The influence of viral protein R amino acid substitutions on clinical outcomes in people living with HIV: A systematic review

The HIV viral protein R (Vpr) is a multifunction protein involved in the pathophysiology of HIV‐1. Recent evidence has suggested that Vpr amino acid substitutions influence the pathophysiology of HIV‐1 and clinical outcomes in people living with HIV (PLWH). Several studies have linked Vpr amino acid substitutions to clinical outcomes in PLWH; however, there is no clear consensus as to which amino acids or amino acid substitutions are most important in the pathophysiology and clinical outcomes in PLWH. We, therefore, conducted a systematic review of studies investigating Vpr amino acid substitutions and clinical outcomes in PLWH.


| INTRODUCTION
Human immunodeficiency virus (HIV), a retrovirus, is the causative agent of acquired immunodeficiency syndrome (AIDS). 1,2 According to the Joint United Nations Programme on HIV/AIDS (UNAIDS), it was approximated in 2021 that 37.7 million people were living with HIV (PLWH), 1.5 million people were newly infected and 680,000 people had AIDS-related deaths, globally. 3 Even with 75% of PLWH having access to antiretroviral therapy (ART) globally and despite effective treatment for PLWH with ART, other complications, especially those of the central nervous system (CNS), persist. 4 Human immunodeficiency virus is a genetically diverse pathogen due to high replication rates, error-prone reverse transcriptase and recombination events that could occur during viral replication. [5][6][7] Two types of HIV exist, HIV-1 and HIV-2, of which HIV-1 is more prevalent [8][9][10] and is categorised into groups M, N, O and P. [11][12][13][14][15][16] As reviewed by Sharp and colleagues, HIV-1 group M is the more geographically abundant group than other HIV-1 groups. 17 Furthermore, HIV-1 group M consists of viral subtypes A, B, C, D, F, G, H, J and K. 11 Furthermore, subtypes A and F have sub-subtypes, namely A1-4 and F1-2, respectively. [18][19][20][21][22] The subtypes predominate in particular regions worldwide: CRF02_AG/Subtype G-West Africa; Subtype A-East Africa, Eastern Europe, and Central Asia; Subtype C-Southern Africa, Ethiopia, and South Asia (India); Subtype B-Western and Central Europe, North America, Caribbean, Latin America and Oceania. 5 Amongst all HIV-1 subtypes, M and O are considered the more genetically diverse subtypes. [12][13][14][15][16] HIV-1 consists of various viral proteins which play important roles during the viral lifecycle. 23 Human immunodeficiency virus encodes for 15 distinct proteins and these include: structural (matrix, capsid, nucleocapsid, p6, surface and transmembrane), Pol enzymes (protease, reverse transcriptase and integrase), regulatory proteins (transactivation of transcription (Tat) and regulator of expression of virion proteins (Rev)) and accessory proteins (negative regulatory factor (Nef), viral infectivity factor (Vif), viral protein R (Vpr) and virus protein U (Vpu)). 23 Traditionally, the HIV-1 accessory Vpr has received less attention despite its relevance to pathophysiological mechanisms and clinical outcomes in PLWH. Viral protein R is a fourteen-kilodalton basic protein comprised of 96 amino acids and is made up of three amphiphilic helices. 24 These bundled αhelices extend to the amino acid regions 17-33, 38-50 and 55-77 and are flanked by unstructured flexible N-and C-terminal domains that are negatively or positively charged, respectively. 24 HIV Vpr is a multifunctional protein involved in the pathophysiology of HIV, more specifically Vpr (1) counteracts the IFN-stimulated macrophage-specific restriction pathway targeting HIV-1 Env, 25 (2) targets Tet Methylcytosine Dioxygenase 2 (TET2) for degradation by CRL4 VprBP E3 Ligase to sustain pro-inflammatory cytokine interleukin-6 (IL-6) expression and enhance HIV-1 replication, 26 (3) counteracts the restriction of lysosomal-associated transmembrane protein 5 (LAPTM5) to promote HIV-1 infection in macrophages, 27 (4) targets cellular proteins to drive system-level proteomic remodelling in HIV-1 infection, 28 and (5) counteracts helicase-like transcription factor (HLTF)-mediated restriction of HIV-1 infection in T cells. 29 The effect that Vpr has on monocytes and infected macrophages is associated with HIV-1-associated neuropathogenesis. 30 Even in the absence of active viral replication and with the effective use of ART, Vpr can still be detected in the periphery and CNS of PLWH. [31][32][33] Therefore, Vpr may have a crucial role in the pathophysiology of HIV-1 in the modern-ART era.
Subtype-specific sequence variation exists between HIV-1 subtypes, and this results in changes in the amino acid sequences of the HIV-1 viral proteins, including Vpr. Multiple research groups have extensively sequenced the HIV-1 genome. [34][35][36][37] HIV-1 mutates rapidly and using a wide range of investigations, the HIV-1 mutation rate is estimated to be 10 −5 -10 −3 errors/bp/cycle 38 with the Env gene having the fastest mutation rate amongst all viral genes. [39][40][41] Even though Vpr is an accessory protein, the Vpr gene is generally well-conserved in all human (HIV-1, HIV-2) and simian (SIV) immunodeficiency viruses [42][43][44] and has lower mutation rates than other viral genes 40 and therefore indicative of its potential importance in the pathogenesis of HIV.
The sequence variation in the HIV-1 viral proteins influences the pathophysiology of HIV-1. 31,[45][46][47][48][49][50][51] When comparing the Vpr protein sequences between HIV-1 subtypes, several variances are noted ( Figure 1) and certain amino acid substitutions have been associated with the pathophysiology of HIV-1. 50,52 This may be due to a change in the structural characteristics of the Vpr protein ( Figure 2). Overall, the structure of Vpr may seem similar between HIV-1 subtypes (Figure 2), it is known that the alteration of specific amino acids or the substitutions of amino acids at specific positions within Vpr may significantly alter its function. 53 As examples of these, mutations (R36W, I64E, L67A, and I70S) in αhelix residues result in a total inability of Vpr to oligomerize at the nuclear envelope. 54 In addition, the deletion of the amino acids of the SRIG sequence (between amino acids 79 and 82) or mutation of all arginine's in the C-terminal domain prevented G2 arrest by Vpr. 55 As reviewed in Fabryova, Strebel, 56 several preclinical studies have investigated the influence of Vpr sequence variation and their contribution to HIV-1 pathophysiology 56 ; however, these amino acid substitutions do not necessarily present the Vpr amino acid substitutions present in PLWH.
Despite several studies investigating Vpr amino acids' variation in PLWH, 53,56 there is no clear consensus on which amino acid substitutions are most commonly associated with the pathophysiology (disease progression, neurological outcomes and treatment efficacy) of HIV-1 in PLWH. Therefore, this systematic review aimed at reviewing all literature on this topic to identify the key Vpr amino acid substitutions associated with HIV-1 pathophysiology in PLWH. Findings from this review may further our understanding of the role of Vpr within various pathophysiological processes.

