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Published in final edited form as: Virology. 2013 Aug 28;446(0):10.1016/j.virol.2013.07.027. doi: 10.1016/j.virol.2013.07.027

Identification and molecular characterization of SIV Vpr R50G mutation associated with long term survival in SIV-infected morphine dependent and control macaques

Ivelisse Rivera 1,+, Yashira García 1,+, Mohitkumar R Gangwani 3, Richard J Noel Jr 2, Lucianette Maldonado 2, Anil Kumar 3, Vanessa Rivera-Amill 1,*
PMCID: PMC3844929  NIHMSID: NIHMS521547  PMID: 24074576

Abstract

Viral protein R (Vpr) is an accessory protein of HIV and SIV involved in the pathogenesis of viral infection. In this study, we monitored SIV evolution in the central nervous system and other organs from morphine-dependent and control animals by sequencing vpr in an attempt to understand the relationship between drug abuse, disease progression, and compartmentalization of viral evolution. Animals in the morphine group developed accelerated disease and died within twenty weeks post-infection. A unique mutation, R50G, was identified in the macaques that survived regardless of morphine exposure. Functional studies revealed that the R50G mutation exhibited altered cellular localization and decreased the expression levels of both IL-6 and IL-8. Our results, therefore, suggest that sequence changes within the SIV/17E-Fr vpr occur regardless of drug abuse but correlate with survival, and that they alter disease progression rates by affecting Vpr functions.

Keywords: HIV, SIV, vpr, viral evolution, morphine, brain, PBMC, proviral, IL-6, IL-8

Introduction

Viral protein R (Vpr) is an accessory gene of 14k-Da and 96 amino acids present in both HIV and SIV (slightly larger at 15k-Da and 101 amino acids) that is produced late in the virus replication and is also found within the viral particle. Vpr is dispensable for viral replication in CD4+ T-cells but is required for infection of primary macrophages (Eckstein et al., 2001; Rey et al., 1998). Vpr is a multifunctional protein that facilitates many processes in HIV-1 infection, such as evasion of the immune system and persistence in the host, thus contributing to the morbidity and mortality of acquired immunodeficiency syndrome (AIDS). Inside infected cells, Vpr is mainly a nuclear protein that has been demonstrated to play various roles in viral infection. Vpr supports nuclear import of the pre-integration complex (PIC) and has also been shown to participate in the transactivation of long terminal repeat –LTR (Andersen et al., 2008; Andersen and Planelles, 2005; Goh et al., 1998; Tungaturthi et al., 2004). Vpr protein induces G2 cell cycle arrest, modulates T-cell apoptosis, co-activates host genes, and regulates nuclear factor kappa B (NF-κB) activity (Ayyavoo et al., 1997; Conti et al., 2000; Li et al., 2010).

Although much effort has been placed in answering major questions related to HIV-1 quasispecies evolution, the effect that these mutations have on the response to antiretroviral treatment, and the interactions of the newly emerged viruses with the immune system remains only partially understood. In addition, few studies have focused on the effect that viral diversity of the non-structural genes (i.e. vif, vpr, vpu, tat, and rev) has on disease outcomes. Disease progression and infection in vivo is attenuated in patients with Vpr defects at the C terminus, indicating the importance of the role that Vpr plays in viral pathogenesis (Caly et al., 2008; Jacquot et al., 2009; Lum et al., 2003; Mologni et al., 2006; Noel and Kumar, 2007; Wang et al., 1996). However, other studies found an association between increased amino acid variation over time and rapid disease progression (Cali et al., 2005). More recently, a picture is emerging wherein mutations in vpr are associated with antiretroviral-experienced patients failing therapy. Viruses harboring a mutation in vpr (E17A) combined with thymidine analog mutations in the reverse transcriptase exhibit decreased susceptibility to didanosine and thus imply a novel role for Vpr in HIV-1 drug resistance (Fourati et al., 2012).

We have followed SIV virus evolution in morphine-dependent and control animals by sequencing env (Rivera-Amill et al., 2010a; Rivera-Amill et al., 2007; Rivera-Amill et al., 2009; Tirado and Kumar, 2006), tat (Noel and Kumar, 2006; Noel et al., 2006a), vpr (Noel and Kumar, 2007) and nef (Noel et al., 2006b) from cell-free virus in plasma and cerebrospinal fluid samples in an attempt to understand the relationship between drug abuse, disease progression and viral evolution. In terms of vpr evolution, our studies have shown an inverse correlation of SIV vpr evolution and disease progression (Noel and Kumar, 2007). In the current study we wanted to examine the evolution of vpr from proviral sources in brain tissue, PBMCs and several peripheral tissue compartments to determine whether there is an association between virus evolution and disease progression. We also wanted to determine whether there is a correlation between disease-progression rate and viral compartmentalization. We have computationally analyzed the structure of SIV Vpr in the context of a change detected in some of the compartments (R50G) that appears to be linked to the progression rate. These studies were complemented with functional analyses of the SIV Vpr R50G in the context of inflammatory cytokine expression.

Results

A unique change, R50G, in Vpr is associated with survival

Initial studies using the SIV/SHIV macaque model revealed a remarkably consistent pattern of inverse correlation between disease progression and SIV vpr evolution in circulating virus from both plasma and cerebrospinal fluid (CSF) (Noel and Kumar, 2007). In the current study, we analyzed proviral vpr gene variants from brain tissue and peripheral blood mononuclear cells (PBMCs) from morphine-dependent rapid progressors, morphine-dependent normal progressors and control macaques. Phylogenetic analyses using SIV vpr sequences derived from brain DNA and PBMC DNA were carried out to determine the relatedness of variants between these compartments in morphine-dependent and control macaques. Five to ten clones from each macaque were sequenced, aligned and subjected to phylogenetic analysis using the distance-based (neighbor-joining) method. As previously reported for vpr clones derived from plasma and CSF, the resulting phylogenetic trees of proviral brain- and PBMC-derived clones show that vpr evolution and the degree of compartmentalization are inversely correlated with disease progression (data not shown) (Noel and Kumar, 2007). In the brain, morphine-dependent rapid progressors, had roughly 20-25% less sequence diversity as the morphine-dependent normal progressors and approximately 10% less sequence diversity as the control animals, although not statistically significant (data not shown).

