Vif Gene Research Articles

Benefits of HIV-1 transmission cluster surveillance: a French retrospective observational study of the molecular and epidemiological co-evolution of recent circulating recombinant forms 94 and 132.

Molecular surveillance is an important tool for detecting chains of transmission and controlling the HIV epidemic. This can also improve our knowledge of molecular and epidemiological factors for the optimization of prevention. Our objective was to illustrate this by studying the molecular and epidemiological evolution of the cluster including the new circulating recombinant form (CRF) 94_cpx of HIV-1, detected in 2017 and targeted by preventive actions in 2018. In June 2022, 32 HIV-1 sequence databases from French laboratories were screened to identify all individuals who had acquired CRF94_cpx or a similar strain, whatever the date of diagnosis. Phylogenetic analyses were performed with the sequences identified, and biological parameters were collected at the time of diagnosis and after the start of treatment to analyse the evolution of the cluster. Full genomes were sequenced to characterize the new strains. We analysed 98 HIV-1 isolates: 63 were CRF94, three were unclassifiable, and the other 32 formed a new cluster containing a new recombinant, CRF132_94B, derived from CRF94 and a subtype B strain. At least 95% of the individuals in both the CRF94 and CRF132 clusters were men who have sex with men (MSM), most of whom had acquired HIV less than 12 months before diagnosis. The number of CRF94 diagnoses declined drastically after 2018, but CRF132 strains spread widely between 2020 and 2022, into a different area of Ile-de-France region and within a younger population nevertheless aware of pre-exposure prophylaxis. Higher viraemia, lower CD4 cell counts and delayed treatment efficacy suggested that CRF94 was more virulent than CRF132, possibly due to the F subtype fragment of the vif gene. These findings highlight the role of the MSM transmission cluster in spreading HIV and new variants. They show also the benefits of cluster surveillance for improving the targeting of preventive interventions, detecting the emergence of new strains and enriching our knowledge on virulence mechanisms. However, these investigations require support with sufficient resources dedicated to a regional or national programme to be responsive and effective.

A computational exploration of global and temporal dynamics of selection pressure on HIV-1 Vif polymorphism

Virion infectivity factor (Vif), an accessory protein of HIV-1 (human immunodeficiency virus type 1), antagonizes host APOBEC3 protein (apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3) or A3 via proteasomal degradation, facilitating viral replication. HLA (Human leukocyte antigens) alleles, host restriction factors, and error-prone reverse transcription contribute to the global polymorphic dynamics of HIV, impacting effective vaccine design. Our computational analysis of over 50,000 HIV-1 M vif sequences from the Los Alamos National Laboratory (LANL) database (1998–2021) revealed positive selection pressure on the vif gene (nonsynonymous to synonymous ratio, dn/ds=1.58) and an average entropy score of 0.372 in protein level. Interestingly, over the years (1998–2021), a decreasing trend of dn/ds (1.68 to 1.47) and an increasing trend of entropy (0.309 to 0.399) was observed. The predicted mutational frequency against Vif consensus sequence decreased over time (slope = -0.00024, p < 0.0001). Sequence conservation was observed in Vif functional motifs F1, F2, F3, G, BC box, and CBF β binding region, while variability was observed mainly in N- and C- terminal and Zinc finger region, which were dominantly under immune pressure by host HLA-I-restricted CD8+ T cell. Computational analysis of ∆∆Gstability through protein stability prediction tools suggested that missense mutation may affect Vif stability, especially in the Vif-A3 binding interface. Notably, mutations R17K and Y44F in F1 and G box were predicted to destabilize the Vif-A3 binding interface by altering bond formations with adjacent amino acids. Therefore, our analysis demonstrates Vif adaptation with host physiology by maintaining sequence conservation, especially in A3 interacting functional motifs, highlighting important therapeutic candidate regions of Vif against HIV-1 infections.

