Regions Of HIV-1 Genome Research Articles

Protein-Protein and Protein-RNA Interaction Assays to Determine Similarity of INI1/SMARCB1 and TAR RNA in Binding to HIV-1 Integrase.

INI1/SMARCB1 is a host protein that interacts with HIV-1 integrase (IN) and influences multiple stages of viral replication. IN is a viral enzyme responsible for integration, and it also binds to HIV-1 genomic RNA. Recent studies from our laboratory demonstrated that IN-interacting Rpt1 (Repeat 1) domain of INI1 and TAR RNA region of HIV-1 genome both bind to the same residues and surface of IN C-terminal domain (CTD). Based on a series of analyses, we found that INI1-Rpt1 and TAR RNA structurally mimic each other and that IN mutants defective for binding to INI1 are also defective for binding to RNA and produce morphologically defective virions. The similarity of INI1-Rpt1 and TAR RNA in binding to IN was established by testing the binding of IN-CTD mutants with INI1-Rpt1 and TAR RNA using the Alpha assay. Here, I describe Alpha assay methods to compare the binding of INI1-Rpt1 protein and HIV-1 TAR RNA to IN-CTD and describe a three-component assay to demonstrate the competition between TAR RNA and INI1-Rpt1 to bind to IN.

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’s and NNRTI’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.   Key words: Non-nucleoside reverse transcriptase inhibitors (NNRTIs), NRTIs, Kenya, cross-resistance, Etravirine (ETR), Rilpivirine (RPV), HIV-1.

SNAPPy: A snakemake pipeline for scalable HIV-1 subtyping by phylogenetic pairing.

Human immunodeficiency virus 1 (HIV-1) genome sequencing is routinely done for drug resistance monitoring in hospitals worldwide. Subtyping these extensive datasets of HIV-1 sequences is a critical first step in molecular epidemiology and evolution studies. The clinical relevance of HIV-1 subtypes is increasingly recognized. Several studies suggest subtype-related differences in disease progression, transmission route efficiency, immune evasion, and even therapeutic outcomes. HIV-1 subtyping is mainly done using web-servers. These tools have limitations in scalability and potential noncompliance with data protection legislation. Thus, the aim of this work was to develop an efficient method for large-scale local HIV-1 subtyping. We designed SNAPPy: a snakemake pipeline for scalable HIV-1 subtyping by phylogenetic pairing. It contains several tasks of phylogenetic inference and BLAST queries, which can be executed sequentially or in parallel, taking advantage of multiple-core processing units. Although it was built for subtyping, SNAPPy is also useful to perform extensive HIV-1 alignments. This tool facilitates large-scale sequence-based HIV-1 research by providing a local, resource efficient and scalable alternative for HIV-1 subtyping. It is capable of analyzing full-length genomes or partial HIV-1 genomic regions (GAG, POL, and ENV) and recognizes more than ninety circulating recombinant forms. SNAPPy is freely available at: https://github.com/PMMAraujo/snappy/releases.

Near Full-Length Genomic Characterization of HIV-1 CRF01_AE/B Recombinant Strains Identified in Hebei, China.

This study reports a novel HIV-1 recombinant form of CRF01_AE/B that was isolated from a 22-year-old male individual (HE150308) residing in Hebei province, China. The near full-length genome (NFLG) phylogenetic tree showed that the strain was clustered with CRF01_AE reference sequences and placed at the peripheral position within the branch of CRF01_AE strains. The bootscaning and similarity plot analysis revealed that the NFLG of this novel recombinant was composed of eight interlaced segments, including four CRF01_AE and four subtype B segments separated by seven breakpoints observed in the gag, pol, and nef regions of HIV-1 genome. Phylogenetic analysis demonstrated that the CRF01_AE subregions were from the previously identified CRF01_AE cluster 5, and the B subregions were correlated with the B strains originated from Europe and America. They were all the lineages widely prevalent in men who have sex with men (MSM) population in China. In recent years, a large number of recombinants between CRF01_AE and B strains are constantly emerging in the MSM population in China. This continual and recurrent recombination between CRF01_AE and B in high-risk group people deserves more attention and further monitoring.

