[Show abstract][Hide abstract] ABSTRACT: The trans-activator Tat protein is a viral regulatory protein essential for HIV-1 replication. Tat trafficks to the nucleoplasm and the nucleolus. The nucleolus, a highly dynamic and structured membrane-less sub-nuclear compartment, is the site of rRNA and ribosome biogenesis and is involved in numerous cellular functions including transcriptional regulation, cell cycle control and viral infection. Importantly, transient nucleolar trafficking of both Tat and HIV-1 viral transcripts are critical in HIV-1 replication, however, the role(s) of the nucleolus in HIV-1 replication remains unclear. To better understand how the interaction of Tat with the nucleolar machinery contributes to HIV-1 pathogenesis, we investigated the quantitative changes in the composition of the nucleolar proteome of Jurkat T-cells stably expressing HIV-1 Tat fused to a TAP tag. Using an organellar proteomic approach based on mass spectrometry, coupled with Stable Isotope Labelling in Cell culture (SILAC), we quantified 520 proteins, including 49 proteins showing significant changes in abundance in Jurkat T-cell nucleolus upon Tat expression. Numerous proteins exhibiting a fold change were well characterised Tat interactors and/or known to be critical for HIV-1 replication. This suggests that the spatial control and subcellular compartimentaliation of these cellular cofactors by Tat provide an additional layer of control for regulating cellular machinery involved in HIV-1 pathogenesis. Pathway analysis and network reconstruction revealed that Tat expression specifically resulted in the nucleolar enrichment of proteins collectively participating in ribosomal biogenesis, protein homeostasis, metabolic pathways including glycolytic, pentose phosphate, nucleotides and amino acids biosynthetic pathways, stress response, T-cell signaling pathways and genome integrity. We present here the first differential profiling of the nucleolar proteome of T-cells expressing HIV-1 Tat. We discuss how these proteins collectively participate in interconnected networks converging to adapt the nucleolus dynamic activities, which favor host biosynthetic activities and may contribute to create a cellular environment supporting robust HIV-1 production.
PLoS ONE 11/2012; 7(11):e48702. DOI:10.1371/journal.pone.0048702 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It has been well established that immunological escape mutations within the hepatitis C virus genotype (gt) 1a non-structural (NS) 3/4A protease are partly prevented by a reduction in viral protease fitness. Surprisingly little is known about whether similar mutations affect proteases from other genotypes. In the present study, we assessed both the HLA-A2-restricted CTL response and gt3a NS3/4A protease fitness. Similar to gt1, the 1073-1081 epitope was immunodominant within the gt3a-specific HLA-A2-restricted CTL response, despite sequence similarity of only 56 % between the gt1a and gt3a genes. However, unlike the gt1a NS3/4A protease, all residues within the gt3a 1073-1081 epitope could be replaced sequentially by alanine while retaining protease activity, at least in part.
Journal of General Virology 05/2012; 93(Pt 8):1680-6. DOI:10.1099/vir.0.043745-0 · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV) is capable of disrupting different facets of lipid metabolism and lipids have been shown to play a crucial role in the viral life cycle. The aim of this study was to examine the effect HCV infection has on the hepatocyte metabolome. Huh-7.5 cells were infected using virus produced by the HCV J6/JFH1 cell culture system and cells were harvested 24, 48, and 72-hours following infection. Metabolic profiling was performed using a non-targeted multiple platform methodology combining ultrahigh performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS(2)) and gas chromatography/mass spectrometry (GC/MS). There was a significant increase in a number of metabolites involved in nucleotide synthesis and RNA replication during early HCV infection. NAD levels were also significantly increased along with several amino acids. A number of lipid metabolic pathways were disrupted by HCV infection, resulting in an increase in cholesterol and sphingolipid levels, altered phospholipid metabolism and a possible disruption in mitochondrial fatty acid transport. Fluctuations in 5'-methylthioadenosine levels were also noted, along with alterations in the glutathione synthesis pathway. These results highlight a number of previously unreported metabolic interactions and give a more in depth insight into the effect HCV has on host cell biochemical processes.
PLoS ONE 08/2011; 6(8):e23641. DOI:10.1371/journal.pone.0023641 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: While hepatitis C virus (HCV)-specific immune responses are attenuated in HCV/HIV co-infected patients compared to those infected with HCV alone, the reasons for this remain unclear. In this study, the proportions of regulatory, naïve, and memory T cells, along with chemokine receptor expression, were measured in co-infected and mono-infected patients to determine if there is an alteration in the phenotypic profile of lymphocytes in these patients. HCV/HIV co-infected patients had increased proportions of CD4(+) naïve cells and decreased proportions of CD4(+) effector cells when compared to HCV mono-infected patients. The proportions of CD4(+) Tregs and CD4(+) CXCR3(+) T cells were also significantly lower in co-infected patients. A decrease in CD4(+) Tregs and subsequent loss of immunosuppressive function may contribute to the accelerated progression to liver disease in co-infected individuals. Dysregulation of immune responses following reduction in the proportions of CD4(+) CXCR3(+) Th-1 cells may contribute to the reduced functional capacity of HCV-specific immune responses in co-infected patients. The findings of this study provide new information on the T-cell immunophenotype in HCV/HIV co-infected patients when compared to those infected with HCV alone, and may provide insight into why cell-mediated immune responses are diminished during HCV infection.
[Show abstract][Hide abstract] ABSTRACT: Coinfection with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) is associated with increased HCV replication and a more rapid progression to severe liver disease, including the development of cirrhosis and hepatocellular carcinoma. In this review, we discuss the current understanding of the pathogenesis of HCV/HIV coinfection and the cellular and molecular mechanisms associated with the accelerated course of liver disease. The strength and breadth of HCV-specific T-cell responses are reduced in HCV/HIV-coinfected patients compared with those infected with HCV alone, suggesting that the immunosuppression induced by HIV compromises immune responses to HCV. HCV is not directly cytopathic, but many of the pathological changes observed in the liver of infected patients are a direct result of the intrahepatic antiviral immune responses. Apoptosis also has a role in HCV-mediated liver damage through the induction of apoptotic pathways involving the host immune response and HCV viral proteins. This review summarises the evidence correlating the role of cell-mediated immune responses and apoptosis with liver disease progression in HCV/HIV-coinfected patients.
Expert Reviews in Molecular Medicine 02/2008; 10(30):e30. DOI:10.1017/S1462399408000847 · 5.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Coinfection with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) is associated with an accelerated course of HCV infection and a faster progression to severe liver disease. We have investigated whether the development of liver disease in coinfected patients is associated with specific chemokine and cytokine production. Four cohorts--HCV/HIV-coinfected patients, HCV-monoinfected patients, HIV-monoinfected patients, and healthy control subjects--were studied. Serum levels of the 10-kDa interferon- gamma -inducible protein (IP-10) were higher in all 3 groups of infected patients than in control subjects (P<.0001). HCV/HIV-coinfected patients had significantly higher IP-10 levels than monoinfected patients. In HCV-monoinfected patients, liver fibrosis scores and liver enzyme levels were positively correlated with IP-10 levels. Elevated IP-10 levels are associated with and may contribute to liver damage in both HCV-monoinfected and HCV/HIV-coinfected patients.
The Journal of Infectious Diseases 11/2007; 196(7):1053-7. DOI:10.1086/520935 · 6.00 Impact Factor