Current HIV Research (v.7, #2)

Vpr-Induced DNA Double-Strand Breaks: Molecular Mechanism and Biological Relevance by Chikako Nakai-Murakami, Yuzuru Minemoto, Yukihito Ishizaka (109-113).
We focus on the role of Vpr in inducing DNA double-strand breaks (DSBs) in the host cell. Based on the summarized findings of Vpr-induced DSBs and the finding of Vpr in the plasma of HIV-1-positive patients, we discuss the roles of Vpr in viral infection, especially viral infection of resting macrophages. We also describe the possible involvement of Vpr in non-AIDS-defining cancers, which represent an emerging crisis in HIV-1-positive patients.

HIV-1 Vpr: A Closer Look at the Multifunctional Protein from the Structural Perspective by Ramesh Pandey, Debduti Datta, Ruma Mukerjee, Alagarsamy Srinivasan, Sundarasamy Mahalingam, Bassel Sawaya (114-128).
The human immunodeficiency virus-1 (HIV-1) Vpr protein plays multiple roles in HIV-1 replication. In early infection, Vpr provides help in the nuclear localization of pre-integration complex. Subsequently, Vpr induces cell cycle arrest of infected cells at G2/M phase. Cell cycle arrest facilitates higher rate of viral gene transcription. Vpr is also capable of activating transcription of viral and heterologous genes. Vpr induces apoptosis in infected cells leading to loss of immune cells and onset of clinical AIDS. Interestingly, Vpr is also considered as a passenger protein in the virus particles as it is incorporated into the virus particles through interaction with Gag. The structure of full length Vpr has been resolved recently through NMR. In this review, we have analysed the functions of Vpr using the available data from structural perspective. Packing of the three helices of Vpr around a core formed by hydrophobic side chains and integrity of helical domains are critical for Vpr functions. The distinct functions of Vpr have been attributed to structural integrity of different domains. The unique distribution of acidic and basic residues in Vpr is an interesting feature. Two hydrophobic pockets on the structure of Vpr are proposed to be important targets for modulating Vpr functions. The inter-relationship between different functions of Vpr is discussed in the context of structure. Based on bioinformatics analysis, we propose new targets for modulating Vpr functions, which need to be validated experimentally.

Murine Models of Vpr-Mediated Pathogenesis by Alexandra Snyder, Michael Ross (129-135).
HIV viral protein r (Vpr) exerts a variety of cellular effects, including modulation of transcription and cytokine production, apoptosis, and cell cycle arrest. Vpr induces these affects by mechanisms that include inhibition of NFkappaB activation, inducing mitochondrial injury, and promoting proteasomal degradation of cellular factor(s) leading to cell cycle arrest. Murine models have provided invaluable contributions to our understanding of HIV pathogenesis, however many of the HIV-1 proteins, including Vpr, differ in their cellular effects depending upon cell type and speciesspecific factors. Since the majority of in vivo studies elucidating the role of Vpr in disease pathogenesis have utilized murine models, it is critical to understand the species-specific factors that may affect Vpr function. In this manuscript, we review the cellular pathways and end organ effects of Vpr that have been studied in murine cell lines and mouse models, and discuss the relevance of these studies to the role of Vpr in disease in persons living with HIV/AIDS.

The replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells, such as terminally differentiated macrophages, critically depends on the import of the viral pre-integration complex (PIC) into the nucleus. Vpr, one of the accessory gene products of HIV-1, plays a key regulatory role in PIC nuclear import in macrophages, although its role in the PIC entry mechanism remains to be clarified. Here, we summarize what is currently known about the nuclear- entry step of HIV-1 replication, mainly focusing on how Vpr functions as the main regulator of HIV-1 nuclear import and how it could facilitate the development of novel inhibitors of this process.