A new look at the immunopathogenesis of infection caused by the human immunodeficiency virus

Thanks to the achievements of biotechnology and genetics, numerous features of reproduction of the human immunodeficiency virus and its immunopathogenesis have been established. Genetic studies have called into question the African origin of the virus. Thus, the number of individuals with mutational changes in the CCR5∆32 allele among the population providing genetic protection against human immunodeficiency virus is decreasing from north to south. The probability of adaptation of the great ape virus in the human population is characterized. New biological effects and their influence on the immunopathogenesis of infection of the well-known human immunodeficiency virus genes Gag (structural proteins), Pol (enzymes) and Env (envelope glycoproteins), the proteins encoded by them, as well as a number of additional proteins have been discovered. Thus, the main task of the Tat gene is to stimulate the transcription of proviral deoxyribonucleic acid and the transport of ribonucleic acid from the nucleus to the cytoplasm of the cell. Rev promotes the synthesis of viral structural proteins and enzymes, ensures the availability of full-sized genomic ribonucleic acid for inclusion in the reproduced viral progeny. Viruses with the absence of the vif gene are about 1000 times less virulent compared to wild strains. The APOBEC3G factor inhibits the reproduction of lentiviruses in primates, but in humans it is characterized by polymorphism of biological effects. Viral protein R affects the rate of reproduction of the virus in T-lymphocytes, contributes to their destruction. It also promotes proteasomal degradation and protein modification. Vpr targets may be a structurally specific endonuclease subunit SLX4, uracil-deoxynucleoacid glycosylase 2, and a helicase-like transcription factor. Vpu is a powerful inhibitor of teterin by type 1 immunodeficiency viruses of group M, and in group N viruses it shows low activity. Vpu, Nef, and Env proteins of most lentiviruses are characterized by higher tropicity to the CD4 receptor compared to teterin, serine incorporation proteins. These proteins are incorporated into viral particles and reduce their infectivity by inhibiting fusion with target cells. The protein containing the tripartite motif 5a, the protein of mixovirus activity 2/B constitute an ancient system of protection against retroviruses and are extremely variable in their effectiveness to disrupt the reproduction of viruses.

Characterization of bovine immunodeficiency virus genes and proteins

The goal of the study was determining the effect of natural selection on the bovine immunodeficiency virus (BIV) through the identification of polymorphisms by comparing a number of genes, open reading frames and proteins and analyzing the effect of nonsynonymous substitutions in the Vif protein of BIV isolates on the Vif conformational parameters. The selection pressure on the genes was estimated by Tajima test, as well as the determination of the ratio of nonsynonymous substitutions (Ka) to synonymous (Ks) Ka/Ks. Ka and Ks coefficients, their ratio (Ka/Ks) were calculated on the basis of alignments of amino acid and nucleotide sequences of the vif gene of BIV isolates. The nonsynonymous (dN) to synonymous (dS) substitution rate ratio per nucleotide site dN/dS was calculated to estimate the effect of selection on the vif gene by SLAC method on Datamonkey server. The number of hydrogen bonds, α-helices, β-sheets, β-turns for the protein tertiary structures of proteins was determined by I-TASSER server. Genes, open reading frames (ORFs) and the U3 regulatory region of BIV genome which are under the influence of selection have been identified. Gag, pol, s, vif genes, ORF W and ORF Y are under the negative (purifying) selection pressure. Env gene and the U3 regulatory region are under positive selection. Shannon entropy analysis (peaks that are specific to the positions of amino acid residues), interpreted as polymorphisms, revealed 16, 8, and 4 nonsynonymous substitutions for Pol, Gag, and Vif BIV proteins, respectively. Significant changes in the number of hydrogen bonds, α-helices, β-layers, β-turns were determined by the analysis of the effect of four nonsynonymous substitutions in the Vif protein on the conformational parameters of Vif of two BIV isolates. The multidirectional effect of selection on BIV genes is shown

Identification of Two Novel HIV-1 Second-Generation Recombinant Forms (CRF01_AE/CRF07_BC) in Men Who Have Sex with Men in Hebei, China.

