Ecent report by Lorenzo-Redondo, et al. [13] describing that viral replication continues
Ecent report by Lorenzo-Redondo, et al. [13] describing that viral replication continues in tissues where antiretroviral drug concentrations are suboptimal. The authors also proposed that under low ART-pressure, the evolution of drug resistance is greatly diminished by replication fitness advantages of wild-type viruses over drug-resistant variants colocalized in the tissue compartments, providing an explanation for why drug-resistant viral variants do not usually emerge in patients on prolonged suppressive therapy. The residual vDNA clones that we have reconstructed from the patient’s plasma vRNA PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/29069523 isolated at G4 (Fig. 1) did not possess any antiretroviral drug-resistant mutation or G-to-A hypermutations, and had all viral ORFs intact. However, the virions released from vDNAtransfected cells could not replicate in activated CD4+ T-cells or monocyte-derived macrophages of normal donors (data not shown). The absence of viral replication in vitro indicates that the reconstructed vDNAs represented replication-defective cell-free RVs that were circulating in patient G’s blood during his 4th visit to the study. Upon investigation, we found that all vDNA clones had a unique GU-to-GC mutation at the 5-MSD motifs of the corresponding viral genomic RNAs, and the reversion of this mutation to wild-type led to significantly increased levels of HIV-p24 production in the transfected TZM-bl cells, validating that this mutation limits HIV expression (Fig. 6a). The 5-MSD motif is one of the four splicing donor (SD) sites in the HIV-1 RNA genome, which remains highly conserved and is constitutively used during the processing of full-length vRNA through splicing to generate over 40 different viral mRNA species [46, 51?3]. There are about 8?1 3-splice acceptor (SA) sites in the viral genome [52, 53]. In the RNA splicing mechanisms, the cellular PD173074MedChemExpress PD173074 spliceosome cleaves at exon-intron junctions through trans-esterification reactions [54?6], typically right before the GU dinucleotide (underlined) at the 5MSD site (CUGGUGAGUA), and right after the AGdinucleotide at the 3-SA sites in vRNA to remove intronic portion (see Fig. 5b). The noncoding exon 1 spanning upstream of the 5-MSD site is always included with all spliced forms of vRNA [53]. Generally, the GU dinucleotide motif at the mRNA splice donor site is well conserved across the species and possessed by 98 of introns [57, 58], but the mutations at this site are known to affect mRNA splicing negatively in vitro [59, 60]. The 5-MSD mutation (GT-to-GC), to our knowledge, has not been previously reported for HIV in the natural context. While we could detect this mutation in patient G’s plasma frequently (5 of 7 occasions), we failed to detect the same mutation in residual plasma vRNAs of three other patients PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27484364 (not shown) on suppressive therapy, suggesting that the 5-MSD mutation in HIV infection may occur rarely. The introduction of the same 5-MSD mutation (i.e., GT-to-GC) in the standard HIV strain (JRCSF) was found to diminish viral RNA splicing (Fig. 6d) and gene expression severely (Fig. 6b), and the viral mutant (JRCSF-MSD) could not replicate in activated CD4+ T-cells of normal donors (Fig. 6c), which strongly suggests that the RV 5-MSD variant remains replication-defective in vivo. Therefore, the 5-MSD mutation should not offer the virus a selective advantage in vivo, except that the down-modulation of viral gene expression caused by the mutation may facilitate the host cells to survive in vivo by avoidin.