Characterization of HIV-1 Accessory vpr Gene From Virologically Controlled HIV-Infected Older Patients on Long-Term Antiretroviral Therapy

Abstract

Human immunodeficiency virus type 1 (HIV) infected individuals are successfully treated by antiretroviral therapy (ART) which reduces the viral load to undetectable levels, improves CD4 T cell counts, reduces opportunistic infections and increases longevity of many people to older age. However, HIV still persists in reservoir cells such as resting T cells and monocytes/ macrophages and continues to cause a low level of inflammation with increased risk of morbidity and mortality. Therefore, genetic characterization of HIV latent viruses, may provide important information for curative strategies. Specifically, genetic characterization of the HIV vpr gene, which is essential for viral replication in resting T cells and monocytes/macrophages and viral persistence in infected individuals, may provide important information for curative strategies. Characterization of the HIV-1 vpr region from 14 HIV-infected older patients’ peripheral blood mononuclear cell (PBMC) DNA on long-term ART with controlled viremia, identified by undetectable viral load and improved CD4 T cell counts were performed by PCR amplification, cloning and sequencing of 182 quasispecies of HIV-1 followed by analysis of phylogenetic relationship, genetic variability, selective pressure and analyzing the biological and immunological domains. Phylogenetic analysis revealed that the vpr gene sequences of each patient formed distinct clusters that were well separated and discriminated from other patients’ sequences. There was a low degree of heterogeneity and lower estimates of genetic diversity in all 14 patients’ vpr sequences. Eight out of 14 patients’ vpr region sequences were found to be under positive selection pressure by showing a dN/dS ratio of >1. In the multiple alignment analysis of deduced Vpr sequences, most patients’ sequences had intact Vpr open reading frames, and some patients’ sequences had stop codons. Several patient-specific amino acid motifs and common amino acid motifs were found in deduced Vpr sequences. The functional domains required for Vpr activity, including virion incorporation, predicted alpha-helix formation, nuclear import of pre-integration complex and cell cycle arrest and differentiation were highly conserved in most of the Vpr sequences, and most of the substitutions were with conservative or compatible amino acids. Analysis of several previously characterized cytotoxic T-lymphocytes (CTL) epitopes within the Vpr showed substantial variation in the patients’ Vpr sequences, suggesting escape mutants. In conclusion, a low degree of genetic variability and maintenance of functional domains and conservative variations in CTL epitopes were the hallmarks of vpr sequences from the 14 virologically controlled HIV-infected older patients on long-term ART, which may provide important information to develop curative and preventive strategies.