HIV-1 infection is characterized by progressive depletion of CD4+ T lymphocytes and immune deficiency. The number of circulating CD4+ T lymphocytes is inversely correlated with the level of viral replication, and persistent high viral loads in plasma predict a rapid disease onset . On the other hand, patients with low or undetectable plasma viraemia exhibit a non-progressive infection .
The progression time from infection with HIV-1 to the development of AIDS is known to be extremely variable. Moreover, a small proportion of HIV-1-infected persons, called long-term non-progressors (LTNP), remain AIDS-free and clinically healthy with relatively normal immunologic function many years after HIV-1 infection .
CD4+ T-cell depletion is mediated by both direct cytopathic viral effects and by apoptosis of uninfected cells [3–6].
Several HIV-1 proteins interact with the cellular regulatory pathways and have a direct impact on the survival of both infected and uninfected cells. Apoptotic and anti-apoptotic effects in vitro have been described for viral proteins such as Env, Tat, Nef and Vpr .
Several factors have been implicated in the lack of disease progression, including genetic, immunologic and virologic characteristics. There is evidence that viral strains in some LTNP may be attenuated and also accessory genes are defective in certain LTNP [8,9].
HIV-1 viral protein R (Vpr) is a 96-amino-acid accessory protein that is expressed late during viral replication . Vpr has pleiotropic effects on viral replication and cellular proliferation, differentiation, cytokine production, nuclear factor κB-mediated transcription and apoptosis [11–14]. During the early phase of the HIV-1 life cycle, Vpr facilitates the nuclear transport of the HIV-1 pre-integration complex across the limiting nuclear pore [15–18]. Moreover, Vpr is able to prevent host cell proliferation by arresting cell division in the G2 phase of the cell cycle [19–23]. This arrest has been shown to increase viral expression in dividing T cells as well as in macrophages [21,24,25].
Vpr is abundant in virions [11,26,27], is detectable in the serum of HIV-1- infected patients, and correlates with the viral load , in addition it is found in the nuclear and membrane fractions  and it can cross the plasma membrane [30–32], and finally it can localize to mitochondria to kill cells by apoptosis [30,32–35].
Lum and colleagues demonstrated that 80% of LTNP present a point mutation in Vpr at position 77 that leads to the substitution of arginine (R) with glutamine (Q); this mutation is far less frequent among patients with progressive disease .
A recent report analysed proviral DNA extracted from peripheral blood mononuclear cells (PBMC) and showed that the Vpr R77Q mutation is equally present in LTNP (four of 11) and in a group of slow-progressors (three of seven) .
The aim of this study was to analyse the prevalence of the R77Q mutation in four groups of HIV-1-infected patients: LTNP; sub-optimal treated patients (STP); patients who were naive to antiretroviral treatment with progressive disease (Pr) and patients with viral rebound after treatment failure (MEP).
Patients and methods
We randomly selected and examined 19 antiretroviral therapy (ART)-naive HIV-1- infected individuals with progressive disease (Pr, progressors), 15 LTNP defined as patients with an infection dating more than 10 years, a CD4+ T-cell count nadir higher than 500/μl and a low or undetectable viral load, 19 patients with detectable viral load failing antiretroviral therapy (MEP) and finally 23 HIV-1+ sub-optimal-treated patients (STP) defined as patients who had a CD4 cell count greater than 300/μl and HIV-RNA < 10 000 copies/ml at time of blood sampling. STP patients had been on dual therapy for a long period of time for personal or compliance reasons or for liver related toxicity. Written informed consent was obtained from all the patients before blood drawing and laboratory analysis.