| Study design
This systematic review aimed at summarising the extant literature on the influence of Vpr amino acid substitutions on clinical outcomes in PLWH. The study has been carried out according to PRISMA guidelines 57 and the reporting of the study conforms to broad EQUATOR guidelines. 58 This study has been approved by the North-West University Health Research Ethics Committee (NWU-HREC): NWU-00203-22-A1.

| Eligibility criteria
The eligibility criteria were HIV-positive treatmentexperienced adults (>18 years old, all medication types included, and no cut-off for treatment duration) with relevant Vpr protein sequence data. All studies had to investigate Vpr protein sequence variations from clinical sample types (blood, cerebrospinal fluid, and post-mortem brain tissue) to investigate HIV-1 proviral DNA and/or viral RNA. For comparability of studies, sequencing had to be carried out by polymerase chain reaction (PCR) based sequencing methods (e.g. Sanger sequencing and/or Next-Generation Sequencing). Studies had to have a control group/comparative arm to be included. Exclusionary criteria were preclinical (animal and cell culture models) studies and reviews. Studies investigating Vpr sequence variation without relation to clinical outcomes were also excluded.

| Data sources
We electronically searched for publications in PubMed, Scopus and Web of Science databases based on all studies published until 06/09/2022. Eligible studies included published studies in English only. The search strategy was executed without publication date limitations. The full search criteria for each database are included in File S1. The following search terms were applied to PubMed: (HIV [mh] OR HIV [tw] OR Acquired Immunodeficiency F I G U R E 1 Schematic presentation of HIV-1 Vpr amino acid sequence alignment comparing (A) Subtype B (P12520 · VPR_HV1N5) (B) Subtype D (P12519 · VPR_HV1Z2) (C) Subtype C (Q75004 · VPR_HV1ET), (D) Subtype F2 (F2 P0C1P4 · VPR_HV1MP), (E) Subtype K (P0C1P2 · VPR_HV196). Amino acids in boxes indicate noteworthy positions. Alpha helical domains are indicated in blue boxes and are at regions 17-33, 38-50 and 55-77. Each subtype Vpr sequence was extracted from UniProt where available. No Vpr sequence was available for subtype A. Only the 'pure subtypes' were presented and the sequences from the circulating recombinant forms (CRFs) were not included due to the high number of known CRFs globally.
F I G U R E 2 Predicted 3D structures of Vpr (Subtype B (P12520 · VPR_HV1N5) generated by Swiss Model webserver. The Vpr sequence was extracted from UniProt and subjected to 3D structure prediction. Noteworthy amino acid positions are indicated as yellow sticks. Helical structures are indicated in red and unstructured regions are indicated in green. In addition, we also (1) reviewed reference sections of eligible articles and manually searched for relevant publications and (2) consulted with the contact authors of the included studies. This search strategy and the retrieved articles are shown in Figure 3.

| Data selection
All articles were retrieved and loaded onto a single database using a reference manager (EndNote X9, Clarivate, PA, USA). Two authors independently identified studies meeting the inclusion criteria. Where there was a discrepancy in article inclusion/exclusion, this was discussed amongst all authors, and a decision was made regarding its suitability.

| Quality assessment of included studies
The quality of the included studies was assessed, and kappa statistics were calculated. The Joanna Briggs Institute (JBI) critical appraisal tools have been adopted for the quality criterion. Here, we have amended the JBI critical appraisal tools by implementing a Likert scale 59 to provide a quantitative measure of study quality. For all included studies, we have only considered the JBI quality questions from the Checklist for Analytical Cross-Sectional Studies which may influence the findings of the reviewed studies. These included (1) cohort information, (2) validity and conditions of measurement and (3) confounding factors and statistical analysis. These were assessed using the following questions (1) Were the criteria for inclusion in the sample clearly defined and were study subjects and the setting described? (2) was the method used to investigate the clinical outcome done in an objective, valid and reliable method (i.e. the inclusion of viral load and CD4 + count)? and (3) did they consider confounding factors, and did they employ relevant statistical analysis? This F I G U R E 3 Preferred reporting items for systematic reviews and meta-analyses (PRISMA) flow diagram for results of search strategy. included a total of three questions. Each question was rated 0-no, 1-partly and 2-yes. Studies that addressed all the above questions and had a total rating of ≥5 were classified as high quality. Studies with a rating between 3 and 4 were considered intermediate-quality and ≤2 as low quality (Table S1).

| Potential confounders
Several factors may influence clinical outcomes in PLWH and therefore may influence the findings reported in the reviewed studies investigating Vpr amino acid substitutions and clinical outcomes. Therefore, we wanted to consider certain confounders when analysing the data. In particular, we considered viral load and CD4 + count concerning the findings reported in the studies. First, we stratified studies according to viral load, which was defined as <400 copies/millilitre (cps/ml) (viral suppression) versus >400 cps/ml (nonviral suppression) [60][61][62] (Table S2). Second, studies were also stratified according to a mean/median CD4 + count of <200 cells/μl or >200 cells/μl (Table S3). The mean values were primarily considered when stratifying studies; however, where the mean data were not available, the median data were used. We wanted to determine whether studies were striated according to viral load and CD4 + counts, do Vpr amino acid substitutions still influence clinical outcomes in PLWH. Lastly, we wanted to investigate whether the HIV-1 subtype influenced the prevalence of studies reporting the association of Vpr amino acid substitutions and clinical outcomes.

