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Virology Jan 2008Azidothymidine (AZT) is a reverse transcriptase (RT) inhibitor that efficiently blocks the replication of spumaretroviruses or foamy viruses (FVs). To more precisely...
Azidothymidine (AZT) is a reverse transcriptase (RT) inhibitor that efficiently blocks the replication of spumaretroviruses or foamy viruses (FVs). To more precisely elucidate the mechanism of action of the FV RT enzyme, we generated an AZT-resistant FV in cell culture. Biologically resistant virus was obtained for simian foamy virus from macaque (SFVmac), which was insensitive to AZT concentrations of 1 mM, but not for FVs derived from chimpanzees. Nucleotide sequencing revealed four non-silent mutations in the pol gene. Introduction of these mutations into an infectious molecular clone identified all changes to be required for the fully AZT-resistant phenotype of SFVmac. The alteration of individual sites showed that AZT resistance in SFVmac was likely acquired by consecutive acquisition of pol mutations in a defined order, because some alterations on their own did not result in an efficiently replicating virus, neither in the presence nor in the absence of AZT. The introduction of the mutations into the RT of the closely related prototypic FV (PFV) did not yield an AZT-resistant virus, instead they significantly impaired the viral fitness.
Topics: Amino Acid Sequence; Animals; Cell Line; Drug Resistance, Viral; Gene Products, pol; Genes, pol; Humans; Microbial Sensitivity Tests; Molecular Sequence Data; Mutation; Reverse Transcriptase Inhibitors; Simian foamy virus; Virus Replication; Zidovudine
PubMed: 17904181
DOI: 10.1016/j.virol.2007.08.025 -
Scientific Reports Dec 2016HIV molecular epidemiology studies analyse viral pol gene sequences due to their availability, but whole genome sequencing allows to use other genes. We aimed to...
HIV molecular epidemiology studies analyse viral pol gene sequences due to their availability, but whole genome sequencing allows to use other genes. We aimed to determine what gene(s) provide(s) the best approximation to the real phylogeny by analysing a simulated epidemic (created as part of the PANGEA_HIV project) with a known transmission tree. We sub-sampled a simulated dataset of 4662 sequences into different combinations of genes (gag-pol-env, gag-pol, gag, pol, env and partial pol) and sampling depths (100%, 60%, 20% and 5%), generating 100 replicates for each case. We built maximum-likelihood trees for each combination using RAxML (GTR + Γ), and compared their topologies to the corresponding true tree's using CompareTree. The accuracy of the trees was significantly proportional to the length of the sequences used, with the gag-pol-env datasets showing the best performance and gag and partial pol sequences showing the worst. The lowest sampling depths (20% and 5%) greatly reduced the accuracy of tree reconstruction and showed high variability among replicates, especially when using the shortest gene datasets. In conclusion, using longer sequences derived from nearly whole genomes will improve the reliability of phylogenetic reconstruction. With low sample coverage, results can be highly variable, particularly when based on short sequences.
Topics: Cohort Studies; Epidemics; Genes, env; Genes, gag; Genes, pol; Genome, Viral; HIV; HIV Infections; Humans; Likelihood Functions; Molecular Epidemiology; Phylogeny; Regression Analysis; Reproducibility of Results; South Africa; United Kingdom
PubMed: 28008945
DOI: 10.1038/srep39489 -
Emerging Microbes & Infections Jun 2018The avian leukosis virus subgroup K (ALV-K), a novel subgroup in Chinese indigenous chicken breeds, has been difficult to isolate in the past due to its poor replication...
The avian leukosis virus subgroup K (ALV-K), a novel subgroup in Chinese indigenous chicken breeds, has been difficult to isolate in the past due to its poor replication ability. However, according to the latest monitoring data, the replication ability and isolation rate of ALV-K have clearly increased, and new strains with mutations in the pol gene have also been found. To determine the effects of such mutations on the biological characteristics of ALV-K, a pair of infectious clones were constructed and rescued. The first virus was an ALV-K Chinese isolate with mutations in its pol gene, named rSDAUAK-11. The second virus was a recuperative rSDAUAK-11 from which mutations in the pol gene were recovered according to the corresponding region of the ALV-K prototype virus JS11C1, named rRSDAUAK-11. In addition, two quantitative real-time polymerase chain reaction assays were developed to specifically detect these virus strains. Using such methods, we observed a marked improvement of the reverse transcriptase activity, replication ability and vertical transmission ability of rSDAUAK-11, which also revealed a formidable competitive advantage in mixed infection with rRSDAUAK-11 and corresponded to the differences between the wild strains SDAUAK-11 and JS11C1. Accordingly, our findings not only show that mutations in the pol gene are an important molecular mechanism contributing to corresponding changes in the biological characteristics of the newest ALV-K but also emphasize the potential future eradication of ALV.
