drug resistance against both Etravirine and Rilpivirine which were classified as ranging from potentially low level resistance to high level resistance despite being exposed to first-generation NNRTIs only. The study thus reveals that cross-resistance was demonstrated between primary and secondary NNRTI drugs. The development of cross resistance for RPV and ETR in patients on EFV and NVP poses a challenge in the use of these drugs as second generation NNRTI drugs. &nbsp; Key words:&nbsp;Non-nucleoside reverse transcriptase inhibitors (NNRTIs), NRTIs, Kenya,&nbsp;cross-resistance, Etravirine (ETR), Rilpivirine (RPV), HIV-1.

The Role of HIV-1 genome on biological mechanisms, Molecular function, Interactions between viral and host cell components, Mechanisms of viral entry, Chromosomal integration, and Transcription as potential target for new Preventive HIV-1 Vaccine Development

The human immunodeficiency virus (HIV) genome encodes a total of three structural proteins, two envelope proteins, three enzymes, and six accessory proteins. Three-dimensional molecular structures can provide detailed information on biological mechanisms and, for cases in which the molecular function affects human health, can significantly aid in the development of therapeutic interventions.

The Expression Level of HIV-1 Vif Is Optimized by Nucleotide Changes in the Genomic SA1D2prox Region during the Viral Adaptation Process.

HIV-1 Vif plays an essential role in viral replication by antagonizing anti-viral cellular restriction factors, a family of APOBEC3 proteins. We have previously shown that naturally-occurring single-nucleotide mutations in the SA1D2prox region, which surrounds the splicing acceptor 1 and splicing donor 2 sites of the HIV-1 genome, dramatically alter the Vif expression level, resulting in variants with low or excessive Vif expression. In this study, we investigated how these HIV-1 variants with poor replication ability adapt and evolve under the pressure of APOBEC3 proteins. Adapted clones obtained through adaptation experiments exhibited an altered replication ability and Vif expression level compared to each parental clone. While various mutations were present throughout the viral genome, all replication-competent adapted clones with altered Vif expression levels were found to bear them within SA1D2prox, without exception. Indeed, the mutations identified within SA1D2prox were responsible for changes in the Vif expression levels and altered the splicing pattern. Moreover, for samples collected from HIV-1-infected patients, we showed that the nucleotide sequences of SA1D2prox can be chronologically changed and concomitantly affect the Vif expression levels. Taken together, these results demonstrated the importance of the SA1D2prox nucleotide sequence for modulating the Vif expression level during HIV-1 replication and adaptation.

Integration features of intact latent HIV-1 in CD4+ T cell clones contribute to viral persistence.

Latent intact HIV-1 proviruses persist in a small subset of long-lived CD4+ T cells that can undergo clonal expansion in vivo. Expanded clones of CD4+ T cells dominate latent reservoirs in individuals on long-term antiretroviral therapy (ART) and represent a major barrier to HIV-1 cure. To determine how integration landscape might contribute to latency, we analyzed integration sites of near full length HIV-1 genomes from individuals on long-term ART, focusing on individuals whose reservoirs are highly clonal. We find that intact proviruses in expanded CD4+ T cell clones are preferentially integrated within Krüppel-associated box (KRAB) domain-containing zinc finger (ZNF) genes. ZNF genes are associated with heterochromatin in memory CD4+ T cells; nevertheless, they are expressed in these cells under steady-state conditions. In contrast to genes carrying unique integrations, ZNF genes carrying clonal intact integrations are down-regulated upon cellular activation. Together, the data suggest selected genomic sites, including ZNF genes, can be especially permissive for maintaining HIV-1 latency during memory CD4+ T cell expansion.

Targeting and Understanding HIV Latency: The CRISPR System against the Provirus.

The presence of latently infected cells and reservoirs in HIV-1 infected patients constitutes a significant obstacle to achieve a definitive cure. Despite the efforts dedicated to solve these issues, the mechanisms underlying viral latency are still under study. Thus, on the one hand, new strategies are needed to elucidate which factors are involved in latency establishment and maintenance. On the other hand, innovative therapeutic approaches aimed at eradicating HIV infection are explored. In this context, advances of the versatile CRISPR-Cas gene editing technology are extremely promising, by providing, among other advantages, the possibility to target the HIV-1 genome once integrated into cellular DNA (provirus) and/or host-specific genes involved in virus infection/latency. This system, up to now, has been employed with success in numerous in vitro and in vivo studies, highlighting its increasing significance in the field. In this review, we focus on the progresses made in the use of different CRISPR-Cas strategies to target the HIV-1 provirus, and we then discuss recent advancements in the use of CRISPR screens to elucidate the role of host-specific factors in viral latency.

