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Theoretical Biology & Medical Modelling Jul 2011Infection with HIV, which culminates in the establishment of a latent proviral reservoir, presents formidable challenges for ultimate cure. Building on the hypothesis...
BACKGROUND
Infection with HIV, which culminates in the establishment of a latent proviral reservoir, presents formidable challenges for ultimate cure. Building on the hypothesis that ex-vivo or even in-vivo abolition or disruption of HIV-gene/genome-action by target mutagenesis or excision can irreversibly abrogate HIV's innate fitness to replicate and survive, we previously identified the isoschizomeric bacteria restriction enzymes (REases) AcsI and ApoI as potent cleavers of the HIV-pol gene (11 and 9 times in HIV-1 and 2, respectively). However, both enzymes, along with others found to cleave across the entire HIV-1 genome, slice (SX) at palindromic sequences that are prevalent within the human genome and thereby pose the risk of host genome toxicity. A long-term goal in the field of R-M enzymatic therapeutics has thus been to generate synthetic restriction endonucleases with longer recognition sites limited in specificity to HIV. We aimed (i) to assemble and construct zinc finger arrays and nucleases (ZFN) with either proviral-HIV-pol gene or proviral-HIV-1 whole-genome specificity respectively, and (ii) to advance a model for pre-clinically testing lentiviral vectors (LV) that deliver and transduce either ZFN genotype.
METHODS AND RESULTS
First, we computationally generated the consensus sequences of (a) 114 dsDNA-binding zinc finger (Zif) arrays (ZFAs or ZifHIV-pol) and (b) two zinc-finger nucleases (ZFNs) which, unlike the AcsI and ApoI homeodomains, possess specificity to >18 base-pair sequences uniquely present within the HIV-pol gene (ZifHIV-polFN). Another 15 ZFNs targeting >18 bp sequences within the complete HIV-1 proviral genome were constructed (ZifHIV-1FN). Second, a model for constructing lentiviral vectors (LVs) that deliver and transduce a diploid copy of either ZifHIV-polFN or ZifHIV-1FN chimeric genes (termed LV- 2xZifHIV-polFN and LV- 2xZifHIV-1FN, respectively) is proposed. Third, two preclinical models for controlled testing of the safety and efficacy of either of these LVs are described using active HIV-infected TZM-bl reporter cells (HeLa-derived JC53-BL cells) and latent HIV-infected cell lines.
CONCLUSION
LV-2xZifHIV-polFN and LV- 2xZifHIV-1FN may offer the ex-vivo or even in-vivo experimental opportunity to halt HIV replication functionally by directly abrogating HIV-pol-gene-action or disrupting/excising over 80% of the proviral HIV DNA from latently infected cells.
Topics: Base Sequence; DNA, Viral; Endonucleases; Genes, pol; Genetic Therapy; Genetic Vectors; Genome, Viral; HIV-1; Humans; Lentivirus; Proviruses; Substrate Specificity; Transduction, Genetic; Zinc Fingers
PubMed: 21781315
DOI: 10.1186/1742-4682-8-26 -
AIDS Research and Human Retroviruses Jun 2022HIV-1 gene sequences were analyzed from 77 HIV-1 positive children infected perinatally and exhibiting virological failure (VF). Viral subtyping, phylogenetic analysis,...
HIV-1 gene sequences were analyzed from 77 HIV-1 positive children infected perinatally and exhibiting virological failure (VF). Viral subtyping, phylogenetic analysis, and genotypic drug resistance analysis were carried out on samples collected before start of anti retroviral treatment (ART) (baseline, BL), and at 12 months post-ART initiation (M12). Subtype C was found to be most predominant, seen in 75 of the 77 (97.4%) children. The level of pretreatment drug resistance (PDR) was 14% among these children. At BL, K103N (5), E138A/G (4), and M184V (3) were the most common mutations. At M12 the prevalence of any resistance-associated mutation (RAM) (acquired drug resistance/ADR) was 81.8% (63/77). Dual class resistance mutations were seen in 64% (49/77) of children. M184V/I, K103N/S, and Y181C were the most commonly occurring mutations, seen in 76%, 51%, and 36% children. RAMs to the second-generation non-nucleoside reverse transcriptase inhibitors (NNRTI), etravirine (ETR) and rilpivirine (RPV), were seen in 40.2% (31/77) and 48.05% (37/77) of the children, respectively. Our findings reveal similar prevalence rates of PDR and ADR in children with VF as reported in other studies. Occurrence of ETR and RPV resistance associated mutations (RAMs) is of concern and highlights the need for timely switch of regimens guided by genotypic resistance testing in perinatally infected children from India.