Topics: Animals; Avian Leukosis; Avian Leukosis Virus; Chickens; DNA Mutational Analysis; Genes, pol; Genome, Viral; Genomic Instability; Infectious Disease Transmission, Vertical; Mutation; Virus Replication
PubMed: 29946141
DOI: 10.1038/s41426-018-0111-4 -
Viruses Jan 2021The ability to efficiently establish a new infection is a critical property for human immunodeficiency virus type 1 (HIV-1). Although the envelope protein of the virus...
The ability to efficiently establish a new infection is a critical property for human immunodeficiency virus type 1 (HIV-1). Although the envelope protein of the virus plays an essential role in receptor binding and internalization of the infecting virus, the structural proteins, the polymerase and the assembly of new virions may also play a role in establishing and spreading viral infection in a new host. We examined Ugandan viruses from newly infected patients and focused on the contribution of the genes to replication capacity. A panel of sequences generated using single genome amplification from incident HIV-1 infections were cloned into a common HIV-1 NL4.3 pol/env backbone and the influence of changes on replication capacity was monitored. Using a novel protein domain approach, we then documented diversity in the functional protein domains across the region and identified differences in the Gag-p6 domain that were frequently associated with higher in vitro replication.
Topics: Adult; Female; HIV Infections; HIV Protease; HIV-1; Humans; Male; Middle Aged; Protein Domains; Uganda; Virus Replication; Young Adult; gag Gene Products, Human Immunodeficiency Virus; pol Gene Products, Human Immunodeficiency Virus
PubMed: 33498793
DOI: 10.3390/v13020171 -
Journal of Virology Nov 1998TED (transposable element D) is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This...
TED (transposable element D) is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This lepidopteran (moth) retroelement contains gag and pol genes that encode proteins capable of forming viruslike particles (VLP) with reverse transcriptase. Since VLP are likely intermediates in TED transposition, we investigated the roles of gag and pol in TED capsid assembly and maturation. By using constructed baculovirus vectors and TED Gag-specific antiserum, we show that the principal translation product of gag (Pr55(gag)) is cleaved to produce a single VLP structural protein, p37(gag). Replacement of Asp436 within the retrovirus-like active site of the pol-encoded protease (PR) abolished Pr55(gag) cleavage and demonstrated the requirement for PR in capsid processing. As shown by expression of an in-frame fusion of TED gag and pol, PR is derived from the Gag-Pol polyprotein Pr195(gag-pol). The PR cleavage site within Pr55(gag) was mapped to a position near the junction of a basic, nucleocapsid-like domain and a C-terminal acidic domain. Once released by cleavage, the C-terminal fragment was not detected. This acidic fragment was dispensable for VLP assembly, as demonstrated by the formation of VLP by C-terminal Pr55(gag) truncation proteins and replacement of the acidic domain with a heterologous protein. In contrast, C-terminal deletions that extended into the adjacent nucleocapsid-like domain of Pr55(gag) abolished VLP recovery and demonstrated that this central region contributes to VLP assembly or stability, or both. Collectively, these data suggest that the single TED protein p37(gag) provides both capsid and nucleocapsid functions. TED may therefore use a simple processing strategy for VLP assembly and genome packaging.
Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Line; DNA Primers; DNA, Viral; Endogenous Retroviruses; Fusion Proteins, gag-pol; Gene Products, gag; Gene Products, pol; Genes, gag; Genes, pol; Kinetics; Models, Biological; Moths; Mutation; Nucleopolyhedroviruses; Protein Processing, Post-Translational; Retroelements; Spodoptera
PubMed: 9765414
DOI: 10.1128/JVI.72.11.8718-8724.1998 -
Frontiers in Immunology 2021Pig to human xenotransplantation is considered to be a possible approach to alleviate the shortage of human allografts. Porcine endogenous retrovirus (PERV) is the most...
