BackgroundCytokines are the hallmark of immune response to different pathogens and often dictate the disease outcome. HIV infection and tuberculosis (TB) are more destructive when confronted together than either alone. Clinical data related to the immune status of HIV-TB patients before the initiation of any drug therapy is not well documented. This study aimed to collect the baseline information pertaining to the immune status of HIV-TB co-infected patients and correlate the same with CD4+T cell levels and viral loads at the time of diagnosis prior to any drug therapy.Methodology/Principal FindingsWe analyzed the cytokines, CD4+T cell levels and viral loads to determine the immune environment in HIV-TB co-infection. The study involved four categories namely, Healthy controls (n = 57), TB infected (n = 57), HIV infected (n = 59) and HIV-TB co-infected (n = 57) patients. The multi-partite comparison and correlation between cytokines, CD4+T-cell levels and viral loads prior to drug therapy, showed an altered TH1 and TH2 response, as indicated by the cytokine profiles and skewed IFN-γ/IL-10 ratio. Inadequate CD4+T cell counts in HIV-TB patients did not correlate with high viral loads and vice-versa. When compared to HIV category, 34% of HIV-TB patients had concurrent high plasma levels of IL-4 and TNF-α at the time of diagnosis. TB relapse was observed in 5 of these HIV-TB co-infected patients who also displayed high IFN-γ/IL-10 ratio.Conclusion/SignificanceWith these studies, we infer (i) CD4+T-cell levels as baseline criteria to report the disease progression in terms of viral load in HIV-TB co-infected patients can be misleading and (ii) co-occurrence of high TNF-α and IL-4 levels along with a high ratio of IFN-γ/IL-10, prior to drug therapy, may increase the susceptibility of HIV-TB co-infected patients to hyper-inflammation and TB relapse.

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BackgroundThe export of intron containing viral RNAs from the nucleus to the cytoplasm is an essential step in the life cycle of Human Immunodeficiency Virus-1 (HIV-1). As the eukaryotic system does not permit the transport of intron containing RNA out of the nucleus, HIV-1 makes a regulatory protein, Rev, that mediates the transportation of unspliced and partially spliced viral mRNA from the nucleus to the cytoplasm, thereby playing a decisive role in the generation of new infectious virus particles. Therefore, the host factors modulating the RNA export activity of Rev can be major determinants of virus production in an infected cell.ResultsIn this study, human Staufen-2 (hStau-2) was identified as a host factor interacting with HIV-1 Rev through affinity chromatography followed by MALDI analyses. Our experiments involving transient expressions, siRNA mediated knockdowns and infection assays conclusively established that hStau-2 is a positive regulator of HIV-1 pathogenesis. We demonstrated that Rev-hStau-2 interactions positively regulated the RNA export activity of Rev and promoted progeny virus synthesis. The Rev-hStau-2 interaction was independent of RNA despite both being RNA binding proteins. hStau-2 mutant, with mutations at Q314R-A318F-K319E, deficient of binding Rev, failed to promote hStau-2 dependent Rev activity and viral production, validating the essentiality of this protein-protein interaction. The expression of this positive regulator was elevated upon HIV-1 infection in both human T-lymphocyte and astrocyte cell lines.ConclusionsWith this study, we establish that human Staufen-2, a host factor which is up-regulated upon HIV-1 infection, interacts with HIV-1 Rev, thereby promoting its RNA export activity and progeny virus formation. Altogether, our study provides new insights into the emerging role of the Staufen family of mRNA transporters in host-pathogen interaction and supports the notion that obliterating interactions between viral and host proteins that positively regulate HIV-1 proliferation can significantly contribute to anti-retroviral treatments.

Changes in circulating microRNAs (miRNAs) in the cerebrospinal fluid (CSF) have been associated with different neurological diseases. Here, we presented results of a pilot study aimed at determining the feasibility of detecting miRNAs in the CSF of Japanese Encephalitis virus (JEV) infected individuals with acute encephalitis syndrome (AES). We demonstrated the circulating miRNA profile in CSF of acute encephalitis patients infected with JEV. Using a quantitative real-time PCR-based miRNA array, we examined the level of 87 miRNAs expressed in human exosomes isolated from CSF. Subsequently, correlation between cytokine level and miRNAs expression in CSF samples was examined. In this study, we identified and validated the upregulated expression of three miRNAs, miR-21-5p, miR-150-5p, and miR-342-3p that were specifically circulated in CSF of acute encephalitis patients infected with JEV. CSF miR-21-5p, miR-150-5p, and miR-342-3p expressions were also elevated in infected mice brain. However, the expression pattern of these miRNAs differed in neuronal cells, microglial cells, and the exosome derived from JEV-infected cell culture supernatant. Interestingly, neuronal cells infected with vaccine strain (SA-14-14) did not lead to any upregulation of these three miRNAs. Further, miR-150-5p expression was found to be negatively correlated(r = -0.5279, p = 0.016) with TNFα level. Pathway analysis of putative target genes of these miRNAs indicated involvement of TGF-β, NGF, axon guidance, and MAPK signaling pathways in JEV/AES patients. This study for the first time represents the circulating miRNA in CSF of AES patients and identified the upregulated miRNAs in JEV-infected patients and offers the basis for future investigation.

