Identification of a circulating long non-coding RNA signature panel in plasma as a novel biomarker for the detection of acute/early-stage HIV-1 infection.

BACKGROUND: Individuals with acute / early HIV-1 infection are often unaware that they are infected with HIV-1 and may be involved in high-risk behavior leading to transmission of HIV-1. Identifying individuals with acute / early HIV-1 infection is critical to prevent further HIV-1 transmission, as diagnosis can lead to several effective HIV-1 prevention strategies. Identification of disease-stage specific non-viral host biomarkers would be useful as surrogate markers to accurately identify new HIV-1 infections. The goal of this study was to identify a panel of host derived plasma long non-coding RNAs (lncRNAs) that could serve as prognostic and predictive biomarkers to detect early/acute HIV-1 infection. METHODS: A total of 84 lncRNAs were analyzed in sixteen plasma samples from HIV-1 infected individuals and four healthy controls using the lncRNA PCR-array. Twenty-one lncRNAs were selected and validated in 80 plasma samples from HIV-1 infected individuals [HIV-1 infected patients in the eclipse stage (n = 20), acute stage (n = 20), post-seroconversion p31 negative stage (n = 20), and post-seroconversion p31 positive stage (n = 20) of infection] and 20 healthy controls. The validation study results were used to develop a plasma lncRNA panel that was evaluated in the panel test phase to detect early/acute HIV-1 infection in 52 independent samples. RESULTS: We identified a lncRNA panel (Pmodel-I) containing eight lncRNAs (DISC2, H19, IPW, KRASP1, NEAT1, PRINS, WT1-AS and ZFAS1) that could distinguish HIV-1 infection from healthy controls with high AUC 0·990 (95% CI 0.972-1.000), sensitivity (98.75%), and specificity (95%). We also found that Pmodel-II and Pmodel-III demonstrates 100% sensitivity and specificity (AUC 1·00; 95%CI:1·00-1·00) and could distinguish eclipse stage and acute stage of HIV-1 infection from healthy controls respectively. Antiretroviral treatment (ART) cumulatively restored the levels of lncRNAs to healthy controls levels. CONCLUSION: lncRNA expression changes significantly in response to HIV-1 infection. Our findings also highlight the potential of using circulating lncRNAs to detect both the eclipse and acute stages of HIV-1 infection, which may help to shorten the window period and facilitate early detection and treatment initiation. Initiating ART treatment at this stage would significantly reduce HIV-1 transmission. The differentially expressed lncRNAs identified in this study could serve as potential prognostic and diagnostic biomarkers of HIV-1 infection, as well as new therapeutic targets.

Modulation of HIV Replication in Monocyte-Derived Macrophages (MDM) by Host Antiviral Factors Secretory Leukocyte Protease Inhibitor and Serpin Family C Member 1 Induced by Steroid Hormones.

The impact of steroid hormones estrogen and progesterone on human immunodeficiency virus type 1 (HIV-1) replication is well documented. However, the exact mechanism involved in the regulation of HIV-1 replication by estrogen and progesterone is still unclear. In the present study, we wanted to elucidate the molecular mechanisms underlying the modulation of HIV-1 replication by estrogen and progesterone. To achieve this goal, we used real-time quantitative PCR arrays (PCR arrays) to identify differentially expressed host genes in response to hormone treatments that are involved in antiviral responses. Our in vitro results suggest that treatment with high doses of estrogen and progesterone promotes the expression of host antiviral factors Secretory leukocyte protease inhibitor (SLPI) and Serpin family C member 1 (SERPIN C1) among others produced in response to HIV-1 infection. SLPI is an enzyme that inhibits human leukocyte elastase, human cathepsin G, human trypsin, neutrophil elastase, and mast cell chymase. SERPIN C1 is a plasma protease inhibitor that regulates the blood coagulation cascade by the inhibition of thrombin and other activated serine proteases of the coagulation system. A dose dependent downmodulation of HIV-1 replication was observed in monocyte-derived macrophages (MDMs) pre-treated with the two proteins SLPI and SERPIN C1. Further investigations suggests that the host antiviral factors, SLPI and SERPIN C1 act at the pre-integration stage, inhibiting HIV-1 viral entry and leading to the observed downmodulation of HIV-1 replication. Our studies would help identify molecular mechanisms and pathways involved in HIV-1 pathogenesis.