Topics: Anti-HIV Agents; Child; Drug Resistance, Viral; Genes, pol; Genotype; HIV Infections; HIV Seropositivity; HIV-1; Humans; Infectious Disease Transmission, Vertical; Mutation; Phylogeny; Pyridazines; Reverse Transcriptase Inhibitors; Rilpivirine
PubMed: 35302390
DOI: 10.1089/AID.2021.0227 -
PloS One 2023Antiretroviral therapy (ART) effectiveness is compromised by the emergence of HIV drug resistance mutations (DRM) and can lead to the failure of ART. Apart from...
BACKGROUND
Antiretroviral therapy (ART) effectiveness is compromised by the emergence of HIV drug resistance mutations (DRM) and can lead to the failure of ART. Apart from intrinsic viral factors, non-compliance with drugs and/or the use of sub-optimum therapy can lead to the emergence of DRMs. In Pakistan HIV currently exists as a concentrated epidemic, however, ART coverage is very low, and drug adherence is poor. ART is selected assuming without baseline genotyping. Pakistan has recently seen a rise in treatment failures, but the country's actual burden of DRM is still unknown. In this study, we perform the genetic and drug resistance analysis of the pol gene from Pakistani HIV-positive ART-naïve and ART-experienced individuals.
METHODS
In this study, HIV-1 pol was sequenced from 146 HIV-1 positive individuals, divided into ART-naïve (n = 37) and ART-experienced (n = 109). The sequences were also used to determine HIV-1 subtypes, the prevalence of DRM, and pol genetic variability.
RESULTS
DRM analysis identified numerous DRMs against reverse transcriptase inhibitors in both ART-naïve and ART-experienced groups, including a few that are classified as rare. Additionally, the ART-experienced group showed mutations associated with resistance to protease inhibitors. Genetic analysis showed negative selection pressure in both groups, but a higher rate of evolution in the ART-naïve group.
CONCLUSION
High prevalence of DRMs, especially against previous first-line treatment in ART- naïve and the accumulation of DRMs in ART-experienced groups is concerning and warrants that a more extensive DRM survey be carried out to inform first-line and second-line ART regimen recommendations.
Topics: Humans; Anti-Retroviral Agents; Drug Resistance, Multiple, Viral; Genes, pol; HIV Infections; HIV Seropositivity; HIV-1; Peptide Hydrolases; RNA-Directed DNA Polymerase
PubMed: 37616294
DOI: 10.1371/journal.pone.0290425 -
Disease Markers 2023This study is to investigate the difference in HIV-1 RNA pol gene expression in AIDS patients before and after antiviral treatment and its effect on the expression level...
OBJECTIVE
This study is to investigate the difference in HIV-1 RNA pol gene expression in AIDS patients before and after antiviral treatment and its effect on the expression level of CD4/CD8 T cells in peripheral blood.
METHODS
The participants included 200 AIDS patients who had undergone antiviral medication, and the quantity of HIV-1 RNA pol gene was determined using nested polymerase chain reaction (nPCR). The levels of CD3+, CD4+, and CD8+ T lymphocytes in peripheral blood were measured by flow cytometry before and after therapy. The receiver operating characteristics (ROC) curve was used to assess the impact of HIV-1 RNA pol gene expression and the CD4+/CD8+ ratio on the prognosis of AIDS patients.
RESULTS
After three months of therapy, the levels of HIV-1 RNA and viral load in the patients showed a drastic decline, while the levels of CD4+/CD8+ were markedly elevated ( < 0.05). Logistic analysis revealed that patients' viral loads were positively correlated with HIV-1 RNA and negatively correlated with CD4+/CD8+ ( < 0.05). The alanine aminotransferase (ALT), white blood cell (WBC) count, Serum creatinine (Cr), total cholesterol (TC), triglyceride (TG), and platelet (PLT) levels significantly increased following a 24-month therapy, while no significant changes were observed in the level of aspartate aminotransferase (AST), red blood cell (RBC), and neutrophil (NEU) (%). ( > 0.05).