Analysis of the Potential for HIV-1 Vpr as an Anti-Cancer Agent by Karrupiah Muthumani, Vance Lambert, Niranjan Sardesai, Jong Kim, Richard Heller, David Weiner, Kenneth Ugen (144-152).
Viral protein R (Vpr) is a 14kD, 96 amino acid accessory protein of the HIV virion that has been demonstrated to have important functions in the viral replication cycle including, among others, the induction of cell cycle arrest and apoptosis in rapidly proliferating cells, which results in immune dysfunction in infected individuals. Several investigators have studied the potential use of the apoptosis inducing and cell cycle arrest effect of Vpr as an anti-tumor therapeutic. In vitro studies have indicated that Vpr is cytotoxic against a large number of different tumor cell types including a number that are p53 independent. Likewise, some in vivo tumor studies using different delivery platforms/methods have indicated an anti-cancer effect mediated by Vpr. Our group has used the aggressive and poorly immunogenic murine melanoma tumor line B16.F10 as a model to deliver, through in vivo electroporation, Vpr expressing plasmids to established tumors and have demonstrated that this treatment regimen can induce growth attenuation and tumor regression in a proportion of the treated mice and appears to be associated with the induction of intratumoral apoptosis. Overall, to date, the data from a number of research groups, including our own, have indicated that Vpr has biological activity against a number of tumors in both in vivo and in vitro models and, as such, may be a potential candidate for testing in human clinical trials. In this report, we summarize the evidence supporting this hypothesis.

HIV-1 Vpr: Regulator of Viral Survival by Khanh Thieu, Matthew Morrow, Devon Shedlock, Kimberly Schoenly, Karthikbabu Mallilankaraman, Andrew Choo, Paolo Fagone, David Weiner, Karuppiah Muthumani (153-162).
The HIV-1 Vpr protein is a viral accessory protein that plays a number of important roles during HIV infection. The activities of Vpr are numerous and include the induction of apoptosis, the modulation of cell cycle arrest, as well as control of viral transcription. Study of HIV clones lacking Vpr in vitro and analysis of HIV variants isolated from longterm nonprogressors in vivo highlight the importance of Vpr for viral replication as well as immune suppression and cell death. Vpr may therefore be considered among the most important accessory proteins encoded by HIV.

HIV-1 Vpr: A Novel Role in Regulating RNA Splicing by Xianfeng Zhang, Yoko Aida (163-168).
Pre-mRNA splicing is a critical step in gene expression for metazoans. Several viral proteins regulate the splicing of pre-mRNAs through complex interactions with the host cell RNA splicing machinery. Here, we focus on a novel function of HIV-1 Vpr, which selectively inhibits cellular and viral pre-mRNA splicing via interactions with components of the spliceosome complex. This review discusses our current knowledge of how RNA splicing regulation is accomplished by Vpr-host factor interactions.

HIV-1 Mediated Immune Pathogenesis: Spotlight on the Role of Viral Protein R (VPR) by Biswanath Majumder, Narasimhan Venkatachari, Alagarsamy Srinivasan, Velpandi Ayyavoo (169-177).
HIV-1 exploits the cellular machinery to replicate in the host cells by targeting a wide range of host factors at different stages of its life cycle. HIV-1 also induces detrimental effects in the infected and uninfected bystander cells resulting in dysregulation including interference in immune effector functions. The latter is specifically linked to the immune evasion strategies of the virus. In addition to the essential roles of structural proteins (Gag, Pol and Env), HIV-1 encoded auxiliary proteins such as Nef, Vif, Vpu, and Vpr through their interaction with the host cellular partners facilitate viral replication and dissemination. HIV-1 Vpr, a virion-associated molecule, has been implicated to play a role in the early events in virus life cycle. Vpr is a pleiotropic protein that exerts a range of effects including inhibition of cell proliferation, induction of apoptosis and modulation of a number of immune molecules. These functions could be in part responsible for Vpr induced immune evasion and virus replication. Appreciating this view is the genetic variation in vpr gene reflected in the form of polymorphisms at the amino acid level that may contribute to the potential CTL escape of the virus. It is likely that Vpr mediated dysregulation of host immune response contributes, in part, to the progression of disease. This review focuses on the recent advances regarding HIV-1 Vpr mediated immunopathogenesis and the mechanistic insight from in vitro and in vivo studies.

HIV-1 Viral Protein R (VPR) and its Interactions with Host Cell by Ge Li, Michael Bukrinsky, Richard Zhao (178-183).
Human immunodeficiency virus type 1 (HIV-1) is engaged in dynamic and antagonistic interactions with host cells. Once infected by HIV-1, host cells initiate various antiviral strategies, such as innate antiviral defense mechanisms, to counteract viral invasion. In contrast, the virus has different strategies to suppress these host responses to infection. The final balance between these interactions determines the outcome of the viral infection and disease progression. Recent findings suggest that HIV-1 viral protein R (Vpr) interacts with some of the host innate antiviral factors, such as heat shock proteins, and plays an active role as a viral pathogenic factor. Cellular heat stress response factors counteract Vpr activities and inhibit HIV replication. However, Vpr overcomes these heat-stress-like responses by preventing heat shock factor-1 (HSF-1) - mediated activation of heat shock proteins. In this review, we will focus on the virus-host interactions involving Vpr. In addition to heat stress response proteins, we will discuss interactions of Vpr with other proteins, such as EF2 and Skp1/GSK3, their involvements in cellular responses to Vpr, as well as strategies to develop novel antiviral therapies aimed at enhancing anti-Vpr responses of the host cell.