Pig to human xenotransplantation is considered to be a possible approach to alleviate the shortage of human allografts. Porcine endogenous retrovirus (PERV) is the most significant pathogen in xenotransplantation. We screened for pigs that consistently did not transmit human-tropic replication competent PERVs (HTRC PERVs), namely, non-transmitting pigs. Then, we conducted whole-genome resequencing and full-length transcriptome sequencing to further investigate the sequence characteristics of one non-transmitting pig. Using transmission assays, we found 5 (out of 105) pigs of the Chinese Wuzhishan minipig inbred line that did not transmit PERV to human cells, i.e., non-transmitting pigs. Whole-genome resequencing and full-length transcriptome sequencing of one non-transmitting pig showed that all of the genes were defective at both the genome and transcript levels. We speculate that the defective PERV genes in this pig might be attributable to the long-term inbreeding process. This discovery is promising for the development of a strain of highly homozygous and genetically stable pigs with defective PERV genes as a source animal species for xenotransplantation.
Topics: Amino Acid Sequence; Animals; Cell Line; Cells, Cultured; China; Endogenous Retroviruses; Gene Expression Profiling; Gene Products, pol; Genes, pol; Genome; Genome, Viral; HEK293 Cells; Humans; Proviruses; Sequence Homology, Amino Acid; Swine; Swine, Miniature; Transcription, Genetic; Transplantation, Heterologous
PubMed: 35126361
DOI: 10.3389/fimmu.2021.797608 -
Scientific Data Jul 2018Accurate classification of HIV-1 group M lineages, henceforth referred to as subtyping, is essential for understanding global HIV-1 molecular epidemiology. Because most...
Accurate classification of HIV-1 group M lineages, henceforth referred to as subtyping, is essential for understanding global HIV-1 molecular epidemiology. Because most HIV-1 sequencing is done for genotypic resistance testing pol gene, we sought to develop a set of geographically-stratified pol sequences that represent HIV-1 group M sequence diversity. Representative pol sequences differ from representative complete genome sequences because not all CRFs have pol recombination points and because complete genome sequences may not faithfully reflect HIV-1 pol diversity. We developed a software pipeline that compiled 6,034 one-per-person complete HIV-1 pol sequences annotated by country and year belonging to 11 pure subtypes and 70 CRFs and selected a set of sequences whose average distance to the remaining sequences is minimized for each subtype/CRF and country to generate a Geographically-Stratified set of 716 Pol Subtype/CRF (GSPS) reference sequences. We provide extensive data on pol diversity within each subtype/CRF and country combination. The GSPS reference set will also be useful for HIV-1 pol subtyping.
Topics: Genes, pol; HIV Infections; HIV-1; Humans; Molecular Epidemiology; Recombination, Genetic
PubMed: 30063225
DOI: 10.1038/sdata.2018.148 -
PloS One 2016HIV-1 subtypes and drug resistance are routinely tested by many international surveillance groups. However, results from different sites often vary. A systematic...
HIV-1 subtypes and drug resistance are routinely tested by many international surveillance groups. However, results from different sites often vary. A systematic comparison of results from multiple sites is needed to determine whether a standardized protocol is required for consistent and accurate data analysis. A panel of well-characterized HIV-1 isolates (N = 50) from the External Quality Assurance Program Oversight Laboratory (EQAPOL) was assembled for evaluation at seven international sites. This virus panel included seven subtypes, six circulating recombinant forms (CRFs), nine unique recombinant forms (URFs) and three group O viruses. Seven viruses contained 10 major drug resistance mutations (DRMs). HIV-1 isolates were prepared at a concentration of 107 copies/ml and compiled into blinded panels. Subtypes and DRMs were determined with partial or full pol gene sequences by conventional Sanger sequencing and/or Next Generation Sequencing (NGS). Subtype and DRM results were reported and decoded for comparison with full-length genome sequences generated by EQAPOL. The partial pol gene was amplified by RT-PCR and sequenced for 89.4%-100% of group M viruses at six sites. Subtyping results of majority of the viruses (83%-97.9%) were correctly determined for the partial pol sequences. All 10 major DRMs in seven isolates were detected at these six sites. The complete pol gene sequence was also obtained by NGS at one site. However, this method missed six group M viruses and sequences contained host chromosome fragments. Three group O viruses were only characterized with additional group O-specific RT-PCR primers employed by one site. These results indicate that PCR protocols and subtyping tools should be standardized to efficiently amplify diverse viruses and more consistently assign virus genotypes, which is critical for accurate global subtype and drug resistance surveillance. Targeted NGS analysis of partial pol sequences can serve as an alternative approach, especially for detection of low-abundance DRMs.