CONCLUSION
Antiviral drugs significantly inhibit the HIV-1 RNA POL gene expression and viral load in AIDS patients but upregulate the expression level of CD4/CD8 T cells in peripheral blood.
Topics: Humans; Antiviral Agents; Acquired Immunodeficiency Syndrome; CD8-Positive T-Lymphocytes; HIV-1; HIV Infections; Viral Load; Genes, pol; RNA; Gene Expression; CD4-Positive T-Lymphocytes; CD4 Lymphocyte Count
PubMed: 37091892
DOI: 10.1155/2023/9910542 -
Virulence Dec 2023Hepatitis B virus (HBV) immune escape and Pol/RT mutations account for HBV immunoprophylactic, therapeutic, and diagnostic failure globally. Little is known about...
Patterns of hepatitis b virus immune escape and pol/rt mutations across clinical cohorts of patients with genotypes a, e and occult hepatitis b infection in Nigeria: A multi-centre study.
Hepatitis B virus (HBV) immune escape and Pol/RT mutations account for HBV immunoprophylactic, therapeutic, and diagnostic failure globally. Little is known about circulating HBV immune escape and Pol/RT mutants in Nigeria. This study focused on narrowing the knowledge gap of the pattern and prevalence of the HBV mutants across clinical cohorts of infected patients in southwestern Nigeria. Ninety-five enrollees were purposively recruited across clinical cohorts of HBV-infected patients with HBsAg or anti-HBc positive serological outcome and occult HBV infection. Total DNA was extracted from patients' sera. HBV S and Pol gene-specific nested PCR amplification was carried out. The amplicons were further sequenced for serotypic, genotypic, phylogenetic, and mutational analysis. HBV S and Pol genes were amplified in 60 (63.2%) and 19 (20%) of HBV isolates, respectively. All the sixty HBV S gene and 14 of 19 Pol gene sequences were exploitable. The ayw4 serotype was predominant (95%) while ayw1 serotype was identified in 5% of isolates. Genotype E predominates in 95% of sequences, while genotype A, sub-genotype A3 was observed in 5%. Prevalence of HBV IEMs in the "a" determinant region was 29%. Commonest HBV IEM was S113T followed by G145A and D144E. The Pol/RT mutations rtV214A and rtI163V among others were identified in this study. This study provided data on the occurrence of existing and new HBV IEMs and Pol gene mutations in Nigeria.
Topics: Humans; Hepatitis B virus; Genes, pol; Phylogeny; Nigeria; Hepatitis B, Chronic; Hepatitis B; Mutation; Genotype; DNA, Viral
PubMed: 37262110
DOI: 10.1080/21505594.2023.2218076 -
Viruses Jun 2021As the global effort to eradicate hepatitis B continues, immune escape mutations (IEMs) and drug resistance mutations (DRMs) affecting its diagnosis, treatment, and...
As the global effort to eradicate hepatitis B continues, immune escape mutations (IEMs) and drug resistance mutations (DRMs) affecting its diagnosis, treatment, and prevention are compromising this goal. However, knowledge about the prevalence and circulation of these mutations in Nigeria is scarce. Serum samples ( = 199) from apparently healthy prospective blood donors, pregnant women, and individuals presenting with fever in southwestern Nigeria were analyzed for the presence of IEMs and DRMs by means of nested PCR in the HBV S (HBs) and HBV polymerase (Pol) genes, followed by phylogenetic and mutational analyses. In total, 25.1% ( = 50/199) of samples were positive for HBV, as measured by PCR. In 41 samples (20.6%), both fragments could be amplified, whereas the HBs gene and the Pol gene fragment alone were detected in 0.5% ( = 1/199) and 4% ( = 8/199) of samples, respectively. Sequences were successfully obtained for all 42 HBs gene fragments but for only 31/49 Pol gene fragments (totaling 73 sequences from 44 individuals). All sequences were identified as HBV genotype E. IEMs were present in 18.2% ( = 8/44) of the sequences of HBV-positive individuals with available sequences. IEM Q129H was detected in eight out of the 44 (18.2%) HBV isolates sequenced in this study; however, no DRMs were observed. This study confirms the circulation of HBV IEMs and reports the presence of Q129H IEM for the first time in Nigeria. Intensified research on the dynamics of IEM is necessary in order to enhance the elimination of HBV.