Components of apoptotic pathways modulate HIV-1 latency in Jurkat cells.

The ability of the human immunodeficiency virus type 1 (HIV-1) to establish latent infections serves as a major barrier for its cure. This process could occur when its host cells undergo apoptosis, but it is uncertain whether the components of the apoptotic pathways affect viral latency. Using the susceptible Jurkat cell line, we investigated the relationship of apoptosis-associated components with HIV-1 DNA levels using the sensitive real-time PCR assay. Here, we found that the expression of proapoptotic proteins, including Fas ligand (FasL), FADD, and p53, significantly decreased HIV-1 viral DNA in cells. In contrast, the expression of antiapoptotic molecules, such as FLIP, Bcl2, and XIAP, increased the levels of viral DNA. Furthermore, promoting cellular antiapoptotic state via the knockdown of Bax with siRNA and FADD with antisense mRNA or the treatment with the Caspase-3 inhibitor, Z-DEVD, also raised viral DNA. We also simultaneously measured viral RNA from supernatants of these cell cultures and found that HIV-1 latency is inversely proportional to viral replication. Furthermore, we demonstrated that HIV-1-infected cells that underwent the transient expression of FLIP- or XIAP-induced viral latency would then produce an increased level of viral RNA upon the reversal of these antiapoptotic effects via PMA treatment compared to LacZ control cells. Taken together, these data suggest that HIV-1 infection could be adapted to employ or even manipulate the cellular apoptotic pathway to its advantage: when the host cell remains in a pro-apoptotic state, HIV-1 favors active replication, while when the host cell prefers an anti-apoptotic state, the virus establishes viral latency and promotes latent reservoir seeding in a way which would enhance viral replication and cytopathogenesis when the cellular conditions shift to encourage the productive infection phase.

Comparison of miRNA Expression Profiles between HIV-1 and HIV-2 Infected Monocyte-Derived Macrophages (MDMs) and Peripheral Blood Mononuclear Cells (PBMCs).

During the progression of HIV-1 infection, macrophage tropic HIV-1 that use the CCR5 co-receptor undergoes a change in co-receptor use to CXCR4 that is predominately T cell tropic. This change in co-receptor preference makes the virus able to infect T cells. HIV-2 is known to infect MDMs and T cells and is dual tropic. The aim of this study was to elucidate the differential expression profiles of host miRNAs and their role in cells infected with HIV-1/HIV-2. To achieve this goal, a comparative global miRNA expression profile was determined in human PBMCs and MDMs infected with HIV-1/HIV-2. Differentially expressed miRNAs were identified in HIV-1/HIV-2 infected PBMCs and MDMs using the next-generation sequencing (NGS) technique. A comparative global miRNA expression profile in infected MDMs and PBMCs with HIV-1 and HIV-2 identified differential expression of several host miRNAs. These differentially expressed miRNAs are likely to be involved in many signaling pathways, like the p53 signaling pathway, PI3K-Akt signaling pathways, MAPK signaling pathways, FoxO signaling pathway, and viral carcinogenesis. Thus, a comparative study of the differential expression of host miRNAs in MDMs and T cell in response to HIV-1 and HIV-2 infection will help us to identify unique biomarkers that can differentiate HIV-1 and HIV-2 infection.

Development and validation of plasma miRNA biomarker signature panel for the detection of early HIV-1 infection.

