PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of viroljBioMed CentralBiomed Central Web Sitesearchsubmit a manuscriptregisterthis articleVirology JournalJournal Front Page
 
Virol J. 2008; 5: 99.
Published online Aug 23, 2008. doi:  10.1186/1743-422X-5-99
PMCID: PMC2553080
A comprehensive analysis of the naturally occurring polymorphisms in HIV-1 Vpr: Potential impact on CTL epitopes
Alagarsamy Srinivasan,corresponding author1 Velpandi Ayyavoo,corresponding author2 Sundarasamy Mahalingam,3 Aarthi Kannan,1,4 Anne Boyd,1 Debduti Datta,3 Vaniambadi S Kalyanaraman,5 Anthony Cristillo,5 Ronald G Collman,6 Nelly Morellet,7 Bassel E Sawaya,8 and Ramachandran Murali9
1Thomas Jefferson University, Department of Microbiology and Immunology, Jefferson Alumni Hall Rm 461, 1020 Locust Street, Philadelphia, PA 19107, USA
2University of Pittsburgh, Department of Infectious Diseases & Microbiology, Parran Hall Rm 439, 130 DeSoto Street, Pittsburgh, PA 15261, USA
3Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India
4Wellesley College, 21 Wellesley College Rd Unit 7430, Wellesley, MA 02481, USA
5Advanced Bioscience Laboratories, Inc., 5510 Nicholson Lane, Kensington, MD 20895, USA
6University of Pennsylvania School of Medicine, 522 Johnson Pavilion, 36th and Hamilton Walk, Philadelphia PA 19104, USA
7Unite de Pharmacologie Chimique et Genetique, INSERM, Avenue de l'Observatoire, Paris Cedex 06, France
8Department of Neuroscience, Center for Neurovirology, Temple University School of Medicine, Philadelphia, PA 19122, USA
9University of Pennsylvania School of Medicine, Dept of Pathology and Laboratory Medicine, 243 John Morgan, Philadelphia PA 19104, USA
corresponding authorCorresponding author.
Alagarsamy Srinivasan: alagarsamy.srinivasan/at/gmail.com; Velpandi Ayyavoo: velpandi/at/pitt.edu; Sundarasamy Mahalingam: Mahalingam/at/iitm.ac.in; Aarthi Kannan: akannan/at/wellesley.edu; Anne Boyd: annekboyd/at/gmail.com; Debduti Datta: debduti.datta/at/gmail.com; Vaniambadi S Kalyanaraman: vs.kaly/at/ablinc.com; Anthony Cristillo: Anthony.cristillo/at/ablinc.com; Ronald G Collman: collmanr/at/mail.med.upenn.edu; Nelly Morellet: Morellet/at/pharmacie.univ-paris5.fr; Bassel E Sawaya: sawaya/at/temple.edu; Ramachandran Murali: murali/at/xray.med.upenn.edu
Received July 7, 2008; Accepted August 23, 2008.
The enormous genetic variability reported in HIV-1 has posed problems in the treatment of infected individuals. This is evident in the form of HIV-1 resistant to antiviral agents, neutralizing antibodies and cytotoxic T lymphocytes (CTLs) involving multiple viral gene products. Based on this, it has been suggested that a comprehensive analysis of the polymorphisms in HIV proteins is of value for understanding the virus transmission and pathogenesis as well as for the efforts towards developing anti-viral therapeutics and vaccines. This study, for the first time, describes an in-depth analysis of genetic variation in Vpr using information from global HIV-1 isolates involving a total of 976 Vpr sequences. The polymorphisms at the individual amino acid level were analyzed. The residues 9, 33, 39, and 47 showed a single variant amino acid compared to other residues. There are several amino acids which are highly polymorphic. The residues that show ten or more variant amino acids are 15, 16, 28, 36, 37, 48, 55, 58, 59, 77, 84, 86, 89, and 93. Further, the variant amino acids noted at residues 60, 61, 34, 71 and 72 are identical. Interestingly, the frequency of the variant amino acids was found to be low for most residues. Vpr is known to contain multiple CTL epitopes like protease, reverse transcriptase, Env, and Gag proteins of HIV-1. Based on this, we have also extended our analysis of the amino acid polymorphisms to the experimentally defined and predicted CTL epitopes. The results suggest that amino acid polymorphisms may contribute to the immune escape of the virus. The available data on naturally occurring polymorphisms will be useful to assess their potential effect on the structural and functional constraints of Vpr and also on the fitness of HIV-1 for replication.
Humoral and cellular responses have been implicated in controlling viral and bacterial infections in addition to the host's innate immune responses. This is, indeed, demonstrated in the context of HIV-1 infection [1-3]. Specifically, CTL responses against the virus have been shown to limit the virus replication at a low level in the infected individuals. This is evident in the inverse correlation of CTL responses vs. virus load observed in acutely infected individuals [4-6]. Utilizing the rhesus macaque/SIV infection model, a suppressive effect on virus replication was shown for CTLs [7]. However, the initial CTL responses are not able to contain the virus at a later stage, possibly due to the emergence of viral variants that evade the immune responses resulting in continued virus replication [8,9]. Hence, an understanding of the CTL escape variants of HIV is important both in natural viral infections and also in the context of vaccine-induced immunity for developing effective CTL based polyvalent vaccines for containing diverse HIV-1 strains [10]. This is an area of research which is actively being pursued by several investigators [11,12].
The genome of HIV-1 has been shown to code for two regulatory proteins (Tat and Rev) and four auxiliary proteins (Vif, Vpr, Vpu and Nef) in addition to the Gag, Pol, and Env structural proteins [13]. The regulatory proteins Tat and Rev are essential for virus replication. Rev is involved in the transport of genomic and partially spliced subgenomic mRNA from the nucleus to the cytoplasm [14]. Tat is known as an activator of transcription of viral and cellular RNA. Vif plays an important role in HIV-1 replication in peripheral blood mononuclear cells (PBMC). Specifically, Vif prevents hypermutation in the newly made viral DNA through its interaction with APOBEC3G [15,16]. Vpr is known for its incorporation into the virus particles. The interaction of Vpr with the Gag enables its incorporation into the virus particle. Vpr is a multifunctional protein and is involved in the induction of apoptosis, cell cycle arrest, and transcriptional activation [17]. Vpu plays a role in the particle release and degradation of CD4 [14,18,19]. The features of Nef include downregulation of cell surface receptors, interference with signal transduction pathways, enhancement of virion infectivity, induction of apoptosis in bystander cells, and protection of infected cells from apoptosis [20-24].
Based on the data reported so far, it is clear that HIV-1 employs multiple strategies to successfully replicate in the infected individuals [14,25,26]. The enormous genetic variation that is generated through errors of reverse transcriptase enzyme may provide a pool of variants to evade the host immune responses against the virus and also result in the emergence of drug resistant viruses during treatment. In addition, it is also likely that the immunosuppressive effects of HIV-1 encoded proteins may attenuate the host immune responses in favor of the virus.
