Modeling and Analysis of HIV-1 Pol Polyprotein as a Case Study for Predicting Large Polyprotein Structures.

Acquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV). HIV protease, reverse transcriptase, and integrase are targets of current drugs to treat the disease. However, anti-viral drug-resistant strains have emerged quickly due to the high mutation rate of the virus, leading to the demand for the development of new drugs. One attractive target is Gag-Pol polyprotein, which plays a key role in the life cycle of HIV. Recently, we found that a combination of M50I and V151I mutations in HIV-1 integrase can suppress virus release and inhibit the initiation of Gag-Pol autoprocessing and maturation without interfering with the dimerization of Gag-Pol. Additional mutations in integrase or RNase H domain in reverse transcriptase can compensate for the defect. However, the molecular mechanism is unknown. There is no tertiary structure of the full-length HIV-1 Pol protein available for further study. Therefore, we developed a workflow to predict the tertiary structure of HIV-1 NL4.3 Pol polyprotein. The modeled structure has comparable quality compared with the recently published partial HIV-1 Pol structure (PDB ID: 7SJX). Our HIV-1 NL4.3 Pol dimer model is the first full-length Pol tertiary structure. It can provide a structural platform for studying the autoprocessing mechanism of HIV-1 Pol and for developing new potent drugs. Moreover, the workflow can be used to predict other large protein structures that cannot be resolved via conventional experimental methods.

HIV-1 pol gene diversity and molecular dating of subtype C from Sri Lanka.

BACKGROUND: The first case of HIV infection in Sri Lanka was reported in 1987 and at the end of 2018 there were 3500 people living with HIV. There have been commendable efforts made towards the detection, treatment, and prevention of HIV in the country. Even though the genetic diversity of HIV has been shown to affect the parameters ranging from detection to vaccine development, there is no data available with respect to the molecular epidemiology of HIV-1 in Sri Lanka. METHODS: In this report we have performed the ancillary analysis of pol gene region sequences (n = 85) obtained primarily for the purpose of HIV-1 drug resistance genotyping. Briefly, dried blood spot specimens (DBS) collected from HIV-1 infected individuals between December 2015 and August 2018 were subjected to pol gene amplification and sequencing. These pol gene sequences were used to interpret the drug resistance mutation profiles. Further, sequences were subjected to HIV-1 subtyping using REGA 3.0, COMET, jPHMM and, RIP online subtyping tools. Moreover, Bayesian phylogenetic analysis was employed to estimate the evolutionary history of HIV-1 subtype C in Sri Lanka. RESULTS: Our analysis revealed that the majority (51.8%) of pol gene sequences were subtype C. Other than subtype C, there were sequences categorized as subtypes A1, B, D and G. In addition to pure subtypes there were sequences which were observed to be circulating recombinant forms (CRFs) and a few of the recombinants were identified as potential unique recombinants (URFs). We also observed the presence of drug resistance mutations in 56 (65.9%) out of 85 sequences. Estimates of the Bayesian evolutionary analysis suggested that the HIV-1 subtype C was introduced to Sri Lanka during the early 1970s (1972.8). CONCLUSION: The findings presented here indicate the presence of multiple HIV-1 subtypes and the prevalence of drug resistance mutations in Sri Lanka. The majority of the sequences were subtype C, having their most recent common ancestor traced back to the early 1970s. Continuous molecular surveillance of HIV-1 molecular epidemiology will be crucial to keep track of drug resistance, genetic diversity, and evolutionary history of HIV-1 in Sri Lanka.

Characterisation of HIV-1 Molecular Epidemiology in Nigeria: Origin, Diversity, Demography and Geographic Spread.

Nigeria has the highest number of AIDS-related deaths in the world. In this study, we characterised the HIV-1 molecular epidemiology by analysing 1442 HIV-1 pol sequences collected 1999-2014 from four geopolitical zones in Nigeria using state-of-the-art maximum-likelihood and Bayesian phylogenetic analyses. The main circulating forms were the circulating recombinant form (CRF) 02_AG (44% of the analysed sequences), CRF43_02G (16%), and subtype G (8%). Twenty-three percent of the sequences represented unique recombinant forms (URFs), whereof 37 (11%) could be grouped into seven potentially novel CRFs. Bayesian phylodynamic analysis suggested that five major Nigerian HIV-1 sub-epidemics were introduced in the 1960s and 1970s, close to the Nigerian Civil War. The analysis also indicated that the number of effective infections decreased in Nigeria after the introduction of free antiretroviral treatment in 2006. Finally, Bayesian phylogeographic analysis suggested gravity-like dynamics in which virus lineages first emerge and expand within large urban centers such as Abuja and Lagos, before migrating towards smaller rural areas. This study provides novel insight into the Nigerian HIV-1 epidemic and may have implications for future HIV-1 prevention strategies in Nigeria and other severely affected countries.