BACKGROUND: Accurate laboratory diagnosis of HIV is essential to reduce the risk of HIV-positive individuals transmitting HIV-1 infection. The goal of this study was to identify and assess a panel of host derived plasma miRNAs that could to serve as a prognostic and predictive biomarker to detect early/acute HIV-1 infection. METHODS: A total of 372 microRNAs were analyzed in nine plasma samples from HIV-1 infected individuals in the early phase of infection and three healthy controls using the miRNA PCR-array. Seventeen microRNAs were selected and validated in 80 plasma samples from HIV-1 infected individuals in the early phase of infection (20 each of eclipse stage, RNA+ stage, Ag + stage, and Ag + Ab+ stage of HIV-1 patients) and 25 healthy controls. Using the validation study results a plasma miRNA panel was developed and evaluated to detect early/acute HIV-1 infection in 49 blinded samples. FINDING: We identified an miRNA panel (PeHIV-1) containing four differentially expressed miRNAs (miR-16-5p, miR-20b-5p, miR-195-5p, and miR-223-3p) that could distinguish early HIV-1 infection from healthy controls with high AUC (1·000[1·00-1·00]), sensitivity (100%), and specificity (100%).We also found that miR-223-3p demonstrates 100% sensitivity and specificity (AUC 1·00[1·00-1·00]) and could distinguish eclipse stage of HIV-1 infection from healthy controls. To detect eclipse stage of HIV-1 infection we also developed a four-miRNA based (miR-16-5p, miR-206, let-7 g-3p, and miR-181c-3p) panel (PE) with AUC 0·999 (0·995-1·000), 100% sensitivity and 95·8% specificity. INTERPRETATION: The miRNA panel, PeHIV-1 is a potential biomarker for detecting early/acute stage of HIV-1infection and could help initiate early antiretroviral treatment, thus preventing the spread of HIV-1 infection.

Differentially expressed host long intergenic noncoding RNA and mRNA in HIV-1 and HIV-2 infection.

Non-coding RNAs and mRNAs have been implicated in replication, pathogenesis and host response in HIV infection. However, the impact of long intergenic non-coding RNAs (lincRNAs) on HIV-1 and HIV-2 infection is not known. In this study, we have analyzed expression profiles of lincRNAs and mRNAs in monocyte derived macrophages (MDMs) infected with HIV-1/HIV-2 using microarrays. Our study identified many differentially expressed lincRNAs and mRNAs in MDMs infected with HIV-1/HIV-2 compared to uninfected MDMs. Genes involved in glutathione metabolism and lysine degradation were differentially regulated only in HIV-1 infected MDMs. In HIV-2 infected MDMs, CUL 2, SFRS9, and RBBP4 genes were differentially expressed. Furthermore, we found that plasma levels of lincRNA: chr2: 165509129-165519404 and lincRNA: chr12: 57761837-57762303 were better indicators of HIV-1 infection while lincRNA: chr10:128586385-128592960, XLOC_001148 and lincRNA: chr5:87580664-87583451, were better indicators of HIV-2 infection. In summary, our study has demonstrated that there is substantial alteration in lincRNA and mRNA expression in response to HIV-1/HIV-2 infection. These differentially expressed lincRNAs and mRNAs could serve as prognostic and diagnostic biomarkers of HIV infection and help in the identification of new targets for therapy.

Modulation of HIV replication in monocyte derived macrophages (MDM) by steroid hormones.

Significant sex specific differences in the progression of HIV/AIDS have been reported. Several studies have implicated steroid hormones in regulating host factor expression and modulating HIV transmission and replication. However, the exact mechanism exerted by steroid hormones estrogen and progesterone in the regulation of HIV-1 replication is still unclear. Results from the current study indicated a dose dependent down regulation of HIV-1 replication in monocyte derived macrophages pre-treated with high concentrations of estrogen or progesterone. To elucidate the molecular mechanisms associated with the down regulation of HIV-1 replication by estrogen and progesterone we used PCR arrays to analyze the expression profile of host genes involved in antiviral responses. Several chemokines, cytokines, transcription factors, interferon stimulated genes and genes involved in type-1 interferon signaling were down regulated in cells infected with HIV-1 pre-treated with high concentrations of estrogen or progesterone compared to untreated HIV-1 infected cells or HIV-1 infected cells treated with low concentrations of estrogen or progesterone. The down regulation of CXCL9, CXCL10 and CXCL11 chemokines and IL-1ß, IL-6 cytokines in response to high concentrations of estrogen and progesterone pre-treatment in HIV-1 infected cells was confirmed at the protein level by quantitating chemokine and cytokine concentrations in the culture supernatant. These results demonstrate that a potent anti-inflammatory response is mediated by pre-treatment with high concentrations of estrogen and progesterone. Thus, our study suggests a strong correlation between the down-modulation of anti-viral and pro-inflammatory responses mediated by estrogen and progesterone pre-treatment and the down regulation of HIV-1 replication. These findings may be relevant to clinical observations of sex specific differences in patient populations and point to the need for further investigation.