Influence of HIV-1 Genetic Variability on Vertical Transmission and Pathogenesis

HIV-1 exists as a genetically heterogeneous population within infected individuals and in infected population. Genetic variability or viral heterogeneity plays an important role in transmission, pathogenesis, immune response, disease progression and therapy as well as is the most effective way for the virus to evade the host immune response and persists in infected individuals. We have used HIV-1 infected motherinfant pairs as a transmitter-recipient model to elucidate the role of the molecular and biological properties of HIV-1 associated with transmission and pathogenesis. HIV-1 sequences derived from infected mothers were more heterogeneous than their younger infected infants’ HIV-1 sequences. However, the infants’ HIV-1 sequences diversified as they grew older. HIV-1 infants’ sequences were different from each other but similar or closer to their mother’s sequences. We found that the minor genotypes of HIV-1 found in mothers were transmitted to their infants, which was initially maintained as a homogeneous population in infants and diversified as the infants grew older. In addition, the phenotype of these minor geno types was macrophage-tropic, nonsyncytium inducing (NSI) referred as R5 HIV-1. We also analyzed several other regions of HIV-1 genome, including env, gag p17 and NC, pol RT, tat, rev, vif, vpr, vpu and nef infected mother-infant pairs and found that there was a high functional conservation of these genes. These findings suggested that HIV-1 heterogenity plays an important role in vertical transmission and pathogenesis. The properties of transmitted viruses and those that are associated with disease progression should be targeted for the development of new strategies for prevention and treatment of HIV-1 infection. Research should focus utilizing these findings in the development of better strategies for prevention and treatment of HIV-1.

In–Depth Characterization of Viral Isolates from Plasma and Cells Compared with Plasma Circulating Quasispecies in Early HIV-1 Infection

BackgroundThe use of in vitro models to unravel the phenotypic characteristics of circulating viral variants is key to understanding HIV-1 pathogenesis but limited by the availability of primary viral isolates from biological samples. However, overall in vivo genetic variability of HIV-1 within a subject may not be reflected in the viable viral population obtained after isolation. Although several studies have tried to determine whether viral populations expanded in vitro are representative of in vivo findings, the answer remains unclear due to the reduced number of clonal sequences analyzed or samples compared. In order to overcome previous experimental limitations, here we applied Deep Pyrosequencing (DPS) technology in combination with phenotypic experiments to analyze and compare with unprecedented detail the composition of viral isolates and in vivo quasispecies.Methodology/Principal FindingsWe amplified by DPS HIV-1 genomic regions covering gag, protease, integrase and env-V3 to characterize paired isolates from plasma and peripheral blood mononuclear cells and compare them with total plasma viral RNA in four recently HIV-1 infected subjects. Our study demonstrated the presence of unique haplotypes scattered between sample types with conservation of major variants. In addition, no differences in intra- and inter-population encoded protein variability were found between the different types of isolates or when these were compared to plasma viral RNA within subjects. Additionally, in vitro experiments demonstrated phenotypic similarities in terms of replicative capacity and co-receptor usage between viral isolates and plasma viral RNA.ConclusionThis study is the first in-depth comparison and characterization of viral isolates from different sources and plasma circulating quasispecies using DPS in recently HIV-1 infected subjects. Our data supports the use of primary isolates regardless of their plasma or cellular origin to define genetic variability and biological traits of circulating HIV-1 quasispecies.

Phylogeographic Analyses Reveal a Crucial Role of Xinjiang in HIV-1 CRF07_BC and HCV 3a Transmissions in Asia

BackgroundChina faces an increasing prevalence of two HIV-1 circulating recombinant forms (CRFs) 07_BC and 08_BC. Both CRFs_BC were previously demonstrated to originate in Yunnan and spread to Liaoning from Yunnan via injection drug use (IDU) in China. Supposing it is true, we are unable to answer why only CRF07_BC, rather than both CRFs_BC together, was transmitted to Xinjiang.Methodology/Principal FindingsWe investigated the phylogeography of CRF07_BC and CRF08_BC using multiple HIV-1 genomic regions with Bayesian phylogeography method. Phylogenetic reconstructions showed that all CRF07_BC sequences were divided into two clades, Yunnan and Xinjiang, and all strains from other regions of mainland China clustered within the Xinjiang clade. Significant geographic diffusion links of Xinjiang with other regions (including Liaoning, Beijing, Jiangsu and Guangdong) were supported by Bayes factor tests. The temporal dynamics analyses showed that CRF07_BC spread from Xinjiang to Liaoning in 1996.10, and to Jiangsu in 2000.9. The analyses of CRF08_BC not only confirmed the previous conclusion on temporal and spatial dynamics of CRF08_BC, but also indicated that the CRF08_BC strains from Guangdong and Shanghai originated from Yunnan. The analyses of HCV 3a showed that it was introduced into Xinjiang in the early 1980s, and spread from Xinjiang to Yunnan in 1990.10 and to Jiangsu in 1999.2, and further from Yunnan to Guangxi in 1995.3. The temporal and spatial dynamics of HCV 3a were similar to some extent to that of HIV-1 CRF07_BC and/or CRF08_BC, suggesting a possible association in migration patterns between HCV and HIV-1 through IDU. In addition, HCV 3a spread from Xinjiang to Pakistan, implying a drug trafficking route linking them.Conclusions/SignificanceXinjiang, as the most important transfer station for drug trafficking from Golden Crescent to other regions of China, plays a very crucial role in the transmission of viruses (e.g., HIV-1 and HCV) through IDU in Asia.