| Study characteristics
Using this criterion and search strategy (Section 2.2), 984 abstracts and titles were screened as indicated in Figure 3. Furthermore, n = 3 clinical studies were added from additional sources. Duplicates (n = 121) were removed, resulting in 866 studies. Thereafter, abstracts and titles were screened and a total of 654 studies were excluded which comprised of: • Review articles/book chapters/conference proceedings (n = 173). • Studies not investigating HIV-1 in general (n = 48). • Studies investigating HIV-2 (n = 8).
• Studies not investigating Vpr in general (n = 414). • Studies only investigating Vpr sequence variation on a genetic level (n = 4).
Full-text articles assessed for eligibility were done for 212 studies, and an additional n = 95 were excluded: • Review articles/book chapters/conference proceedings (n = 1). • Studies investigating HIV-2 (n = 6).
• Studies not investigating Vpr in general (n = 1). • Studies only investigating HIV-1 sequence variation of multiple viral genes on a genetic level on (n = 5). • Studies investigating Vpr but not investigating Vpr amino acid substitutions (n = 74). • Paper retraction (n = 1). • Investigated Vpr amino acid substitutions but no correlation with clinical outcomes (n = 5). • Database sequences (n = 1). • A study that was speculative regarding correlations of Vpr amino acids and clinical outcomes (n = 1).
Using this criterion (Sections 2.1-2.3), a total of 117 studies resulted which included n = 95 preclinical/fundamental studies. The 95 preclinical studies were excluded as this is part of an ongoing study looking at fundamental Vpr amino acid substitutions. Therefore, a total of 22 studies were included for data extraction, and from these studies, n = 14 (63.64%) were cross-sectional and n = 8 (36.36%) were longitudinal in design. Of all of the studies, n = 5 studies were case studies. [63][64][65][66][67] Across all studies, a total sample size of n = 2929 PLWH and n = 0 HIVnegative controls.

| Quality of assessment of included studies
The quality of the studies was rated. The Kappa statistic for interrater agreement and reliability was .757, indicating substantial agreement. 68 Most studies were rated as intermediate (54%-64%), followed by high (27%-36%) and low-quality (8%) ( Table S1). Based on these findings, recommendations are made in the latter part of this review.
The overall findings show that certain Vpr amino acid substitutions were consistently associated with certain clinical outcomes as reported by more than one independent study. However, for this review, we considered an amino acid substitution finding to be 'noteworthy' when (1) two or more independent studies investigated a particular Vpr amino acid substitution and (2) when 50% or more of the studies investigating a particular Vpr amino acid substitution found it to be consistently associated with the same clinical outcome. 69 In other words, if >50% of studies that investigated a particular amino acid substitution found it to be associated with the same positive/negative/ no significant contribution clinical outcome, we considered it noteworthy for future investigation ( Figure 4).

| Neurological outcomes
Certain amino acid substitutions were associated with positive clinical outcomes (nonstroke and decreased neurocognitive deficits) or negative clinical outcomes (stroke and increased neurocognitive deficits). The Vpr amino acid substitutions 22L, 37I, 41G, 41S, and 77R were associated with positive clinical outcomes and were more prevalent in nonstroke participants 81 and lower neurocognitive deficits 49,52 in PLWH ( Table 1). The Vpr amino acid substitutions 21I, 41 S, 41N, 55A and 77Q were associated with negative clinical outcomes and were more prevalent with ischaemic stroke 81 and pronounced neurocognitive deficits 49,52 in PLWH ( Table 1). The Vpr amino acid substitution 41S was investigated by two or more independent studies, therefore, meeting our first criteria as a noteworthy finding ( Figure 4B). The findings for 41S were mixed as one of the two studies linked it to improved neurological outcomes, whereas the second one linked it to ischaemic stroke. Therefore, none of the Vpr amino acid substitutions investigated met our criteria as noteworthy for neurological outcomes (Table 1 and Figure 4B).

| Treatment status
Of all amino acid substitutions investigated, several were associated with treatment status by higher prevalence in PLWH that were using ART ( 85P Stable CD4 count [76] No contribution to LTNP [80] No contribution to disease progression [65] 86G Stable CD4 count [76] 86R Associated with LTNP [70] 87P No contribution to disease progression [65] 89G Stable CD4 count [76] 89T Associated with LTNP [70] T A B L E 1 (Continued)  with positive clinical outcomes and were more prevalent in treated PLWH 63,82 ( Table 1). The findings for these amino acid substitutions were only reported by one independent study; therefore, none of the Vpr amino acid substitutions investigated met our criteria as noteworthy for treatment status (Table 1 and Figure 4C).