Progesterone augments cell susceptibility to HIV-1 and HIV-1/HSV-2 co-infections.

In human immunodeficiency virus type 1 (HIV-1)-infected women, oral or injectable progesterone containing contraceptive pills may enhance HIV-1 acquisition in vivo, and the mechanism by which this occurs is not fully understood. In developing countries, Herpes simplex virus type-2 (HSV-2) co-infection has been shown to be a risk for increase of HIV-1 acquisition and, if co-infected women use progesterone pills, infections may increase several fold. In this study, we used an in vitro cell culture system to study the effects of progesterone on HIV-1 replication and to explore the molecular mechanism of progesterone effects on infected cells. In our in vitro model, CEMss cells (lymphoblastoid cell line) were infected with either HIV-1 alone or co-infected with HSV-2. HIV-1 viral load was measured with and without sex hormone treatment. Progesterone-treated cells showed an increase in HIV-1 viral load (1411.2 pg/mL) compared with cells without progesterone treatment (993.1 pg/mL). Increased cell death was noted with HSV-2 co-infection and in progesterone-treated cells. Similar observations were noted in peripheral blood mononuclear cells (PBMC) cells derived from three female donors. Progesterone-treated cells also showed reduced antiviral efficacy. Inflammatory cytokines and associations with biomarkers of disease progression were explored. Progesterone upregulated inflammatory cytokines and chemokines conversely and downregulated anti-apoptotic Bcl-2 expression. Nuclear protein analysis by electrophoretic mobility shift assay showed the association of progesterone with progesterone response element (PRE), which may lead to downregulation of Bcl-2. These data indicate that progesterone treatment enhances HIV-1 replication in infected cells and co-infection with HSV-2 may further fuel this process.

Identification of Host Micro RNAs That Differentiate HIV-1 and HIV-2 Infection Using Genome Expression Profiling Techniques.

While human immunodeficiency virus type 1 and 2 (HIV-1 and HIV-2) share many similar traits, major differences in pathogenesis and clinical outcomes exist between the two viruses. The differential expression of host factors like microRNAs (miRNAs) in response to HIV-1 and HIV-2 infections are thought to influence the clinical outcomes presented by the two viruses. MicroRNAs are small non-coding RNA molecules which function in transcriptional and post-transcriptional regulation of gene expression. MiRNAs play a critical role in many key biological processes and could serve as putative biomarker(s) for infection. Identification of miRNAs that modulate viral life cycle, disease progression, and cellular responses to infection with HIV-1 and HIV-2 could reveal important insights into viral pathogenesis and provide new tools that could serve as prognostic markers and targets for therapeutic intervention. The aim of this study was to elucidate the differential expression profiles of host miRNAs in cells infected with HIV-1 and HIV-2 in order to identify potential differences in virus-host interactions between HIV-1 and HIV-2. Differential expression of host miRNA expression profiles was analyzed using the miRNA profiling polymerase chain reaction (PCR) arrays. Differentially expressed miRNAs were identified and their putative functional targets identified. The results indicate that hsa-miR 541-3p, hsa-miR 518f-3p, and hsa-miR 195-3p were consistently up-regulated only in HIV-1 infected cells. The expression of hsa-miR 1225-5p, hsa-miR 18a* and hsa-miR 335 were down modulated in HIV-1 and HIV-2 infected cells. Putative functional targets of these miRNAs include genes involved in signal transduction, metabolism, development and cell death.

Novel Time-Resolved Fluorescence Europium Nanoparticle Immunoassay for Detection of Human Immunodeficiency Virus-1 Group O Viruses Using Microplate and Microchip Platforms.