In recent years, men who have sex with men (MSM) have been identified as the primary source of HIV-1 transmission in Hebei Province, China. Co-circulation of multiple subtypes in HIV-1 positive MSM populations may contribute to the emergence of the second generation of recombinant HIV-1 strains, indicating the occurrence of dual infections or superinfections in MSM populations. Thus, the discovery of new recombinant strains is important to indicate the appearance of multiple infected individuals and the prevalence caused by changes in the parent strains. In this study, we present two new unique recombinant forms (URFs) from two HIV-1-positive subjects (HB070052 and HB070056) infected through homosexual contact in Hebei Province, China. The near full-length genome of the two URFs revealed that HB070052 was divided into seven segments by six breakpoints in the gag, pol, vif, and vpr genes; HB070056 was separated into five fragments by four breakpoints, with two regions of CRF07_BC inserted into a CRF01_AE backbone's gag, pol regions. The subregion tree showed CRF01_AE segments were traced back to the cluster 4 and 6 of the CRF01_AE phylogenetic tree, which were prevalent among HIV-1 infections through MSM in China. The continued emergence of the novel CRF01_AE/CRF07_BC recombinant forms indicates the HIV-1 epidemic is complex and long-term surveillance of recombinant strains is necessary among MSM in this region.

A Novel Vpu Adaptive Mutation of HIV-1 Degrades Tetherin in Northern Pig-Tailed Macaques (Macaca leonina) Mainly via the Ubiquitin-Proteasome Pathway and Increases Viral Release.

Tetherin prevents viral cross-species transmission by inhibiting the release of multiple enveloped viruses from infected cells. With the evolution of simian immunodeficiency virus of chimpanzees (SIVcpz), a pandemic human immunodeficiency virus type 1 (HIV-1) precursor, its Vpu protein can antagonize human tetherin (hTetherin). Macaca leonina (northern pig-tailed macaque [NPM]) is susceptible to HIV-1, but host-specific restriction factors limit virus replication in vivo. In this study, we isolated the virus from NPMs infected with strain stHIV-1sv (with a macaque-adapted HIV-1 env gene from simian-human immunodeficiency virus SHIV-KB9, a vif gene replaced by SIVmac239, and other genes originating from HIV-1NL4.3) and found that a single acidic amino acid substitution (G53D) in Vpu could increase its ability to degrade the tetherin of macaques (mTetherin) mainly through the proteasome pathway, resulting in an enhanced release and resistance to interferon inhibition of the mutant stHIV-1sv strain, with no influence on the other functions of Vpu. IMPORTANCE HIV-1 has obvious host specificity, which has greatly hindered the construction of animal models and severely restricted the development of HIV-1 vaccines and drugs. To overcome this barrier, we attempted to isolate the virus from NPMs infected with stHIV-1sv, search for a strain with an adaptive mutation in NPMs, and develop a more appropriate nonhuman primate model of HIV-1. This is the first report identifying HIV-1 adaptations in NPMs. It suggests that while tetherin may limit HIV-1 cross-species transmission, the Vpu protein in HIV-1 can overcome this species barrier through adaptive mutation, increasing viral replication in the new host. This finding will be beneficial to building an appropriate animal model for HIV-1 infection and promoting the development of HIV-1 vaccines and drugs.

Development of a novel Macaque-Tropic HIV-1 adapted to cynomolgus macaques.

Macaque-tropic HIV-1 (HIV-1mt) variants have been developed to establish preferable primate models that are advantageous in understanding HIV-1 infection pathogenesis and in assessing the preclinical efficacy of novel prevention/treatment strategies. We previously reported that a CXCR4-tropic HIV-1mt, MN4Rh-3, efficiently replicates in peripheral blood mononuclear cells (PBMCs) of cynomolgus macaques homozygous for TRIMCyp (CMsTC). However, the CMsTC challenged with MN4Rh-3 displayed low viral loads during the acute infection phase and subsequently exhibited short-term viremia. These virological phenotypes in vivo differed from those observed in most HIV-1-infected people. Therefore, further development of the HIV-1mt variant was needed. In this study, we first reconstructed the MN4Rh-3 clone to produce a CCR5-tropic HIV-1mt, AS38. In addition, serial in vivo passages allowed us to produce a highly adapted AS38-derived virus that exhibits high viral loads (up to approximately 106 copies ml-1) during the acute infection phase and prolonged periods of persistent viremia (lasting approximately 16 weeks postinfection) upon infection of CMsTC. Whole-genome sequencing of the viral genomes demonstrated that the emergence of a unique 15-nt deletion within the vif gene was associated with in vivo adaptation. The deletion resulted in a significant increase in Vpr protein expression but did not affect Vif-mediated antagonism of antiretroviral APOBEC3s, suggesting that Vpr is important for HIV-1mt adaptation to CMsTC. In summary, we developed a novel CCR5-tropic HIV-1mt that can induce high peak viral loads and long-term viremia and exhibits increased Vpr expression in CMsTC.