| Potential confounders: Viral
load/CD4 + count and HIV-1 subtype Several findings may influence the reported association of Vpr amino acid substitutions and clinical outcomes.
In particular, viral load and CD4 + count are well-known determinants of clinical outcomes in PLWH. 83 Therefore, we wanted to determine the potential influence of these confounders on the reported associations between Vpr amino acid substitutions and clinical outcomes in the reviewed studies. Therefore, studies were stratified according to the mean/median viral suppression (<400 cps/ml) or nonviral suppression (>400 cps/ml) (Table S2). Studies reported viral load (VL) as a group mean (SD), median [IQR], or percentage distribution. For studies only reporting percentage distribution for virally suppressed participants, we considered viral suppression when more than 50% of participants were virally suppressed (i.e. suppression (<400 cps/ml) within the respective study). We do acknowledge that a fraction of the overall population may have been nonvirally suppressed and therefore, findings reported for these studies should be interpreted with caution.
For longitudinal studies that have reported multiple measurements of viral load, the last recordings of viral load were used for stratification. If treatment-experienced participants were virally suppressed at baseline, we considered follow-up as virally suppressed due to continued use of ART. For studies that reported nonviral suppression at baseline, we then considered follow-up viral loads where available.
Of all studies reviewed (n = 22), n = 9 studies had not provided sufficient information to stratify participant groups according to viral suppression. 49,65,67,70,[76][77][78]80,84 Of the remaining 13 studies, the majority (n = 12) investigated cohorts with a group of nonviral suppression (>400 cps/ml), and approximately 38.46% of these studies investigated Vpr amino acid substitutions that had an association with negative clinical outcomes in PLWH (Table S2). One study had participants with a group viral suppression of <400 cps/ml, and this study reported an association between Vpr amino acid substitutions and negative clinical outcomes in PLWH. It is also relevant to note that n = 3 studies were case studies and the findings reported were from three or fewer participants and these findings may not be representative of larger populations. 63