We next examined the deduced amino acid sequences for all vpr clones to identify specific changes that could lead to altered disease progression. As shown in Figure 1, a unique mutation, R50G, was consistently detected as the predominant form in brain-derived sequences of macaques that survived 20 weeks or longer post-infection. This change was not present in the inoculum, nor did it constitute a major form in the rapid progressors.

Figure 1. An R50G change was detected in macaques that survived. Panel A: The vpr coding sequences were translated to amino acids using BioEdit.

Figure 1

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SIV/17E-Fr serves as the reference (Genbank #AY033146). Each sequence represents the consensus sequence in brain and PBMC for each group of animals. A minimum of eight clones were analyzed for each macaque and compartment. Positions of identity are indicated by a dot (.). Panel B: The mean frequency of the R50G change in brain tissue was calculated as the total number of changes per total number of clones for a given animal. Statistically significant differences were assessed by ANOVA followed by a post hoc Tukey-Kramer test. Error bars indicate the mean standard error. Panel C: Correlation of the frequency of R50G mutation in brain tissue with survival time. Correlation was statistically analyzed using a Spearman correlation test.

Analyses of proviral sequences form other compartments including kidney, lung, spleen, stomach, and testes revealed that the R50G change was markedly decreased in macaques that died within 19 weeks post-infection (Figure 2). Analysis of the frequency of amino acid mutations in all compartments revealed a consistent pattern of fewer R50G mutations in the morphine-dependent rapid progressors as compared to the morphine-dependent normal progressors and control macaques, reaching significance in all compartments analyzed. These results suggest that among proviral forms, SIV vpr evolution is independent of the presence of morphine, inversely related to the disease progression rate and correlates with survival. This trend agrees with our previous study of circulating virus in plasma and CSF (Noel and Kumar, 2007).

Figure 2. Mean frequency of SIV Vpr R50G mutation in morphine dependent and control macaques.

Figure 2

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The mean frequency R50G mutation was calculated as the total number of changes per the total number of clones for a given animal. A minimum of eight clones were analyzed for each macaque and compartment. Error bars indicate the mean of standard error. Statistically significant differences were assessed by ANOVA and a post hoc Tukey-Kramer test. p < 0.01 when comparing rapid progressors to normal progrssors and control.

The R50G mutation results in structural and cellular distribution differences

The 3D models were initially built based on the amino acid sequences for wild type (WT) SIV Vpr and SIV Vpr R50G using multiple-threading alignments and assembly simulations within I-TASSER. Protein function insights were then derived by matching the predicted models with protein function database (Roy et al., 2010; Zhang, 2008). Figure 3 shows the 3D models with the highest confidence (C-score) and TM score, estimates of the quality of the predicted models, for SIV Vpr and SIV Vpr R50G as determined by I-TASSER. The C-score is calculated based on the significance of threading template alignments and the convergence parameters of the structure assembly simulations. C-score is typically in the range of [−5, 2], where a C-score of higher value signifies a model with a high confidence and vice-versa. The TM-score is the estimated accuracy of the model where a TM-score >0.5 indicates a model of correct topology and a TM-score <0.17 means a random similarity. On comparison of the C-scores and TM-scores of predicted 3D structures, both SIV Vpr WT and SIV Vpr R50G exhibit a similar 3D model (Zhang and Skolnick, 2005). In context with the secondary structure, both the SIV Vpr and SIV Vpr R50G are composed of three α-helices separated by loops of exposed residues as is observed for HIV Vpr (Morellet et al., 2003). The R50G change falls within the loop that separates α-helices two and three and is predicted to disrupt the spatial relationships of the three helices and both the N- and C-termini.

Figure 3. The three-dimensional (3D) model for SIV Vpr R50G reveals global structural differences.

Figure 3

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Panels: A, WT SIV Vpr (in blue) superimposed on SIV Vpr R50G (in red); B, WT SIV Vpr (in blue) superimposed on SIV Vpr E29GR50G (in red); C, SIV Vpr (in blue) superimposed on SIV Vpr L24Q (in red). The 3D models were generated using the iterative threading assembly refinement (I-TASSER) server. I-TASSER is an integrated platform for automated protein structure and function prediction based on the sequence-to-structure-to-function paradigm. Starting from an amino acid sequence, I-TASSER first generates three-dimensional (3D) atomic models from multiple threading alignments and iterative structural assembly simulations. The amino- and carboxy-terminals of the proteins are denoted by a yellow (N) and (C), respectively.

We next wanted to determine whether R50G mutation results in differences in SIV Vpr localization. Using the 3D models generated in I-TASSER, COFACTOR threaded the structure through three comprehensive function libraries by local and global structure matches to identify functional sites and homologies. Based on the gene ontology (GO) terms in I-TASSER server, the putative cellular localization of modeled SIV Vpr R50G differs from SIV Vpr WT (data not shown). In order to corroborate these findings V5-Vpr mammalian cell expression vectors were used to transfect U87 MG astrocytoma cells (Figure 4, panel A) to investigate Vpr's subcellular localization (Figure 4, panel B). Wild-type SIV Vpr localized to both the nucleus and the cytoplasm of transfected cells, whereas SIV Vpr R50G was localized mainly in the nucleus. To determine whether this difference in cellular distribution was specific to the R50G mutation, we also analyzed other SIV Vpr mutants. SIV Vpr L24Q had a similar distribution as WT Vpr and Vpr E29G, that also contains the R50G, localized mainly to the cell nucleus. These results corroborate that the R50G mutation alters the cellular distribution of SIV Vpr.