Renewing Felsenstein's phylogenetic bootstrap in the era of big data.

Felsenstein's application of the bootstrap method to evolutionary trees is one of the most cited scientific papers of all time. The bootstrap method, which is based on resampling and replications, is used extensively to assess the robustness of phylogenetic inferences. However, increasing numbers of sequences are now available for a wide variety of species, and phylogenies based on hundreds or thousands of taxa are becoming routine. With phylogenies of this size Felsenstein's bootstrap tends to yield very low supports, especially on deep branches. Here we propose a new version of the phylogenetic bootstrap in which the presence of inferred branches in replications is measured using a gradual 'transfer' distance rather than the binary presence or absence index used in Felsenstein's original version. The resulting supports are higher and do not induce falsely supported branches. The application of our method to large mammal, HIV and simulated datasets reveals their phylogenetic signals, whereas Felsenstein's bootstrap fails to do so.

HIV-1 transmitted drug resistance in Slovenia and its impact on predicted treatment effectiveness: 2011-2016 update.

HIV-positive individuals that have a detected transmitted drug resistance (TDR) at baseline have a higher risk of virological failure with antiretroviral therapy (ART). This study offers an update on the prevalence of TDR in Slovenia, looks for onward transmission of TDR, and reassesses the need for baseline drug resistance testing. Blinded questionnaires and partial pol sequences were obtained from 54.5% (168/308) of all of the patients diagnosed with HIV-1 from 2011 to 2016. Subtype B was detected in 82.7% (139/168) of patients, followed by subtype A (8.3%), subtype C (2.4%), and CRF01_AE (1.8%). Surveillance drug resistance mutations (SDRMs) were found in four individuals (2.4%), all of them men who have sex with men (MSM) and infected with subtype B. K103N was detected in two patients and T68D and T215D in one person each, corresponding to a prevalence of 0%, 1.2%, and 1.2% of TDR to protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs), and non-NRTIs (NNRTIs), respectively. The impact of mutations on drug susceptibility was found to be most pronounced for NNRTIs. No forward spread of TDR within the country was observed; however, phylogenetic analysis revealed several new introductions of HIV into Slovenia in recent years, possibly due to increased risky behavior by MSM. This was indirectly confirmed by a substantial increase in syphilis cases and HIV-1 non-B subtypes during the study period. A drug-resistant HIV variant with good transmission fitness is thus more likely to be imported into Slovenia in the near future, and so TDR should be closely monitored.

Phylodynamic and Phylogeographic Profiles of Subtype B HIV-1 Epidemics in South Spain.

BACKGROUND: Since 1982, HIV-1 epidemics have evolved to different scenarios in terms of transmission routes, subtype distribution and characteristics of transmission clusters. We investigated the evolutionary history of HIV-1 subtype B in south Spain. PATIENTS & METHODS: We studied all newly diagnosed HIV-1 subtype B patients in East Andalusia during the 2005-2012 period. For the analysis, we used the reverse transcriptase and protease sequences from baseline resistance, and the Trugene® HIV Genotyping kit (Siemens, Barcelona, Spain). Subtyping was done with REGA v3.0. The maximum likelihood trees constructed with RAxML were used to study HIV-1 clustering. Phylogeographic and phylodynamic profiles were studied by Bayesian inference methods with BEAST v1.7.5 and SPREAD v1.0.6. RESULTS: Of the 493 patients infected with HIV-1 subtype B, 234 grouped into 55 clusters, most of which were small (44 clusters ≤ 5 patients, 31 with 2 patients, 13 with 3). The rest (133/234) were grouped into 11 clusters with ≥ 5 patients, and most (82%, 109/133) were men who have sex with men (MSM) grouped into 8 clusters. The association with clusters was more frequent in Spanish (p = 0.02) men (p< 0.001), MSM (p<0.001) younger than 35 years (p = 0.001) and with a CD4+ T-cell count above 350 cells/ul (p<0.001). We estimated the date of HIV-1 subtype B regional epidemic diversification around 1970 (95% CI: 1965-1987), with an evolutionary rate of 2.4 (95%CI: 1.7-3.1) x 10-3 substitutions/site/year. Most clusters originated in the 1990s in MSMs. We observed exponential subtype B HIV-1 growth in 1980-1990 and 2005-2008. The most significant migration routes for subtype B went from inland cities to seaside locations. CONCLUSIONS: We provide the first data on the phylodynamic and phylogeographic profiles of HIV-1 subtype B in south Spain. Our findings of transmission clustering among MSMs should alert healthcare managers to enhance preventive measures in this risk group in order to prevent future outbreaks.