Upon infection of target cells by the virus, viral proteins are synthesized for carrying out the functions related to the virus replication and also exert effect on specific host cell functions. In addition, viral proteins are also targeted to the proteosomal degradation pathway. This process results in the generation of peptides, which are then translocated to the ER through TAP and are presented on the cell surface in association with human leukocyte antigen (HLA) class I molecules. The genetic variability present in the coding sequences of the virus may result in viral proteins with alterations in the CTL epitopes, which may lead to defective processing, presentation or lack of recognition of the epitope by the reactive CTLs. This is the likely mechanism of the CTL escape by HIV-1 and other viruses. The presence of multiple CTL epitopes has been demonstrated in HIV-1 proteins including Gag, Pol, Vif, Vpr, Tat, Rev, Vpu, Env and Nef. Though the characterization of the epitopes with respect to the viral proteins is achievable in individual cases, such an analysis at a population level is difficult to carry out for the following reasons: i) HIV-1 exhibits high genetic variation in different regions of the genome. The extent of heterogeneity among circulating HIV-1 strains is described to be in the range of 20% or more in relatively conserved proteins and up to 35% for Env protein [11]. In addition, there is also extensive diversity among HIV-1 within a subtype, ii) There are multiple subtypes of HIV-1, and iii) There are variables at the HLA loci. On the other hand, this limitation can be overcome to some extent by utilizing alternative approaches where information about CTL epitopes and their variants can be inferred from the sequences available for HIV-1 [27-29]. The HIV sequence database has information about the viral isolates from different parts of the world. This information can be used as a source to assess the extent of naturally occurring polymorphisms and their potential impact on CTL epitopes. We hypothesize that mutations or alterations in the residues which are part of the CTL epitope in the Vpr molecule are likely to affect the epitope at multiple levels (processing and recognition of the epitope). Recently, studies have addressed this issue using full length or partial HIV-1 genome sequences [30]. This has prompted us to carry out a comprehensive analysis of the extent of variation at the amino acid level in the auxiliary gene product Vpr of HIV-1.
The underlying reasons for the selection of Vpr for a comprehensive analysis are the following: i) Vpr is a virion associated protein, ii) Vpr plays a critical role for the replication of virus in macrophages, iii) Vpr is a transcriptional activator of HIV-1 and heterologous cellular genes, iv) Vpr arrests cells at G2/M, v) Vpr induces apoptosis in diverse cell types, vi) Vpr exhibits immune suppressive effect, vii) Vpr is present in the body fluids as an extracellular protein, viii) Vpr is highly immunogenic, ix) Vpr is a small protein comprising only 96 amino acids and x) Structural information for the whole Vpr molecule is available through NMR [17,31-34]. These features enable a detailed analysis of the polymorphisms in Vpr with respect to CTL epitopes, structure-function of the protein, and fitness of the virus for replication.
In this study, we have analyzed the predicted amino acid sequences of Vpr from global HIV-1 isolates available through the HIV database. Specifically, the extent of genetic variation in Vpr in the form of polymorphisms at the individual amino acid level was comprehensively analyzed. Several of the amino acid polymorphisms were found to be part of the experimentally verified and predicted CTL epitopes. The location and nature of the variant amino acid were found to affect the CTL epitope considerably. Hence, our results provide a glimpse into the genetic footprints of immune evasion in Vpr.
The goal of our studies is to assess the nature and extent of polymorphisms at the level of individual residues in the Vpr molecule. The sequences considered here comprise Vpr sequences derived from all the major subtypes of HIV-1. The details regarding the subtypes and the number of sequences from each subtype are presented in Table Table11 and are taken from the HIV database http://www.hiv.lanl.gov[35-38]. In addition, we have included Vpr sequences derived from HIV-1 positive long term non-progressors (McKeithen et al., unpublished data). It should be noted that we have also included Vpr from SIV isolated from chimpanzees, as this is likely the progenitor virus for HIV-1. Vpr sequences from the database were accessed in January of 2007. The deletions in the Vpr molecule were excluded from our analysis. The alignment of Vpr sequences (which is available from the authors upon request) was analyzed manually for variant amino acids at the level of individual residue in Vpr from global and distinct subtypes of HIV-1.
Table 1
Table 1
Vpr sequences used for the analysis of amino acid polymorphisms
Characteristics of Vpr sequences selected for this study
The alignment of Vpr sequences has enabled us to analyze the differences at the level of each residue from diverse HIV-1 isolates. A total of 976 Vpr sequences have been used for alignment. The polymorphisms, with respect to the length, have been noted in Vpr by several investigators [17,39]. As this may pose problem for our analysis, our alignment does not take into account both deletions and insertions. The Vpr alleles are from diverse subtypes and include 67, 294, 185 and 44 Vpr sequences representing subtype A, B, C, and D, respectively (Table (Table1).1). The O, AE, AG, and cpx groups represent 39, 45, 39 and 28 Vpr sequences, respectively. Since the Vpr sequences are derived from different sources such as viral RNA, cloned viral DNA and proviral DNA from tissues, we have not made attempts to classify them in our analysis.
Amino acid polymorphisms in the predicted Vpr sequences
Recently, the structure of full length Vpr has been resolved by NMR [40]. According to this study, Vpr consists of a flexible N-terminal domain (amino acids 1–16), helical domain I (HI) (residues 17–33), turn (residues 34–37), helical domain II (HII) (residues 38–50), turn (residues 51–54), helical domain III (HIII) (residues 55–77), and a flexible C-terminal domain (residues 78–96). Based on this structure, the polymorphisms observed in Vpr are presented with respect to the individual domain.
N-terminus of Vpr (residues 1–16)
The results presented in Table Table22 regarding the N-terminal domain of Vpr show that all the residues excluding the initiator methionine are susceptible for alterations. The altered amino acids or polymorphisms at each residue are indicated as variant amino acids or substitutions. For convenience, we have used Vpr from NL4-3 proviral DNA as a reference sequence. The amino acid sequence of NL4-3 Vpr is similar to HIV-1 subtype B consensus Vpr except for residues 28(S), 77(Q) and 83(I). Interestingly, the residue 9, which is G, has only one variant amino acid. In an earlier study, it was noted that a change in residue 3 from Q to R was not associated with cytopathic effect [41]. In our analysis, variant amino acids H, L, M, and P were also noted for Q. Studies involving synthetic peptides corresponding to the N-terminus and also the full-length Vpr molecule have shown that the Vpr sequence (residues PHN) have the ability to form a γ-turn. The residue 15(H) exhibits eleven, residue 16 (N) shows ten and residue 14 (P) shows four variant amino acids. While residue 2 has two, residues 5 and 12 register three variant amino acids. Residues 3, 4, 6, 7, 8, 10, 11, and 13 contain multiple variant amino acids ranging from five to eleven. The N-terminal domain contains a total of 79 variant amino acids. Of these, non-conserved substitutions correspond to about 80% of the residues.
Table 2
Table 2
The polymorphisms in the N-Terminus of Vpr (residues 1–16)
The impact of the majority of the polymorphisms on Vpr functions is not clear. Substitution of alanine for proline at residue 5 and 10 showed less or increased virion incorporation of Vpr, respectively [42]. Similarly, substitution of alanine for residue 12 reduced the cell cycle arrest function of Vpr [43]. On the other hand, substitution at residue 13 and 14 showed an increase in cell cycle arrest [42,44]. Hence, the naturally occurring polymorphisms are likely to affect the functions of Vpr.
Helical domain I (HI residues 17–33)
NMR studies of full length Vpr show that a region comprising the residues 17–33 adapt a helical structure. This was also predicted by several algorithms. The polymorphisms observed for the residues 17–33 are presented in Table Table3.3. The characteristics of the residues with respect to the variant amino acids are the following: residues 18, 23 and 26 show two substitutions; residue 20 has three substitutions; residues 25, and 29 show four substitutions; residues 21, 24, 27, 30 and 32 show five substitutions; and residues 17, 22, and 31 register six substitutions and residue 19 has eight substitutions. Interestingly, residue 28 exhibits the highest number of substitutions and residue 33 has only one substitution. This domain exhibits a total of 80 variant amino acids and 61 of them are of non-conservative in nature.
Table 3
Table 3
The polymorphisms in Helical Domain I of Vpr (residues 17–33)
Several laboratories including ours have reported on the importance of residues in the helical domain I for Vpr functions. Substitution of a proline residue for glutamic acid (residue 17, 21, 24, 25, and 29) has a drastic effect on the stability, subcellular localization, and virion incorporation of Vpr [44-49]. The variant amino acids noted in this domain have the potential to destabilize and disrupt the function of Vpr. Similarly, substitution of alanine for leucine residue affected the stability and virion incorporation of Vpr [45,48,50-53]. Based on the studies reported, varying amino acid arginine for histidine at residue 33 will affect the subcellular localization and virion incorporation of Vpr [54].