Topics: Drug Resistance, Viral; Genes, pol; Genotype; HIV Infections; HIV-1; High-Throughput Nucleotide Sequencing; Humans; Molecular Sequence Data; Mutation; Phylogeny; Recombination, Genetic
PubMed: 27314585
DOI: 10.1371/journal.pone.0157340 -
Scientific Reports Feb 2019Background noise in metagenomic studies is often of high importance and its removal requires extensive post-analytic, bioinformatics filtering. This is relevant as...
Background noise in metagenomic studies is often of high importance and its removal requires extensive post-analytic, bioinformatics filtering. This is relevant as significant signals may be lost due to a low signal-to-noise ratio. The presence of plasmid residues, that are frequently present in reagents as contaminants, has not been investigated so far, but may pose a substantial bias. Here we show that plasmid sequences from different sources are omnipresent in molecular biology reagents. Using a metagenomic approach, we identified the presence of the (pol) of equine infectious anemia virus in human samples and traced it back to the expression plasmid used for generation of a commercial reverse transcriptase. We found fragments of multiple other expression plasmids in human samples as well as commercial polymerase preparations. Plasmid contamination sources included production chain of molecular biology reagents as well as contamination of reagents from environment or human handling of samples and reagents. Retrospective analyses of published metagenomic studies revealed an inaccurate signal-to-noise differentiation. Hence, the plasmid sequences that seem to be omnipresent in molecular biology reagents may misguide conclusions derived from genomic/metagenomics datasets and thus also clinical interpretations. Critical appraisal of metagenomic data sets for the possibility of plasmid background noise is required to identify reliable and significant signals.
Topics: Computational Biology; DNA Contamination; DNA, Viral; Diagnostic Errors; Genes, pol; High-Throughput Nucleotide Sequencing; Humans; Indicators and Reagents; Infectious Anemia Virus, Equine; Metagenomics; Plasmids; Retrospective Studies; Sequence Analysis, DNA
PubMed: 30733546
DOI: 10.1038/s41598-019-38733-1 -
Journal of Virology Sep 2008During acute human immunodeficiency virus type 1 (HIV-1) infection, early host cellular immune responses drive viral evolution. The rates and extent of these mutations,...
Marked epitope- and allele-specific differences in rates of mutation in human immunodeficiency type 1 (HIV-1) Gag, Pol, and Nef cytotoxic T-lymphocyte epitopes in acute/early HIV-1 infection.
During acute human immunodeficiency virus type 1 (HIV-1) infection, early host cellular immune responses drive viral evolution. The rates and extent of these mutations, however, remain incompletely characterized. In a cohort of 98 individuals newly infected with HIV-1 subtype B, we longitudinally characterized the rates and extent of HLA-mediated escape and reversion in Gag, Pol, and Nef using a rational definition of HLA-attributable mutation based on the analysis of a large independent subtype B data set. We demonstrate rapid and dramatic HIV evolution in response to immune pressures that in general reflect established cytotoxic T-lymphocyte (CTL) response hierarchies in early infection. On a population level, HLA-driven evolution was observed in approximately 80% of published CTL epitopes. Five of the 10 most rapidly evolving epitopes were restricted by protective HLA alleles (HLA-B*13/B*51/B*57/B*5801; P = 0.01), supporting the importance of a strong early CTL response in HIV control. Consistent with known fitness costs of escape, B*57-associated mutations in Gag were among the most rapidly reverting positions upon transmission to non-B*57-expressing individuals, whereas many other HLA-associated polymorphisms displayed slow or negligible reversion. Overall, an estimated minimum of 30% of observed substitutions in Gag/Pol and 60% in Nef were attributable to HLA-associated escape and reversion events. Results underscore the dominant role of immune pressures in driving early within-host HIV evolution. Dramatic differences in escape and reversion rates across codons, genes, and HLA restrictions are observed, highlighting the complexity of viral adaptation to the host immune response.
Topics: Acute Disease; Alleles; Amino Acid Sequence; Epitopes, T-Lymphocyte; Evolution, Molecular; Genes, gag; Genes, nef; Genes, pol; HIV Infections; HIV-1; HLA-B Antigens; Human Immunodeficiency Virus Proteins; Humans; Molecular Sequence Data; Mutation; T-Lymphocytes, Cytotoxic
PubMed: 18614631
DOI: 10.1128/JVI.01041-08