Synthetic consensus HIV-1 DNA induces potent cellular immune responses and synthesis of granzyme B, perforin in HIV infected individuals.

This study evaluated the safety and immunogenicity of PENNVAX-B in 12 HIV infected individuals. PENNVAX-B is a combination of three optimized synthetic plasmids encoding for multiclade HIV Gag and Pol and a consensus CladeB Env delivered by electroporation. HIV infected individuals whose virus was effectively suppressed using highly active antiretroviral therapy (HAART) received PENNVAX-B DNA followed by electroporation with CELLECTRA-5P at study weeks 0, 4, 8, and 16. Local administration site and systemic reactions to PENNVAX-B were recorded after each treatment along with any adverse events. Pain of the treatment procedure was assessed using a Visual Analog Scale. Whole PBMCs were isolated for use in IFN ELISpot and Flow Cytometric assays. PENNVAX-B was generally safe and well tolerated. Overall, the four dose regimen was not associated with any serious adverse events or severe local or systemic reactions. A rise in antigen-specific SFU was detected in the INFγ ELISpot assay in all 12 participants. T cells from 8/12 participants loaded with both granzyme B and perforin in response to HIV antigen, an immune finding characteristic of long-term nonprogressors (LTNPs) and elite controllers (ECs). Thus administration of PENNVAX-B may prove useful adjunctive therapy to ART for treatment and control of HIV infection.

[Molecular characterization of complex recombinant HIV-1 CRF06_cpx subtype detected in Turkey]. / Türkiye'de saptanan kompleks rekombinant HIV-1 CRF06_cpx alttipinin moleküler karakterizasyonu.

A major proportion of the global HIV infections is caused by group M of HIV-1 genotype and to date approximately nine subtypes (A, B, C, D, F, G, H, J, K) and 50 circulating recombinant forms (CRFs) have been recognized. Recombinants between different HIV-1 group M subtypes are designated as CRF. The extension 'cpx', for complex, is given if the CRF consists of contributions from three or more different subtypes but the composition of the subtype is not given. The objective of this study was to present, for the first time an HIV-1 positive married couple infected with CRF06_cpx subtype in Izmir, Turkey. A 39-year-old male patient who admitted to hospital with the complaints of oral candidiasis and zona, was found to be anti-HIV positive. CD4+ T lymphocyte count was 21 cells/mm3 and plasma HIV-1 RNA level was 56.380 copies/ml. He reported unprotected heterosexual contact with multiple partners including African women during his stay in Saudi Arabia between 1996 and 2002. After his diagnosis, his 37-year-old wife was screened for HIV infection and she was also found anti-HIV positive, with CD4+ T cell count of 122 cells/mm3. However, her results of basal plasma HIV-1 RNA could not be obtained because of an internal control error. HIV-1 strains were analysed for subtyping, recombination and drug resistance mutations with pol gene region sequencing. HIV-1 sequences were subtyped as CRF06_cpx after phylogenetic analysis using neighbor-joining method. According to the recombination analysis, HIV-1 pol gene regions consisted of group M subtype G, A, D, and B in the male patient and G K, A, F, and D in the female patient. While L10I + L33F mutation associated with protease inhibitor (PI) resistance was detected in both of the patients, K219N mutation associated with nucleoside reverse transcriptase inhibitor (NRTI) resistance was detected only in the male patient. In conclusion, HIV-1 molecular epidemiology studies are important tools for tracking transmission patterns and the spread of CRF. Global monitoring of CRF subtypes is also important to supply data for HIV vaccine development studies. On the other hand, the detection of HIV-1 primary resistance mutations in antiretroviral naive patients suggested that the resistance testing should be an integral part of the management of HIV infection.