Viral RNA isolation and reverse transcription
HIV-1 RNA was extracted from 0.5–1.0 ml of blood plasma using the Nuclisens Extraction Kit (BioMérieux bv, Boxtel, The Netherlands). One-quarter of the isolated viral RNA was reverse transcribed using AMV reverse transcriptase (Finnzymes Oy, Espoo, Finland), VprI R primer (5′-ATGTAATGCAACCT-3′) for the Vpr gene and P8 primer (5′-TAAATCTGACTTGCCCAATTCAATTTT-3′) for the Pol gene. The reaction was first activated at 70°C for 10 min, then incubated at 42°C for 60 min and heat inactivated for 5 min at 95°C.
The Pol gene was amplified with a nested polymerase chain reaction (PCR): in the first step we used 10 μl of cDNA product with P7 (5′-AGACCAGAGCCAACAGCCCCA-3′) and P8 primers. The reaction conditions were one cycle at 95°C for 9 min; 35 cycles at 94°C for 30 s, at 62°C for 1 min and at 72°C for 2 min; and an extension cycle at 72°C for 6 min. Twenty microlitres of the first step product (1218 bp) were added to the second step mix to amplify two distinct Pol regions codifying HIV-1 reverse transcriptase. The first region was amplified using primers no. 3 (5′-TGTAAAACGACGGCCAGTGTATTAGTAGGACCTACACCT-3′) and SEQ1 (5′-CAGGAAACAGCTATGACCGCACGATATCTAATCCTGGTGTCTCA-3′), while the second region was amplified using primers PIA3 (5′-TGTAAAACGACGGCCAGTATTTTTCAGTTCCCTTAG-3′) and no. 4 (5′-CAGGAAACAGCTATGACCTCAGTCCAGCTGTCTTTTTCTGGC-3′). Reaction conditions were one cycle at 95°C for 9 min; 2 cycles at 98°C for 50 s, at 45°C for 50 s and at 72°C for 2 min; followed by 38 cycles at 94°C for 30 s, at 55°C for 50 s and at 72°C for 2 min; and an extension cycle at 72°C for 10 min. The secondary PCR products (511 bp for the first region and 418 bp for the second) were purified using the QIAquick PCR purification kit (QIAGEN, Toronto, Ontario, Canada) and directly sequenced with the PRISM Big Dye Terminator cycle sequencing kit and an ABI 3100 automatic sequencer (Applied Biosystems, Inc, Foster City, California, USA). Sequences were analysed using Sequence Navigator software (Applied Biosystems, Inc.).
The Vpr region was amplified with nested PCR: in the first step we used one-sixth of the cDNA product with VprI F (5′-GAGACTGGCATTTGGGTCA-3′) and VprI R primers. The reaction conditions were one cycle at 50°C for 30 min; one cycle at 95°C for 15 min; 30 cycles at 95°C for 1 min, at 50°C for 1 min and at 72°C for 1.5 min; and an extension cycle at 70°C for 7 min. Three microlitres of the first step product (800 bp) were added to the second step mix with primers VprII F (5′-GCAGGACATAACAAGGTAGGA-3′) and VprII R (5′-GAAGCGGAGACAGCGAC-3′). Reaction conditions were one cycle at 95°C for 15 min; 30 cycles at 95°C for 1 min, at 50°C for 1 min and at 72°C for 1.5 min; and an extension cycle at 70°C for 10 min. The secondary PCR product (547 bp) was purified using the QIAquick PCR Purification kit (QIAGEN) and cloned into the pGEM-T Easy vector (Promega, Madison, Wisconsin, USA). Blue-white screening identified recombinant clones and 10 clones per patient were sequenced as described above for Pol sequencing.
Vpr Phylogenetic analysis
Cycle-sequencing dideoxychain termination chemistry with sequence-specific primers was used to obtain an approximately 290 bp sequence on an ABI 3100 automated sequencer.
