Viral infections in individuals generally lead to a scenario where the virus is confronted by the host immune system involving both innate and adaptive immune responses. Regarding the latter, cellular and humoral immune responses have been shown to play a role in the control of infections of viruses including HIV-1 [70, 71]. It has been suggested that an understanding of the correlates of protective immunity is an important requirement for the development of vaccines against HIV-1. Several studies have been published on this subject [71–73]. These studies point out a role for CD8+ and CD4+ T cell responses and neutralizing antibodies in the control of HIV-1 replication. For example, it has been reported that CD8+ cells control HIV-1 in the acutely infected individuals [4–6]. The relevance of CD8+ T cells for the control of virus infection was also shown in the case of SIV infected rhesus macaques [74, 75]. Recently, the published data on CD8+ T cells in acute and chronic HIV-1 infection revealed that CTL epitopes are present in all of the proteins encoded by HIV-1. Virus replication, however, is not completely contained due to the emergence of CTL escape variant viruses. Based on this, it is suggested that vaccine efforts to control HIV-1 should take into account the high genetic variability noted among HIV-1.
The continued emergence of genetic variants is a characteristic feature of RNA viruses. RNA dependent RNA polymerase and reverse transcriptase are error-prone enzymes and have been implicated as a cause for the generation of variants [76, 77]. The mutational changes in the protease and reverse transcriptase, depending on their location, may impact on their binding inhibitors targeting these enzymes. The viruses containing alterations may then be able to evade the inhibitory activities of the agents and are designated as drug-resistant variants. Similarly, the mutations in Env, Tat, and possibly other proteins can also evade the neutralizing antibody, CTL and T-helper cell responses [12, 71]. The emergence of escape variants eventually repopulates the body in the face of immune responses against the virus. It has been suggested that immune escape may be a key step in the evolution of HIV-1 [30, 78–80].
In an effort to understand the overall polymorphisms in a HIV-1 gene product, we undertook a comprehensive analysis of the predicted amino acid sequences of Vpr from diverse HIV-1 subtypes. Considering the genetic variation noted in diverse HIV-1 , our hypothesis is that the differences in Vpr and other viral proteins may enable the viruses to escape the host immunological pressures. To address this issue, we have initially compiled the polymorphisms in Vpr at the level of individual amino acid. Vpr contains only 96 amino acids. Hence, the small size of the protein is an advantage for a comprehensive analysis. For this purpose, we have turned to the Vpr sequences which are available in the HIV database and also sequences from specific groups such as HIV-1 positive long-term non-progressors. A total of 976 predicted Vpr amino acid sequences were used for our studies. The analysis revealed several characteristic features with respect to the individual amino acids in the Vpr. Of the 96 amino acids, all the amino acids except the initiator methionine have the propensity to change. This indicates that Vpr molecule is highly flexible in nature. The frequency of the variant amino acids, calculated for subtype B Vpr at the level of individual residue, revealed that substitution is very low for most of the residues. This suggests that many of the substitutions in Vpr may compromise the function and possibly the fitness of the virus. Interestingly, there are several amino acids that can accommodate ten or more alterations. We designate such amino acids as hot spots in Vpr which include residues 15, 16, 28, 36, 37, 48, 55, 58, 77, 84, 86 and 89. The underlying basis for the extensive genetic changes in specific regions of Vpr is not clear. It is likely that the error-prone reverse transcriptase, the secondary structure of RNA and other factors, either alone and/or in combination may play a role in the generation of genetic variants. In this regard, Yusim et al.  have noted that Integrase (IN) exhibits the least variability and Vpu exhibits the highest variability. Boutwell and Essex  also showed that the proportion of polymorphic amino acids ranged from a low of 55% (RT, IN) to a high of 94% (Vpu). In our analysis, Vpr variability is high which may likely be due to the inclusion of diverse isolates including the HIV-1 progenitor virus SIVcpz.