Figure 4. SIV Vpr R50G mutation alters the localization of V5-tagged SIV Vpr.

Figure 4

Figure 4

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U87 cells were transfected with 1 mg of plasmid DNA of the empty expression vector or expression constructs for 24 hours. Panel A: Proteins were extracted using the QIAGEN Allprep kit and 35 mg of proteins of each sample were analyzed by western blot using antibodies against the V5 tag for detection of Vpr proteins and b-actin (loading control). Lanes: 1, mock transfection; 2, empty vector; 3, wild type Vpr; 4, Vpr R50G; 5, Vpr E29G; 6, Vpr L24Q. Panel B: The cells were transfected for 24 hours and then processed for immunofluorescence using an antibody against the V5 tag. Additional controls consisted of omitting the primary antibody from the staining procedure (last panel in each row, merged no primary ab). Cell nuclei were visualized with DAPI. The results are representative of three independent experiments.

Differential regulation of pro-inflammatory factors in WT SIV vpr, R50G and mock transfected astrocytes

Astrocytes are abundant cells in the central nervous system and their physiological roles are essential for normal brain function. In the setting of HIV infection, these cells play a central role in the generation of inflammatory mediators in response to HIV-1 presence in the brain (Cota et al., 2000; Dou et al., 2006; Minagar et al., 2002; Williams et al., 2009). Viral proteins, among them Vpr, lead to the activation of transcription factors that can result in increased expression of IL-6 and IL-8 cytokines (Roux et al., 2000). Increases in the levels of these two inflammatory cytokines have also been reported in HIV-1 infected patients with neurocognitive impairment suggesting a link between cytokines levels and HIV-associated neurocognitive disorders (Airoldi et al., 2012; Yuan et al., 2013).

We next wanted to determine whether the R50G mutation results in altered expression of IL-6 and IL-8 in astrocytes. Compared to mock (empty vector control), WT SIV Vpr stimulated the expression of both IL-6 and IL-8 (Figure 5, panels A-B). Whereas SIV Vpr R50G induced mRNA levels comparable to the empty vector control. SIV Vpr L24Q exhibited levels that where either comparable or higher than WT SIV Vpr. Whereas the SIV Vpr E29G, that also carries the R50G change, partially rescued the R50G phenotype. To further examine whether the differences noted in the inflammatory cytokines at the transcriptional level were also present at the protein level, the supernatants from mock transfected, WT SIV Vpr and Vpr mutants were analyzed. Consistent with the data on the transcript levels, the presence of the R50G change resulted in a significantly reduced release of Il-6 and IL-8. Whereas the presence of the E29G in the background of the R50G change led to a partial rescue of the secretion levels of both cytokines as compared to SIV Vpr R50G (Figure 5, panels C-D).

Figure 5. SIV Vpr R50G mutation alters the expression of IL-6 and IL-8 in SVGA cells.

Figure 5

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Cells were transfected with plasmids encoding SIV Vpr WT, SIV Vpr R50G, SIV Vpr L24Q, SIV Vpr E29G and empty vector. The cells were harvested after 6 hours for RNA extraction followed by real time RT-PCR to measure the expression of IL-6 (Panel A) and IL-8 (Panel B). The cell supernatants were collected after 48 hours for the determination of protein concentration of IL-6 (Panel C) and IL-8 (Panel D) using multiple cytokine assay. Statistical significance was determined using ANOVA followed by a post-hoc Tukey-Kramer test.

Discussion

We have been studying the SIV/SHIV macaque model of drug dependency and AIDS for over ten years- Reviewed in: (Noel et al., 2008; Rivera-Amill et al., 2010b). Our studies have demonstrated the multiple effects that morphine may have on both the host and the virus, and raised the possible relationship between viral evolution and rapid disease progression. The work described herein represents the first attempt to directly link how viral evolution may affect disease progression by focusing on vpr.

Vpr has been shown to be dispensable for viral growth in various systems, (Aldrovandi and Zack, 1996; Hoch et al., 1995; Le Rouzic and Benichou, 2005). However, it has also been shown to influence the viral efficiency of replication in others. In a study by Lang et al., macaques were infected with SIVmac239 which contained a mutation in the initiation codon for vpr. The animals progressed to AIDS at a much slower rate, thus revealing that vpr is important for maximal SIV replication, persistence in vivo, and disease progression (Lang et al., 1993). Regardless, the emergence of this ‘accessory protein' which have no counterparts in other retroviral groups, strongly suggest that Vpr fulfills specific needs in the context of natural infection.

In the present study we analyzed the extent of vpr sequence variation and molecular characterization using the morphine-dependence SIV/SHIV infection macaque model. The work presented here is an extension of our previous study (Noel and Kumar, 2007) in an attempt to further understand the evolution of viral genes and their contribution to disease progression. As previously observed in plasma and CSF, evolution of vpr appears to exhibit compartmentalization as the macaques survive the infection regardless of the morphine status. This prompted us to examine more closely the vpr changes that may influence this outcome. We analyzed PBMC and various tissue samples at time of death. While in our previous study no differences were readily evident among the three groups in terms of amino acid distribution, examination of the deduced amino acid sequences consistently revealed an R50G change in the majority of the clones obtained from macaques that survived beyond 20 weeks. The R50G change maybe the result of an increased time of infection in morphine-dependent normal progressors and control macaques, as compared to the rapid progressors that died within 20 weeks of infection. However, the mutation was not detected in any of the PBMC samples, some of which were also end-point samples for normal progressor macaques (i.e., 1/56L, 2/02P, 2/AC42) arguing that vpr mutation rate differs within each compartment and is not merely the result of increased time for mutations to occur. The fact that the change is so strongly correlated to survival is also a suggestive factor that the change occurred in the surviving macaques in the same time frame that the rapid progressors died.