IN VITRO INFECTIVITY ANALYSIS OF SIMIAN-HUMAN IMMUNODEFICIENCY VIRUS IN PERIPHERAL BLOOD MONONUCLEAR CELL MACACA FASCICULARIS AND M. NEMESTRINA

Development of human immunodeficiency virus type 1 (HIV-1) vaccines and anti-retroviral treatmentis currently hindered by the lack of models representing prominent symptoms of HIV-1 infections seen in humans. Simian-human immunodeficiency virus (SHIV) was constructed to resolve the limitations of SIVmac model and has been used in nonhuman primate model ofviral infections, particularly infections by the close relatives of HIV-1. Macaca fascicularis and M. nemestrina are being developed as model HIV/AIDS, by using chimeric virus SHIV produced by replacing the nucleotide structure of cyclophilin A binding region, vif gene and nef of HIV-1 with cyclophilin A binding region, vif gene and nef from SIV. The research aims to study the model of HIV/AIDS on nonhuman primates PBMC in vitro using SHIV. In particular, the study aims to obtain information about the capability of SHIV replication in PBMC of M. fascicularis and M. nemestrina. Results showed a cytopathic effect (CPE) in the form of multinucleated giant cells and expression of p24 protein in PHA-stimulated PBMC cultures of M. fascicularis and M. nemestrina after SHIV infection. The conclusion of this study is that SHIV can infect PBMC M. fascicularis and M. nemestrina in vitro based on CPE and expression of p24 protein.

Expression and Characterization of Two DNA Constructs Derived from HIV-1-vif in Escherichia coli and Mammalian Cells

Background:Acquired immunodeficiency syndrome (HIV/AIDS) is still a major global concern and no effective therapeutic vaccine has been produced to prevent the problem. Among HIV-1 proteins, vif as a basic cytoplasmic protein of HIV-1 is involved in late stages of viral generation and plays important role in HIV-1 virion replication. It also increases the stability of virion cores, which probably inhibits early degradation of viral entry. Therefore, it seems rational to apply this protein as a vaccine based on its impact on HIV-1 life cycle. This study aimed at cloning, expression and production of vif protein as an HIV-1 vaccine candidate.Methods:In this study, vif sequence was amplified from pLN4-3 plasmid including HIV-1 vif gene and then cloned in pET23a to generate the recombinant plasmids of pET23a/vif with hexahistidine tags. BL21 competent cells were transformed to obtain the protein of interest. Ni-NTA column was used to purify the protein of interest and western blotting confirmed vif protein using anti-His tag antibody. In order to express the gene of interest in eukaryotic cells, vif was sub-cloned into pEGFP plasmids and HEK 293-T cells were transfected. Flow cytometry was then applied to evaluate GFP expression.Results:vif protein was expressed in BL21(DE3) strain and identified as a23 kDa band in SDS-PAGE and confirmed by anti-His antibody in western blotting. The purified protein concentration was 173.3 μg/ml using Bradford assay. HEK-293T cells were successfully transfected by recombinant pEGFP plasmids and flow cytometry confirmed the cell transfection.Conclusion:vif protein can be expressed in mammalian cells and may be a proper protein subunit vaccine candidate against HIV-1.