Structure-Function Relationship of Vpr: Biological Implications by Nelly Morellet, Bernard Roques, Serge Bouaziz (184-210).
Vpr, incorporated into the HIV-1 virion, shows multiple activities including nuclear transport of the preintegration complex to the nucleus, activation of the transcription, cell cycle arrest at the G2/M transition and induction of apoptosis. Vpr controls many host cell functions through a variety of biological activities and by interaction with cellular biochemical pathways. Nuclear import of Vpr may be due to its interaction with nuclear transport factors and components of the nuclear pore complex. Cell cycle arrest has been correlated with the binding to DCAF1, a cullin 4A-associated factor and apoptosis may be facilitated by interaction with mitochondrial proteins, in a caspase-dependent mechanism. The structure of Vpr(1-96) and various fragments have been determined by NMR in diverse solvents. The different functions of Vpr can be classified according to their relationship with the different structural domains of the protein and appear to correlate with the partners interacting with these domains. Thus, virion packaging seems to be mediated by the first and#945;- helix (17-33), activation of the transcription, regulation of apoptosis and subcellular transport appear to be dependent on the second and#945;-helix (38-50) and cell cycle arrest seems to be induced by the carboxyl terminal and#945;-helix (55-77). Mutational analysis performed by several groups have provided a strong basis to understand the structure-function relationship of Vpr. The aim of this review is to run through these mutations using the available information on sequences and discuss their effect on the functions of Vpr from the point of view of its structure.

Higher Placental Anti-Inflammatory IL-10 Cytokine Expression in HIV-1 Infected Women Receiving Longer Zidovudine Prophylaxis Associated with Nevirapine by Sakorn Pornprasert, Jean-Yves Mary, Albert Faye, Pranee Leechanachai, Aram Limtrakul, Sungwal Rugpao, Pannee Sirivatanapa, Vorapin Gomuthbutra, Wanmanee Matanasaravoot, Sophie Coeur, Marc Lallemant, Francoise Barre-Sinoussi, Elisabeth Menu, Nicole Ngo-Giang-Huong (211-217).
Placental cytokine balance may be critical for the control of mother-to-child transmission (MTCT) of HIV. We assessed whether the type and duration of antiretrovirals used for prevention of HIV-1-MTCT modified the inflammatory cytokine profile. We investigated the levels of cytokine expression in the placentas of 61 HIV-1-infected women who received zidovudine (ZDV) plus single dose nevirapine (SD-NVP) or ZDV only for prevention of MTCT. Placentas of 38 HIV-1-uninfected women were included as controls. All placentas were obtained after vaginal delivery. Levels of mRNA and cytokine expression were quantified using real-time PCR and ELISA, respectively, in placental explants and 24-hour culture supernatants and analyzed in relation to the women's characteristics and the type and duration of antiretroviral prophylaxis. HIV-1-infected and uninfected women did not show any differences in the expression of placental cytokine secretion except for a trend toward lower TNF-and#945; mRNA levels in HIV-1-infected women. Within the HIV-1-infected group, women who were exposed to a long duration of ZDV ( > 72 days) or received SD-NVP less than 5h prior to delivery, more frequently expressed detectable levels of IL-10 in their placentas (32and#x25; versus 7and#x25; (p = 0.01) and 32and#x25; versus 5and#x25; (p = 0.02), respectively). No infant was found to be HIV-1-infected. Our results showed a normalization of the placental cytokine balance in HIV-1-infected women receiving antiretroviral prophylaxis. Furthermore, the type and duration of antiretroviral prophylaxis have an impact on the placental anti-inflammatory IL-10 expression level, which may contribute to controlling HIV replication at the placental level, thus reducing MTCT of HIV-1.