We evaluated whether the R50G mutation results in altered protein function or localization as compared to the WT Vpr. Our approach was to deduce the three-dimensional structure of the Vpr R50G and to compare it with WT SIV Vpr as an initial study to understand the functional characteristics of the protein and precise molecular details that could facilitate experimental characterization of the altered function, if any. I-TASSER (Iterative Threading ASSEmbly Refinement), a computational method that has been successful in accurately modeling protein structures, was used to determine whether there were any differences between WT SIV Vpr and Vpr R50G. I-TASSER predictions indicated that SIV Vpr R50G had different structure and potential functions as compared to the wild type. While both WT SIV Vpr and R50G were associated with tetrapyrrole binding activity, SIV Vpr R50G was also predicted to have intramolecular lyase activity and was not predicted to have ion channel, oxidoreductase, and heme-copper terminal oxidase activity, among others. Previous studies have demonstrated that Vpr forms ion channels in vitro via its hydrophobic segment (amino acids 55–83) penetrating phospholipid bilayers (Chen et al., 2010; Piller et al., 1996; Wecker et al., 2002) and thus it is easy to assume, as has been demonstrated, that Vpr exerts an effect at the level of nuclear and cell membranes (de Noronha et al., 2001). Changes in Vpr that alter any of the Vpr-specific activities would have a negative impact on viral survival and thus could potentially slow down disease progression. Whether the R50G mutation results altered ion channel activity or localization as predicted, remain to be elucidated. Given the structural observations and potential functional disparity, we analyzed the functional capabilities of SIV Vpr R50G to better understand the intricate correlation between SIV vpr mutation and long-term survival.

Induction of an inflammatory response in the central nervous system is considered to be a key insult in HIV-related neurodegenerative disease (Gonzalez-Scarano and Martin-Garcia, 2005). Analysis of astrocyte cytokine expression revealed that that R50G mutation led to decreased levels of IL-6 and IL-8 mRNA and protein as compared to WT SIV Vpr. These cytokines have been shown to be increased in HAND patients despite HAART treatment suggesting a specific role as mediators of neuroinflammation during chronic HIV inflammation (Airoldi et al., 2012; Yuan et al., 2013). The presence of the Vpr R50G mutation coupled with a decreased production of inflammatory cytokines by astrocytes partially accounts for the long term survival of the macaques harboring the change in Vpr.

In conclusion, these results suggest that the genetic variation observed in the brain sequences from morphine-dependent normal progressors and control macaques was a consequence of the difference in length of survival rather than a result of a higher rate of accumulation of mutations and perhaps morphine does not alter selection. Functional studies comparing WT SIV Vpr to Vpr R50G revealed structure and localization differences that translated to an altered production of two key cytokines, IL-6 and IL-8. Further studies are needed to define the role of vpr and other viral genes in the development of rapid disease progression and neuroAIDS in the setting of drug abuse.

Materials and Methods

Animal Model

This study used a previously described rhesus macaque model of drug abuse and AIDS (Kumar et al., 2004). The clinical disease parameters of the six morphine-dependent and three control macaques used in this study have been published previously (Kumar et al., 2006; Rivera-Amill et al., 2010a). Briefly, half of the morphine-dependent macaques developed rapid disease progression (1/04L, 1/42N and 1/28Q) as compared to infected control macaques. The rapid progressors maintained high viral loads both in plasma and CSF, and low CD4+ T cell counts. In contrast, morphine-dependent normal progressors and control macaques exhibited lower viral loads, and higher CD4+ T cell counts. All macaques were infected with a mixture of SlV/17-EFr, SHIV ku-1b and SHIV89.6P (Kumar et al., 2006).

Tissue collection and processing

Tissue specimens were obtained from our archived collection of rhesus macaque tissues at Ponce School of Medicine. DNA from tissues was isolated using the QIAmp DNA Blood and Tissue Kit (Qiagen, Inc., Valencia, CA) according to the manufacture's recommendations.

Amplification, cloning and sequencing of SIV vpr

Genomic DNA from brain, lung, kidney, PBMC, stomach, spleen, and testes was extracted using QIAmp DNA Mini kit (Qiagen, Inc., Valencia, CA). Primers (specific for SIV 17E-Fr) and PCR conditions were as previously described (Noel and Kumar, 2007). The amplified PCR product was confirmed by agarose gel electrophoresis prior to cloning into pCR2.1 (Invitrogen, Carslbad, CA). Positive clones were identified by colony screening with restriction enzyme digest (EcoRI, Promega) and agarose gel electrophoresis. Six to 10 clones of each sample were sent for sequencing to MCLAB (South San Francisco, CA).

Sequence analysis and statistics

All sequence files were first manually verified and edited as necessary using the software ChromasLite 2.0 (Technelysium Pty Ltd, Australia). Edited sequences were aligned using BioEdit version 7.0.5.2 (Hall TA,) and Clustal W (Thompson et al., 1994). The Clustal W program (runs within BioEdit) was set to perform multiple sequence alignments using the default penalties. Sequences were then subjected to phylogenetic analysis, including tree construction, divergence and diversity calculations using MEGA5 (Tamura et al., 2011). Finally, sequences were translated to amino acids using BioEdit. All phylogenetic analyses used the SIV/17E-Fr inoculum sequence as a reference. Statistical comparisons were done using single factor analysis of variance (ANOVA) followed by the post hoc Tukey test. Values of p < 0.05 were considered to be statistically significant.

Protein Structure and Function Prediction

Protein structure and function were predicted using I-TASSER server after translating the nucleotide sequence into amino acid. 3D models were built based on multiple-threading alignments and I-TASSER assembly simulations; function insights were then derived by matching the predicted models with protein function database (Roy et al., 2010; Zhang, 2008). I-TASSER server predicted functions of modeled proteins based on global and local similarity to template proteins in Protein Data Bank (PDB) with known structure and functions. The resultant global and local similarity scores were used to rank the template proteins (functional homologs) and transfer the annotation (EC numbers and gene ontology terms) based on the top scoring hits.

