Genetic Analysis of HIV-1 vpr Sequences from HIV-Infected Older Patients on Long-Term Antiretroviral Therapy

Background: Many HIV-infected individuals have achieved undetectable viral load and increased CD4 T cell counts due to the success of antiretroviral therapy (ART). However, HIV persists in resting T cells, monocytes/macrophages and other quiescent cells. Furthermore, the HIV-1 vpr accessory gene may play an important role in the persistence of HIV in these infected patients. Objectives: Therefore, we characterized the HIV-1 vpr gene from PBMC DNA of 14 HIV-infected older patients on long-term ART with mostly undetectable viral load and increased CD4 T cell counts. Methods: Peripheral blood mononuclear cells (PBMC) were isolated from 14 HIV-infected individuals, followed by extraction of genomic DNA, amplification of HIV-1 vpr gene by polymerase chain reaction (PCR), cloning of vpr gene in TOPO vector and characterization of correct size recombinant inserts containing vpr genes. An average of 13 clones were sequenced from each patient, followed by sequence analysis by bioinformatic tools. Results: Phylogenetic analysis of 182 vpr sequences demonstrated that the vpr sequences of each patient were well separated and discriminated from other patients’ sequences and formed distinct clusters. The vpr sequences showed a low degree of viral heterogeneity, lower estimates of genetic diversity and about half of the patients’ sequences were under positive selection pressure. While the majority of the Vpr deduced amino acid sequences from most patients contained intact open reading frames, several sequences, mostly from two patients, had stop codons. Numerous patient-specific and common amino acid motifs were found in deduced Vpr sequences. The functional domains required for Vpr activity, including virion incorporation, nuclear import of pre-integration complex and cell cycle arrest, were generally conserved in most Vpr sequences. Several of the known cytotoxic T-lymphocytes (CTL) epitopes in Vpr showed variation in our patients’ sequences. Conclusion: In summary, a low degree of genetic variability, conservation of functional domains and variations in CTL epitopes were the features of vpr sequences from the 14 HIV-infected older patients with controlled viremia on long-term ART.