In-House RT-PCR Assay for Detection of Human Immunodeficiency Virus Type 1 Infection

Serologic assays are commonly used for screening (ELISA) and for confirmation (Western blot) of HIV-1 infection; however, both assays have potentially yielded the false-positive or false-negative results. In this study, a diagnostic RT-PCR assay as an alternative test for detection of HIV-1 was developed. Forty-six plasma specimens from highly risky groups, who visited a voluntary counseling and testing for HIV (VCT) in Sanglah Clinic of General Hospital, Denpasar, Bali, were tested by RT-PCR assay with specific primers for Pol region of HIV-1 genome. The results of the RT-PCR tests were then compared with those of serologic tests to obtain the sensitivity and specificity of RT-PCR assay. The results of this study showed that the RT-PCR assay could detect 17 (sensitivity: 65.4%) of 26 serologically positive specimens and was unexpectedly able to detect 2 (specificity: 90%) of 20 serologically negative specimens. Thus, the RT-PCR assay developed in this study is potential to be used as an alternative test, even though there are numerous aspects, particularly the sensitivity, that need to be improved in further research.

P16-39. T cell recognition of autologous and non-autologous HIV-1 protease peptides by HIV-1 infected patients undergoing PI therapy

Methods Seventy-two HIV-1+ patients undergoing protease inhibitor (PI) therapy were recruited. We synthesized three peptides from HXB2 strain protease (wild-type) and 32 additional peptides from the same regions, which encompassed the most frequent PI-induced mutations in Brazil. PBMC from those patients were analyzed by the CFSE dilution assay to detect proliferating CD4+ and/or CD8+ T cell responses against each of the 35 protease peptides. Pol protease HIV-1 genomic regions were sequenced using internal primers with the ABI Prism Dye Terminator Cycle Sequencing Reaction Kit in an automated sequencer.

P.016 Diagnosis of mumps using detection of specific immunoglobulins M, A, and G in a predominantly vaccinated population

Background: Current guidelines for antiretroviral (ARV) therapy recommend at least triple-drug combination, the so-called highly active antiretroviral therapy (HAART). Not all patients respond to HAART and the development of drug resistance remains one of the most serious obstacles to sustained suppression of HIV. Objective: In an attempt to correlate the HIV therapeutic failure with reverse transcriptase (RT) and protease resistance mutations, we describe the ARV resistance profile in patients failing HAART in Brazil. We studied 267 Brazilian HIV-1 infected patients failing HAART looking for mutations in RT and protease genes. The mutation profile of the viruses infecting these individuals were deduced and correlated to laboratorial parameters. Study Design: Two different HIV-1 genomic regions were targeted for PCR amplification, the protease (pro) and pol RT (palm finger region) genes. The mutations related to drug resistance in RT gene was analyzed using a line probe assay (LIPA®) and pro amino acids positions 82 and 90 were screened through RFLP using HincII restriction digestion. Results: There was strong correlation between the mutation in the pro and RT genes and therapeutic failure. The main mutation found in RT gene was the M184V (48%) followed by T69D/N (47%), T215Y/F (46%), M41L (39%), and L74V (7%). In the pro gene the main mutation found was L90M (26%) followed by dual substitution in L90M and V82A (6%). All mutations profiles matched very well with the patients drug regimen. Conclusions: This study has shown that 84.7% of HIV infected subjects failing HAART for more than 3 months presented viral genomic mutations associated with drug resistance.