Accurate detection and quantification of HIV-1 group O viruses have been challenging for currently available HIV assays. We have developed a novel time-resolved fluorescence (TRF) europium nanoparticle immunoassay for HIV-1 group O detection using a conventional microplate enzyme-linked immunosorbent assay (ELISA) and a microchip platform. We screened several antibodies for optimal reactivity with several HIV-1 group O strains and identified antibodies that can detect all the strains of HIV-1 group O that were available for testing. The antibodies were used to develop a conventional ELISA format assay and an in-house developed europium nanoparticle-based assay for sensitivity. The method was evaluated on both microwell plate and microchip platforms. We identified two specific and sensitive antibodies among the six we screened. The antibodies, C65691 and ANT-152, were able to quantify 15 and detect all 17 group O viruses, respectively, as they were broadly cross-reactive with all HIV-1 group O strains and yielded better signals compared with other antibodies. We have developed a sensitive assay that reflects the actual viral load in group O samples by using an appropriate combination of p24 antibodies that enhance group O detection and a highly sensitive TRF-based europium nanoparticle for detection. The combination of ANT-152 and C65690M in the ratio 3:1 was able to give significantly higher signals in our europium-based assay compared with using any single antibody.

Pandemic Influenza A (H1N1) Virus Infection Increases Apoptosis and HIV-1 Replication in HIV-1 Infected Jurkat Cells.

Influenza virus infection has a significant impact on public health, since it is a major cause of morbidity and mortality. It is not well-known whether influenza virus infection affects cell death and human immunodeficiency virus (HIV)-1 replication in HIV-1-infected patients. Using a lymphoma cell line, Jurkat, we examined the in vitro effects of pandemic influenza A (H1N1) virus (pH1N1) infection on cell death and HIV-1 RNA production in infected cells. We found that pH1N1 infection increased apoptotic cell death through Fas and Bax-mediated pathways in HIV-1-infected Jurkat cells. Infection with pH1N1 virus could promote HIV-1 RNA production by activating host transcription factors including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB), nuclear factor of activated T-cells (NFAT) and activator protein 1 (AP-1) through mitogen-activated protein kinases (MAPK) pathways and T-cell antigen receptor (TCR)-related pathways. The replication of HIV-1 latent infection could be reactivated by pH1N1 infection through TCR and apoptotic pathways. These data indicate that HIV-1 replication can be activated by pH1N1 virus in HIV-1-infected cells resulting in induction of cell death through apoptotic pathways.

Analysis of Host Gene Expression Profile in HIV-1 and HIV-2 Infected T-Cells.

HIV replication is closely regulated by a complex pathway of host factors, many of them being determinants of cell tropism and host susceptibility to HIV infection. These host factors are known to exert a positive or negative influence on the replication of the two major types of HIV, HIV-1 and HIV-2, thereby modulating virus infectivity, host response to infection and ultimately disease progression profiles characteristic of these two types. Understanding the differential regulation of host cellular factors in response to HIV-1 and HIV-2 infections will help us to understand the apparent differences in rates of disease progression and pathogenesis. This knowledge would aid in the discovery of new biomarkers that may serve as novel targets for therapy and diagnosis. The objective of this study was to determine the differential expression of host genes in response to HIV-1/HIV-2 infection. To achieve this, we analyzed the effects of HIV-1 (MN) and HIV-2 (ROD) infection on the expression of host factors in PBMC at the RNA level using the Agilent Whole Human Genome Oligo Microarray. Differentially expressed genes were identified and their biological functions determined. Host gene expression profiles were significantly changed. Gene expression profiling analysis identified a subset of differentially expressed genes in HIV-1 and HIV-2 infected cells. Genes involved in cellular metabolism, apoptosis, immune cell proliferation and activation, cytokines, chemokines, and transcription factors were differentially expressed in HIV-1 infected cells. Relatively few genes were differentially expressed in cells infected with HIV-2.

HIV-1 induced nuclear factor I-B (NF-IB) expression negatively regulates HIV-1 replication through interaction with the long terminal repeat region.