[APOBEC3 hypermutations in HIV-1 infected cases in Turkey]. / Türkiye'de HIV-1 ile enfekte olgularda APOBEC3 hipermutasyonlari

Host genetic factors may play an effective role on the human immunodeficiency virus (HIV) pathogenesis. APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypeptide like-3) proteins are cellular antiviral proteins which inhibits HIV replication in the absence of vif (virion infectivity factor). In this study, we aimed to determine the APOBEC 3G/F hypermutations in HIV-1 strains isolated in Turkey. A total of 515 HIV-1 infected patients between June 2009 - February 2012 were included in the study. Three hundred ninety four cases were newly diagnosed antiretroviral-naive patients [349 male, 45 female; median age (range): 37.1 (2-69) years; median CD4+ T-cell count (range): 340 (1-1660) mm3; median HIV-RNA load (range): 5.76 + E5 (8.7 + E2-9.4 + E6) IU/ml] and 121 were under HAART therapy [99 male, 22 female; median age (range): 40.7 (20-70) years; median CD4+ T-cell count (range): 195 (6-720) mm3; median HIV-RNA load (range): 5.4 + E5 (1.37 + E3-1.07 + E7) IU/ml]. APOBEC 3G/F hypermutations in HIV-1 pol sequences (reverse transcriptase; codons 41-238 and protease; codons 1-99) analysed by nested RT-PCR and direct sequencing techniques. APOBEC 3G/F hypermutations have been determined by using of HIVdb-Stanford algorithm. The prevalence of overall APOBEC 3G/F hypermutations was 2.5% (13/515) in HIV-1 pol gene sequences in study group, and the rates were 2% (8/394) and 4.1% (5/121) in antiretroviral naive and treatment groups, respectively. However, the location and marker hypermutations of determined APOBEC in the HIV-1 pol gene sequences were RT and 3G in the Turkish patients. The hypermutated HIV-1 strains identified in HIV-1 infected patients may facilitate our understanding the nature and the consequences of HIV-1 infections. Moreover, investigations of the motif and frequency of APOBEC 3G/F hypermutations in HIV-1 proviral DNA samples and understanding their relationships with HIV-1 subtypes in Turkish patients would be beneficial.

Successful isolation of infectious and high titer human monocyte-derived HIV-1 from two subjects with discontinued therapy.

BACKGROUND: HIV-1 DNA in blood monocytes is considered a viral source of various HIV-1 infected tissue macrophages, which is also known as "Trojan horse" hypothesis. However, whether these DNA can produce virions has been an open question for years, due to the inability of isolating high titer and infectious HIV-1 directly from monocytes. RESULTS: In this study, we demonstrated successful isolation of two strains of M-HIV-1 (1690 M and 1175 M) from two out of four study subjects, together with their in vivo controls, HIV-1 isolated from CD4+ T-cells (T-HIV-1), 1690 T and 1175 T. All M- and T- HIV-1 isolates were detected CCR5-tropic. Both M- HIV-1 exhibited higher levels of replication in monocyte-derived macrophages (MDM) than the two T- HIV-1. Consistent with our previous reports on the subject 1175 with late infection, compartmentalized env C2-V3-C3 sequences were identified between 1175 M and 1175 T. In contrast, 1690 M and 1690 T, which were isolated from subject 1690 with relatively earlier infection, showed homogenous env C2-V3-C3 sequences. However, multiple reverse transcriptase (RT) inhibitor resistance-associated variations were detected in the Gag-Pol region of 1690 M, but not of 1690 T. By further measuring HIV DNA intracellular copy numbers post-MDM infection, 1690 M was found to have significantly higher DNA synthesis efficiency than 1690 T in macrophages, indicating a higher RT activity, which was confirmed by AZT inhibitory assays. CONCLUSIONS: These results suggested that the M- and T- HIV-1 are compartmentalized in the two study subjects, respectively. Therefore, we demonstrated that under in vitro conditions, HIV-1 infected human monocytes can productively release live viruses while differentiating into macrophages.

Molecular epidemiology of human immunodeficiency virus type 1 in Guangdong province of southern China.