Full-Length HIV Type 1 Proviral Sequencing of 10 Highly Exposed Women from Nairobi, Kenya Reveals a High Proportion of Intersubtype Recombinants

Phylogenetic analysis has revealed that the current HIV/AIDS pandemic consists of a multitude of different viral clades and recombinant viruses. The predominant circulating HIV-1 clade in Kenya is A1; however, Kenya borders countries where different subtypes are prominent, making Kenya a likely location for recombination. Previous studies have reported significant differences in the proportions of sequences in Kenya that are intersubtype recombinants. Studies that performed sequence-based typing on multiple HIV-1 genomic regions or full-length sequences found higher rates of recombination than those that examined a single gene or gene fragment. In this study, we describe full-length HIV-1 proviral sequence-based genotyping after limited peripheral blood mononuclear cell (PBMC) coculture. Ten subjects from a highly exposed cohort located in Nairobi, Kenya were examined. Pairwise comparison found minimal difference between sequences generated directly from patient PBMC DNA compared to sequences from cocultured PBMC DNA. Of the 10 full-length HIV-1 sequences examined, five were nonrecombinant clade A1, while the other five were unique intersubtype recombinants. Although this frequency of recombination is higher than previously described in Kenya, this finding is in agreement with previous full-length sequence data. Interestingly, although all the nonrecombinant sequences were clade A1, not all the recombinant sequences contained a clade A1 sequence.

Intragenic HIV-1 env sequences that enhance gag expression

Expression of HIV-1 genes is regulated at multiple levels including the complex RNA splicing and transport mechanisms. Multiple cis-acting elements involved in these regulations have been previously identified in various regions of HIV-1 genome. Here we show that another cis-acting element was present in HIV-1 env region. This element enhanced the expression of Gag when inserted together with Rev response element (RRE) into a truncated HIV-1 genome in the presence of Rev. The enhancing activity was mapped to a 263-bp fragment in the gp41 region downstream to RRE. RNA analysis showed that it might function by promoting RNA stability and Rev-dependent RNA export. The enhancement was specific to Rev-dependent expression, since it did not enhance Gag expression driven by Sam68, a cellular protein that has been shown to be able to substitute for Rev in RNA export function.

Prevalence of mutations related to HIV-1 antiretroviral resistance in Brazilian patients failing HAART.

Current guidelines for antiretroviral (ARV) therapy recommend at least triple-drug combination, the so-called highly active antiretroviral therapy (HAART). Not all patients respond to HAART and the development of drug resistance remains one of the most serious obstacles to sustained suppression of HIV. In an attempt to correlate the HIV therapeutic failure with reverse transcriptase (RT) and protease resistance mutations, we describe the ARV resistance profile in patients failing HAART in Brazil. We studied 267 Brazilian HIV-1 infected patients failing HAART looking for mutations in RT and protease genes. The mutation profile of the viruses infecting these individuals were deduced and correlated to laboratorial parameters. Two different HIV-1 genomic regions were targeted for PCR amplification, the protease (pro) and pol RT (palm finger region) genes. The mutations related to drug resistance in RT gene was analyzed using a line probe assay (LIPA(R)) and pro amino acids positions 82 and 90 were screened through RFLP using HincII restriction digestion. There was strong correlation between the mutation in the pro and RT genes and therapeutic failure. The main mutation found in RT gene was the M184V (48%) followed by T69D/N (47%), T215Y/F (46%), M41L (39%), and L74V (7%). In the pro gene the main mutation found was L90M (26%) followed by dual substitution in L90M and V82A (6%). All mutations profiles matched very well with the patients drug regimen. This study has shown that 84.7% of HIV infected subjects failing HAART for more than 3 months presented viral genomic mutations associated with drug resistance.

Selection of appropriate HIV-1 genomic regions for single-strand conformation polymorphism analysis of the diversity, modification, and transmission of HIV-1 quasispecies.

Single-strand conformation polymorphism (SSCP) analysis is a useful tool for studying viral quasispecies. Four regions within the HIV-1 genome were studied by means of SSCP analysis with the aim of determining which regions were the most informative for the study of HIV-1 transmission or for detection of changes in HIV-1 quasispecies populations. Nested polymerase chain reaction (PCR) was used to amplify V1, V2, V3 of the env gene, and the p2 region in the gag gene. In total, 114 plasma specimens from 79 individuals were tested, including serial specimens from 10 mother-infant pairs that were provided by the Women and Infants Transmission Study (WITS). HIV-1 in specimens that were PCR-positive with primer pair SK38/SK39 showed different percentages of positive signals with primer pairs for the four regions: V1, 63%; V2, 83%; V3, 88%, and p2, 100%. HIV-1 sequences in the p2 target region displayed the greatest degree of polymorphism. Analysis of serial specimens showed that the V1 target region was the most variable of the four regions studied and was the most appropriate region for monitoring changes in quasispecies populations. Of the four regions studied, p2 was the most informative for the study of HIV transmission, as shown by analysis of samples from documented cases of mother to infant HIV-1 transmission.