Microglia are the primary resident immune cells of the central nervous system that maintain physiological homeostasis in the brain and contribute to the pathogenesis of many psychiatric disorders and neurodegenerative diseases. Due to the lack of appropriate human cellular models, it is difficult to study the basic pathophysiological processes linking microglia to brain diseases. In this study, we adopted a microglia-like cellular model derived from peripheral blood monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-34 (IL-34). We characterized and validated this in vitro cellular model by morphology, immunocytochemistry, gene expression profiles, and functional study. Our results indicated that the iMG cells developed typical microglial ramified morphology, expressed microglial specific surface markers (P2RY12 and TMEM119), and possessed phagocytic activity. Principal component analyses and multidimensional scaling analyses of RNA-seq data showed that iMG cells were distinct from monocytes and induced macrophages (iMacs) but clustered closer to human microglia and hiPSC-induced microglia. Heatmap analyses also found that iMG cells, but not monocytes, were closely clustered with human primary microglia. Further pathway and relative expression analysis indicated that unique genes from iMG cells were involved in the regulation of the complement system, especially in the synapse and ion transport. Overall, our data demonstrated that the iMG model mimicked many features of the brain resident microglia, highlighting its utility in the study of microglial function in many brain diseases, such as schizophrenia and Alzheimer's disease (AD).

CNS associated cells are permissive to HIV-1 infection, but poor in virus production due to attenuated Rev activity. The temporal and the spatial distribution of Rev in human astrocyte 1321N1 and glioblastoma GO-G-CCM were monitored for explaining the reduced Rev activity and low viral production during HIV-1 infection. Rev remained localized to the nuclei of these cells upon infection, attenuating its export activity, as manifested by low copy number of RRE-containing viral-mRNA in the cytoplasm of these cells. In contrast to infection, when Rev alone was transiently expressed, it localized in the cytoplasm of 1321N1. The localization changed to the nucleus when Rev was expressed in the presence of other viral proteins through pro-viral DNA pNL4-3. This study, for the first time, revealed the impact of other HIV-1 proteins apart from host factors in regulating the subcellular localization of Rev in astrocytes and hence the fate of HIV-1 infection in these cells.

A single-step amino acid-catalyzed diastereoselective three-component synthesis of optically pure highly functionalized spiro[5,5]undecane-1,5,9-triones preferentially over the four stereoisomers was accomplished in very good yields with >99% ee/de. Preliminary cell culture-based in vivo screening on these molecules revealed that cis-1aca and cis-1jca are better lead compounds for HIV-1 treatment than the known antiretroviral drug azidothymidine (AZT).

Dengue virus (DENV) infection causes dengue fever in humans, which can lead to thrombocytopenia showing a marked reduction in platelet counts, and dengue hemorrhagic fever. The virus may cause thrombocytopenia either by destroying the platelets or by interfering with their generation via the process of megakaryopoiesis. MEG-01 is the human megakaryoblastic leukemia cell line that can be differentiated in vitro by phorbol-12-myristate-13-acetate (PMA) treatment to produce platelet-like-particles (PLPs). We have studied DENV infection of MEG-01 cells to understand its effect on megakaryopoiesis and the generation of PLPs. We observed that DENV could infect only naive MEG-01 cells, and differentiated cells were refractory to virus infection/replication. However, DENV-infected MEG-01 cells, when induced for differentiation with PMA, supported an enhanced viral replication. Following the virus infection, the MEG-01 cells showed a marked reduction in the surface expression of platelet markers (CD41, CD42a, and CD61), a decreased polyploidy, and significantly reduced PLP counts. DENV infection caused an enhanced Notch signaling in MEG-01 cells where the virus envelope protein was shown to interact with TAL-1, a host protein important for megakaryopoiesis. These observations provide new insight into the role of DENV in modulating the megakaryopoiesis and platelet production process.