A phylogenetic study of Feline Immunodeficiency Virus (FIV) among domestic cats in Turkey

Feline Immunodeficiency Virus (FIV) is the most prominent retrovirus in cats. Molecular studies on FIV are of great importance to enable further studies, for example, understanding the pathogenesis and developing improved vaccines. We aimed to elucidate the molecular status of FIV and provide a detailed characterization of FIV in Turkey because at present there is very limited information available in the literature. We also evaluated a potential link between clinical symptoms and FIV subtypes according to results obtained from molecular tests.Whole blood was collected from 200 client-owned domestic cats and molecular diagnosis and characterization was performed. The env, gag and vif gene regions were amplified and sequenced for phylogenetic analysis. We obtained specific amplicons based on bothenvand gag for FIV in 21 cats; only 2 of the 21 positive samples could also be characterized based on the vif gene region. Separate clusters were identified according to previously determined genotyping strategies; however, they were observed in FIV subtype B. The molecular findings of some individual cats were evaluated in conjunction with their clinical symptoms in an attempt to determine potential relationships between the genetic characteristics of FIV and symptoms of disease. As a result, overexpression of the vif gene could be important in leading to serious clinical symptoms.Our results emphasize the necessity of considering FIV in diagnosis and performing the neccesary diagnostics to confirm or rule out FIV infection. The molecular dynamics of FIV should be periodically updated by further analyses to establish a successful prevention strategy.

Is the tryptophan codon of gene vif the Achilles' heel of HIV-1?

To evaluate the impact of hypermutation on the HIV-1 dissemination at the population level we studied 7072 sequences HIV-1 gene vif retrieved from the public databank. From this dataset 854 sequences were selected because they had associated values of CD4+ T lymphocytes counts and viral loads and they were used to assess the correlation between clinical parameters and hypermutation. We found that the frequency of stop codons at sites 5, 11 and 79 ranged from 2.8x10-4 to 4.2x10-4. On the other hand, at codons 21, 38, 70, 89 and 174 the frequency of stop codons ranged from 1.4x10-3 to 2.5x10-3. We also found a correlation between clinical parameters and hypermutation where patients harboring proviruses with one or more stop codons at the tryptophan sites of the gene vif had higher CD4+ T lymphocytes counts and lower viral loads compared to the population. Our findings indicate that A3 activity potentially restrains HIV-1 replication because individuals with hypermutated proviruses tend to have lower numbers of RNA copies. However, owing to the low frequency of hypermutated sequences observed in the databank (44 out of 7072), it is unlikely that A3 has a significant impact to curb HIV-1 dissemination at the population level.

The frequency of defective genes in vif and vpr genes in 20 hemophiliacs is associated with Korean Red Ginseng and highly active antiretroviral therapy: the impact of lethal mutations in vif and vpr genes on HIV-1 evolution

BackgroundWe have reported that internal deletions in the nef, gag, and pol genes in HIV-1–infected patients are induced in those treated with Korean Red Ginseng (KRG). KRG delays the development of resistance mutations to antiretroviral drugs. MethodsThe vif-vpr genes over 26 years in 20 hemophiliacs infected with HIV-1 from a single source were sequenced to investigate whether vif-vpr genes were affected by KRG and KRG plus highly active antiretroviral therapy (ART) (hereafter called GCT) and compared the results with our previous data. ResultsA significantly higher number of in-frame small deletions were found in the vif-vpr genes of KRG-treated patients than at the baseline, in control patients, and in ART-alone patients (p < 0.001). These were significantly reduced in GCT patients (p < 0.05). In contrast, sequences harboring a premature stop codon (SC) were more significant in GCT patients (10.1%) than in KRG-alone patients, control (p < 0.01), and ART-alone patients (p = 0.078 for peripheral blood mononuclear cells). The proportion of SC in Vpr was similar to that in Vif, whereas the proportion of sequences revealing SC in the env-nef genes was significantly lower than that in the pol-vif-vpr genes (p < 0.01). The genetic distance was 1.8 times higher in the sequences harboring SC than in the sequences without SC (p < 0.001). Q135P in the vif gene is significantly associated with rapid progression to AIDS (p < 0.01). ConclusionOur data show that KRG might induce sΔ in the vif-vpr genes and that vif-vpr genes are similarly affected by lethal mutations.