Topics: Adolescent; Adult; Child; DNA, Viral; Female; Gene Products, pol; Genotype; Hepatitis B; Hepatitis B Surface Antigens; Hepatitis B virus; Humans; Immune Evasion; Male; Middle Aged; Mutation; Nigeria; Phylogeny; Pregnancy; Prevalence; Prospective Studies; Young Adult
PubMed: 34210073
DOI: 10.3390/v13071273 -
Virology Journal Feb 2020Anhui Province in China is facing a severe HIV epidemic with an increasing number of newly diagnosed cases.
BACKGROUND
Anhui Province in China is facing a severe HIV epidemic with an increasing number of newly diagnosed cases.
METHODS
In this study, HIV genetic characteristics in the province were investigated. Newly reported HIV-positive individuals from 15 districts of Anhui Province were enrolled and interviewed. Total viral RNA was extracted from plasma isolated from blood samples. We amplified and sequenced an HIV pol fragment of the 1062 bp. The sequences were used for determination of HIV subtypes and the presence of drug resistance mutations. Transmission networks were constructed to explore possible relationships. And all of assembled partial pol genes were submitted to the Stanford HIV Drug Resistance Database website to find the transmitted drug resistance.
RESULTS
Partial pol gene sequences were obtained from 486 cases. The results showed that MSM was the most dominant transmission route (253, 52.06%), followed by heterosexual transmission (210, 43.21%) and blood-borne transmission (1, 0.21%). Many subtypes were identified, including CRF01_AE (226, 46.50%), CRF07_BC (151, 31.07%), subtype B (28, 5.76%), CRF08_BC (20, 4.12%), CRF55_01B (15, 3.09%), CRF68_01B (7, 1.44%), CRF67_01B (3, 0.62%), CRF57_BC (2, 0.41%), CRF59_01B (2, 0.41%), CRF79_0107 (2, 0.41%), subtype C (2, 0.41%), CRF64_BC (1, 0.21%), and circulating recombinant forms (URFs) (27, 5.55%). Four transmission subnetworks containing high transmission risk individuals (with degree ≥4) were identified based on CRF01_AE and CRF07_BC sequences, including two CRF01_AE transmission subnetworks constituted by elderly people with average ages of 67.9 and 61.5 years. Infection occurred most likely through heterosexual transmission, while the other two CRF07_BC transmission subnetworks consist mainly of MSMs with average ages of 31.73 and 34.15. The level of HIV-transmitted drug resistance is 3.09%.
CONCLUSIONS
The simultaneous spread of multiple HIV subtypes in Anhui province underscores that close surveillance of the local HIV epidemic is necessary. Furthermore, the elderly people were frequently involved, arguing for behaviour intervention in this specific population besides the MSM risk group.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antiviral Agents; Child; China; Drug Resistance, Viral; Epidemics; Female; HIV Infections; HIV-1; Humans; Male; Middle Aged; Mutation; Phylogeny; RNA, Viral; Sequence Analysis, DNA; Sexual Behavior; Young Adult; pol Gene Products, Human Immunodeficiency Virus
PubMed: 32014042
DOI: 10.1186/s12985-020-1281-y -
Journal of Virology Jun 1984The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains... (Comparative Study)
Comparative Study
The nucleotide sequence of the gag gene of feline leukemia virus and its flanking sequences were determined and compared with the corresponding sequences of two strains of feline sarcoma virus and with that of the Moloney strain of murine leukemia virus. A high degree of nucleotide sequence homology between the feline leukemia virus and murine leukemia virus gag genes was observed, suggesting that retroviruses of domestic cats and laboratory mice have a common, proximal evolutionary progenitor. The predicted structure of the complete feline leukemia virus gag gene precursor suggests that the translation of nonglycosylated and glycosylated gag gene polypeptides is initiated at two different AUG codons. These initiator codons fall in the same reading frame and are separated by a 222-base-pair segment which encodes an amino terminal signal peptide. The nucleotide sequence predicts the order of amino acids in each of the individual gag-coded proteins (p15, p12, p30, p10), all of which derive from the gag gene precursor. Stable stem-and-loop secondary structures are proposed for two regions of viral RNA. The first falls within sequences at the 5' end of the viral genome, together with adjacent palindromic sequences which may play a role in dimer linkage of RNA subunits. The second includes coding sequences at the gag-pol junction and is proposed to be involved in translation of the pol gene product. Sequence analysis of the latter region shows that the gag and pol genes are translated in different reading frames. Classical consensus splice donor and acceptor sequences could not be localized to regions which would permit synthesis of the expected gag-pol precursor protein. Alternatively, we suggest that the pol gene product (RNA-dependent DNA polymerase) could be translated by a frameshift suppressing mechanism which could involve cleavage modification of stems and loops in a manner similar to that observed in tRNA processing.