T A B L E 1 (Continued)
F I G U R E 4 (A) Frequency an amino acid substitution has been investigated in relation to disease progression/nonprogression. Blue, Orange and grey bars indicate positive, negative and no contribution to clinical outcomes, respectively. Criteria 1 (red line cut-off) was ≥2 independent studies that had to investigate a particular substitution. The percentage indicates the number of studies that reported the same clinical outcome for a particular substitution in relation to the number of studies that investigated the substitution. Criteria 2 was that the percentage needed to be >50% to be considered a noteworthy finding. Vpr substitutions 63T, 77H and 85P were considered noteworthy. (B) Frequency an amino acid substitution has been investigated in relation to neurological outcomes. Blue, Orange and grey bars indicate positive, negative and no contribution to clinical outcomes, respectively. Criteria 1 (red line cut-off) was ≥2 independent studies that had to investigate a particular substitution. The percentage indicates the number of studies that reported the same clinical outcome for a particular substitution in relation to the number of studies that investigated the substitution. Criteria 2 was that the percentage needed to be >50% to be considered a noteworthy finding. Vpr substitution 41S met criteria one, however, no substitutions were considered noteworthy. (C) Frequency an amino acid substitution has been investigated in relation to treatment status. Blue, Orange and grey bars indicate positive, negative and no contribution to clinical outcomes, respectively. Criteria 1 (red line cut-off) was >2 independent studies that had to investigate a particular substitution. Criteria 2 was that the percentage needed to be >50% to be considered a noteworthy finding. None of the substitutions met criteria one or two and therefore none were considered noteworthy. an association between Vpr amino acid substitutions and negative clinical outcomes in PLWH. For the current CD4 + count, not all studies used the same measure of CD4 + count. Therefore, the studies within this review were stratified into one of two categories depending on which was provided by the studies; group mean/median CD4 + count of <200 cells/μl or >200 cells/μl. For longitudinal studies with multiple measures of CD4 + count, the CD4 + count used for stratification of studies was the time point when the correlation between Vpr amino acid substitutions and clinical outcome was done. When study participants are stratified according to CD4 + count, most studies included participants with a higher CD4 + count (>200 cells/μl), and 26.67% of these studies reported an association between Vpr amino acid substitutions and negative clinical outcomes in PLWH. Certain studies included participants with defined LTNP or non-LTNP status 64,67,77 and therefore for these studies, we also considered the LTNP status when stratifying according to CD4 + count (Table S3). Of all studies reviewed, n = 4 studies had not provided sufficient information to stratify participant groups according to the current CD4 + count. 49,76,80,84 Of the remaining 18 studies, n = 3 studies could be stratified according to LTNP status. For these studies, LTNP status was linked with CD4 + counts of >200 cells/μl and in studies that had LTNP, Vpr amino acid substitutions were not associated with negative clinical outcomes. 64,67,77 In studies with participants with progressive HIV, the findings for the association of Vpr amino acid substitutions with negative clinical outcomes were mixed. 64,67,77 Of the remaining n = 15 studies, n = 12 studies investigated cohorts with a mean/median >200 cells/μl, and approximately 33% of these studies investigated Vpr amino acid substitutions with an association with negative clinical outcomes in PLWH (Table S3). Three studies had participants with a group mean/median of <200 cells/μl, and two studies (66%) reported significant associations between Vpr amino acid substitutions and negative clinical outcomes in PLWH. From the studies reporting findings for CD4 + count, certain studies were from case studies and/or single-participant data. [63][64][65][66][67] Therefore, findings from these studies may not necessarily be representative of larger cohorts.
Most of the studies (64%) reported the HIV-1 subtype of which subtype B was most common (71%), followed by subtype C (14%), subtype A (7%), and circulating recombinant form (CRF)01-AE (7%). For the remaining studies that did not report the HIV-1 subtype, 18% of the total studies reported the geographical region, with one study each conducted in the USA, Canada, Portugal and a combination of the USA and Australia, respectively ( Table 2). The remaining studies (17%) did not report the subtype or geographical region. Considering the findings for studies that reported an association of Vpr amino acids with negative clinical outcomes (n = 8) (Table 1) and studies with a confirmed HIV-1 subtype, (Table 2), it was noted that studies with subtypes B and C equally reported findings of Vpr amino acid substitutions with negative clinical outcomes. All key findings are summarised in Table 2.