BACKGROUND: Retroviruses rely on host factors for cell entry, replication, transcription, and other major steps during their life cycle. Human Immunodeficiency Virus-1 (HIV-1) is well known for utilizing a plethora of strategies to evade the host immune response, including the establishment of latent infection within a subpopulation of susceptible cells. HIV-1 also manipulates cellular factors in latently infected cells and persists for long periods of time, despite the presence of successful highly active antiretroviral therapy (HAART). RESULTS: In this study we demonstrate that Nuclear Factor-IB (NF-IB) is induced during HIV-1 infection and its expression negatively impacts viral replication. During HIV-1 infection in peripheral blood mononuclear cells (PBMCs), and the T cell line, Jurkat or during induction of virus replication in latently infected cells, ACH2 and J1.1, we observed a time-dependent alteration in NF-IB expression pattern that correlated with HIV-1 viral expression. Using the Chip assay, we observed an association of NF-IB with the long terminal repeat region of HIV-1 (LTR) (-386 to -453 nt), and this association negatively correlated with HIV-1 transcription. Furthermore, knock-down of NF-IB levels in J1.1 cells resulted in an increase of HIV-1 levels. Knock-down of NF-IB levels in J-Lat-Tat-GFP (A1), (a Jurkat cell GFP reporter model for latent HIV-1 infection) resulted in an increase in GFP levels, indicating a potential negative regulatory role of NF-IB in HIV-1 replication. CONCLUSION: Overall, our results suggest that NF-IB may play a role in intrinsic antiretroviral defenses against HIV-1. These observations may offer new insights into the correlation of the latently infected host cell types and HIV-1, and help to define new therapeutic approaches for triggering the switch from latency to active replication thereby eliminating HIV-1 latent infection.

Some mechanisms of FLIP expression in inhibition of HIV-1 replication in Jurkat cells, CD4+ T cells and PBMCs.

HIV-1 infection and replication are affected by host factors. Recent studies demonstrate that molecules from apoptotic pathways regulate HIV-1 replication. Therefore, studies on effects of host factors that maintain host cell survival and influence HIV-1 replication are critical to understanding the mechanisms of HIV-1 replicative cycle. Using the susceptible Jurkat cell line, CD4(+) T cells, and peripheral blood mononuclear cells (PBMCs), we studied the role of FLIP, an inhibitor of caspase-8, in HIV-1 production. Full length cellular FLIP (cFLIP) inhibited HIV-1 replication in these cells. cFLIP upregulated the expression of viral restriction factors, such as TRIM5, Apobec3G, and Bst2/tetherin, decreased nuclear factor 1C expression and inactivated ERK and p38 induced by HIV-1 in Jurkat cells. cFLIP blocked the trafficking of gp120 and Gag p24 capsid protein into lipid rafts with inhibition of Tsg101 and Alix in ESCRT signaling pathway. cFLIP also promoted Bst2/tetherin trafficking into lipid rafts. These results indicate that cFLIP may inhibit the HIV-1 replication cycle at multiple steps, including viral RNA release, transcription, traffic and assembly. We also found that cFLIP expression downregulated Fas expression and inactivated FADD in the Fas-mediated apoptotic pathway. The inactivated FADD also inhibited HIV-1 replication.

Effect of sex steroid hormones on replication and transmission of major HIV subtypes.