HIV-1 Genomes Are Enriched in Memory CD4+ T-Cells with Short Half-Lives.

ABSTRACTFuture HIV-1 curative therapies require a thorough understanding of the distribution of genetically-intact HIV-1 within T-cell subsets during antiretroviral therapy (ART) and the cellular mechanisms that maintain this reservoir. Therefore, we sequenced near-full-length HIV-1 genomes and identified genetically-intact and genetically-defective genomes from resting naive, stem-cell memory, central memory, transitional memory, effector memory, and terminally-differentiated CD4+ T-cells with known cellular half-lives from 11 participants on ART. We find that a higher infection frequency with any HIV-1 genome was significantly associated with a shorter cellular half-life, such as transitional and effector memory cells. A similar enrichment of genetically-intact provirus was observed in these cells with relatively shorter half-lives. We found that effector memory and terminally-differentiated cells also had significantly higher levels of expansions of genetically-identical sequences, while only transitional and effector memory cells contained genetically-intact proviruses that were part of a cluster of identical sequences. Expansions of identical sequences were used to infer cellular proliferation from clonal expansion. Altogether, this indicates that specific cellular mechanisms such as short half-life and proliferative potential contribute to the persistence of genetically-intact HIV-1.

Small-molecule Inhibitors of HIV-1 Reverse Transcriptase-Associated Ribonuclease H Function: Challenges and Recent Developments.

The reverse transcriptase-associated ribonuclease H (RNase H) hydrolyzes the HIV genome to allow synthesizing viral DNA. Currently, no RNase H inhibitors (RHIs) have reached the clinical phase. Therefore, RNase H can be defined as an attractive target for drug design. Despite the wealth of information available for RNase H domain, the development of RHIs with high specificity and low cellular toxicity has been disappointing. However, it is now becoming increasingly evident that reverse transcriptase is a highly versatile enzyme, undergoing major structural alterations to complete its catalysis, and that exists a close spatial and temporal interplay between reverse transcriptase polymerase and RNase H domains. This review sums up the present challenges in targeting RNase H encompassing the challenges in selectively inhibiting RNase H vs polymerase and/or HIV-1 integrase and the weak antiviral activity of active site inhibitors, probably for a substrate barrier that impedes small molecules to reach the targeted site. Moreover, the focus is given on the most recent progress in the field of medicinal chemistry that has led to the identification of several small molecules as RHIs in the last few years. RHIs could be a new class of drugs with a novel mechanism of action highly precious for the treatment of resistant HIV strains.

5′-Cap sequestration is an essential determinant of HIV-1 genome packaging

HIV-1 selectively packages two copies of its 5'-capped RNA genome (gRNA) during virus assembly, a process mediated by the nucleocapsid (NC) domain of the viral Gag polyprotein and encapsidation signals located within the dimeric 5' leader of the viral RNA. Although residues within the leader that promote packaging have been identified, the determinants of authentic packaging fidelity and efficiency remain unknown. Here, we show that a previously characterized 159-nt region of the leader that possesses all elements required for RNA dimerization, high-affinity NC binding, and packaging in a noncompetitive RNA packaging assay (ΨCES) is unexpectedly poorly packaged when assayed in competition with the intact 5' leader. ΨCES lacks a 5'-tandem hairpin element that sequesters the 5' cap, suggesting that cap sequestration may be important for packaging. Consistent with this hypothesis, mutations within the intact leader that expose the cap without disrupting RNA structure or NC binding abrogated RNA packaging, and genetic addition of a 5' ribozyme to ΨCES to enable cotranscriptional shedding of the 5' cap promoted ΨCES-mediated RNA packaging to wild-type levels. Additional mutations that either block dimerization or eliminate subsets of NC binding sites substantially attenuated competitive packaging. Our studies indicate that packaging is achieved by a bipartite mechanism that requires both sequestration of the 5' cap and exposure of NC binding sites that reside fully within the ΨCES region of the dimeric leader. We speculate that cap sequestration prevents irreversible capture by the cellular RNA processing and translation machinery, a mechanism likely employed by other viruses that package 5'-capped RNA genomes.