Vpr is known as a highly immunogenic protein. The presence of CTL epitopes verified through experimental approaches has been reported by several groups . These include the region encompassing residues 9–70 of Vpr. Of the 96 residues, 62 (65%) have been shown to be associated with experimentally defined CTL epitopes. The data presented in Table 16 show that there are polymorphisms with respect to the experimentally verified CTL epitopes. The presence of variant amino acids at distinct locations within the epitope is likely to impact the CTL epitope. Further, we have also evaluated the effect of Vpr polymorphisms on CTL epitopes using the bioinformatics approach by calculating the estimate of half time of disassociation of the molecule containing the epitope. Such an analysis predicted several CTL epitopes all over Vpr including the C-terminus with respect to specific HLA class 1 molecules. The detailed analysis was carried out for different HLA alleles (HLA-A2, Cw-4, HLA-B7 and HLA-B2705) involving a total of 12 epitopes. The polymorphisms have also been analyzed for three predicted epitopes corresponding to residues 18–26, 38–46, and 66–74. The substitution of the variant amino acids for the residues comprising the epitope resulted in a drastic reduction in the value corresponding to the half time of the disassociation of the molecule containing the epitope. It should, however be noted that additional in vitro binding studies are necessary to confirm the predicted values.
Based on the data presented here, the amino acid polymorphisms noted in Vpr have the potential to contribute to the escape of the virus along with the epitopes present in other HIV-1 proteins . It is also likely that the information regarding the polymorphisms at the CTL epitope will provide an opportunity to create an epitope-based vaccine that will exert control over viral isolates from different parts of the world. It is important to mention that the extensive HLA-associated amino acid polymorphisms noted here may also impact on the structure/function of Vpr and fitness of the virus [10, 81–85]. The biological sources used for generating the sequence information of vpr include tissues from infected individuals, plasma viral RNA, and cloned viral DNA. For this reason, the Vpr sequences considered here for the analysis may be derived from both infectious and non-infectious viral genomes. Hence, there is a possibility that the amino acid polymorphisms noted here may or may not have a chance to be acted upon by CTL and T-helper cell pressures. It is known that amino acids in the proximal region of the epitope can also influence their immunogenic potential. The amino acid polymorphisms noted in the putative CTL epitopes can have an effect at a single and/or multiple levels in the generation of immune response: i) The mutations may eliminate the binding of the peptide to the appropriate HLA molecule, which will be presented on the cell surface. ii) Mutations may also disrupt the interaction with the T-cell receptors. iii) Mutations may disrupt the intracellular processing of the peptides. This results in the escape of the cells expressing the viral proteins from the surveillance of CD8+ T cells. The variant amino acids present in the proximal or far away from the epitope could influence through interference with the processing of the peptide from the protein. With regard to the latter, the variant amino acids may be either independent or compensatory in relation to changes in specific residues of Vpr. In addition, variant amino acids, which are part of overlapping epitopes presented by different HLA molecule, can also exert an influence on the epitope .
HIV variability is an important factor that should be taken into account in the efforts directed towards the development of vaccines against HIV-1. In order for the vaccines to be effective against diverse HIV-1, strategies that are being considered include consensus sequence approaches and polyvalent vaccines in the form of a mixture of genes/proteins from different subtypes of HIV-1. Despite the extensive variability reported for HIV-1, the nature and extent of variation has not been systematically investigated. Such an analysis is difficult to carry out for HIV-1 Gag, Pol or Env protein due to its size. It is for this reason that we have selected Vpr, a small protein. The results presented for Vpr here are interesting and novel as they describe genetic variation involving global HIV-1. Surprisingly, the frequency of the variant amino acids for most of the residues is low. This suggests that majority of the residues cluster around a sequence shared by HIV-1 isolates of different subtypes. It is likely that the influence of the residues on the fitness of the virus counters the variability, thus limiting the genetic variation. The information on Vpr polymorphisms will be of value for the development of vaccines based on the auxiliary genes of HIV-1.