Cell Transfections

U87 MG (human astrocytoma) cells were obtained through the AIDS Research and Reference Reagent Program, Division, NIAID, NIH from Dr. Bruce Chesebro (Ponten and Macintyre, 1968). Cells were maintained in Eagle's minimal essential medium (EMEM, BioWhittaker) supplemented with Earl's balanced salt solution, L-glutamine and 10% fetal bovine serum (FBS, HyClone) at 37 °C in a 5% CO2 environment. For immunoblot and immunofluorescence analyses, 2 × 105 cells were seeded per well in a 6-well plate or collagen type I cell ware 4-well culture slide (BD BioCoat), respectively. Cells were transiently transfected with 1 μg of plasmid using Effectene transfection reagent as recommended by the manufacturer (QIAGEN). After 24 hours, cells were processed for immunoblot and immunofluorescence analyses as described below.

SVGA (clone of a human fetal astrocyte cell line) cells were maintained in Dulbecco's modified Eagle medium (DMEM, Cellgro) supplemented with 10% FBS, 1% L-glutamine, 1% Nonessential amino acids, 1% sodium bicarbonate, and gentamycin (50 μg/ml) at 37 °C in 5% CO2 environment. SVGA cells were transiently transfected with the lipofectamine as per manufacturer's protocol and as published previously (Shah and Kumar, 2010; Shah et al., 2011a; Shah et al., 2011b). Briefly, the 0.275 × 106 cells in each well of 12 well plates were incubated with 0.5 μg of plasmid and 2 μl of lipofectamine in 500 μl serum-free medium for 5 hours after which cell were allowed to grow in complete DMEM for 6 and 48 hours for cytokine/chemokine RNA and protein, respectively.

Immunoblot and Immunofluorescence analyses

For immunoblot, extracted proteins were analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentration was determined via direct measurement using NanoDrop 2000 (Thermo Fisher). Thirty five μg of proteins were diluted 1:1 with Laemmli sample buffer, boiled for 5 minutes and then loaded onto a 12.0% SDS-PAGE minigel. Proteins were transferred onto a Polyvinylidene difluoride (PVDF) membrane and blocked overnight in 5% non-fat milk, 0.05% Tween-20 in 1× PBS. Immunoblot analyses were performed with a 1:3500 dilution of the anti-V5 antibody (Invitrogen, Life Technologies) and 1:5000 dilution of the anti- -actin antibody (Santa Cruz Biotechnology, Inc.). Bound antibodies were detected using 1:5000 dilution of peroxidase-conjugated rabbit anti-mouse (Sigma-Aldrich) and Amersham ECL western blotting detection reagents (GE Healthcare) coupled with autoradiography film (Kodak X-OMAT LS).

For immunofluorescence, cells were rinsed with 1× phosphate buffer saline (PBS), fixed with 2% paraformaldehyde (PFA) and then treated with 100% methanol. Cells were then washed with 1× PBS and permeabilized with 0.1% Triton X-100/PBS. The chambers were blocked with 1%BSA/PBS for 30 minutes. Primary (anti-V5) and secondary (Aleaxa fluor 488 goat-anti-mouse, Invitrogen) antibodies were prepared in blocking solution and exposed to cells for 1 hour each at room temperature. Washes were performed with 1× PBS. Cell nuclei were stained with 4',6-diamino-2-phenylindole, dihydrochloride (DAPI) for 10 minutes at room temperature. After removing the chamber walls and excess PBS, 1 drop of mounting medium was added and a coverslip was placed on top. Samples were visualized using an Olympus BX60/ X-CITE series 120Q microscope and images were acquired and processed using Nikon NIS elements imaging software.

Real time RT-PCR

Total RNA was extracted from the SVGA cells using RNeasy mini kits (QIAGEN, Valencia, CA) and 150ng of mRNA was amplified using Bio-Rad iCycler. The primer sequences were commercially synthesized. Primers sequence for IL-6, Il-8 and RANTES and PCR conditions have been previously published (Shah and Kumar, 2010; Shah et al., 2011a; Shah et al., 2011b). HPRT (hypoxanthine-guanine phosphoribosyl transferase) was used as house keeping control and 2-ΔΔCt method was used to calculate the expression levels of cytokine/chemokines. Results were expressed as mean ± SEM for three experiments. The data were analyzed using ANOVA followed by a post-hoc Tukey-Kramer test.

Multiple cytokine assays

The cell supernatants were centrifuged twice at 3000rpm to remove the cell debris and stored at -80°C. The concentration of extracellular cytokines in the supernatants was measured by Bio-Plex multiple cytokine assay kits (Bio-Rad, Hercules, CA) using manufacturer's protocol and previously published method (Shah and Kumar, 2010; Shah et al., 2011a; Shah et al., 2011b). The protein concentration was determined based on 5PL standard curve using Bio-Plex manager 5.0 software. Results were expressed as mean ± SEM of three experiments. The data were analyzed using ANOVA followed by a post-hoc Tukey-Kramer test.

Sequence Data

Sequences in this report are available from GenBank with accession numbers from KC213324 to KC213473.

Highlights.

  • We examined SIV vpr evolution from control and morphine-dependent macaques

  • We detected a change (R50G) in Vpr that was present in macaques that survived

  • The R50G change in Vpr results in altered protein structure, localization and function

Acknowledgments

This work was supported by NCRR-RCMI (RR003050)/NIMHD (G12-MD007579) and the National Institute on Drug Abuse (DA015013). We also acknowledge the support of RCMI Molecular Biology Core and Publications Office.

