Used in the laboratoryTo investigate the underlying reason for the limited
Used in the laboratoryTo investigate the underlying reason for the limited viral production as well as the replication-defect of the virions produced from the cloned vDNAs, we sought to examine whether these clones possess G-to-A hypermutations at statistically significant levels compared with the SB 202190 price standard HIVNL4? clone. G-to-A hypermutations (or transitions) occur in proviral sequences due to the mutational pressure exerted by the APOBEC-induced cytosine deamination on nascent viral cDNA during reverse-transcription in the infected cells [37?9]. An excess of such mutations, including premature stop PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/29069523 codon generation in the viral ORFs [40, 41], results in viral replication defects. Although the viral Vif counters the negative effects of APOBEC proteins on the virus in host cells [42], G-to-A hypermutations still occur in patients’ PBMC-associated proviral sequences [43]. Upon analyses of the full-length residual vDNA sequences using the Hypermut 2.0 program [44] available in the Los Alamos National Laboratory (LANL) website, we found that none of the eight clones had G-to-A hypermutations when compared with the standard HIVNL4? sequence (Fisher’s exact P-values range from 0.91 to 0.95, where P < 0.05 would indicate hypermutation). These data indicate that the cloned RVs were devoid of hypermutations; this observation is in agreement with a previously published report [40]. By using the QC tool available in the LANL, we found that all the viral open reading frames (ORFs) in these full-length clones were also intact. Overall, these data suggest that the detrimental mutations did not accumulate to account for HIV clones' low viral productivity or replication defects in vitro. Since the vDNA clones were derived from vRNA isolated from plasma during suppressive therapy, an obvious question arises whether the cloned RVs possessed ART-resistant mutations for which they were able to somehow replicate in vivo at low levels during therapy. We found none of the viral pol ORFs possessed any of the drug-resistant mutations listed in the Stanford University's HIV Drug Resistance Database, suggesting that the cloned RVs represent drug-sensitive viruses circulating in plasma during therapy.Rassler et al. Virology Journal (2016) 13:Page 6 ofFig. 4 Limited levels of HIV-p24 production from all residual vDNA clones. TZM-bl cells were transfected separately with eight reconstructed vDNA clones in duplicate, and after 48 h, HIV-p24 levels in culture media were quantified by ELISA (Advanced Bioscience, Inc.). A standard HIV (JRCSF) DNA and our previously published residual vDNA clone (C1P) were used as positive controls for virus production in these assays. Error bars represent standard deviations (s.d.). Results were compared with the positive control C1P PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27484364 using unpaired t-test; asterisk (*) indicates p < 0.A novel mutation identified in the conserved 5-MSD site of vRNA caused limited HIV-p24 production from residual vDNA clonesWe sought to compare residual vDNA sequences with those of three standard strains (HIV213, HIVNL4? and HIVJRCSF) in the CLUSTAL-W program [45] to identifyany unique mutations present in the residual vDNA clones, which could potentially be associated with the low p24-production phenotypes of these clones. Among many sequence changes, strikingly, we observed a single nt mutation (GT-to-GC) present in the 5-MSD motif of each RV clone (Fig. 5a), which was not previouslyFig. 5 Analyses of viral genomic region containing 5-MSD motif. a The.