Interhelical domain I (residues 34–37)
This region is present between helical domains I and II and comprises only four residues. It has been shown that residues in this region have the ability to form a γ-turn. The naturally occurring polymorphisms in this region are presented in Table Table4.4. Site-specific mutagenesis studies have shown an important role for residues in subcellular localization, cell cycle arrest, apoptosis and virion incorporation of Vpr [42,44,51,55,56]. Residues 34 and 35 show only three substitutions. On the other hand, residue 36 and 37 register 10 and 16 substitutions, respectively. The variant amino acids reach a total of 31 and 21 of them are of non-conservative in nature.
Table 4
Table 4
The polymorphisms in the Interhelical Domain 1 of Vpr (residues 34 – 37)
Helical domain II (residues 38–50)
Studies with peptide (1–50 amino acids) and full-length Vpr have shown that residues 38–50 correspond to helical domain II of Vpr. The naturally occurring polymorphisms corresponding to the residues in this region are presented in Table Table5.5. The characteristics of the substitution are the following: residues 39 and 47 exhibit a single substitution; residues 43, 46 and 50 record two substitutions; residue 38 shows four substitutions; residues 42, 45 and 49 show five substitutions; and residues 40 and 44 have eight substitutions. Nine and thirteen substitutions were noted for residues 41 and 48, respectively. This domain contains 64 variant amino acids and non-conservative substitutions correspond to 41 residues. Several laboratories have carried out experiments addressing the role of residues in this region by utilizing site-specific mutagenesis. The alteration of hydrophobic residues severely affected the virion incorporation and transcriptional activation of Vpr [43,44,50,56].
Table 5
Table 5
The polymorphisms in Helical Domain II of Vpr (residues 38 – 50)
Interhelical domain II (residues 51–54)
This region is located between helical domains II and III. Of the four residues which are part of this domain, only the residue G51 has been shown to reduce G2/M cell cycle arrest through alanine substitution [44]. The naturally occurring polymorphisms corresponding to the residues in this region are presented in Table Table6.6. The characteristics of the substitutions are the following: residue 54 shows two substitutions; residue 51 shows three substitutions; residue 52 shows four substitutions and residue 53 shows five substitutions. The variant amino acids reach a total of fourteen and the majority of them are non-conservative substitutions.
Table 6
Table 6
The polymorphisms in Interhelical Domain II of Vpr (residues 51 – 54)
Helical domain III (residues 55–77)
The presence of helical domain III has been demonstrated by NMR [40]. Several laboratories including ours have shown the importance of this domain for the function of Vpr. The naturally occurring polymorphisms noted for the residues in this region are presented in Table Table7.7. The characteristics of the substitutions are the following: residues 56, 64, 65, 71 and 75 exhibit two substitutions; residues 69, 70, 72, 73 and 76 register three substitutions; residues 57, 66 and 68 show four substitutions; residues 60, 61 and 67 show six substitutions; residues 62 and 63 have seven substitutions; residue 74 has eight substitutions; residues 58, 59, and 77 exhibit ten substitutions; and residue 55 shows eleven substitutions. While the variant amino acids reach a total of 108, 65 of them are of non-conservative nature. This region comprises LXXLL motif which is important for subcellular localization and also influences the virion incorporation of Vpr [44,57-62]. Additionally the LXXLL domain is also involved in Vpr-GR interaction and its subsequent role in virus replication [63,64].
Table 7
Table 7
The polymorphisms in Helical Domain III of Vpr (residues 54–77)
C-terminus of Vpr (residues 78–96)
The naturally occurring polymorphisms corresponding to the residues in the C-terminus of Vpr are presented in Table Table8.8. The characteristics of the substitutions for the residues in this region are the following: residue 80 has only two substitutions; residues 78, 79, 82 and 92 have three substitutions; residues 81 and 90 have four substitutions; residues 91 and 96 have five substitutions. All of the other residues have substitutions ranging from six to thirteen. Of the 124 variant amino acids in this domain, 100 of them are of non-conservative nature.
Table 8
Table 8
The polymorphisms in the Carboxy-Terminal Region of Vpr (residues 78–96)
This domain contains multiple arginine and serine residues. It has been reported that the arginine residues are important for the cell cycle arrest and subcellular localization [65,66]. Vpr is known to undergo post-translational modification and the serine residues located at 28, 79, 94, and 96 positions of the protein serve as substrates for the phosphorylation [67]. Vpr, devoid of phosphorylation through site-specific mutagenesis, severely affects replication of HIV-1 in macrophages [68]. Residue 28 contains equivalent proportion of amino acids N (44%) and S (48%) and Vpr of SIV cpz contains N or T at this position. On the other hand, serine residues at 79, 94, and 96 are conserved in SIV cpz Vpr.
The naturally occurring polymorphisms for the whole Vpr molecule reach a total of 498 substitutions. The non-conservative variant amino acids correspond to 72%. It is important to note that all the residues in Vpr have the propensity to accept variant amino acids. The data presented here also reveal that the variant amino acids noted with respect to some residues are identical. These include residues 60(I), 61(I), 34(F), 71(H) and 72(F). We have carried out a detailed analysis of the variant amino acids noted in distinct subtypes (A, B, C, and D) of HIV-1. Such an analysis could not be carried out for several groups because of the limited information available regarding Vpr alleles. The data generated for subtype B Vpr alleles are presented in Tables Tables9,9, ,10,10, ,11,11, ,12,12, ,13,13, ,14,14, ,15.15. The analysis of subtype B involves a total of 275 Vpr alleles. As expected, the extent of polymorphisms in subtype B is less in comparison to the total polymorphisms noted with all the Vpr alleles. Interestingly, there are several residues that did not have any variant amino acids. These include residues 9, 18, 26, 34, 35, 38, 42, 46, 64, 66, and 79. On the other hand, the residues without variant amino acids in subtype C are different from that of subtype B except for 9, 26, and 64. In addition, the frequency of variant amino acids at the level of each residue was also determined for subtype B Vpr. The results indicate that the frequency of variant amino acids is low in most cases (0.4–1.1%) except for the residues 7, 19, 37, 41, 45, 55, 60, 63, 77, 80, 84, 85, 86, 89, and 93. Analysis involving a large number of Vpr alleles also showed frequency patterns consistent with the data presented in Tables Tables9,9, ,10,10, ,11,11, ,12,12, ,13,13, ,14,14, ,15.15. With respect to the N-terminus domain (Table (Table9),9), the residue 7 (D) has residue N substitution with a frequency of 6.2%. Also, while the reference Vpr allele has Y at position 15, which is the predominant amino acid (85%), the variant amino acid F occurs to a limited extent (6.9%). Similar scenario is also applicable to the residues 28, 77, and 83 (Tables (Tables1010 and and15).15). The residue R 80, which has been implicated in cell cycle arrest function of Vpr, exhibits substitution of A with a frequency of 5.1%.
Table 9
Table 9
The frequency of variant amino acids in the N-Terminus of Vpr (Residues 1–16)
Table 10
Table 10
The frequency of variant amino acids in Helical Domain I of Vpr (Residues 17–33)
Table 11
Table 11
The frequency of variant amino acids in the Interhelical Domain 1 of Vpr (Residues 34–37)
Table 12
Table 12
The frequency of variant amino acids in Helical Domain II of Vpr (Residues 38–50)
Table 13
Table 13
The frequency of variant amino acids in Interhelical Domain II of Vpr (Residues 51 – 54)
Table 14
Table 14
The frequency of variant amino acids in Helical Domain III of Vpr (Residues 55–77)
Table 15
Table 15
The frequency of variant amino acids in the Carboxy-Terminal Region of Vpr (Residues 78–96)
Impact of amino acid polymorphisms on defined and predicted CTL epitopes in Vpr
It has been shown that a single amino acid change in the epitope enables the virus to evade the T cell surveillance [9,69]. Hence, it is of interest to analyze the polymorphisms in the context of both experimentally verified and predicted CTL epitopes. As Vpr is a highly immunogenic protein, several CTL epitopes have been already defined [12]. CD8+ epitopes are contiguous and nine amino acids long. The experimentally verified CTL epitopes in Vpr are presented in Table Table1616 with their location in the protein. We have presented the overall amino acid polymorphisms for each of the epitope. The experimentally verified CTL epitopes cluster in the region covering 1–70 residues of Vpr. The total amino acid polymorphisms range from 36 to 107 for the individual epitopes. For example, the CTL epitope comprising the residues REPHNEWTL contains 53 variant amino acids. Residues at position 1 to 9 of the epitope show 3, 6, 4, 11, 10, 6, 2, 8, and 3 variant amino acids, respectively.