Topics: Amino Acid Sequence; Animals; Antigens, Viral; Base Sequence; Cats; DNA Restriction Enzymes; Gene Products, gag; Genes; Genes, Viral; Leukemia Virus, Feline; Retroviridae; Species Specificity; Viral Proteins
PubMed: 6328019
DOI: 10.1128/JVI.50.3.884-894.1984 -
Journal of Virology May 1991Duck hepatitis B virus mutants containing frameshift or stop codon mutations in a portion of the viral pol gene separating the terminal protein and reverse transcriptase...
Duck hepatitis B virus mutants containing frameshift or stop codon mutations in a portion of the viral pol gene separating the terminal protein and reverse transcriptase domains had a leaky phenotype and, depending on the location and type of mutation, synthesized up to 10% as much viral DNA as did the wild type. This region of the pol gene had previously been reported to be refractory to missense mutations; in fact, the leakiness of most of our mutants appeared attributable to translational suppression, which would also be expected to introduce amino acid changes. However, at least one mutant (pH1093 + 2), which was ca. 10% as active as the wild type, appeared to use a novel pathway to express the viral pol gene. Our analyses indicated that pH1093 + 2 synthesized the viral reverse transcriptase as a fusion protein with the amino-terminal portion of the pre-S envelope protein. Thus, in this case, the products of the terminal-protein and reverse transcriptase domains of the pol gene would function as separate protein species, though perhaps noncovalently joined in a dimeric structure during assembly of DNA replication complexes. Evidence was also obtained that was consistent with the idea that the wild-type pol gene may, at least in certain instances, be expressed as functional, subgenic polypeptides.
Topics: Base Sequence; Cells, Cultured; Codon; DNA, Viral; Frameshift Mutation; Gene Products, pol; Genes, pol; Genetic Complementation Test; Hepatitis B Virus, Duck; Humans; Molecular Sequence Data; Phenotype; Protein Biosynthesis; RNA-Directed DNA Polymerase; Recombinant Fusion Proteins; Transfection
PubMed: 1707980
DOI: 10.1128/JVI.65.5.2155-2163.1991 -
Electrophoresis Dec 2012Nanopores have emerged as a prominent single-molecule analytic tool with particular promise for genomic applications. In this review, we discuss two potential... (Review)
Review
Nanopores have emerged as a prominent single-molecule analytic tool with particular promise for genomic applications. In this review, we discuss two potential applications of the nanopore sensors: First, we present a nanopore-based single-molecule DNA sequencing method that utilizes optical detection for massively parallel throughput. Second, we describe a method by which nanopores can be used as single-molecule genotyping tools. For DNA sequencing, the distinction among the four types of DNA nucleobases is achieved by employing a biochemical procedure for DNA expansion. In this approach, each nucleobase in each DNA strand is converted into one of four predefined unique 16-mers in a process that preserves the nucleobase sequence. The resulting converted strands are then hybridized to a library of four molecular beacons, each carrying a unique fluorophore tag, that are perfect complements to the 16-mers used for conversion. Solid-state nanopores are then used to sequentially remove these beacons, one after the other, leading to a series of photon bursts in four colors that can be optically detected. Single-molecule genotyping is achieved by tagging the DNA fragments with γ-modified synthetic peptide nucleic acid probes coupled to an electronic characterization of the complexes using solid-state nanopores. This method can be used to identify and differentiate genes with a high level of sequence similarity at the single-molecule level, but different pathology or response to treatment. We will illustrate this method by differentiating the pol gene for two highly similar human immunodeficiency virus subtypes, paving the way for a novel diagnostics platform for viral classification.
Topics: Electronic Data Processing; Genes, pol; Genotyping Techniques; HIV; Humans; Molecular Diagnostic Techniques; Nanopores; Nanotechnology; Peptide Nucleic Acids; Sequence Analysis, DNA; Spectrometry, Fluorescence
PubMed: 23109189
DOI: 10.1002/elps.201200266