The raw nucleic acid sequences were edited, assembled to generate the consensus sequences and trimmed to equivalent length (265 bp) using the Sequence Navigator program. The 78 sequences were aligned with 90 Vpr representative sequences available in the Los Alamos database (http://hiv-web.lan1.gov) using the CLUSTAL algorithm implemented in BioEdit version 5.0.9 (http://www.mbio.ncsu.edu/BioEdit/page2.html). From two to five strains were chosen as representative of each of the nine pure subtypes (A–D, F–H, J and K), the five sub-subtypes (A1, A2, A3, and F1 and F2) and the 16 known circulating recombinant forms (CRFs).
Subtype or CRF assignment was performed using the PHYLIP software package, version 3.57 (http://evolution.genetics.washington.edu/phylip.html) . Evolutionary distances were estimated using DNADIST with the Kimura two-parameter method and a transition/transversion ratio of 2.0. Phylogenetic relationships were inferred using neighbour-joining method. Reproducibility of branching patterns was evaluated with SEQBOOT (bootstrap method; 1000 replicates), and the consensus tree was generated with CONSENSE.
Comparisons between categorical (group, sex) variables were assessed by means of chi-squared and Fisher exact tests. Continuous variables (viral load and CD4 cell count) were analysed utilizing analyses of variance (ANOVA) models; in case of non-normal distribution of variables (HIV-RNA), Kruskal–Wallis non-parametric test was performed. Multivariate relative risks for potential predictors of presenting R77Q mutation were estimated using a logistic regression model; 95% confidence intervals (CI) for each estimated risk were calculated from standard errors. All tests were two-tailed and performed at the conventional level of statistical significance of 0.05.
The general characteristics of the patients enrolled in the study are summarized in Table 1. No statistically significant difference emerged among the studied groups with respect to sex. As expected, patients in the Pr group were younger (P = 0.032 by ANOVA) and had a shorter time of infection (P < 0.001). The LTNP had higher CD4+ T-cell count (P = 0.001). Subjects in the MEP group showed the highest HIV-RNA viraemia (P = 0.001).
Patients on sub-optimal therapy were on the following antiretroviral combinations: stavudine + lamivudine, stavudine + didanosine, zidovudine + lamivudine, zidovudine + didanosine, zidovudine + zalcitabine. These patients had been on a dual NRTI therapy for at least 6 years with a median time on therapy of 8.1 years.
MEP were patients failing highly active antiretroviral therapy (HAART) with detectable viral load and receiving several different combination of drugs; these patients were analysed as a control group of STP considering the similar pattern of genotypic resistance in the Pol region.
Drug-related genotypic mutation in RT HIV-1 RNA
Due to low levels of plasma viraemia, only 14 of 23 STP could be sequenced for RT HIV-1 regions. All the subjects exhibited a certain degree of genotypic resistance to NRTI.
Eleven of 14 subjects treated with zidovudine or stavudine presented a variable number of thymidine analogue mutations (TAMs). Moreover, all sequences obtained from patients treated with lamivudine showed the M184V mutation (12 of 14). Of note, the virus from a single patient remained completely wild type in the RT region. Neither K65R mutation nor 69 insertion nor Q151M complex emerged; furthermore no evidence of mutations conferring a multi-drug resistance profile towards the NRTI was present in this group of patients.
Genotypic changes were evidenced in 18 of 19 patients who failed antiretroviral therapy (MEP). All 18 patients presented amino acid substitutions that conferred resistance to one or more compounds. Sixteen of 18 patients had been treated with lamivudine and showed the M184I/V mutation. Seven subjects presented the TAM-1 profile; namely mutations at codons 41+210+215 (a troublesome NRTI resistance feature), and five subjects featured the TAM-2 profile; namely mutations at codons 67+70+219. Of note, no patient presented both profiles at the same time and no K65R mutation occurred. Q151M was evidenced in one subject together with mutations at codons 69 and 116. Eight of 18 subjects presented various degrees of non-nucleoside reverse transcriptase inhibitor resistance, the most frequent mutations being at codons 103, 181 and 190, thus confirming the persistence of these mutations over time.