Table 16
Table 16
The extent of amino acid polymorphisms in experimentally defined CTL epitopes
In addition, we have also utilized bioinformatics approach to assess the effect of polymorphisms on CTL epitope http://Bimas.dcrt.nih.gov/molbio/hla-bind. The predicted CTL epitopes with respect to several HLA class I alleles are presented in Table Table17.17. The impact of polymorphisms on the CTL epitope was assessed by determining the estimate of half-time of disassociation of the molecule containing the epitope. For this purpose, we have considered 3, 1, 2, and 6 epitopes corresponding to HLA-A2, Cw-4, HLA B-7 and HLA B-2705, respectively. The influence of variant amino acids on the CTL epitope is presented in Table Table18,18, ,19,19, ,2020 with respect to HLA-A2 molecule. The epitopes considered for analysis correspond to residues 18–26, 38–46, and 66–74 of Vpr. While the reference peptide of the epitope located at residues 18–26 (Table (Table18)18) of Vpr shows the estimate of half time of disassociation value of 1213.356, the variant amino acid at position 1–9 in the epitope predicted a lower value. The substitution of variant amino acids at residue position 2 of the epitope affected the half-time value considerably. Interestingly, substitution of R lowered the value to 0.233. Similarly, the substitution of F for L at position 9 of the epitope also lowered the value to 4.233. The analysis of the epitope corresponding to the residues 38–46 is shown in Table Table19.19. The variant amino acids at residue 39 and 41 drastically lowered the value. The residue 46 showed contrasting values based on the nature of the variant amino acid present. The impact of polymorphisms on the epitope corresponding to the residues 66–74 is shown in Table Table20.20. The results show that both the location and nature of the amino acid have an effect on the half-time disassociation of the molecule, which may lead to defective processing, presentation, and recognition of the epitope.
Table 17
Table 17
The predicted HLA Class 1 CTL epitopes in HIV-1 Vpr
Table 18
Table 18
Effect of variant amino acids on CTL epitope corresponding to residues 18–26 of Vpr
Table 19
Table 19
Effect of variant amino acids on CTL Epitope corresponding to residues 38–46 of Vpr
Table 20
Table 20
Effect of variant amino acids on CTL Epitope corresponding to residues 66–74 of Vpr
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 [39], 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. [28] have noted that Integrase (IN) exhibits the least variability and Vpu exhibits the highest variability. Boutwell and Essex [27] 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 [12]. 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 Table1616 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 [30]. 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 [30].
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.
Authors' contributions
AS, VA, AK, AB, VS, RC and AC participated in the analysis of the predicted amino acid sequences of Vpr. SM, DD and BS provided information regarding the structure-function of Vpr. NM and RM contributed to the analysis of polymorphisms in Vpr from the structural angle. AS, VA, SM, VS, AC, and RC were involved in the preparation of the manuscript. All authors read and approved the final manuscript.
Acknowledgements
This work was supported in part by grant R56-AI50463 from NIAID, National Institute of Health to VA.
  • Alter G, Altfeld M. NK cell function in HIV-1 infection. Curr Mol Med. 2006;6:621–629. doi: 10.2174/156652406778195035. [PubMed] [Cross Ref]
  • Benito JM, Lopez M, Soriano V. The role of CD8+ T-cell response in HIV infection. AIDS Rev. 2004;6:79–88. [PubMed]
  • Maecker HT, Maino VC. T cell immunity to HIV: defining parameters of protection. Curr HIV Res. 2003;1:249–259. doi: 10.2174/1570162033485294. [PubMed] [Cross Ref]
  • Koup RA. Virus escape from CTL recognition. J Exp Med. 1994;180:779–782. doi: 10.1084/jem.180.3.779. [PMC free article] [PubMed] [Cross Ref]
  • Ogg GS, Jin X, Bonhoeffer S, Dunbar PR, Nowak MA, Monard S, Segal JP, Cao Y, Rowland-Jones SL, Cerundolo V, et al. Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science. 1998;279:2103–2106. doi: 10.1126/science.279.5359.2103. [PubMed] [Cross Ref]
  • Borrow P, Lewicki H, Hahn BH, Shaw GM, Oldstone MB. Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection. J Virol. 1994;68:6103–6110. [PMC free article] [PubMed]
  • Matano T, Kobayashi M, Igarashi H, Takeda A, Nakamura H, Kano M, Sugimoto C, Mori K, Iida A, Hirata T, et al. Cytotoxic T lymphocyte-based control of simian immunodeficiency virus replication in a preclinical AIDS vaccine trial. J Exp Med. 2004;199:1709–1718. doi: 10.1084/jem.20040432. [PMC free article] [PubMed] [Cross Ref]
  • O'Connor D, Friedrich T, Hughes A, Allen TM, Watkins D. Understanding cytotoxic T-lymphocyte escape during simian immunodeficiency virus infection. Immunol Rev. 2001;183:115–126. doi: 10.1034/j.1600-065x.2001.1830110.x. [PubMed] [Cross Ref]
  • Allen TM, O'Connor DH, Jing P, Dzuris JL, Mothe BR, Vogel TU, Dunphy E, Liebl ME, Emerson C, Wilson N, et al. Tat-specific cytotoxic T lymphocytes select for SIV escape variants during resolution of primary viraemia. Nature. 2000;407:386–390. doi: 10.1038/35036559. [PubMed] [Cross Ref]
  • Goulder PJ, Watkins DI. HIV and SIV CTL escape: implications for vaccine design. Nat Rev Immunol. 2004;4:630–640. doi: 10.1038/nri1417. [PubMed] [Cross Ref]
  • Fischer W, Perkins S, Theiler J, Bhattacharya T, Yusim K, Funkhouser R, Kuiken C, Haynes B, Letvin NL, Walker BD, et al. Polyvalent vaccines for optimal coverage of potential T-cell epitopes in global HIV-1 variants. Nat Med. 2007;13:100–106. doi: 10.1038/nm1461. [PubMed] [Cross Ref]
  • Korber BTM, Brander C, Hayens BF, Koup R, Moore JP, Walker BD, Watkins DI. HIV Molecular Immunology. Los Alamos National Laboratory, Los Alamos, NM; 2007.
  • Freed EO. HIV-1 replication. Somat Cell Mol Genet. 2001;26:13–33. doi: 10.1023/A:1021070512287. [PubMed] [Cross Ref]
  • Freed EO, Martin MA. HIVs and their replication. In: Knipe DM, Howley PM, editor. "Fields' Virology". Lippincott Williams & Wilkins, Philadelphia; 2001. pp. 1971–2041.
