Replication-independent expression of anti-apoptosis marker genes in human peripheral blood mononuclear cells infected with the wild-type HIV-1 and reverse transcriptase variants

Abstract

Clinical trials with highly-active antiretroviral therapy (HAART) have shown that a substantial number of patients continue to show a decrease in viral load and/or increase or stable CD4(+) T-cell numbers even in the presence of multidrug resistant (MDR) viruses. We compared replication capacity (RC) and expression of anti-apoptosis marker genes (AAMGs) in human peripheral blood mononuclear (PBM) cells infected with NL4-3 (wild-type; WT) and mutant viruses. Replication kinetics assays showed a significant decrease in RC of all mutant viruses in comparison to the WT virus. The viruses containing patient-derived MDR RT without the K65R mutation (PSD5.2) replicated efficiently in comparison to the viruses with MDR RT containing the K65R mutation (PSD5.1), or the single mutations K65R and M184V. Compared with WT, a significant decrease in RCs of viruses: K65R (RC=0.39±0.02; p≤0.0001), M184V (RC=0.72±0.04; p≤0.0001), PSD5.1 (RC=0.32±0.04; p≤0.0001), and PSD5.2 (RC=0.90±0.04; p=0.002) was observed on day 10. RT-PCR-based apoptosis array was performed on total cellular RNA. Recombinant virus PSD5.2 showed a 1.5- to 6-fold upregulation in 8 AAMGs (AKT1, BAG3, BCL2A1, BFAR, BIRC2, BNIP1, BNIP3, and CFLAR) on day 1 and day 7 post-infection with respect to WT virus. PSD5.1 showed upregulation of only one gene (BAG1) on day 1 (1.75-fold) and day 7 (1.97-fold). Point mutant K65R showed a 1.5- to 4-fold upregulation of six AAMGs on day 7. Viruses with the M184V mutation showed upregulation of only one gene (BAG1). These observations indicate that the upregulation of specific AAMGs may not be dependent on the RCs of HIV-I variants, and that the possible interaction among mutated RT residues and viral and/or host proteins may induce CD4(+) T-cell-protective anti-apoptosis proteins.

FIG. 1.

Replication kinetics of HIV RT variants. PHA-stimulated PBM cells (10×10⁶) were infected with each virus containing 100 TCID₅₀/1×10⁶ PBM cells, and culture supernatants were collected at various time points to measure replication efficiencies of mutant viruses. Both HIV-1 antigen p24 (A) and RT activity (B) were measured at various time points. Replication plot shows that all of the mutants were attenuated in comparison to WT virus, but PSD5.2 replicated efficiently and similarly to WT virus. The results show the mean of three independent assays. Three replicates for each dilution at different time points were used in antigen p24 ELISA, and all RT assays included two replicates for each time point. Color images available online at www.liebertonline.com/vim