BACKGROUND: The HIV epidemic is expanding worldwide with an increasing number of distinct viral subtypes and circulating recombinant forms (CRFs). Out of 34 million adults living with HIV and AIDS, women account for one half of all HIV-1 infections worldwide. These gender differences in HIV pathogenesis may be attributed to sex hormones. Little is known about the role of sex hormone effects on HIV Subtypes pathogenesis. The aim of our study was to determine sex hormone effects on replication and transmissibility of HIV subtypes. METHODS: Peripheral blood mononuclear cells (PBMC) and monocyte derived dendritic cells (MDDC) from male and female donors were infected with HIV subtypes A-D and CRF02_AG, CRF01_AE, MN (lab adapted), Group-O, Group-N and HIV-2 at a concentration of 5ng/ml of p24 or p27. Virus production was evaluated by measuring p24 and p27 levels in culture supernatants. Similar experiments were carried out in the presence of physiological concentrations of sex steroid hormones. R5/X4 expressions measured by flow cytometry and transmissibility was evaluated by transfer of HIV from primary dendritic cells (DC) to autologous donor PBMC. RESULTS: Our results from primary PBMC and MDDC from male and female donors indicate in the absence of physiological concentrations of hormone treatment virus production was observed in three clusters; high replicating virus (subtype B and C), moderate replicative virus (subtype A, D, CRF01_AE, Group_N) and least replicative virus (strain MN). However, dose of sex steroid hormone treatment influenced HIV replication and transmission kinetics in PBMC, DCs and cell lines. Such effects were inconsistent between donors and HIV subtypes. Sex hormone effects on HIV entry receptors (CCR5/CXCR4) did not correlate with virus production. CONCLUSIONS: Subtypes B and C showed higher replication in PBMC from males and females and were transmitted more efficiently through DC to male and female PBMC compared with other HIV-1 subtypes, HIV-1 Group O and HIV-2. These findings are consistent with increased worldwide prevalence of subtype B and C compared to other subtypes. Sex steroid hormones had variable effect on replication or transmission of different subtypes. These findings suggest that subtype, gender and sex hormones may play a crucial role in the replication and transmission of HIV.

Identification of XMRV infection-associated microRNAs in four cell types in culture.

INTRODUCTION: XMRV is a gammaretrovirus that was thought to be associated with prostate cancer (PC) and chronic fatigue syndrome (CFS) in humans until recently. The virus is culturable in various cells of human origin like the lymphocytes, NK cells, neuronal cells, and prostate cell lines. MicroRNAs (miRNA), which regulate gene expression, were so far not identified in cells infected with XMRV in culture. METHODS: Two prostate cell lines (LNCaP and DU145) and two primary cells, Peripheral Blood Lymphocytes [PBL] and Monocyte-derived Macrophages [MDM] were infected with XMRV. Total mRNA was extracted from mock- and virus-infected cells at 6, 24 and 48 hours post infection and evaluated for microRNA profile in a microarray. RESULTS: MicroRNA expression profiles of XMRV-infected continuous prostate cancer cell lines differ from that of virus-infected primary cells (PBL and MDMs). miR-193a-3p and miRPlus-E1245 observed to be specific to XMRV infection in all 4 cell types. While miR-193a-3p levels were down regulated miRPlus-E1245 on the other hand exhibited varied expression profile between the 4 cell types. DISCUSSION: The present study clearly demonstrates that cellular microRNAs are expressed during XMRV infection of human cells and this is the first report demonstrating the regulation of miR193a-3p and miRPlus-E1245 during XMRV infection in four different human cell types.

HIV-1 and HIV-2 infections induce autophagy in Jurkat and CD4+ T cells.

Autophagy plays important roles during innate and adaptive immune responses to pathogens, including virus infection. Viruses develop ways to subvert the pathway for their own benefit in order to escape restriction by autophagy, leading to increased viral replication and/or control over apoptosis of their host cells. The effects of HIV infection on the autophagic pathway in host cells have been little documented. Using the susceptible Jurkat cell line and CD4(+) T cells, we studied the relationship of HIV-1 and -2 infections with autophagy. We found that HIV infections significantly increase transcription of ULK1, a member of the autophagy-initiated complex. Two ubiquitin-like conjugation systems, the Atg12 conjugation system and the microtubule-associated protein L chain 3 (LC3) conjugation system that control the elongation of the autophore to form the autophagosome, were activated after HIV infection, with upregulation of Atg12-Atg5 complex and increased transcription of LC3, and formed more autophagosome in infected cells detected using an EM assay. We also found that HIV-1 induced more autophagic death in Jurkat cells relative to HIV-2, and the inhibition of autophagy with 3MA and Beclin-1 knockdown decreased HIV-1 replication significantly. The results indicate that HIV is able to induce the autophagic signaling pathway in HIV-infected host cells, which may be required for HIV infection-mediated apoptotic cell death.

Absence of detectable XMRV and other MLV-related viruses in healthy blood donors in the United States.