  • Sheehy AM, Gaddis NC, Choi JD, Malim MH. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature. 2002;418:646–650. doi: 10.1038/nature00939. [PubMed] [Cross Ref]
  • Zhang H, Yang B, Pomerantz RJ, Zhang C, Arunachalam SC, Gao L. The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA. Nature. 2003;424:94–98. doi: 10.1038/nature01707. [PMC free article] [PubMed] [Cross Ref]
  • Tungaturthi PK, Sawaya BE, Ayyavoo V, Murali R, Srinivasan A. HIV-1 Vpr: genetic diversity and functional features from the perspective of structure. DNA Cell Biol. 2004;23:207–222. doi: 10.1089/104454904773819798. [PubMed] [Cross Ref]
  • Varthakavi V, Smith RM, Bour SP, Strebel K, Spearman P. Viral protein U counteracts a human host cell restriction that inhibits HIV-1 particle production. Proc Natl Acad Sci USA. 2003;100:15154–15159. doi: 10.1073/pnas.2433165100. [PubMed] [Cross Ref]
  • Willey RL, Maldarelli F, Martin MA, Strebel K. Human immunodeficiency virus type 1 Vpu protein induces rapid degradation of CD4. J Virol. 1992;66:7193–7200. [PMC free article] [PubMed]
  • James CO, Huang MB, Khan M, Garcia-Barrio M, Powell MD, Bond VC. Extracellular Nef protein targets CD4+ T cells for apoptosis by interacting with CXCR4 surface receptors. J Virol. 2004;78:3099–3109. doi: 10.1128/JVI.78.6.3099-3109.2004. [PMC free article] [PubMed] [Cross Ref]
  • Arold ST, Baur AS. Dynamic Nef and Nef dynamics: how structure could explain the complex activities of this small HIV protein. Trends Biochem Sci. 2001;26:356–363. doi: 10.1016/S0968-0004(01)01846-1. [PubMed] [Cross Ref]
  • Geleziunas R, Xu W, Takeda K, Ichijo H, Greene WC. HIV-1 Nef inhibits ASK1-dependent death signalling providing a potential mechanism for protecting the infected host cell. Nature. 2001;410:834–838. doi: 10.1038/35071111. [PubMed] [Cross Ref]
  • Okada H, Takei R, Tashiro M. HIV-1 Nef protein-induced apoptotic cytolysis of a broad spectrum of uninfected human blood cells independently of CD95(Fas) FEBS Lett. 1997;414:603–606. doi: 10.1016/S0014-5793(97)01080-6. [PubMed] [Cross Ref]
  • Das SR, Jameel S. Biology of the HIV Nef protein. Indian J Med Res. 2005;121:315–332. [PubMed]
  • Cohen OJ, Fauci AS. "Fields' Virology". Lippincott Williams & Wilkins, Philadelphia; 2001. Pathogenesis and medical aspects of HIV-1 infection; pp. 2043–2094.
  • Levy JA. "HIV and the Pathogenesis of AIDS". ASM Press, Washington, DC; 1998.
  • Boutwell CL, Essex M. Identification of HLA class I-associated amino acid polymorphisms in the HIV-1C proteome. AIDS Res Hum Retroviruses. 2007;23:165–174. doi: 10.1089/aid.2006.0131. [PubMed] [Cross Ref]
  • Yusim K, Kesmir C, Gaschen B, Addo MM, Altfeld M, Brunak S, Chigaev A, Detours V, Korber BT. Clustering patterns of cytotoxic T-lymphocyte epitopes in human immunodeficiency virus type 1 (HIV-1) proteins reveal imprints of immune evasion on HIV-1 global variation. J Virol. 2002;76:8757–8768. doi: 10.1128/JVI.76.17.8757-8768.2002. [PMC free article] [PubMed] [Cross Ref]
  • Rousseau CM, Birditt BA, McKay AR, Stoddard JN, Lee TC, McLaughlin S, Moore SW, Shindo N, Learn GH, Korber BT, et al. Large-scale amplification, cloning and sequencing of near full-length HIV-1 subtype C genomes. J Virol Methods. 2006;136:118–125. doi: 10.1016/j.jviromet.2006.04.009. [PubMed] [Cross Ref]
  • Rousseau CM, Daniels MG, Carlson JM, Kadie C, Crawford H, Prendergast A, Matthews P, Payne R, Rolland M, Raugi DN, et al. HLA Class-I Driven Evolution of Human Immunodeficiency Virus Type 1 Subtype C Proteome: Immune Escape and Viral Load. J Virol. 2008;82:6434–46. doi: 10.1128/JVI.02455-07. [PMC free article] [PubMed] [Cross Ref]
  • Azad AA. Could Nef and Vpr proteins contribute to disease progression by promoting depletion of bystander cells and prolonged survival of HIV-infected cells? Biochem Biophys Res Commun. 2000;267:677–685. doi: 10.1006/bbrc.1999.1708. [PubMed] [Cross Ref]
  • Emerman M. HIV-1, Vpr and the cell cycle. Curr Biol. 1996;6:1096–1103. doi: 10.1016/S0960-9822(02)00676-0. [PubMed] [Cross Ref]
  • Bukrinsky M, Adzhubei A. Viral protein R of HIV-1. Rev Med Virol. 1999;9:39–49. doi: 10.1002/(SICI)1099-1654(199901/03)9:1<39::AID-RMV235>3.0.CO;2-3. [PubMed] [Cross Ref]
  • Emerman M, Malim MH. HIV-1 regulatory/accessory genes: keys to unraveling viral and host cell biology. Science. 1998;280:1880–1884. doi: 10.1126/science.280.5371.1880. [PubMed] [Cross Ref]
  • Luk KC, Holzmayer V, Yamaguchi J, Swanson P, Brennan CA, Ngansop C, Mbanya D, Gayum H, Djuidje MN, Ndembi N, et al. Near full-length genome characterization of three additional HIV type 1 CRF13_cpx strains from Cameroon. AIDS Res Hum Retroviruses. 2007;23:297–302. doi: 10.1089/aid.2006.0177. [PubMed] [Cross Ref]
  • Reinis M, Weiser B, Kuiken C, Dong T, Lang D, Nachman S, Zhang Y, Rowland-Jones S, Burger H. Genomic analysis of HIV type 1 strains derived from a mother and child pair of long-term nonprogressors. AIDS Res Hum Retroviruses. 2007;23:309–315. doi: 10.1089/aid.2006.0180. [PMC free article] [PubMed] [Cross Ref]
  • Bell CM, Connell BJ, Capovilla A, Venter WD, Stevens WS, Papathanasopoulos MA. Molecular characterization of the HIV type 1 subtype C accessory genes vif, vpr, and vpu. AIDS Res Hum Retroviruses. 2007;23:322–330. doi: 10.1089/aid.2006.0181. [PubMed] [Cross Ref]
  • Shen C, Gupta P, Wu H, Chen X, Huang X, Zhou Y, Chen Y. Molecular Characterization of the HIV Type 1 vpr Gene in Infected Chinese Former Blood/Plasma Donors at Different Stages of Diseases. AIDS Res Hum Retroviruses. 2008;24:661–666. doi: 10.1089/aid.2007.0270. [PubMed] [Cross Ref]
  • Kuiken CL, Cornelissen MT, Zorgdrager F, Hartman S, Gibbs AJ, Goudsmit J. Consistent risk group-associated differences in human immunodeficiency virus type 1 vpr, vpu and V3 sequences despite independent evolution. J Gen Virol. 1996;77:783–792. doi: 10.1099/0022-1317-77-4-783. [PubMed] [Cross Ref]
  • Morellet N, Bouaziz S, Petitjean P, Roques BP. NMR structure of the HIV-1 regulatory protein VPR. J Mol Biol. 2003;327:215–227. doi: 10.1016/S0022-2836(03)00060-3. [PubMed] [Cross Ref]
  • Somasundaran M, Sharkey M, Brichacek B, Luzuriaga K, Emerman M, Sullivan JL, Stevenson M. Evidence for a cytopathogenicity determinant in HIV-1 Vpr. Proc Natl Acad Sci USA. 2002;99:9503–9508. doi: 10.1073/pnas.142313699. [PubMed] [Cross Ref]
  • Votteler J, Studtrucker N, Sorgel S, Munch J, Rucker E, Kirchhoff F, Schick B, Henklein P, Fossen T, Bruns K, et al. Proline 35 of human immunodeficiency virus type 1 (HIV-1) Vpr regulates the integrity of the N-terminal helix and the incorporation of Vpr into virus particles and supports the replication of R5-tropic HIV-1 in human lymphoid tissue ex vivo. J Virol. 2007;81:9572–9576. doi: 10.1128/JVI.02803-06. [PMC free article] [PubMed] [Cross Ref]
  • Iijima S, Nitahara-Kasahara Y, Kimata K, Zhong Zhuang W, Kamata M, Isogai M, Miwa M, Tsunetsugu-Yokota Y, Aida Y. Nuclear localization of Vpr is crucial for the efficient replication of HIV-1 in primary CD4+ T cells. Virology. 2004;327:249–261. doi: 10.1016/j.virol.2004.06.024. [PubMed] [Cross Ref]
  • Di Marzio P, Choe S, Ebright M, Knoblauch R, Landau NR. Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeficiency virus type 1 Vpr. J Virol. 1995;69:7909–7916. [PMC free article] [PubMed]