BACKGROUND: Although the outbreak of human immunodeficiency virus type 1 (HIV-1) in Guangdong has been documented for more than a decade, the molecular characteristics of such a regional HIV-1 epidemic remained unknown. METHODOLOGY/PRINCIPAL FINDINGS: By sequencing of HIV-1 pol/env genes and phylogenetic analysis, we performed a molecular epidemiologic study in a representative subset (n  = 200) of the 508 HIV-1-seropositive individuals followed up at the center for HIV/AIDS care and treatment of Guangzhou Hospital of Infectious Diseases. Of 157 samples (54.1% heterosexual acquired adults, 20.4% needle-sharing drug users, 5.7% receivers of blood transfusion, 1.3% men who have sex with men, and 18.5% remained unknown) with successful sequencing for both pol and env genes, 105 (66.9%) HIV-1 subtype CRF01_AE and 24 (15.3%) CRF07_BC, 9 (5.7%) B', 5 (3.2%) CRF08_BC, 5 (3.2%) B, 1 (0.6%) C, 3 (1.9%) CRF02_AG, and 5 (3.2%) inter-region recombinants were identified within pol/env sequences. Thirteen (8.3%) samples (3 naïves, 6 and 5 received with antiretroviral treatment [ART] 1-21 weeks and ≥24 weeks respectively) showed mutations conferring resistance to nucleoside/nonnucleoside reverse transcriptase inhibitors or protease inhibitors. Among 63 ART-naïve patients, 3 (4.8%) showed single or multiple drug resistant mutations. Phylogenetic analysis showed 8 small clusters (2-3 sequences/cluster) with only 17 (10.8%) sequences involved. CONCLUSION/SIGNIFICANCE: This study confirms that sexual transmission with dominant CRF01_AE strain is a major risk for current HIV-1 outbreak in the Guangdong's general population. The transmission with drug-resistant variants is starting to emerge in this region.

[Comparison between different gene and near full-length genome on the phylogenetic analyses of HIV-1 subtype B' in China].

This study aims to compare the influence of different genes to the results of HIV-1 subtype B' phylogenetic analyses. We first split 47 near full-length genome sequences of subtype B' into different regions (gag, pol, vif, vpr, vpu, env, nef), which derived from various risk populations and geographic regions from Thailand, Myanmar and China from published studies. Phylogenetic analyses were performed to each region obtained. The phylogenetic results of different regions were compared to that of the near full-length genome sequences. The pol gene was found to have the lowest diversity and evolutionary rate, and could repeat the phylogenetic results by using near full-length genome sequences. Although the env gene has the highest diversity and evolutionary rate, it could not achieve the similar results. This study compared the influence to the results of HIV-1 subtype B' phylogenetic analyses by using different genes and laid foundation for further molecular survey and analyses of the transmission of subtype B' in China.

Terminal interface conformations modulate dimer stability prior to amino terminal autoprocessing of HIV-1 protease.

The HIV-1 protease (PR) mediates its own release (autoprocessing) from the polyprotein precursor, Gag-Pol, flanked by the transframe region (TFR) and reverse transcriptase at its N- and C-termini, respectively. Autoprocessing at the N-terminus of PR mediates stable dimer formation essential for catalytic activity, leading to the formation of infectious virus. An antiparallel ß-sheet interface formed by the four N- and C-terminal residues of each subunit is important for dimer stability. Here, we present the first high-resolution crystal structures of model protease precursor-clinical inhibitor (PI darunavir or saquinavir) complexes, revealing varying conformations of the N-terminal flanking (S(-4)FNF(-1)) and interface residues (P(1)QIT(4)). A 180° rotation of the T(4)-L(5) peptide bond is accompanied by a new Q(2)-L(5) hydrogen bond and complete disengagement of PQIT from the ß-sheet dimer interface, which may be a feature for intramolecular autoprocessing. This result is consistent with drastically lower thermal stability by 14-20 °C of PI complexes of precursors and the mature PR lacking its PQIT residues (by 18.3 °C). Similar to the TFR-PR precursor, this deletion also results in a darunavir dissociation constant (2 × 10(4))-fold higher and a markedly increased dimer dissociation constant relative to the mature PR. The terminal ß-sheet perturbations of the dimeric structure likely account for the drastically poorer inhibition of autoprocessing of TFR-PR relative to the mature PR, even though significant differences in active site-PI interactions in these structures were not observed. The novel conformations of the dimer interface may be exploited to target selectively the protease precursor prior to its N-terminal cleavage.