Comparison of the frequency of the R77Q mutation in LTNP, Pr, STP and MEP
The sequence analysis of Vpr showed a significantly higher prevalence of the R77Q mutation both in LTNP (86.7%) and STP (73.9%) in comparison with Pr (42.1%) and MEP (42.1%). The differences were statistically significant among the different groups (P = 0.007, by ANOVA). Direct comparison of the Vpr sequence in 19 progressors and in 15 LTNP showed that eight progressors had glutamine (Q) in position 77 (42.1%), whereas eight patients had arginine (R) (42.1%) and three patients had histidine (H) (15.8%); on the contrary, 13 LTNP had glutamine (86.7%), one LTNP had arginine (6.65%) and one LTNP had histidine (6.65%). Statistical analysis revealed significant differences in the frequency of the R77Q mutation in LTNP compared with progressors (P < 0.05), whereas there was no statistical relevance in the presence of R77H mutation in these two groups of patients (P = 0.41) (Fig. 1).
We compared the Vpr sequence of 19 progressors with 23 STP. Our analysis showed that 17 two-ART drug-treated patients had glutamine (73.9%), four had arginine (17.4%) and two had histidine (8.7%). Statistical analysis revealed significant differences in the frequency of the R77Q mutation in STP compared with progressors (P < 0.05), although there was no statistical relevance in the presence of R77H mutation in these two groups of patients (P = 0.48) (Fig. 2).
The prevalence of the mutation at position 77 in patients with a controlled infection despite sub-optimal therapy (STP) was also analysed in comparison with patients with treatment failure during HAART (MEP). In the second group glutamine was represented in eight (42.1%), arginine in 10 (52.6%) and histidine in one patient (5.3%). Statistical analysis revealed significant differences in the frequency of the R77Q mutation in STP in comparison with MEP (P < 0.05), whereas there was no statistical relevance in the presence of the R77H mutation in these two different groups of patients (P = 0.67) (Fig. 3).
No correlation was observed between particular RT mutations and Vpr 77 mutation either in STP or in MEP.
Moreover, when comparing groups of patients with progressive disease (Pr + MEP) and groups with non-progressive disease (LTNP + STP) the probability of harbouring the R77Q mutation was significantly higher in non-progressors (odds ratio, 5.16; 95% CI, 1.88–14.18; P = 0.001).
Vpr Phylogenetic analysis
Phylogenetic analysis revealed that all but five sequences clustered into subtype B with bootstrap values higher than 80%. The non-B isolates were significantly related with reference strains of sub-subtypes A3, F1 and CRF02_AG in one, two and two cases, respectively.
A unique group of HIV-1-infected individuals remain clinically healthy and do not experience a decline in CD4+ T-cell counts. A recent report has shown that a point mutation, involving the substitution from arginine to glutamine at position 77 in the Vpr gene, affects viral pathogenesis and is involved in the mechanism of long-term non-progression of HIV-1 infection. Similarly to our results, they found that 80% of LTNP had the mutation, whereas only 33% of progressors presented the R77Q .
On the contrary another study did not confirm a correlation between the frequency of the R77Q substitution and different measures of disease intensity .
We analysed the cohort of LTNP enrolled in the Resistant Host Prospective Study (rHoPeS) in Italy and our data support the relevance of the point mutation in preventing disease progression as 86.7% of LTNP bear the substitution compared with only 42.1% of progressors. Both our analysis and the study by Lum et al were performed on HIV-RNA, while the study failing to detect the relevance of the mutation in altering disease progression was done only on HIV-DNA from PBMC.
It is possible that the different results of the studies are related to the different technical approach. HIV-RNA reflects the viral strain currently proliferating and affecting CD4+ T-cell depletion, whereas integrated DNA is not the main replicating strain and represents the archived virus.
As demonstrated for antiretroviral therapy-induced mutations in the Pol gene, HIV mutations in PBMC may persist for a long time, possibly lifelong and re-emerge in the absence of selective pressure [40,41].
