Phosphoproteomic analysis of dengue virus infected U937 cells and identification of pyruvate kinase M2 as a differentially phosphorylated phosphoprotein.

Dengue virus (DENV) is an arthropod-borne Flavivirus that can cause a range of symptomatic disease in humans. There are four dengue viruses (DENV 1 to 4) and infection with one DENV only provides transient protection against a heterotypic virus. Second infections are often more severe as the disease is potentiated by antibodies from the first infection through a process known as antibody dependent enhancement (ADE) of infection. Phosphorylation is a major post-translational modification that can have marked effects on a number of processes. To date there has been little information on the phosphorylation changes induced by DENV infection. This study aimed to determine global phosphoproteome changes induced by DENV 2 in U937 cells infected under an ADE protocol. A 2-dimensional electrophoretic approach coupled with a phosphoprotein-specific dye and mass spectroscopic analysis identified 15 statistically significant differentially phosphorylated proteins upon DENV 2 infection. One protein identified as significantly differentially phosphorylated, pyruvate kinase M2 (PKM2) was validated. Treatment with a PKM2 inhibitor modestly reduced levels of infection and viral output, but no change was seen in cellular viral protein levels, suggesting that PKM2 acts on exocytic virus release. While the effect of inhibition of PKM2 was relatively modest, the results highlight the need for a greater understanding of the role of phosphoproteins in DENV infection.

High microbial translocation limits gut immune recovery during short-term HAART in the area with high prevalence of foodborne infection.

BACKGROUND: Individuals residing in areas with high prevalence of foodborne infection could have a higher risk of gut microbial translocation which may affect monocyte activation, gut immune recovery and intestinal epithelial cell damage. We aimed to measure alterations in microbial translocation, monocyte activation, gut immune recovery, and intestinal epithelial cell damage in HAART treated individuals. METHODS: A prospective, single-arm, longitudinal, cohort study was conducted among antiretroviral naïve HIV-1 infected Thai participants. All participants were in chronic stage of HIV-1 infection before starting HAART. Data and samples were collected prior to initiation of HAART and then after 24 and 48 weeks of HAART. Plasma biomarkers for microbial translocation (16S rDNA and LBP), monocyte activation (sCD14) and intestinal epithelial cell damage (I-FABP) were evaluated. We measured circulating gut-homing CD4+ T cells and circulating gut-homing Th17 cells to assess recoveries of gut immunity and gut immunity to microbial pathogens. RESULTS: The kinetic studies showed no reduction in the levels of plasma 16S rDNA, sCD14 or I-FABP, significant decrease of plasma LBP level, and slow but significant increases in the frequencies of circulating gut-homing CD4+ T cells and circulating gut-homing Th17 cells during 48 weeks of HAART. Dividing participants into low and high microbial translocation (low and high MT) groups at baseline, both groups showed persistent plasma levels of 16S rDNA, sCD14 and I-FABP, and significantly decreased plasma level of LBP. The low MT group had significantly increased frequencies of circulating gut-homing CD4+ T cells and circulating gut-homing Th17 cells during 48 weeks of HAART but this was not observed in the high MT group. CONCLUSIONS: We demonstrated persistent high microbial translocation, monocyte activation and intestinal epithelial cell damage with slow gut immune recovery during successful short-term HAART. Additionally, gut immune recovery was apparently limited by high microbial translocation. Our findings emphasize the adverse impact of high microbial translocation on gut immune recovery and the necessity of establishing a novel therapeutic intervention to inhibit microbial translocation.

Broad-Spectrum Antiviral Activity of an Ankyrin Repeat Protein on Viral Assembly against Chimeric NL4-3 Viruses Carrying Gag/PR Derived from Circulating Strains among Northern Thai Patients.

Certain proteins have demonstrated proficient human immunodeficiency virus (HIV-1) life cycle disturbance. Recently, the ankyrin repeat protein targeting the HIV-1 capsid, AnkGAG1D4, showed a negative effect on the viral assembly of the HIV-1NL4-3 laboratory strain. To extend its potential for future clinical application, the activity of AnkGAG1D4 in the inhibition of other HIV-1 circulating strains was evaluated. Chimeric NL4-3 viruses carrying patient-derived Gag/PR-coding regions were generated from 131 antiretroviral drug-naïve HIV-1 infected individuals in northern Thailand during 2001⁻2012. SupT1, a stable T-cell line expressing AnkGAG1D4 and ankyrin non-binding control (AnkA32D3), were challenged with these chimeric viruses. The p24CA sequences were analysed and classified using the K-means clustering method. Among all the classes of virus classified using the p24CA sequences, SupT1/AnkGAG1D4 demonstrated significantly lower levels of p24CA than SupT1/AnkA32D3, which was found to correlate with the syncytia formation. This result suggests that AnkGAG1D4 can significantly interfere with the chimeric viruses derived from patients with different sequences of the p24CA domain. It supports the possibility of ankyrin-based therapy as a broad alternative therapeutic molecule for HIV-1 gene therapy in the future.