Identification of folding preferences of cleavage junctions of HIV-1 precursor proteins for regulation of cleavability.

Human immunodeficiency virus type 1 protease (HIV-1 PR) cleaves two viral precursor proteins, Gag and Gag-Pol, at multiple sites. Although the processing proceeds in the rank order to assure effective viral replication, the molecular mechanisms by which the order is regulated are not fully understood. In this study, we used bioinformatics approaches to examine whether the folding preferences of the cleavage junctions influence their cleavabilities by HIV-1 PR. The folding of the eight-amino-acid peptides corresponding to the seven cleavage junctions of the HIV-1(HXB2) Gag and Gag-Pol precursors were simulated in the PR-free and PR-bound states with molecular dynamics and homology modeling methods, and the relationships between the folding parameters and the reported kinetic parameters of the HIV-1(HXB2) peptides were analyzed. We found that a folding preference for forming a dihedral angle of Cß (P1)-Cα (P1)- Cα (P1')-Cß (P1') in the range of 150 to 180 degrees in the PR-free state was positively correlated with the 1/K(m) (R = 0.95, P = 0.0008) and that the dihedral angle of the O (P2)-C (P2)- C (P1)- O (P1) of the main chains in the PR-bound state was negatively correlated with k(cat) (R = 0.94, P = 0.001). We further found that these two folding properties influenced the overall cleavability of the precursor protein when the sizes of the side chains at the P1 site were similar. These data suggest that the dihedral angles at the specific positions around the cleavage junctions before and after binding to PR are both critical for regulating the cleavability of precursor proteins by HIV-1 PR.

Substrate recognition in HIV-1 protease: a computational study.

HIV-1 protease is a crucial enzyme for the life cycle of the human immunodeficiency virus, the retrovirus that triggers AIDS. It is well documented that HIV-1 protease mediates the cleavage of Gag, Gag-Pol, and Nef precursor polyproteins and is highly selective concerning the set of 12 different amino acid sequences that cleaves. However, the governing principles and physical parameters, which determine substrate recognition and specificity, remain poorly understood despite the many speculative proposals that abound in the literature. In fact, it has been difficult so far to circumvent the fact that protease's substrates share little sequence identity and lack an obvious consensus binding motif. We have used microsecond time scale MD simulations to quantitatively show that some sequences of the polyprotein Gag-Pol that are not cleaved (nonsubstrates) have in fact a higher affinity to the active site of HIV-1 protease than a substrate; i.e., recognition is not governed by affinity to the active site. On the basis of a detailed analysis of the results and experimental data, we propose that the recognition is based on the geometric specificity of PR:Gag and PR:Gag-Pol multiprotein complex, that selects which residues lie in the specific position that makes them accessible to the active site for cleavage.

Uncoupling human immunodeficiency virus type 1 Gag and Pol reading frames: role of the transframe protein p6* in viral replication.

Apart from its regulatory role in protease (PR) activation, little is known about the function of the human immunodeficiency virus type 1 transframe protein p6* in the virus life cycle. p6* is located between the nucleocapsid and PR domains in the Gag-Pol polyprotein precursor and is cleaved by PR during viral maturation. We have recently reported that the central region of p6* can be extensively mutated without abolishing viral infectivity and replication in vitro. However, mutagenesis of the entire p6*-coding sequence in the proviral context is not feasible without affecting the superimposed frameshift signal or the overlapping p1-p6(gag) sequences. To overcome these limitations, we created a novel NL4-3-derived provirus by displacing the original frameshift signal to the 3' end of the gag gene, thereby uncoupling the p6* gene sequence from the p1-p6(gag) reading frame. The resulting virus (AL) proved to be replication competent in different cell cultures and thus represents an elegant tool for detailed analysis of p6* function. Hence, extensive deletions or substitutions were introduced into the p6* gene sequence of the AL provirus, and effects on particle release, protein processing, and viral infectivity were evaluated. Interestingly, neither the deletion of 63% of all p6* residues nor the partial substitution by a heterologous sequence affected virus growth and infectivity, suggesting that p6* is widely dispensable for viral in vitro replication. However, the insertion of a larger reporter sequence interfered with virus production and maturation, implying that the length or conformation of this spacer region might be critical for p6* function.