BACKGROUND: Preliminary studies in chronic fatigue syndrome (CFS) patients and XMRV infected animals demonstrated plasma viremia and infection of blood cells with XMRV, indicating the potential risk for transfusion transmission. XMRV and MLV-related virus gene sequences have also been detected in 4-6% of healthy individuals including blood donors in the U.S. These results imply that millions of persons in the U.S. may be carrying the nucleic acid sequences of XMRV and/or MLV-related viruses, which is a serious public health and blood safety concern. METHODOLOGY/PRINCIPAL FINDINGS: To gain evidence of XMRV or MLV-related virus infection in the U.S. blood donors, 110 plasma samples and 71 PBMC samples from blood donors at the NIH blood bank were screened for XMRV and MLV-related virus infection. We employed highly sensitive assays, including nested PCR and real-time PCR, as well as co-culture of plasma with highly sensitive indicator DERSE cells. Using these assays, none of the samples were positive for XMRV or MLV-related virus. CONCLUSIONS/SIGNIFICANCE: Our results are consistent with those from several other studies, and demonstrate the absence of XMRV or MLV-related viruses in the U.S. blood donors that we studied.

XMRV: usage of receptors and potential co-receptors.

BACKGROUND: XMRV is a gammaretrovirus first identified in prostate tissues of Prostate Cancer (PC) patients and later in the blood cells of patients with Chronic Fatigue Syndrome (CFS). Although XMRV is thought to use XPR1 for cell entry, it infects A549 cells that do not express XPR1, suggesting usage of other receptors or co-receptors. METHODS: To study the usage of different receptors and co- receptors that could play a role in XMRV infection of lymphoid cells and GHOST (GFP- Human osteosarcoma) cells expressing CD4 along with different chemokine receptors including CCR1, CCR2, etc., were infected with XMRV. Culture supernatants and cells were tested for XMRV replication using real time quantitative PCR. RESULTS: Infection and replication of XMRV was seen in a variety of GHOST cells, LNCaP, DU145, A549 and Caski cell lines. The levels of XMRV replication varied in different cell lines showing differential replication in different cell lines. However, replication in A549 which lacks XPR1 expression was relatively higher than DU145 but lower than, LNCaP. XMRV replication varied in GHOST cell lines expressing CD4 and each of the co- receptors CCR1-CCR8 and bob. There was significant replication of XMRV in CCR3 and Bonzo although it is much lower when compared to DU145, A549 and LNCaP. CONCLUSION: XMRV replication was observed in GHOST cells that express CD4 and each of the chemokine receptors ranging from CCR1- CCR8 and BOB suggesting that infectivity in hematopoietic cells could be mediated by use of these receptors.

Absence of detectable xenotropic murine leukemia virus-related virus in plasma or peripheral blood mononuclear cells of human immunodeficiency virus type 1-infected blood donors or individuals in Africa.

BACKGROUND: Since the identification of xenotropic murine leukemia virus-related virus (XMRV) in prostate cancer patients in 2006 and in chronic fatigue syndrome patients in 2009, conflicting findings have been reported regarding its etiologic role in human diseases and prevalence in general populations. In this study, we screened both plasma and peripheral blood mononuclear cells (PBMNCs) collected in Africa from blood donors and human immunodeficiency virus Type 1 (HIV-1)-infected individuals to gain evidence of XMRV infection in this geographic region. STUDY DESIGN AND METHODS: A total of 199 plasma samples, 19 PBMNC samples, and 50 culture supernatants from PBMNCs of blood donors from Cameroon found to be infected with HIV-1 and HIV-1 patients from Uganda were screened for XMRV infection using a sensitive nested polymerase chain reaction (PCR) or reverse transcription (RT)-PCR assay. RESULTS: Using highly sensitive nested PCR or RT-PCR and real-time PCR assays capable of detecting at least 10 copies of XMRV plasmid DNA per reaction, none of the 268 samples tested were found to be XMRV DNA or RNA positive. CONCLUSIONS: Our results failed to demonstrate the presence of XMRV infection in African blood donors or individuals infected with HIV-1. More studies are needed to understand the prevalence, epidemiology, and geographic distribution of XMRV infection worldwide.