Moving Toward a Functional Cure for HIV-1

Abstract Despite the recent success of suppressing human immunodeficiency virus 1 (HIV-1) replication in the peripheral blood with antiretroviral therapy, elimination of the provirus from infected cells remains challenging because the virus can integrate into the host genome and become a latent proviral reservoir. The removal of these latent viral reservoirs from tissue containing infected resting memory CD4+ T cells remains a major barrier to developing a cure for HIV-1. Several novel strategies have recently been developed for curing HIV infection. These include stem cell transplantation with CCR5-Δ32 mutated cells, initial antiretroviral treatment in the very early stage of HIV infection, gene editing to excise the HIV genome or generate CCR5-mutated HIV-1-resistant cells, “shock-and-kill” to reactivate latent HIV using latency reversal agents combined with enhanced immune clearance methods, and an emerging “block-and-lock” strategy to reactivate latent HIV using latency reversal agents combined with enhanced immune clearance methods. However, the high risks and limitations to scale-up in clinics, as well as poor performance in clinical trials, have hampered these efforts. This review aims to describe the present efforts that are being made to combat HIV latency, and critically discuss the limitations and future of the cure for HIV-1.

Potential Achilles heels of SARS-CoV-2 are best displayed by the base order-dependent component of RNA folding energy

The base order-dependent component of folding energy has revealed a highly conserved region in HIV-1 genomes that associates with RNA structure. This corresponds to a packaging signal that is recognized by the nucleocapsid domain of the Gag polyprotein. Long viewed as a potential HIV-1 "Achilles heel," the signal can be targeted by a new antiviral compound. Although SARS-CoV-2 differs in many respects from HIV-1, the same technology displays regions with a high base order-dependent folding energy component, which are also highly conserved. This indicates structural invariance (SI) sustained by natural selection. While the regions are often also protein-encoding (e. g. NSP3, ORF3a), we suggest that their nucleic acid level functions can be considered potential "Achilles heels" for SARS-CoV-2, perhaps susceptible to therapies like those envisaged for AIDS. The ribosomal frameshifting element scored well, but higher SI scores were obtained in other regions, including those encoding NSP13 and the nucleocapsid (N) protein.

Coevolved Multidrug-Resistant HIV-1 Protease and Reverse Transcriptase Influences Integrase Drug Susceptibility and Replication Fitness.

Integrase strand transfer inhibitors (InSTIs) are recommended agents in first-line combination antiretroviral therapy (cART). We examined the evolution of drug resistance mutations throughout HIV-1 pol and the effects on InSTI susceptibility and viral fitness. We performed single-genome sequencing of full-length HIV-1 pol in a highly treatment-experienced patient, and determined drug susceptibility of patient-derived HIV-1 genomes using a phenotypic assay encompassing full-length pol gene. We show the genetic linkage of multiple InSTI-resistant haplotypes containing major resistance mutations at Y143, Q148 and N155 to protease inhibitor (PI) and reverse transcriptase inhibitor (RTI) resistance mutations. Phenotypic analysis of viruses expressing patient-derived IN genes with eight different InSTI-resistant haplotypes alone or in combination with coevolved protease (PR) and RT genes exhibited similar levels of InSTI susceptibility, except for three haplotypes that showed up to 3-fold increases in InSTI susceptibility (p ≤ 0.032). The replicative fitness of most viruses expressing patient-derived IN only significantly decreased, ranging from 8% to 56% (p ≤ 0.01). Interestingly, the addition of coevolved PR + RT significantly increased the replicative fitness of some haplotypes by up to 73% (p ≤ 0.024). Coevolved PR + RT contributes to the susceptibility and viral fitness of patient-derived IN viruses. Maintaining patients on failing cART promotes the selection of fitter resistant strains, and thereby limits future therapy options.