| DISCUSSION
This review summarises the key findings for the association between Vpr substitutions and clinical outcomes in PLWH. We reported the Vpr amino acid substitutions that were associated with specific clinical outcomes including disease progressions, neurological outcomes and treatment status in PLWH. Certain Vpr amino acid substitutions reported consistent associations with clinical outcomes in PLWH and were therefore considered noteworthy within this study. (1) The Vpr substitution 63T was associated with characteristics of slower disease progression, whereas 77H and 85P were associated with no significant contribution to disease progression, (2) preliminary evidence suggests that Vpr amino acid substitutions may have implications in neurological outcomes and treatment efficacy in PLWH, despite limited investigation, and (3) regardless of higher viral load and CD4 + counts, studies still report an association of Vpr amino acid substitutions with negative clinical outcomes in PLWH.
First, Vpr amino acid substitutions are associated with clinical outcomes in PLWH. Nearly, a third (31.82%) of all studies reported an association between Vpr amino acid substitutions and negative clinical outcomes in PLWH. Much of our understanding of the role of Vpr amino acid substitutions in HIV-1 pathophysiology has been derived from fundamental studies. However, these amino acid substitutions do not necessarily present the Vpr amino acid substitutions present in PLWH. To our best knowledge, this is the first study that systematically reviewed the Vpr amino acid substitutions found in PLWH and their association with clinical outcomes. More specifically, here, we provide a summary of Vpr amino acid substitutions associated with clinical outcomes, particularly, disease progressions, neurological outcomes, and treatment status.
Second, we considered the following findings as noteworthy: (1) 63T was more commonly associated with positive clinical outcomes including higher CD4 counts and lower viral load, and Vpr 77H and 85P were more commonly associated with no significant contribution to disease progression. Limited studies have reported findings for the Vpr 63T substitution in clinical or preclinical studies, and no findings for 63T have been reported in previous reviews performed on this topic. 53   3. LTNP compared to progressors, there were significant differences in the frequency of 77Q amino acid substitution.
4. In sub-optimal treated PLWH and PLWH with viral rebound after treatment failure, there were significant differences in the frequency of 77Q.
5. 77H had no significant difference in LTNP and progressors, and in sub-optimal treated PLWH and PLWH with viral rebound after treatment failure.
6. In nonprogressors, 77Q was significantly higher. 3. Blood-derived sequences: No amino acid changes distinguished HAD and ND in blood were not evident.
4. Synonymous amino acid substitutions within clinical groups: no difference within the blood-or brain-derived sequences.
5. The frequencies of non-synonymous amino acid substitutions were significantly reduced within the HAD brain-derived sequences compared with the HAD blood-derived sequences.
6. dN/dS rates: No significant difference 7. The amount of amino acid differences were significantly reduced in the HAD brain-derived sequences than in HAD blood-derived sequences.
8. The frequency of detection of Vpr transcripts were significantly higher in the brain amongst PLWH with HAD (59%) than in PLWH with ND (31%). It was detected in all blood-derived samples examined.  3. 73R and 80R were highly conservative in all PLWH. 4. 77Q amino acid substitution was present in all PBD PLWH irrespective of disease stages.
5. All the predicted binding motifs were highly conserved with slight changes seen in LTNP and AIDS patients.
6. All Vpr sequences were tightly clustered with, firstly, Chinese B and, secondly, Thailand B and intermingled together regardless of disease stage.
7. There was no difference between the asymptomatic PLWH and AIDS participants.
8. Analysis of the dS/dN ratio and potential glycosylation sites: No significant difference in the dS/dN ratio and glycosylation sites of Vpr between patients at different stages of the disease.
