Study On Variation Of HIV-1Gag, Env And Pol Genes

To further explore the variation of HIV-1gag, env and pol genes, we studied the variation characteristics of HIV-1gag, env and pol gene sequences to understand the HIV epidemics, the quasispecies characteristics of gag, env and pol gene, the genetic characteristics of env gene and drug resistance mutation of pol gene. The purpose of this study is contribute to the basic research of HIV-1genetic variation and prevalence and disease progression to AIDS.In our study,60whole blood samples were collected from antiviral-treated HIV-infected individuals among former paid blood donors in Shanxi province. HIV-1gag gene pl7-p24regions, env gene V3-V5regions and pol gene PR-RT regions were amplified by nested polymerase chain reaction. The purified PCR products were cloned into T cloning vectors, and the positive clones were randomly picked out, then the T cloning vectors were amplified by colony polymerase chain reaction. The products of nested-PCR and colony-PCR were sequenced. A total of45gag,39env and47pol nested-PCR sequences and827cloned gag sequences,798cloned env sequences and812cloned pol sequences were obtained, respectively.The phylogenetic tree analysis showed that the45gag PCR sequences were clustered with B.FR.HXB2, B.CN.RL42and B.TH.92TH014C g, except one strains clustered with CRF07_BC reference strains. The genetic distances and homology analyses showed that the gene-types of these samples were subtype B or B’. That variation of different gene fragments was not identical with statistical analyses of the genetic distances and Shannon entropy score of gag, env and pol genes using SPSS13.0software. We founded that variation of different gene fragments were significant difference (P=0.02;P=0.033) in one strains sample through further analysis on29samples that all three gene fragments were amplified. There was significant difference between the env and gag or pol genes, whereas no significant difference between the gag and pol genes. The gag and pol genes were subject to negative selection and the V3region was subject to positive selection through the calculation of ds/dn to analyses the selection pressure, suggesting that escape the antibody recognition was a major factor to drive the selection pressure in V3region.A total of4in8CTL antigen epitopes appeared9mutations in consensus sequence of subtypes B and B’. There were no antigen epitope mutations detected in the PCR sequences, whereas a few mutations were detected in clone sequences in5antigen epitopes in p24 region. The results suggested that the antigen epitopes locate in p24region displayed more conservative than the p17. The minority mutations of antigen epitopes locate in p24region were detected.There were6forms of V3loop tip motifs in PCR sequences (GPGR, GPGQ, GQGR, GPGA, GLGR,GPGK) and4forms of them in clone sequences (GPGL, GPGG, RPGR, GPAR) in env gene. This result indicated that the variate of V3loop tip motif was rare in quasispecies. This suggested that V3loop tip motif was conservative in Shanxi area after more than10years of the prevalence of HIV-1. HIV-1V3region sequences were submitted into Geno2pheno [co-receptor] online tool to predict the co-receptor usage. The results revealed that69.23%of V3region sequences used CCR5and30.77%used CXCR4as co-receptors, respectively. Based on the predictions of co-receptor usage, we determined the relationship between co-receptor usage (CXCR4or CCR5) and N-glycosylation sites. Nine significant difference sites were observed in two group. The site located within the V3loop at position N300of HXB2, which was present in all R5group, whereas7/27(25.93%) in X4group, suggesting this site to be associated with co-receptor usage. These sites of386,396,398in V4region and460,461in V5region of the R5group were significantly higher than that of the X4group. These sites of362in C3region and398,413in V4region of the X4group were significantly higher than that of the R5group. This result showed that the N-glycosylation patterns were differences between R5strains and X4strains. There were different lost rates of N-glycosylation sites in env gene, and the N-glycosylation sites that comprise the2G12epitope were relatively conserved:N295(17.95%), N332(2.56%) and N392(2.56%).Twenty-eight protease inhibitors-related mutations were detected in PCR sequences and clone sequences in pol gene region, in which4PIs primary resistance mutations (N88D, L76V, N88S and I50V)were detected. Six of39PCR sequences were identified to be resistant to one or more reverse transcriptase inhibitors. Thirty-one PCR sequences were detected RTIs-related drug resistance mutations in clone sequences. The89.29%of PIs-related mutations,72.72%of NRTIs-related mutations and50.00%of NNRTIs-related mutations were found only in the clone sequences, respectively, which were low-frequency: PIs-related mutations in the range of0.12～6.47%, NRTIs-related mutations in the range of0.12～0.47%, NNRTIs-related mutations in the range of0.12～1.86%. In this study, the5most prevalent NRTIs-related mutations were M41L (1.98%), M184V (1.63%), L210W (1.16%) and T215Y (1.98%); the6most prevalent NNRTIs-related mutations were K103R (3.14%), K103E (0.35%), V108I (1.63%), V179E (3.61%), G190S (1.98%) and G190A (1.28%). In this study, the analyses of drug resistance showed that most of the drug resistance mutations was resistant to the AZT/DDI/NVP regime, the prevalence of HIV-1drug resistance strains at a higher level(12.77%), and there were a large amount of low-frequency HIV-1drug resistance mutations in the HIV-1infections. We suggested it was necessary to genotyping resistance testing for blood donors.