HIV-1 molecular diversity in Brazil unveiled by 10\xa0years of sampling by the national genotyping network

HIV-1 has diversified into several subtypes and recombinant forms that are heterogeneously spread around the world. Understanding the distribution of viral variants and their temporal dynamics can help to design vaccines and monitor changes in viral transmission patterns. Brazil has one of the largest HIV-1 epidemics in the western-world and the molecular features of the virus circulating in the country are still not completely known. Over 50,000 partial HIV-1 genomes sampled between 2008 and 2017 by the Brazilian genotyping network (RENAGENO) were analyzed. Sequences were filtered by quality, duplicate sequences per patient were removed and subtyping was performed with online tools and molecular phylogeny. Association between patients’ demographic data and subtypes were performed by calculating the relative risk in a multinomial analysis and trends in subtype prevalence were tested by Pearson correlation. HIV-1B was found to be the most prevalent subtype throughout the country except in the south, where HIV-1C prevails. An increasing trend in the proportion of HIV-1C and F1 was observed in several regions of the country, while HIV-1B tended to decrease. Men and highly educated individuals were more frequently infected by HIV-1B and non-B variants were more prevalent among women with lower education. Our results suggest that socio-demographic factors partially segregate HIV-1 diversity in Brazil while shaping viral transmission networks. Historical events could explain a preferential circulation of HIV-1B among men who have sex with men (MSM) and non-B variants among heterosexual individuals. In view of an increasing male/female ratio of AIDS cases in Brazil in the last 10–15 years, the decrease of HIV-1B prevalence is surprising and suggests a greater penetrance of non-B subtypes in MSM transmission chains.

HIVIntact: a python-based tool for HIV-1 genome intactness inference

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.

B-CePs as cross-linking probes for the investigation of RNA higher-order structure.

Elucidating the structure of RNA and RNA ensembles is essential to understand biological functions. In this work, we explored the previously uncharted reactivity of bis-chloropiperidines (B-CePs) towards RNA. We characterized at the molecular level the different adducts induced by the fast reacting compound B-CeP 1 with RNA. Following an approach based on solution thermal melting coupled with ESI mass spectrometry (STHEM-ESI), we proved the ability of B-CePs to induce inter-molecular cross-links between guanines in double stranded RNA. These results open the possibility of using B-CePs as structural probes for investigating higher-order structures, such as the kissing loop complex established by the dimerization initiation site (DIS) of the HIV-1 genome. We confirmed the potential of B-CePs to reveal the identity of RNA structures involved in long-range interactions, expecting to benefit the characterization of samples that are not readily amenable to traditional high-resolution techniques, and thus promoting the elucidation of pertinent RNA systems associated with old and new diseases.

APOBEC3G rescues cells from the deleterious effects of DNA damage.

Human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (hA3G), a member of the APOBEC family, was described as an anti-HIV-1 restriction factor, deaminating reverse transcripts of the HIV-1 genome. Several types of cancer cells that express high levels of A3G, such as diffuse large B-cell lymphoma cells and glioblastomas, show enhanced cell survival after ionizing radiation and chemotherapy treatments. Previously, we showed that hA3G promotes (DNA) double-strand breaks repair in cultured cells and rescues transgenic mice from a lethal dose of ionizing radiation. Here, we show that A3G rescues cells from the detrimental effects of DNA damage induced by ultraviolet irradiation and by combined bromodeoxyuridine and ultraviolet treatments. The combined treatments stimulate the synthesis of cellular proteins, which are exclusively associated with A3G expression. These proteins participate mainly in nucleotide excision repair and homologous recombination DNA repair pathways. Our results implicate A3G inhibition as a potential strategy for increasing tumor cell sensitivity to genotoxic treatments.

Genetic Characterization of a New HIV-1 Sub-Subtype A in Cabo Verde, Denominated A8.

Previous molecular characterization of Human immunodeficiency virus (HIV-1) samples from Cabo Verde pointed out a vast HIV-1 pol diversity, with several subtypes and recombinant forms, being 5.2% classified as AU-pol. Thus, the aim of the present study was to improve the characterization of these AU sequences. The genomic DNA of seven HIV-1 AU pol-infected individuals were submitted to four overlapping nested-PCR fragments aiming to compose the full-length HIV-1 genome. The final classification was based on phylogenetic trees that were generated using the maximum likelihood and bootscan analysis. The genetic distances were calculated using Mega 7.0 software. Complete genome amplification was possible for two samples, and partial genomes were obtained for the other five. These two samples grouped together with a high support value, in a separate branch from the other sub-subtypes A and CRF26_A5U. No recombination was verified at bootscan, leading to the classification of a new sub-subtype A. The intragroup genetic distance from the new sub-subtype A at a complete genome was 5.2%, and the intergroup genetic varied from 8.1% to 19.0% in the analyzed fragments. Our study describes a new HIV-1 sub-subtype A and highlights the importance of continued molecular surveillance studies, mainly in countries with high HIV molecular diversity.