Identification and characterization of 2 HIV-1 Gag immunodominant epitopes restricted by Asian HLA allele HLA-B*4801.

HLA-B*4801 is frequently found in Asian populations but rarely in Caucasian or African populations. Although HLA-B*4801-restricted human immunodeficiency virus-1 (HIV-1) epitopes would be useful for acquired immune deficiency syndrome (AIDS) vaccine development in Asia, they have not been reported so far. In the present study, we sought to identify HLA-B*4801-restricted HIV-1 epitopes by using 17-mer overlapping peptides derived from HIV-1 Gag, Pol, and Nef as well as 8- to 11-mer truncated peptides, and thereby identified two HLA-B*4801-restricted Gag epitopes. These epitope-specific CD8(+) T cells strongly responded to HIV-1-infected cells expressing HLA-B*4801, confirming that these Gag epitopes were endogenously presented by HLA-B*4801. These epitope-specific CD8(+) T cells were elicited in five of the seven tested chronically HIV-1-infected individuals with HLA-B*4801, suggesting them to be immunodominant epitopes. These epitopes will be useful for the studies of AIDS immunopathogenesis and the development of an HIV-1 vaccine in Asia.

HIV type 1 pol gene diversity and antiretroviral drug resistance mutations in Santos, Brazil.

HIV-1 antiretroviral drug resistance mutations in subtype B, F, and recombinants B/F in Santos, Brazil were characterized. We studied 83 samples from individuals enrolled at the Brazilian HIV/AIDS programs from Santos. These patients have been treated with multiantiretroviral therapy. Samples were collected in 2006; RNA was extracted from plasma and used as a target to amplify the pol gene of HIV-1. PCR products were sequenced on both strands, phylogenetic analyses were performed by neighbor-joining, and recombination was evaluated by bootscan. pol gene sequencing of the samples revealed that 54 strains belonged to subtype B, 4 were subtype F, 1 was subtype C, and 24 were B/F recombinants. Recombinant break points in 20 samples are the same identified in CRF28_BF and CRF29_BF. Drug resistance mutations identified in common to subtypes B, F, and recombinants B/F were protease inhibitors M46I/L (29%), I54V (24%), A71V (22%), and V82A/F (31%); reverse transcriptase nucleoside resistance mutations M41L (52%), D67N (30%), K70R (26%), M184V (88%), L210W (29%), T215Y/I/F (65%), and K219Q/E/N (28%); and reverse transcriptase nonnucleoside resistance mutation K103N (52%). Our results suggest that, in general, the same amino acids are emerging in both subtypes B, F, and recombinant forms BF due to the selective pressure of antiretrovirals. Recombinant break points in samples are the same as identified in CRF28_BF and CRF29_BF and are recognized as important for the evolution of the local epidemic.

Novel cytotoxic T-lymphocyte escape mutation by a three-amino-acid insertion in the human immunodeficiency virus type 1 p6Pol and p6Gag late domain associated with drug resistance.

Cytolytic T lymphocytes (CTL) play a major role in controlling human immunodeficiency virus type 1 (HIV-1) infection. To evade immune pressure, HIV-1 is selected at targeted CTL epitopes, which may consequentially alter viral replication fitness. In our longitudinal investigations of the interplay between T-cell immunity and viral evolution following acute HIV-1 infection, we observed in a treatment-naïve patient the emergence of highly avid, gamma interferon-secreting, CD8(+) CTL recognizing an HLA-Cw*0102-restricted epitope, NSPTRREL (NL8). This epitope lies in the p6(Pol) protein, located in the transframe region of the Gag-Pol polyprotein. Over the course of infection, an unusual viral escape mutation arose within the p6(Pol) epitope through insertion of a 3-amino-acid repeat, NSPT(SPT)RREL, with a concomitant insertion in the p6(Gag) late domain, PTAPP(APP). Interestingly, this p6(Pol) insertion mutation is often selected in viruses with the emergence of antiretroviral drug resistance, while the p6(Gag) late-domain PTAPP motif binds Tsg101 to permit viral budding. These results are the first to demonstrate viral evasion of immune pressure by amino acid insertions. Moreover, this escape mutation represents a novel mechanism whereby HIV-1 can alter its sequence within both the Gag and Pol proteins with potential functional consequences for viral replication and budding.