Point mutant frequencies in the pol gene of human immunodeficiency virus type 1 are two- to threefold lower than those of env.

Nucleotide sequences have been determined for the HIV-1 genomic regions encoding codons 41-108 and 181-219 of reverse transcriptase (RT) (pol gene), and codons 198-331 of gp120 (the C2V3 domains; env gene). Eighty-one HIV-1 samples from patients treated or untreated with RT inhibitors were used to sequence pol and 28 HIV-1 samples were used to sequence env. Several individual quasispecies have also been analyzed. All HIV-1 isolates belonged to subtype B. Point mutant frequencies and nucleotide diversities for pol were at most two- to threefold lower than env, with the proportion of nonsynonymous mutations ranging from 33 to 67%. A new variability index that takes into account the type of amino acid substitution as well as genetic distances between the compared sequences is introduced, and its main features for sequence comparisons emphasized. Extension of the calculations to gag, pol, and env sequences contained in the current HIV-1 database confirmed the high mutant frequencies for all HIV-1 genomic regions. The results indicate that although env is more tolerant to insertions and deletions than pol, point mutant frequencies for HIV-1 regions encoding nonstructural proteins are only two- to threefold lower than for regions encoding structural proteins. This implies the occurrence of frequent changes in HIV-1 phenotypes that are dependent on amino acid substitutions in viral enzymes.

Solution structure of a conserved DNA sequence from the HIV-1 genome: restrained molecular dynamics simulation with distance and torsion angle restraints derived from two-dimensional NMR spectra.

The three-dimensional solution structure of a trisdecamer DNA duplex sequence, d(AGCTTGCCTTGAG).d(CTCAAGGCAAGCT), from a conserved region of HIV-1 genome's long terminal repeat, has been investigated using NMR spectroscopy and restrained molecular dynamics calculations. Interproton distances derived from two-dimensional nuclear Overhauser enhancement (2D NOE) experiments, using the iterative complete relaxation matrix algorithm MARDIGRAS, and torsion angles for sugar rings, estimated from stimulated fitting of double-quantum-filtered correlation (2QF-COSY) spectra, were obtained [Mujeeb, A., Kerwin, S. M., Egan, W. M., Kenyon, G. L., & James, T. L. (1992) Biochemistry 31, 9325-38]. These structural restraints have now been employed as the basis for structure refinement using restrained molecular dynamics (rMD) to search conformational space for structures consistent with the experimental restraints. Specifically, upper and lower bounds on the restraints were incorporated into the AMBER (version 4.0) total potential energy function of the system, the bounds being used to define the width of a flat-well penalty term in the AMBER force field. Confidence in the time-averaged structure obtained is engendered by convergence to essentially the same structure (root-mean-square deviation approximately 0.9 A) when two quite different DNA models, A-DNA and B-DNA (RMSD approximately 6.5 A), were employed as starting structures and when various initial trajectories were used for the rMD runs. The derived structure is further supported by the total energy calculated, the restraint violation energy, the restraint deviations, and the fit with experimental data. For the latter, the sixth-root residual index indicated a good fit of the determined structure with experimental 2D NOE spectral intensities (R1x < 0.07), and the RMS difference between vicinal proton coupling constants calculated for the derived structure and experimental coupling constants were also in reasonable agreement (JRMS = 0.9 Hz). While the structure of the trisdecamer is basically in the B-DNA family, some structural parameters manifest interesting local variations. The helix parameters indicate that, compared with classical B-DNA, the structure is longitudinally more compressed. Local structural variations at the two TG steps in particular together create bending into the major groove of the duplex. Comparison of the two-CTTG-tetrads in the duplex reveals that they have similar structures, with the TT moieties being almost identical; however, the -CTTG-pur sequence has a larger roll and slide for the -TG- step than for the -CTTG-pyr sequence, in accord with published X-ray crystallographic conclusions.

Cyclic set-based analytic technique to find specific origomers which characterize a region in genome: Application to hypervariable regions of HIV-1 genome

Cyclic set-based analytic technique to find specific origomers which characterize a region in genome: Application to hypervariable regions of HIV-1 genome