Footnotes

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References

  1. Airoldi M, Bandera A, Trabattoni D, Tagliabue B, Arosio B, Soria A, Rainone V, Lapadula G, Annoni G, Clerici M, Gori A. Neurocognitive impairment in HIV-infected naive patients with advanced disease: the role of virus and intrathecal immune activation. Clinical & developmental immunology. 2012;2012:467154. doi: 10.1155/2012/467154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aldrovandi GM, Zack JA. Replication and pathogenicity of human immunodeficiency virus type 1 accessory gene mutants in SCID-hu mice. Journal of virology. 1996;70:1505–1511. doi: 10.1128/jvi.70.3.1505-1511.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Andersen JL, Le Rouzic E, Planelles V. HIV-1 Vpr: mechanisms of G2 arrest and apoptosis. Experimental and molecular pathology. 2008;85:2–10. doi: 10.1016/j.yexmp.2008.03.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Andersen JL, Planelles V. The role of Vpr in HIV-1 pathogenesis. Current HIV research. 2005;3:43–51. doi: 10.2174/1570162052772988. [DOI] [PubMed] [Google Scholar]
  5. Ayyavoo V, Mahboubi A, Mahalingam S, Ramalingam R, Kudchodkar S, Williams WV, Green DR, Weiner DB. HIV-1 Vpr suppresses immune activation and apoptosis through regulation of nuclear factor kappa B. Nature medicine. 1997;3:1117–1123. doi: 10.1038/nm1097-1117. [DOI] [PubMed] [Google Scholar]
  6. Cali L, Wang B, Mikhail M, Gill MJ, Beckthold B, Salemi M, Jans DA, Piller SC, Saksena NK. Evidence for host-driven selection of the HIV type 1 vpr gene in vivo during HIV disease progression in a transfusion-acquired cohort. AIDS research and human retroviruses. 2005;21:728–733. doi: 10.1089/aid.2005.21.728. [DOI] [PubMed] [Google Scholar]
  7. Caly L, Saksena NK, Piller SC, Jans DA. Impaired nuclear import and viral incorporation of Vpr derived from a HIV long-term non-progressor. Retrovirology. 2008;5:67. doi: 10.1186/1742-4690-5-67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen CP, Kremer C, Henklein P, Schubert U, Fink RH, Fischer WB. Modulating the activity of the channel-forming segment of Vpr protein from HIV-1. European biophysics journal : EBJ. 2010;39:1089–1095. doi: 10.1007/s00249-009-0518-x. [DOI] [PubMed] [Google Scholar]
  9. Conti L, Matarrese P, Varano B, Gauzzi MC, Sato A, Malorni W, Belardelli F, Gessani S. Dual role of the HIV-1 vpr protein in the modulation of the apoptotic response of T cells. Journal of immunology. 2000;165:3293–3300. doi: 10.4049/jimmunol.165.6.3293. [DOI] [PubMed] [Google Scholar]
  10. Cota M, Kleinschmidt A, Ceccherini-Silberstein F, Aloisi F, Mengozzi M, Mantovani A, Brack-Werner R, Poli G. Upregulated expression of interleukin-8, RANTES and chemokine receptors in human astrocytic cells infected with HIV-1. Journal of neurovirology. 2000;6:75–83. doi: 10.3109/13550280009006384. [DOI] [PubMed] [Google Scholar]
  11. de Noronha CM, Sherman MP, Lin HW, Cavrois MV, Moir RD, Goldman RD, Greene WC. Dynamic disruptions in nuclear envelope architecture and integrity induced by HIV-1 Vpr. Science. 2001;294:1105–1108. doi: 10.1126/science.1063957. [DOI] [PubMed] [Google Scholar]
  12. Dou H, Morehead J, Bradley J, Gorantla S, Ellison B, Kingsley J, Smith LM, Chao W, Bentsman G, Volsky DJ, Gendelman HE. Neuropathologic and neuroinflammatory activities of HIV-1-infected human astrocytes in murine brain. Glia. 2006;54:81–93. doi: 10.1002/glia.20358. [DOI] [PubMed] [Google Scholar]
  13. Eckstein DA, Sherman MP, Penn ML, Chin PS, De Noronha CM, Greene WC, Goldsmith MA. HIV-1 Vpr enhances viral burden by facilitating infection of tissue macrophages but not nondividing CD4+ T cells. The Journal of experimental medicine. 2001;194:1407–1419. doi: 10.1084/jem.194.10.1407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fourati S, Malet I, Guenzel CA, Soulie C, Maidou-Peindara P, Morand-Joubert L, Wirden M, Sayon S, Peytavin G, Simon A, Katlama C, Benichou S, Calvez V, Marcelin AG. E17A mutation in HIV-1 Vpr confers resistance to didanosine in association with thymidine analog mutations. Antiviral research. 2012;93:167–174. doi: 10.1016/j.antiviral.2011.11.008. [DOI] [PubMed] [Google Scholar]
  15. Goh WC, Rogel ME, Kinsey CM, Michael SF, Fultz PN, Nowak MA, Hahn BH, Emerman M. HIV-1 Vpr increases viral expression by manipulation of the cell cycle: a mechanism for selection of Vpr in vivo. Nature medicine. 1998;4:65–71. doi: 10.1038/nm0198-065. [DOI] [PubMed] [Google Scholar]
  16. Gonzalez-Scarano F, Martin-Garcia J. The neuropathogenesis of AIDS. Nature reviews. Immunology. 2005;5:69–81. doi: 10.1038/nri1527. [DOI] [PubMed] [Google Scholar]
  17. Hoch J, Lang SM, Weeger M, Stahl-Hennig C, Coulibaly C, Dittmer U, Hunsmann G, Fuchs D, Muller J, Sopper S, et al. vpr deletion mutant of simian immunodeficiency virus induces AIDS in rhesus monkeys. Journal of virology. 1995;69:4807–4813. doi: 10.1128/jvi.69.8.4807-4813.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jacquot G, Le Rouzic E, Maidou-Peindara P, Maizy M, Lefrere JJ, Daneluzzi V, Monteiro-Filho CM, Hong D, Planelles V, Morand-Joubert L, Benichou S. Characterization of the molecular determinants of primary HIV-1 Vpr proteins: impact of the Q65R and R77Q substitutions on Vpr functions. PloS one. 2009;4:e7514. doi: 10.1371/journal.pone.0007514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kumar R, Orsoni S, Norman L, Verma AS, Tirado G, Giavedoni LD, Staprans S, Miller GM, Buch SJ, Kumar A. Chronic morphine exposure causes pronounced virus replication in cerebral compartment and accelerated onset of AIDS in SIV/SHIV-infected Indian rhesus macaques. Virology. 2006;354:192–206. doi: 10.1016/j.virol.2006.06.020. [DOI] [PubMed] [Google Scholar]