Different viral quasispecies variations in the Vpr gene may exist within a single person and it is possible that an epitope-specific immune response is able to maintain the replicating virus in a less cytopathic asset blocking the replication of more aggressive strains that may be archived in PBMC. Moreover other studies have related the C-terminus region of Vpr to disease progression [42,43].
We also analysed the frequency of the Vpr mutation in a peculiar group of patients that, despite dual NRTI therapy, are able to control HIV-1 replication and maintain a stable CD4+ T-cell count for a long period of time and compared them to patients failing antiretroviral therapy and showing a progressive depletion of CD4+ T cells (MEP).
STP patients display a high percentage of R77Q mutation similar to LTNP and significantly higher than progressors and MEP.
The C-terminal region of Vpr induces apoptosis by binding to the adenine nucleotide translocator (ANT) component of the mitochondrial permeability transition pore complex . Mitochondrial membrane permeabilization (MMP) is a key event of apoptotic cell death [45–51]. The MMP-inducing activity of Vpr resides in its COOH-terminal moiety (Vpr 52-96), within an α-helical motif of 12 amino acids (Vpr 71–82) containing several critical arginine (R) residues (R73, R77, R80) strongly conserved among different pathogenic HIV-1 isolates [32–34]. These residues participate in the physical interaction with the first loop of ANT exposed to the mitochondrial intermembrane space. This complex forms a composite ion channel, which dissipates the ΔΨm and thus favors MMP and subsequent apoptosis . The interaction of Vpr with ANT is abrogated by the R77Q mutation and such mutation impairs Vpr capability to induce apoptosis of T lymphocytes .
Among patients in sub-optimal therapy not only patients with viraemia < 50 copies/ml but also patients with persistent low levels of viral replication do not experience CD4+ T-cell depletion confirming a potential role for the Vpr R77Q mutation in impeding CD4+ T-cell apoptosis.
It is noteworthy that the large majority of patients with dual NRTI therapy also present the mutation M184V that has been associated to reduction of viral fitness [52–54]. Thus the co-existence with the R77Q mutation in the Vpr gene might further contribute to allow a long-term control of HIV-1 infection in STP. This is not the case in MEP, further suggesting the potential contribution of a specific immune response to Vpr.
A recent report observed a significantly higher prevalence of the R77Q mutation in subtype A than in subtype B viruses (84 versus 32%) ; our data based mainly on subtype B in all the different groups of patients confirm a similar frequency in patients with progressive disease (42.1%), but a much higher frequency in LTNP (86.7%).
The standard of care of HIV-1 treatment consists of continuous HAART, the criteria for initiation of therapy are not univocal, therefore the identification of a laboratory parameter such as the R77Q Vpr point mutation able to predict disease progression might help addressing such issues in conjunction with CD4+ T-cell count and HIV-RNA.
Clinicians are currently facing the problem of drug toxicity, lack of adherence to therapy and the requests of patients to interrupt therapy. Therefore, treatment interruptions are entering clinical practice, but clear criteria for interruption, monitoring and re-introduction of therapy are not available. Treatment interruptions expose patients to the risk of brisk CD4+ T-cell decline and disease progression, but allow reduction in drug toxicities and preservation of future treatment options. Monitoring the R77Q Vpr mutation might constitute a parameter to stop therapy and decide re-initiation of treatment; clinical trials to evaluate the clinical predictive role of such mutation are to be considered.
We are grateful to Bianca Ghisi for excellent editorial assistance, to all the patients participating in the study and to the staff at the Institute of Infectious Diseases and Tropical Medicine, ‘L. Sacco’ Hospital, who cared for the patients.
Sponsorship: This study was supported in part by E.L.V.I.S. (Evaluation of Long Term Non-Progressors Viro-Immunological Italian Studies) with the grants 40F39, 30C32 and 30D34 from the Istituto Superiore di Sanità, Italy.
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