HIV-1 genetic diversity and recombinant forms among men who have sex with men at a sentinel surveillance site in Xi'an City, China

HIV-1 genetic distribution and recombinant patterns are important in understanding the HIV epidemic among men who have sex with men (MSM). In this study, 83 HIV-positive MSM infections were confirmed at a sentinel surveillance site in Xi'an city, China in 2018. HIV-1 genotypes were determined by phylogenetic analyses of HIV-1 gag, pol and env gene fragments, including CRF07_BC (51.8%), CRF01_AE (30.1%), subtype B (3.6%), CRF55_01B (3.6%), CRF104_0107 (1.2%) and unique recombinant forms (URFs) (9.6%). Transmitted drug resistance mutations were detected in 2.4% (2/82) of HIV-infected MSM individuals. The phylogenetic analyses of near full-length genome (NFLG) of HIV-1 URFs were performed. A new circulating recombinant form (CRF), designated as CRF104_0107, was found in three epidemiologically unlinked individuals in Shaanxi province, China. The CRF104_0107 is composed of genomes CRF01_AE and CRF07_BC, with six recombinant breakpoints in the gag, pol, vif and vpr genes. This second-generation CRF has a breakpoint (HXB2 nt 3011) in common with CRF07_BC. The emergence of novel CRF and multiple URFs reflected HIV-1 genetic complexity among the local key populations in Xi'an city, China.

Expression Level of HIV-1 Vif Can Be Fluctuated by Natural Nucleotide Variations in the vif-Coding and Regulatory SA1D2prox Sequences of the Proviral Genome.

Vif is required for HIV-1 replication in natural target cells by counteracting host restriction factors, APOBEC3 (A3) proteins. We recently demonstrated that Vif expression level can be changed by naturally occurring single-nucleotide variations within SA1D2prox of the HIV-1 genome. We also found that levels for vif/vpr mRNAs are inversely correlated. While amino acid sequence per se is critical for functionality, Vif expression level modulated by signal sequences in its coding region is likely to be important as well. There are two splicing sites in the region involved in vpr expression. To reveal possible fluctuations of Vif-expression level, we examined SA1D2prox and vif gene by chimeric approaches using HIV-1 subtypes B and C with distinct anti-A3 activity. In this report, recombinant clones in subtype B backbone carrying chimeric sequences with respect to SA1D2prox/vif and those within the vif-coding region were generated. Of these, clones containing vif-coding sequence of subtype C, especially its 3′ region, expressed vif/Vif at a decreased level but did at an increased level for vpr/Vpr. Clones with reduced vif/Vif level grew similarly or slightly better than a parental clone in weakly A3G-positive cells but more poorly in highly A3G-expressing cells. Three clones with this property were also tested for their A3-degrading activity. One of the clones appeared to have some defect in addition to the poor ability to express vif/Vif. Taken all together, our results show that natural variations in the SA1D2prox and vif-coding region can change the Vif-expression level and affect the HIV-1 replication potential.

A viral genome wide association study and genotypic resistance testing in patients failing first line antiretroviral therapy in the first large countrywide Ethiopian HIV cohort

BackgroundAntiretroviral therapy (ART) was rolled-out in Ethiopia in 2005, but there are no reports on outcome of ART and human immunodeficiency virus drug resistance (HIVDR) at national level. We described acquired drug resistance mutations in pol gene and performed a viral genome wide association study in virologic treatment failure patients who started first line ART during 2009–2011 in the first large countrywide HIV cohort in Ethiopia.MethodsThe outcome of tenofovir (TDF)- and zidovudine (ZDV)-based ART was defined in 874 ART naïve patients using the on-treatment (OT) and intention-to-treat (ITT) analyses. Genotypic resistance testing was done in patients failing ART (> 1000 copies/ml) at month 6 and 12. Near full-length genome sequencing (NFLG) was used to assess amino acid changes in HIV-1 gag, pol, vif, vpr, tat, vpu, and nef genes between paired baseline and month 6 samples.ResultsHigh failure rates were found in ITT analysis at month 6 and 12 (23.3%; 33.9% respectively). Major nucleoside and non-nucleoside reverse transcriptase (NRTI/NNRTI) drug resistance mutations were detected in most failure patients at month 6 (36/47; 77%) and month 12 (20/30; 67%). A high rate of K65R was identified only in TDF treated patients (35.7%; 50.0%, respectively). No significant difference was found in failure rate or extent of HIVDR between TDF- and ZDV- treated patients. All target regions of interest for HIVDR were described by NFLG in 16 patients tested before initiation of ART and at month 6.ConclusionIn this first Ethiopian national cohort, a high degree of HIVDR was seen among ART failure patients, independent on whether TDF- or ZDV was given. However, the major reason to ART failure was lost-to-follow-up rather than virologic failure. Our NFLG assay covered all relevant target genes for antiretrovirals and is an attractive alternative for HIVDR surveillance.