  20. Kumar R, Torres C, Yamamura Y, Rodriguez I, Martinez M, Staprans S, Donahoe RM, Kraiselburd E, Stephens EB, Kumar A. Modulation by morphine of viral set point in rhesus macaques infected with simian immunodeficiency virus and simian-human immunodeficiency virus. Journal of virology. 2004;78:11425–11428. doi: 10.1128/JVI.78.20.11425-11428.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lang SM, Weeger M, Stahl-Hennig C, Coulibaly C, Hunsmann G, Muller J, Muller-Hermelink H, Fuchs D, Wachter H, Daniel MM, et al. Importance of vpr for infection of rhesus monkeys with simian immunodeficiency virus. Journal of virology. 1993;67:902–912. doi: 10.1128/jvi.67.2.902-912.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Le Rouzic E, Benichou S. The Vpr protein from HIV-1: distinct roles along the viral life cycle. Retrovirology. 2005;2:11. doi: 10.1186/1742-4690-2-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li G, Park HU, Liang D, Zhao RY. Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R. Retrovirology. 2010;7:59. doi: 10.1186/1742-4690-7-59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lum JJ, Cohen OJ, Nie Z, Weaver JG, Gomez TS, Yao XJ, Lynch D, Pilon AA, Hawley N, Kim JE, Chen Z, Montpetit M, Sanchez-Dardon J, Cohen EA, Badley AD. Vpr R77Q is associated with long-term nonprogressive HIV infection and impaired induction of apoptosis. The Journal of clinical investigation. 2003;111:1547–1554. doi: 10.1172/JCI16233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Minagar A, Shapshak P, Fujimura R, Ownby R, Heyes M, Eisdorfer C. The role of macrophage/microglia and astrocytes in the pathogenesis of three neurologic disorders: HIV-associated dementia, Alzheimer disease, and multiple sclerosis. Journal of the neurological sciences. 2002;202:13–23. doi: 10.1016/s0022-510x(02)00207-1. [DOI] [PubMed] [Google Scholar]
  26. Mologni D, Citterio P, Menzaghi B, Zanone Poma B, Riva C, Broggini V, Sinicco A, Milazzo L, Adorni F, Rusconi S, Galli M, Riva A, rHoPe SG. Vpr and HIV-1 disease progression: R77Q mutation is associated with long-term control of HIV-1 infection in different groups of patients. Aids. 2006;20:567–574. doi: 10.1097/01.aids.0000210611.60459.0e. [DOI] [PubMed] [Google Scholar]
  27. Morellet N, Bouaziz S, Petitjean P, Roques BP. NMR structure of the HIV-1 regulatory protein VPR. Journal of molecular biology. 2003;327:215–227. doi: 10.1016/s0022-2836(03)00060-3. [DOI] [PubMed] [Google Scholar]
  28. Noel RJ, Jr, Kumar A. Virus replication and disease progression inversely correlate with SIV tat evolution in morphine-dependent and SIV/SHIV-infected Indian rhesus macaques. Virology. 2006;346:127–138. doi: 10.1016/j.virol.2005.10.026. [DOI] [PubMed] [Google Scholar]
  29. Noel RJ, Jr, Kumar A. SIV Vpr evolution is inversely related to disease progression in a morphine-dependent rhesus macaque model of AIDS. Virology. 2007;359:397–404. doi: 10.1016/j.virol.2006.09.043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Noel RJ, Jr, Marrero-Otero Z, Kumar R, Chompre-Gonzalez GS, Verma AS, Kumar A. Correlation between SIV Tat evolution and AIDS progression in cerebrospinal fluid of morphine-dependent and control macaques infected with SIV and SHIV. Virology. 2006a;349:440–452. doi: 10.1016/j.virol.2006.03.032. [DOI] [PubMed] [Google Scholar]
  31. Noel RJ, Jr, Rivera-Amill V, Buch S, Kumar A. Opiates, immune system, acquired immunodeficiency syndrome, and nonhuman primate model. Journal of neurovirology. 2008;14:279–285. doi: 10.1080/13550280802078209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Noel RJ, Toro-Bahamonde A, Marrero-Otero Z, Orsini S, Verma AS, Kumar R, Kumar A. Lack of correlation between SIV-Nef evolution and rapid disease progression in morphine-dependent nonhuman primate model of AIDS. AIDS research and human retroviruses. 2006b;22:817–823. doi: 10.1089/aid.2006.22.817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Piller SC, Ewart GD, Premkumar A, Cox GB, Gage PW. Vpr protein of human immunodeficiency virus type 1 forms cation-selective channels in planar lipid bilayers. Proceedings of the National Academy of Sciences of the United States of America. 1996;93:111–115. doi: 10.1073/pnas.93.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ponten J, Macintyre EH. Long term culture of normal and neoplastic human glia. Acta pathologica et microbiologica Scandinavica. 1968;74:465–486. doi: 10.1111/j.1699-0463.1968.tb03502.x. [DOI] [PubMed] [Google Scholar]
  35. Rey F, BouHamdan M, Navarro JM, Agostini I, Willetts K, Bouyac M, Tamalet C, Spire B, Vigne R, Sire J. A role for human immunodeficiency virus type 1 Vpr during infection of peripheral blood mononuclear cells. The Journal of general virology. 1998;79(Pt 5):1083–1087. doi: 10.1099/0022-1317-79-5-1083. [DOI] [PubMed] [Google Scholar]