Polymorphic Receptors of the Innate Immune System (MBL/MASP-2 and TLR2/4) and Susceptibility to Pneumococcal Bacteremia in HIV-Infected Patients: A Case-Control Study by Juan Horcajada, Francisco Lozano, Ana Munoz, Belen Suarez, Concepcion Farinas-Alvarez, Manuel Almela, Alex Smithson, Esteban Martinez, Josp Mallolas, Josep Mensa, Josep Gatell (218-223).
Some deficient genetic polymorphisms of the innate immune system have been correlated to a higher susceptibility to different infections, especially in immunocompromised patients. The possible association between an increased incidence of pneumococcal bacteremia in HIV-infected patients, and deficient polymorphisms of the mannose-binding lectin (MBL), MBL-associated serine protease 2 (MASP-2), and toll-like receptors (TLR) 2 and 4 is analyzed by means of a case-control study. Cases: HIV-infected patients with pneumococcal bacteremia. Controls: HIV-infected patients without previous pneumococcal bacteremia matched with cases by sex and CD4 count in a 2:1 ratio. Fifty-seven cases and 114 controls were studied. Demographics, HIV infection status, antiretroviral therapy, risk factors for pneumococcal disease, and genotypes of MBL2, MASP2, TLR2 and TLR4 were analyzed. The prevalence of the MBL2, MASP2, TLR2 and TLR4 gene polymorphisms was similar in both groups. No statistical significance was found (OR 0.77, IC95and#x25; 0.27 - 2.13) when analyzing the possible association of MBL2 deficient polymorphisms with pneumococcal bacteremia. In HIV infected patients, no association between the presence of deficient polymorphisms of MBL2, MASP2, TLR2 and TLR4 and the incidence of pneumococcal bacteremia was found.

Delayed Diagnosis of HIV Infection in a Multicenter Cohort: Prevalence, Risk Factors, Response to HAART and Impact on Mortality by Paz Sobrino-Vegas, Lucia Miguel, Ana Caro- Murillo, Jose Miro, Pompeyo Viciana, Cristina Tural, Maria Saumoy, Ignacio Santos, Julio Sola, Julia Amo, Santiago Moreno, CoRIS (224-230).
To study the prevalence of Delayed HIV Diagnosis (DHD) and its associated risk factors, to evaluate the effect of DHD on virological and immunological responses to HAART and to estimate the impact of DHD on all-causes mortality. Prospective cohort of 2, 564 HIV-positive HAART-naive subjects attending 19 hospitals in Spain, 2004-2006. Estimations were made by logistic regression and survival analyses by Cox regression models. Prevalence of DHD was 37.3and#x25; (35.0-39.6). DHD was related to low educational level (OR:1.31, 95and#x25;CI:1.0-1.7). Compared to men who have sex with men (MSM), DHD was more frequent in heterosexuals (OR:1.9 95and#x25;CI:1.5-2.5) and injection drug users (IDUs) (OR:2.0 95and#x25;CI:1.5-2.8).. An interaction between age and sex was found. Although risk of having DHD did not increase after age 30 in women, it increased linearly with age in men. No differences in virological (OR 1.2 95and#x25;CI: 0.8-1.8) and CD4 T cell (OR 1.1 95and#x25;CI: 0.7-1.8) responses to HAART were seen. The adjusted hazard ratio for death in patients with DHD was 5.2, (95and#x25;CI: 1.9-14.5). DHD is very common, especially in older men, heterosexuals and IDUs. Although we did not find differences in virological and immunological responses to HAART, we did observe higher mortality in people with DHD. Increased efforts to early diagnose HIV infection are urgently needed.

To explore the hypotheses that HIV-1 infected patients receiving suppressive antiretroviral therapy believe that they cannot transmit their virus, that this translates to increasing rates of unprotected anal intercourse and new infections, and that sexual practice may be influenced by the HIV-1 serostatus of one's sexual partner. We developed a Bernoulli model for HIV-1 transmission amongst a cross-sectional cohort of 119 HIV-1 positive treated and untreated men having sex with men attending a metropolitan HIV-1 clinic. The model included transmission probabilities for different types of anal intercourse, the local HIV-1 seroprevalence rate, and behavioural data from a validated sexual practice questionnaire and scales of treatment optimism-scepticism and sexual beliefs pertaining to the previous 3 months. Subjects reported partner serostatus as HIV-1 negative or unknown. There were no differences in optimism-scepticism score (p = 0.295) and total sexual belief scores (p = 0.211) according to antiretroviral therapy status. Sex with unknown serostatus partners was higher risk for transmission than sex with HIV-1 negative partners. Most unprotected anal intercourse was practiced by a minority of men, independent of antiretroviral treatment status. Compared to subjects with HIV-1 negative partners, subjects with unknown serostatus partners had more UAI, a 2-fold higher probability of transmitting HIV-1 and were responsible for 2.6 as many new infections. Overall, there were 4 times as many infections predicted to occur amongst unknown serostatus men cf. HIV-1 negative men. A sub-group reported frequent unprotected anal intercourse with their partners. Our model may be used to quantify risk behavior during individual counselling or to develop targeted prevention programmes at the public health level.