  • Mahalingam S, Khan SA, Jabbar MA, Monken CE, Collman RG, Srinivasan A. Identification of residues in the N-terminal acidic domain of HIV-1 Vpr essential for virion incorporation. Virology. 1995;207:297–302. doi: 10.1006/viro.1995.1081. [PubMed] [Cross Ref]
  • Mahalingam S, Khan SA, Murali R, Jabbar MA, Monken CE, Collman RG, Srinivasan A. Mutagenesis of the putative alpha-helical domain of the Vpr protein of human immunodeficiency virus type 1: effect on stability and virion incorporation. Proc Natl Acad Sci USA. 1995;92:3794–3798. doi: 10.1073/pnas.92.9.3794. [PubMed] [Cross Ref]
  • Paxton W, Connor RI, Landau NR. Incorporation of Vpr into human immunodeficiency virus type 1 virions: requirement for the p6 region of gag and mutational analysis. J Virol. 1993;67:7229–7237. [PMC free article] [PubMed]
  • Mueller SM, Lang SM. The first HxRxG motif in simian immunodeficiency virus mac239 Vpr is crucial for G(2)/M cell cycle arrest. J Virol. 2002;76:11704–11709. doi: 10.1128/JVI.76.22.11704-11709.2002. [PMC free article] [PubMed] [Cross Ref]
  • Zhao Y, Chen M, Wang B, Yang J, Elder RT, Song XQ, Yu M, Saksena NK. Functional conservation of HIV-1 Vpr and variability in a mother-child pair of long-term non-progressors. Virus Res. 2002;89:103–121. doi: 10.1016/S0168-1702(02)00127-2. [PubMed] [Cross Ref]
  • Thotala D, Schafer EA, Tungaturthi PK, Majumder B, Janket ML, Wagner M, Srinivasan A, Watkins S, Ayyavoo V. Structure-functional analysis of human immunodeficiency virus type 1 (HIV-1) Vpr: role of leucine residues on Vpr-mediated transactivation and virus replication. Virology. 2004;328:89–100. doi: 10.1016/j.virol.2004.07.013. [PubMed] [Cross Ref]
  • Jacquot G, Le Rouzic E, David A, Mazzolini J, Bouchet J, Bouaziz S, Niedergang F, Pancino G, Benichou S. Localization of HIV-1 Vpr to the nuclear envelope: impact on Vpr functions and virus replication in macrophages. Retrovirology. 2007;4:84. doi: 10.1186/1742-4690-4-84. [PMC free article] [PubMed] [Cross Ref]
  • Forget J, Yao XJ, Mercier J, Cohen EA. Human immunodeficiency virus type 1 vpr protein transactivation function: mechanism and identification of domains involved. J Mol Biol. 1998;284:915–923. doi: 10.1006/jmbi.1998.2206. [PubMed] [Cross Ref]
  • Yao XJ, Subbramanian RA, Rougeau N, Boisvert F, Bergeron D, Cohen EA. Mutagenic analysis of human immunodeficiency virus type 1 Vpr: role of a predicted N-terminal alpha-helical structure in Vpr nuclear localization and virion incorporation. J Virol. 1995;69:7032–7044. [PMC free article] [PubMed]
  • Chen M, Elder RT, Yu M, O'Gorman MG, Selig L, Benarous R, Yamamoto A, Zhao Y. Mutational analysis of Vpr-induced G2 arrest, nuclear localization, and cell death in fission yeast. J Virol. 1999;73:3236–3245. [PMC free article] [PubMed]
  • Vodicka MA, Koepp DM, Silver PA, Emerman M. HIV-1 Vpr interacts with the nuclear transport pathway to promote macrophage infection. Genes Dev. 1998;12:175–185. doi: 10.1101/gad.12.2.175. [PubMed] [Cross Ref]
  • Singh SP, Tomkowicz B, Lai D, Cartas M, Mahalingam S, Kalyanaraman VS, Murali R, Srinivasan A. Functional role of residues corresponding to helical domain II (amino acids 35 to 46) of human immunodeficiency virus type 1 Vpr. J Virol. 2000;74:10650–10657. doi: 10.1128/JVI.74.22.10650-10657.2000. [PMC free article] [PubMed] [Cross Ref]
  • Kamata M, Aida Y. Two putative alpha-helical domains of human immunodeficiency virus type 1 Vpr mediate nuclear localization by at least two mechanisms. J Virol. 2000;74:7179–7186. doi: 10.1128/JVI.74.15.7179-7186.2000. [PMC free article] [PubMed] [Cross Ref]
  • Bolton DL, Lenardo MJ. Vpr cytopathicity independent of G2/M cell cycle arrest in human immunodeficiency virus type 1-infected CD4+ T cells. J Virol. 2007;81:8878–8890. doi: 10.1128/JVI.00122-07. [PMC free article] [PubMed] [Cross Ref]
  • Sherman MP, de Noronha CM, Eckstein LA, Hataye J, Mundt P, Williams SA, Neidleman JA, Goldsmith MA, Greene WC. Nuclear export of Vpr is required for efficient replication of human immunodeficiency virus type 1 in tissue macrophages. J Virol. 2003;77:7582–7589. doi: 10.1128/JVI.77.13.7582-7589.2003. [PMC free article] [PubMed] [Cross Ref]
  • Mahalingam S, Ayyavoo V, Patel M, Kieber-Emmons T, Weiner DB. Nuclear import, virion incorporation, and cell cycle arrest/differentiation are mediated by distinct functional domains of human immunodeficiency virus type 1 Vpr. J Virol. 1997;71:6339–6347. [PMC free article] [PubMed]
  • Tan L, Ehrlich E, Yu XF. DDB1 and Cul4A are required for human immunodeficiency virus type 1 Vpr-induced G2 arrest. J Virol. 2007;81:10822–10830. doi: 10.1128/JVI.01380-07. [PMC free article] [PubMed] [Cross Ref]
  • Hrecka K, Gierszewska M, Srivastava S, Kozaczkiewicz L, Swanson SK, Florens L, Washburn MP, Skowronski J. Lentiviral Vpr usurps Cul4-DDB1[VprBP] E3 ubiquitin ligase to modulate cell cycle. Proc Natl Acad Sci USA. 2007;104:11778–11783. doi: 10.1073/pnas.0702102104. [PubMed] [Cross Ref]
  • Kino T, Tsukamoto M, Chrousos G. Transcription factor TFIIH components enhance the GR coactivator activity but not the cell cycle-arresting activity of the human immunodeficiency virus type-1 protein Vpr. Biochem Biophys Res Commun. 2002;298:17–23. doi: 10.1016/S0006-291X(02)02442-7. [PubMed] [Cross Ref]
  • Sherman MP, de Noronha CM, Pearce D, Greene WC. Human immunodeficiency virus type 1 Vpr contains two leucine-rich helices that mediate glucocorticoid receptor coactivation independently of its effects on G(2) cell cycle arrest. J Virol. 2000;74:8159–8165. doi: 10.1128/JVI.74.17.8159-8165.2000. [PMC free article] [PubMed] [Cross Ref]
  • Zhou Y, Ratner L. A novel inducible expression system to study transdominant mutants of HIV-1 Vpr. Virology. 2001;287:133–142. doi: 10.1006/viro.2001.1031. [PubMed] [Cross Ref]
  • Kitayama H, Miura Y, Ando Y, Hoshino S, Ishizaka Y, Koyanagi Y. Human immunodeficiency virus type 1 Vpr inhibits axonal outgrowth through induction of mitochondrial dysfunction. J Virol. 2008;82:2528–2542. doi: 10.1128/JVI.02094-07. [PMC free article] [PubMed] [Cross Ref]
  • Zhou Y, Lu Y, Ratner L. Arginine residues in the C-terminus of HIV-1 Vpr are important for nuclear localization and cell cycle arrest. Virology. 1998;242:414–424. doi: 10.1006/viro.1998.9028. [PubMed] [Cross Ref]
  • Agostini I, Popov S, Hao T, Li JH, Dubrovsky L, Chaika O, Chaika N, Lewis R, Bukrinsky M. Phosphorylation of Vpr regulates HIV type 1 nuclear import and macrophage infection. AIDS Res Hum Retroviruses. 2002;18:283–288. doi: 10.1089/088922202753472856. [PubMed] [Cross Ref]
  • Barouch DH, Kunstman J, Glowczwskie J, Kunstman KJ, Egan MA, Peyerl FW, Santra S, Kuroda MJ, Schmitz JE, Beaudry K, et al. Viral escape from dominant simian immunodeficiency virus epitope-specific cytotoxic T lymphocytes in DNA-vaccinated rhesus monkeys. J Virol. 2003;77:7367–7375. doi: 10.1128/JVI.77.13.7367-7375.2003. [PMC free article] [PubMed] [Cross Ref]
  • Emini EA, Fan HY. Immunological and pharmacological approaches to the control of retroviral infections. In: Coffin JM, Hughes SH, Varmus HE, editor. "Retroviruses". Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; 1997. pp. 637–706.