  36. Rivera-Amill V, Noel RJ, Jr, Garcia Y, Rivera I, Iszard M, Buch S, Kumar A. Accelerated evolution of SIV env within the cerebral compartment in the setting of morphine-dependent rapid disease progression. Virology. 2010a;398:201–207. doi: 10.1016/j.virol.2009.11.046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rivera-Amill V, Noel RJ, Jr, Orsini S, Tirado G, Garcia JM, Buch S, Kumar A. Variable region 4 of SIV envelope correlates with rapid disease progression in morphine-exposed macaques infected with SIV/SHIV. Virology. 2007;358:373–383. doi: 10.1016/j.virol.2006.08.039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rivera-Amill V, Noel RJ, Jr, Roman IR, Flores YG, Buch S, Kumar A. Analysis of the V1V2 region of the SIV envelope in the brains of morphine-dependent and control SIV/SHIV-infected macaques. AIDS research and human retroviruses. 2009;25:531–534. doi: 10.1089/aid.2008.0279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rivera-Amill V, Silverstein PS, Noel RJ, Jr, Kumar S, Kumar A. Morphine and rapid disease progression in nonhuman primate model of AIDS: inverse correlation between disease progression and virus evolution. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology. 2010b;5:122–132. doi: 10.1007/s11481-009-9184-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Roux P, Alfieri C, Hrimech M, Cohen EA, Tanner JE. Activation of transcription factors NF-kappaB and NF-IL-6 by human immunodeficiency virus type 1 protein R (Vpr) induces interleukin-8 expression. Journal of virology. 2000;74:4658–4665. doi: 10.1128/jvi.74.10.4658-4665.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Roy A, Kucukural A, Zhang Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nature protocols. 2010;5:725–738. doi: 10.1038/nprot.2010.5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Shah A, Kumar A. HIV-1 gp120-mediated increases in IL-8 production in astrocytes are mediated through the NF-kappaB pathway and can be silenced by gp120-specific siRNA. J Neuroinflammation. 2010;7:96. doi: 10.1186/1742-2094-7-96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shah A, Singh DP, Buch S, Kumar A. HIV-1 envelope protein gp120 up regulates CCL5 production in astrocytes which can be circumvented by inhibitors of NF-kappaB pathway. Biochem Biophys Res Commun. 2011a;414:112–117. doi: 10.1016/j.bbrc.2011.09.033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Shah A, Verma AS, Patel KH, Noel R, Rivera-Amill V, Silverstein PS, Chaudhary S, Bhat HK, Stamatatos L, Singh DP, Buch S, Kumar A. HIV-1 gp120 induces expression of IL-6 through a nuclear factor-kappa B-dependent mechanism: suppression by gp120 specific small interfering RNA. PLoS One. 2011b;6:e21261. doi: 10.1371/journal.pone.0021261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular biology and evolution. 2011;28:2731–2739. doi: 10.1093/molbev/msr121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic acids research. 1994;22:4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tirado G, Kumar A. Evolution of SIV envelope in morphine-dependent rhesus macaques with rapid disease progression. AIDS research and human retroviruses. 2006;22:114–119. doi: 10.1089/aid.2006.22.114. [DOI] [PubMed] [Google Scholar]
  48. Tungaturthi PK, Sawaya BE, Ayyavoo V, Murali R, Srinivasan A. HIV-1 Vpr: genetic diversity and functional features from the perspective of structure. DNA and cell biology. 2004;23:207–222. doi: 10.1089/104454904773819798. [DOI] [PubMed] [Google Scholar]
  49. Wang B, Ge YC, Palasanthiran P, Xiang SH, Ziegler J, Dwyer DE, Randle C, Dowton D, Cunningham A, Saksena NK. Gene defects clustered at the C-terminus of the vpr gene of HIV-1 in long-term nonprogressing mother and child pair: in vivo evolution of vpr quasispecies in blood and plasma. Virology. 1996;223:224–232. doi: 10.1006/viro.1996.0471. [DOI] [PubMed] [Google Scholar]
  50. Wecker K, Morellet N, Bouaziz S, Roques BP. NMR structure of the HIV-1 regulatory protein Vpr in H20/trifluoroethanol. Comparison with the Vpr N-terminal (1-51) and C-terminal (52-96) domains. European journal of biochemistry/FEBS. 2002;269:3779–3788. doi: 10.1046/j.1432-1033.2002.03067.x. [DOI] [PubMed] [Google Scholar]
  51. Williams R, Dhillon NK, Hegde ST, Yao H, Peng F, Callen S, Chebloune Y, Davis RL, Buch SJ. Proinflammatory cytokines and HIV-1 synergistically enhance CXCL10 expression in human astrocytes. Glia. 2009;57:734–743. doi: 10.1002/glia.20801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yuan L, Qiao L, Wei F, Yin J, Liu L, Ji Y, Smith D, Li N, Chen D. Cytokines in CSF correlate with HIV-associated neurocognitive disorders in the post-HAART era in China. Journal of neurovirology. 2013;19:144–149. doi: 10.1007/s13365-013-0150-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zhang Y. I-TASSER server for protein 3D structure prediction. BMC bioinformatics. 2008;9:40. doi: 10.1186/1471-2105-9-40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zhang Y, Skolnick J. TM-align: a protein structure alignment algorithm based on the TM-score. Nucleic acids research. 2005;33:2302–2309. doi: 10.1093/nar/gki524. [DOI] [PMC free article] [PubMed] [Google Scholar]

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