The study goal is to identify predictors of HIV infection late detection in an European city with increased immigration, and determine the effects of HAART era in HIV infection detection. We used Barcelona city AIDS registry (1987 - 2006). Late testers were those diagnosed of AIDS defining illness within less than 3 months from time of testing positive for HIV infection. Independent variables were: date of birth, sex, country of origin, HIV transmission category, prison history, city district of residence, AIDS diagnostic disease and HAART era when diagnosed. The statistical methods were based on logistic regression (Odds Ratio, OR and 95and#x25; confidence interval, CI). Among the 6186 AIDS patients, 43.9and#x25; (n=2741) were late testers. Being a male (OR: 1.57, 95and#x25; CI: 1.35-1.83), either andlt; 30 years (OR: 1.21, 95and#x25; CI: 1.06-1.38) or > 40 years (OR: 1.20, 95and#x25; CI: 1.03-1.40), with heterosexual (OR: 3.07, 95and#x25; CI: 2.59-3.63) routes of transmission or men who have sex with men (OR: 2.20, 95and#x25; CI: 1.89-2.57) and with Pneumocystis jiroveci pneumoniae (OR: 1.71, 95and#x25; CI: 1.47-2.00) or tuberculosis (OR: 1.57, 95and#x25; CI: 1.36-1.82) were all independent risk factors for being a late tester. Conversely, injecting drug use (IDU) was associated with early detection (OR: 0.36, 95and#x25; CI: 0.33-0.40). Being migrant was associated with late testing only in the univariate analysis. Individuals with the detected factors (male, having any sexual risk behaviour and being > 50 years) should be in the main focus for HIV testing to further ensure continuous decrease in the slope of late detected HIV infections overall.

Relationship Between Polymerase Gamma (POLG) Polymorphisms and Antiretroviral Therapy-Induced Lipodystrophy in HIV-1 Infected Patients: A Case-Control Study by Franck Chiappini, Elina Teicher, Raphael Saffroy, Brigitte Debuire, Daniel Vittecoq, Antoinette Lemoine (244-253).
Nucleoside Reverse Transcriptase Inhibitors (NRTIs) used for the treatment of HIV-1 inhibit the replication of mitochondrial DNA (mtDNA), which may contribute to severe mitochondrial toxicity including lipodystrophy, through the inhibition of polymerase gamma (POLG). Polymorphisms of POLG could explain the variation in mitochondrial toxicity in HIV-1-infected patients. We explored the relationship between selected polymorphisms of POLG and lipodystrophy related to NRTIs. We studied single nucleotide polymorphisms (SNP) at three amino acid residues (R1142, E1143 and R1146) and the CAG repeats of POLG in a case-control study including HIV-1 treated patients with lipodystrophy (n=69) and 2 controls (without lipodystrophy) per case matched by age, race and sex (n=138). Compared with matched controls, the polymorphisms in E1143 were significantly more frequent in case patients with lipodystrophy (aOR=4.7; p=0.048), and this was associated with a significant decrease of mtDNA in PBMC. In addition, among the parameters tested, the conditional logistic regression showed that the lipodystrophy has a strong link with E1143 polymorphisms, associated with D4T treatment (aOR=9.29, p=0.002). In conclusion, patients harbouring the changes of E1143 in the catalytic site of POLG exhibit a 4-fold increased risk to develop lipodystrophy than HIV-1 treated patients who do not have changes in E1143 and this risk can increase if the patient presenting the SNP received D4T. These could be due to decreased content of mtDNA in PBMC in these patients. Therefore, the toxicity of NRTIs leading to lipodystrophy in some HIV-1 infected patients could be explained in part by the occurrence of POLG polymorphisms.