Characterization of a Novel HIV-1 CRF01_AE/CRF07_BC Recombinant Virus Among Men Who Have Sex with Men in Tianjin, China.

We report here a novel human immunodeficiency virus type 1 (HIV-1) second-generation recombinant form comprising CRF01_AE and CRF07_BC isolated from an HIV-positive male subject infected among men who have sex with men (MSM) in Tianjin in north China. The phylogenetic analysis of the near full-length genome of TJIH0345 reveals that three regions of CRF01_AE were inserted into the CRF07_BC backbone with five recombinant breakpoints observed in the pol, vif, vpu, and env gene regions whose genome structure is distinctly different from other circulating recombinant forms and unique recombinant forms previously reported. The continued emergence of the novel recombinant strain in Tianjin, China, highlights the increasing complexity of the HIV-1 epidemic among the MSM population, and further molecular epidemiological investigation should be done to track the genetic evolution of HIV-1 strains to prevent HIV transmission.

Near-Full-Length Genetic Characterization of a Novel HIV-1 Unique Recombinant with Similarities to A1, CRF01_AE, and CRFO2_AG Viruses in Yaoundé, Cameroon.

Variations in the HIV genome influence HIV/AIDS epidemiology. We report here a novel HIV-1 unique recombinant form (URF) isolated from an HIV-infected female (NACMR092) in Cameroon, based on the analyses of near-full-length viral genome (partial gag, full-length pol, env, tat, rev, vif, vpr, vpu, and nef genes, and partial 3'-long terminal repeat). Phylogeny, recombination breakpoints, and recombination map analyses showed that NACMR092 was infected with a mosaic URF that had eight breakpoints (two in gag, one in pol, one in vpr, two in env, and two in the nef regions), nine subgenomic regions, and included fragments that had important similarities with HIV-1 subtypes A1, CRF02_AG, and CRF01_AE. This novel mosaic URF underscores complex recombination events occurring between HIV-1 subtypes circulating in Cameroon. Continued monitoring and detection of such recombinants and accurate classification of HIV genotype is important for tracking viral molecular epidemiology and antigenic diversity.

Production of breast cancer cell lines expressing viral gene VIF

Introduction: Virion Infectivity factor (Vif) is a protein required for lentiviruses successful infection of human target cells, through the degradation of host restriction factors, APOBEC3 [1]. In the last years, the activity of APOBEC3 deaminases has also been associated with mutation patterns found in cancer cells, including breast cancer cells [2]. We hypothesize that Vif protein can be used to inhibit APOBEC3 avoiding breast cancer progression and resistance to chemotherapy. This hypothesis should be tested in breast cancer cells expressing Vif proteins from HIV-1 (Vif1), and HIV-2 (Vif2) treated with chemotherapy agents. The aim of this work was the production of breast cell lines expressing constitutively Vif1 and Vif 2 proteins, using lentiviruses as a gene-delivering system. Materials and methods: Vif1 and Vif2 delivering lentivirus vectors were obtained using the fourth generation lentiviral packaging systems (Clontech). Briefly, Vif 1 and Vif 2 genes were cloned in a bicistronic lentiviral expression vector (pLVX-IRES-zsGreen1) which allowed the co-expression of Vif and the reporter gene (ZsGreen) from a single mRNA transcript. VSV-G pseudotyped lentiviruses were produced by transfection of packaging cells, Lenti-X 293T cells (Clontech). Viruses were then used to transduce MCF10A and HCC1806 breast cells. Transduced cells stably expressing fluorescent marker were isolated by Fluorescence Activated Cell Sorting (FACS-AriaIII). The isolated cells were expanded and characterized. RNA was extracted from the cell lines MCF10A-Vif and HCC1806-Vif, and cDNA synthesis was performed. Expression of Vif was confirmed by PCR amplification of cDNA and APOBEC3G, 3B and 3C expression levels were also confirmed by qRT-PCR (TaqMan Technology). Results: We obtained high-titer of Vif delivering lentiviruses used to transduce the breast cells. FACS allowed us to obtain sorting populations with highly purified transduced cells (sorting populations with 97 to 99% purity). Characterization of the new cell populations showed that lentiviral constructs integrated either Vif1 or Vif2 genes into genomic DNA. Moreover, Vif genes are expressed in these populations with no significant effect on the mRNA levels of APOBEC3G, 3C, and 3B (∼1.5 to 2 fold increases). Discussion and conclusions: The genomic DNA of MCF10A (normal breast cell line) and HCC1806 (breast cancer cell line) were modified with Vif gene (and ZsGreen gene) integration generating new stable breast cell lines. In the near future, we will be able to confirm or not that inhibition of APOBEC3 by Vif sensitize to chemotherapy the breast cancer cells, and hopefully decrease recurrent relapses. We believe that the use of Vif to inhibit APOBEC3 is a promising therapeutic tool model with application in breast cancer disease.