9. All Vpr sequences in our study had an intact ORF, with 96 amino acids regardless of disease stages.   Case studies. [63][64][65][66][67] Studies that reported sample types other than blood. 49  in this review, 63T should be investigated for its potential influence on positive clinical outcomes in PLWH. In particular, the 63T has been associated with characteristics of slower disease progression. 75,76 When comparing the Vpr sequences between subtypes (Figure 1), subtype C was the only subtype that presented with the 63T substitution. Considering the fact that subtype C has been associated with slower disease progression and replication fitness, [85][86][87][88] it is plausible to speculate that 63T may have a functional role in this outcome. However, too few studies have investigated this particular mutation to make a conclusion and a need exists to further investigate on both clinical and fundamental levels to elucidate the role of 63T in HIV-1 pathogenesis. Furthermore, Vpr 77H was more commonly associated with no significant contribution to disease progression. 66,73 In general, 77H is a less investigated substitution in contrast to Vpr 77Q. It is relevant to note that the Vpr 77Q substitution was also the most widely investigated amino acid substitution across all studies. However, as reported here, there is no clear consensus on whether 77Q influences disease progression or nonprogression in PLWH as the findings were mixed. Therefore, as reported here, 77H may have no influence on disease progression; however, further investigation is needed to ascertain the findings summarised in this review. The findings for 85P were also associated with no significant contribution to disease progression. However, fundamental studies have associated 85P with characteristics of LTNP 89 and with a reduction in the apoptosisinducing ability. 90 This provides some evidence that the 85P amino acid substitution may be an indication of slower disease progression and be investigated as a marker thereof. However, these findings for 85P were reported in fundamental investigations and similar findings may not be reflected in clinical studies. Third, studies investigating the association between Vpr amino acid substitutions and neurological outcomes in PLWH remain limited and therefore no conclusions could be drawn from these studies. However, preliminary studies reviewed here suggest that Vpr amino acid substitutions may influence neurological outcomes. Here, we included two studies that reported findings for Vpr amino acid substitutions and neurological outcomes. 52,81 Although the amino acid substitutions investigated within this study were not deemed noteworthy, it does provide some evidence of an association between Vpr and neurological outcomes (ischaemic stroke, non-ischaemic stroke, neuroprotection and non-neuroprotection). In general, limited studies have investigated the neurological implications of Vpr amino acid substitutions in PLWH; however, the findings here have highlighted the potential role of amino acid substitutions 22I, 22L, 37I, 41N, 41S, 41G, 55A, 77Q and 77R in neurological outcomes in PLWH. A recent study (in press) has also highlighted that the presence of signature 45Y in subtype C infection was associated with a 3.66 times higher global deficit score (GDS), 525 times higher plasma viral load, 15.84 times higher proviral load and 60% lower absolute CD4-T cell count compared those without the signature. 91 This highlights the potential value of investigating the implication of Vpr amino acid substitutions in the development of neurocognitive impairments.
Fourth, studies investigating the association between Vpr amino acid substitutions and treatment status in PLWH remain limited and therefore no conclusions could be drawn from these studies. However, preliminary studies reviewed here suggest that Vpr amino acid substitutions may influence treatment efficacy. Here, we included two studies that reported findings for Vpr amino acid substitutions and treatment efficacy. Even though studies investigating this remain limited for Vpr, previous studies of other viral proteins (e.g. gp120 and group specific antigen; gag) have shown that sequence substitutions L116P, A204D, S375H/M/T, M426L, M434I and M475I in gp120 have shown to affect the efficacy of treatment, fostemsavir. 92 Despite S375T being the most predominant, it had a less substantial role in the reduction in fostemsavir efficacy than other sequence substitutions. 92 For gag, cleavage site amino acid substitutions A431V and I437V, found in the NC-SP2-p6 region, contributed to protease inhibition resistance, 93 whereas adding N374S, E428 and A431V to major resistance-associated protease substitutions results in increased phenotypic resistance to RTV. 94 Therefore, if Vpr amino acid substitutions follow a similar trend as reported for other HIV-1 viral proteins, we hypothesise that Vpr amino acid substitutions may also influence treatment efficacy and should be investigated as potential indicators thereof.
Last, certain studies have also shown that despite higher CD4 + count and lower viral loads, Vpr amino acid substitutions may still influence clinical outcomes and therefore these findings are relevant considering the modern ART treatment era.