  • Montefiori D, Sattentau Q, Flores J, Esparza J, Mascola J. Antibody-based HIV-1 vaccines: recent developments and future directions. PLoS Med. 2007;4:e348. doi: 10.1371/journal.pmed.0040348. [PMC free article] [PubMed] [Cross Ref]
  • Altfeld M, Allen TM. Hitting HIV where it hurts: an alternative approach to HIV vaccine design. Trends Immunol. 2006;27:504–510. doi: 10.1016/j.it.2006.09.007. [PubMed] [Cross Ref]
  • Burton DR, Desrosiers RC, Doms RW, Koff WC, Kwong PD, Moore JP, Nabel GJ, Sodroski J, Wilson IA, Wyatt RT. HIV vaccine design and the neutralizing antibody problem. Nat Immunol. 2004;5:233–236. doi: 10.1038/ni0304-233. [PubMed] [Cross Ref]
  • Jin X, Bauer DE, Tuttleton SE, Lewin S, Gettie A, Blanchard J, Irwin CE, Safrit JT, Mittler J, Weinberger L, et al. Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodeficiency virus-infected macaques. J Exp Med. 1999;189:991–998. doi: 10.1084/jem.189.6.991. [PMC free article] [PubMed] [Cross Ref]
  • Schmitz JE, Kuroda MJ, Santra S, Sasseville VG, Simon MA, Lifton MA, Racz P, Tenner-Racz K, Dalesandro M, Scallon BJ, et al. Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes. Science. 1999;283:857–860. doi: 10.1126/science.283.5403.857. [PubMed] [Cross Ref]
  • Malim MH, Emerman M. HIV-1 sequence variation: drift, shift, and attenuation. Cell. 2001;104:469–472. doi: 10.1016/S0092-8674(01)00234-3. [PubMed] [Cross Ref]
  • Overbaugh J, Bangham CR. Selection forces and constraints on retroviral sequence variation. Science. 2001;292:1106–1109. doi: 10.1126/science.1059128. [PubMed] [Cross Ref]
  • Bhattacharya T, Daniels M, Heckerman D, Foley B, Frahm N, Kadie C, Carlson J, Yusim K, McMahon B, Gaschen B, et al. Founder effects in the assessment of HIV polymorphisms and HLA allele associations. Science. 2007;315:1583–1586. doi: 10.1126/science.1131528. [PubMed] [Cross Ref]
  • Moore JP. AIDS vaccines: on the trail of two trials. Nature. 2002;415:365–366. doi: 10.1038/415365a. [PubMed] [Cross Ref]
  • Brumme ZL, Brumme CJ, Heckerman D, Korber BT, Daniels M, Carlson J, Kadie C, Bhattacharya T, Chui C, Szinger J, et al. Evidence of differential HLA class I-mediated viral evolution in functional and accessory/regulatory genes of HIV-1. PLoS Pathog. 2007;3:e94. doi: 10.1371/journal.ppat.0030094. [PMC free article] [PubMed] [Cross Ref]
  • Crawford H, Prado JG, Leslie A, Hue S, Honeyborne I, Reddy S, Stok M van der, Mncube Z, Brander C, Rousseau C, et al. Compensatory mutation partially restores fitness and delays reversion of escape mutation within the immunodominant HLA-B*5703-restricted Gag epitope in chronic human immunodeficiency virus type 1 infection. J Virol. 2007;81:8346–8351. doi: 10.1128/JVI.00465-07. [PMC free article] [PubMed] [Cross Ref]
  • Yang OO, Sarkis PT, Ali A, Harlow JD, Brander C, Kalams SA, Walker BD. Determinant of HIV-1 mutational escape from cytotoxic T lymphocytes. J Exp Med. 2003;197:1365–1375. doi: 10.1084/jem.20022138. [PMC free article] [PubMed] [Cross Ref]
  • Peyerl FW, Bazick HS, Newberg MH, Barouch DH, Sodroski J, Letvin NL. Fitness costs limit viral escape from cytotoxic T lymphocytes at a structurally constrained epitope. J Virol. 2004;78:13901–13910. doi: 10.1128/JVI.78.24.13901-13910.2004. [PMC free article] [PubMed] [Cross Ref]
  • Leslie A, Kavanagh D, Honeyborne I, Pfafferott K, Edwards C, Pillay T, Hilton L, Thobakgale C, Ramduth D, Draenert R, et al. Transmission and accumulation of CTL escape variants drive negative associations between HIV polymorphisms and HLA. J Exp Med. 2005;201:891–902. doi: 10.1084/jem.20041455. [PMC free article] [PubMed] [Cross Ref]
  • Friedrich TC, Frye CA, Yant LJ, O'Connor DH, Kriewaldt NA, Benson M, Vojnov L, Dodds EJ, Cullen C, Rudersdorf R, et al. Extraepitopic compensatory substitutions partially restore fitness to simian immunodeficiency virus variants that escape from an immunodominant cytotoxic-T-lymphocyte response. J Virol. 2004;78:2581–2585. doi: 10.1128/JVI.78.5.2581-2585.2004. [PMC free article] [PubMed] [Cross Ref]
  • Altfeld M, Allen TM, Kalife ET, Frahm N, Addo MM, Mothe BR, Rathod A, Reyor LL, Harlow J, Yu XG, et al. The majority of currently circulating human immunodeficiency virus type 1 clade B viruses fail to prime cytotoxic T-lymphocyte responses against an otherwise immunodominant HLA-A2-restricted epitope: implications for vaccine design. J Virol. 2005;79:5000–5005. doi: 10.1128/JVI.79.8.5000-5005.2005. [PMC free article] [PubMed] [Cross Ref]
  • Kiepiela P, Ngumbela K, Thobakgale C, Ramduth D, Honeyborne I, Moodley E, Reddy S, de Pierres C, Mncube Z, Mkhwanazi N, et al. CD8+ T-cell responses to different HIV proteins have discordant associations with viral load. Nat Med. 2007;13:46–53. doi: 10.1038/nm1520. [PubMed] [Cross Ref]
  • Altfeld M, Addo MM, Eldridge RL, Yu XG, Thomas S, Khatri A, Strick D, Phillips MN, Cohen GB, Islam SA, et al. Vpr is preferentially targeted by CTL during HIV-1 infection. J Immunol. 2001;167:2743–2752. [PubMed]
  • Addo MM, Yu XG, Rosenberg ES, Walker BD, Altfeld M. Cytotoxic T-lymphocyte (CTL) responses directed against regulatory and accessory proteins in HIV-1 infection. DNA Cell Biol. 2002;21:671–678. doi: 10.1089/104454902760330219. [PubMed] [Cross Ref]
  • Addo MM, Yu XG, Rathod A, Cohen D, Eldridge RL, Strick D, Johnston MN, Corcoran C, Wurcel AG, Fitzpatrick CA, et al. Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load. J Virol. 2003;77:2081–2092. doi: 10.1128/JVI.77.3.2081-2092.2003. [PMC free article] [PubMed] [Cross Ref]