Host Restriction Factors APOBEC3G/3F and Other Interferon-Related Gene Expressions Affect Early HIV-1 Infection in Northern Pig-Tailed Macaque (Macaca leonina).

The northern pig-tailed macaques (NPMs) lack TRIM5α, an antiviral restriction factor, and instead have TRIM5-CypA. In our previous study, we demonstrated that HIV-1NL4−3 successfully infected NPMs and formed a long-term viral reservoir in vivo. However, the HIV-1-infected NPMs showed relatively high viremia in the first 6 weeks of infection, which declined thereafter suggesting that HIV-1 NL4−3 infection in these animals was only partly permissive. To optimize HIV-1 infection in NPMs therefore, we generated HIV-1NL4−R3A and stHIV-1sv, and infected NPMs with these viruses. HIV-1NL4−R3A and stHIV-1sv can replicate persistently in NPMs during 41 weeks of acute infection stage. Compared to the HIV-1NL4−R3A, stHIV-1sv showed a notably higher level of replication, and the NPMs infected with the latter induced a more robust neutralizing antibody but a weaker cellular immune response. In addition, IFN-I signaling was significantly up-regulated with the viral replication, and was higher in the stHIV-1sv infected macaques. Consequently, the sequences of pro-viral env showed fewer G-A hyper-mutations in stHIV-1sv, suggesting that vif gene of SIV could antagonize the antiviral effects of APOBEC3 proteins in NPMs. Taken together, NPMs infected with HIV-1NL4−R3A and stHIV-1sv show distinct virological and immunological features. Furthermore, interferon-related gene expression might play a role in controlling primary HIV-1NL4−R3A and stHIV-1sv replication in NPMs. This result suggests NPM is a potential HIV/AIDS animal model.

Absence of A3Z3-Related Hypermutations in the env and vif Proviral Genes in FIV Naturally Infected Cats.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3) proteins comprise an important family of restriction factors that produce hypermutations on proviral DNA and are able to limit virus replication. Vif, an accessory protein present in almost all lentiviruses, counteracts the antiviral A3 activity. Seven haplotypes of APOBEC3Z3 (A3Z3) were described in domestic cats (hap I–VII), and in-vitro studies have demonstrated that these proteins reduce infectivity of vif-defective feline immunodeficiency virus (FIV). Moreover, hap V is resistant to vif-mediated degradation. However, studies on the effect of A3Z3 in FIV-infected cats have not been developed. Here, the correlation between APOBEC A3Z3 haplotypes in domestic cats and the frequency of hypermutations in the FIV vif and env genes were assessed in a retrospective cohort study with 30 blood samples collected between 2012 and 2016 from naturally FIV-infected cats in Brazil. The vif and env sequences were analyzed and displayed low or undetectable levels of hypermutations, and could not be associated with any specific A3Z3 haplotype.