| LIMITATIONS
The studies included in this review presented several limitations which we would briefly like to discuss. The studies presented in this review were diverse and heterogeneous. The heterogeneity of studies can be explained as follow: studies employed different design (longitudinal and cross-sectional) and different measures of clinical outcomes (e.g. neurological and disease progression). Therefore, a meta-analysis could not be conducted. This is particularly related to key cohort information, including the clear description of inclusion/exclusion definition as well as the setting or subtype of the included participant. Furthermore, studies do not clearly define potential confounding factors nor relevant statistical analysis to control for these variances. Subtype variation is associated with specific amino acid substitutions within the HIV-1 viral proteins, including Vpr, therefore the clear reporting of this may assist in contextualising the findings. Limited studies have reported viral load and CD4 + count and both factors have clear influences on clinical outcomes in PLWH 83 and this should be clearly defined in studies investigating subtype-specific protein variation and clinical outcomes. Therefore, it is not clear how these findings will reflect in the modern ART era. Sample sizes/cohort sizes varied significantly across studies. This potentially had an influence on the level and/or associations of genetic diversity observed. Case studies or studies including small cohort size present genetic variation that may not necessarily be representative of larger cohorts. Therefore, the association of genetic variation with clinical outcomes from such studies should be interpreted with caution.
It should also be noted that most of the studies included in this review were more than 10 years old and consideration should be taken when interpreting the reported findings. Considering the significant development of HIV-1 research and genome sequencing over the past 10 years, this highlights the need for newer studies related to this topic. This will aid in the further understanding of the correlation between Vpr amino acid substitutions and clinical outcomes in PLWH.

| CONCLUSIONS
Studies have collectively reported that Vpr amino acid substitutions are most likely associated with clinical outcomes including disease progressions, neurological outcomes and treatment status. The Vpr amino acid substitutions, 63T were more commonly associated with positive clinical outcomes, and 77H and 85P were associated with no contribution to disease progression. Preliminary evidence suggests that Vpr amino acid substitutions may have implications for neurological outcomes and treatment efficacy in PLWH. The Vpr amino acid substitutions highlighted in this review should be investigated further to determine their potential role as predictive indicators of disease progression or anchor points to better understand their implications in HIV-1 pathophysiology.