  • Liu HW, Hong KX, Ma J, Yuan L, Liu S, Chen JP, Zhang YZ, Ruan YH, Xu JQ, Shao YM. Identification of HIV-1 specific T lymphocyte responses in highly exposed persistently seronegative Chinese. Chin Med J (Engl) 2006;119:1616–1621. [PubMed]
  • Cao J, McNevin J, Holte S, Fink L, Corey L, McElrath MJ. Comprehensive analysis of human immunodeficiency virus type 1 (HIV-1)-specific gamma interferon-secreting CD8+ T cells in primary HIV-1 infection. J Virol. 2003;77:6867–6878. doi: 10.1128/JVI.77.12.6867-6878.2003. [PMC free article] [PubMed] [Cross Ref]
  • Lichterfeld M, Yu XG, Le Gall S, Altfeld M. Immunodominance of HIV-1-specific CD8(+) T-cell responses in acute HIV-1 infection: at the crossroads of viral and host genetics. Trends Immunol. 2005;26:166–171. doi: 10.1016/j.it.2005.01.003. [PubMed] [Cross Ref]
  • Frahm N, Linde C, Brander C. "HIV Molecular Immunology 2006". Los Alamos National Laboratory, Theoritical Biology and Biophysics, Los Alamos, New Mexico; 2007. Identification of HIV-derived HLA class I restricted CTL epitope: Insights into TCR repertoire, CTL escape and viral fitness; p. 3.
  • Feeney ME, Tang Y, Pfafferott K, Roosevelt KA, Draenert R, Trocha A, Yu XG, Verrill C, Allen T, Moore C, et al. HIV-1 viral escape in infancy followed by emergence of a variant-specific CTL response. J Immunol. 2005;174:7524–7530. [PubMed]
  • Novitsky V, Cao H, Rybak N, Gilbert P, McLane MF, Gaolekwe S, Peter T, Thior I, Ndung'u T, Marlink R, et al. Magnitude and frequency of cytotoxic T-lymphocyte responses: identification of immunodominant regions of human immunodeficiency virus type 1 subtype C. J Virol. 2002;76:10155–10168. doi: 10.1128/JVI.76.20.10155-10168.2002. [PMC free article] [PubMed] [Cross Ref]
  • Frahm N, Yusim K, Suscovich TJ, Adams S, Sidney J, Hraber P, Hewitt HS, Linde CH, Kavanagh DG, Woodberry T, et al. Extensive HLA class I allele promiscuity among viral CTL epitopes. Eur J Immunol. 2007;37:2419–2433. doi: 10.1002/eji.200737365. [PMC free article] [PubMed] [Cross Ref]
  • Altfeld MA, Livingston B, Reshamwala N, Nguyen PT, Addo MM, Shea A, Newman M, Fikes J, Sidney J, Wentworth P, et al. Identification of novel HLA-A2-restricted human immunodeficiency virus type 1-specific cytotoxic T-lymphocyte epitopes predicted by the HLA-A2 supertype peptide-binding motif. J Virol. 2001;75:1301–1311. doi: 10.1128/JVI.75.3.1301-1311.2001. [PMC free article] [PubMed] [Cross Ref]
  • Corbet S, Nielsen HV, Vinner L, Lauemoller S, Therrien D, Tang S, Kronborg G, Mathiesen L, Chaplin P, Brunak S, et al. Optimization and immune recognition of multiple novel conserved HLA-A2, human immunodeficiency virus type 1-specific CTL epitopes. J Gen Virol. 2003;84:2409–2421. doi: 10.1099/vir.0.19152-0. [PubMed] [Cross Ref]
  • Yu XG, Addo MM, Rosenberg ES, Rodriguez WR, Lee PK, Fitzpatrick CA, Johnston MN, Strick D, Goulder PJ, Walker BD, Altfeld M. Consistent patterns in the development and immunodominance of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T-cell responses following acute HIV-1 infection. J Virol. 2002;76:8690–8701. doi: 10.1128/JVI.76.17.8690-8701.2002. [PMC free article] [PubMed] [Cross Ref]
  • Bernardin F, Kong D, Peddada L, Baxter-Lowe LA, Delwart E. Human immunodeficiency virus mutations during the first month of infection are preferentially found in known cytotoxic T-lymphocyte epitopes. J Virol. 2005;79:11523–11528. doi: 10.1128/JVI.79.17.11523-11528.2005. [PMC free article] [PubMed] [Cross Ref]
  • Kiepiela P, Leslie AJ, Honeyborne I, Ramduth D, Thobakgale C, Chetty S, Rathnavalu P, Moore C, Pfafferott KJ, Hilton L, et al. Dominant influence of HLA-B in mediating the potential co-evolution of HIV and HLA. Nature. 2004;432:769–775. doi: 10.1038/nature03113. [PubMed] [Cross Ref]
  • Allen TM, Yu XG, Kalife ET, Reyor LL, Lichterfeld M, John M, Cheng M, Allgaier RL, Mui S, Frahm N, et al. De novo generation of escape variant-specific CD8+ T-cell responses following cytotoxic T-lymphocyte escape in chronic human immunodeficiency virus type 1 infection. J Virol. 2005;79:12952–12960. doi: 10.1128/JVI.79.20.12952-12960.2005. [PMC free article] [PubMed] [Cross Ref]
  • Liu F, Bergami PL, Duval M, Kuhrt D, Posner M, Cavacini L. Expression and functional activity of isotype and subclass switched human monoclonal antibody reactive with the base of the V3 loop of HIV-1 gp120. AIDS Res Hum Retroviruses. 2003;19:597–607. doi: 10.1089/088922203322230969. [PubMed] [Cross Ref]
  • Propato A, Schiaffella E, Vicenzi E, Francavilla V, Baloni L, Paroli M, Finocchi L, Tanigaki N, Ghezzi S, Ferrara R, et al. Spreading of HIV-specific CD8+ T-cell repertoire in long-term nonprogressors and its role in the control of viral load and disease activity. Hum Immunol. 2001;62:561–576. doi: 10.1016/S0198-8859(01)00245-2. [PubMed] [Cross Ref]
  • Goulder PJ, Addo MM, Altfeld MA, Rosenberg ES, Tang Y, Govender U, Mngqundaniso N, Annamalai K, Vogel TU, Hammond M, et al. Rapid definition of five novel HLA-A*3002-restricted human immunodeficiency virus-specific cytotoxic T-lymphocyte epitopes by elispot and intracellular cytokine staining assays. J Virol. 2001;75:1339–1347. doi: 10.1128/JVI.75.3.1339-1347.2001. [PMC free article] [PubMed] [Cross Ref]
Articles from Virology Journal are provided here courtesy of
BioMed Central