Maarit Sillanpää

Hepatitis C virus NS2 and NS3/4A proteins are potent inhibitors of host cell cytokine/chemokine gene expression

BackgroundHepatitis C virus (HCV) encodes several proteins that interfere with the host cell antiviral response. Previously, the serine protease NS3/4A was shown to inhibit IFN-β gene expression by blocking dsRNA-activated retinoic acid-inducible gene I (RIG-I) and Toll-like receptor 3 (TLR3)-mediated signaling pathways.ResultsIn the present work, we systematically studied the effect of all HCV proteins on IFN gene expression. NS2 and NS3/4A inhibited IFN gene activation. NS3/4A inhibited the Sendai virus-induced expression of multiple IFN (IFN-α, IFN-β and IFN-λ1/IL-29) and chemokine (CCL5, CXCL8 and CXCL10) gene promoters. NS2 and NS3/4A, but not its proteolytically inactive form NS3/4A-S139A, were found to inhibit promoter activity induced by RIG-I or its adaptor protein Cardif (or IPS-1/MAVS/VISA). Both endogenous and transfected Cardif were proteolytically cleaved by NS3/4A but not by NS2 indicating different mechanisms of inhibition of host cell cytokine production by these HCV encoded proteases. Cardif also strongly colocalized with NS3/4A at the mitochondrial membrane, implicating the mitochondrial membrane as the site for proteolytic cleavage. In many experimental systems, IFN priming dramatically enhances RNA virus-induced IFN gene expression; pretreatment of HEK293 cells with IFN-α strongly enhanced RIG-I expression, but failed to protect Cardif from NS3/4A-mediated cleavage and failed to restore Sendai virus-induced IFN-β gene expression.ConclusionHCV NS2 and NS3/4A proteins were identified as potent inhibitors of cytokine gene expression suggesting an important role for HCV proteases in counteracting host cell antiviral response.

Severe Acute Respiratory Syndrome Coronavirus Fails To Activate Cytokine-Mediated Innate Immune Responses in Cultured Human Monocyte-Derived Dendritic Cells

ABSTRACT Activation of host innate immune responses was studied in severe acute respiratory syndrome coronavirus (SCV)-infected human A549 lung epithelial cells, macrophages, and dendritic cells (DCs). In all cell types, SCV-specific subgenomic mRNAs were seen, whereas no expression of SCV proteins was found. No induction of cytokine genes (alpha interferon [IFN-α], IFN-β, interleukin-28A/B [IL-28A/B], IL-29, tumor necrosis factor alpha, CCL5, or CXCL10) or IFN-α/β-induced MxA gene was seen in SCV-infected A549 cells, macrophages, or DCs. SCV also failed to induce DC maturation (CD86 expression) or enhance major histocompatibility complex class II expression. Our data strongly suggest that SCV fails to activate host cell cytokine gene expression in human macrophages and DCs.

Hepatitis C virus NS2 protease inhibits host cell antiviral response by inhibiting IKKε and TBK1 functions

Hepatitis C virus (HCV) encodes for several proteins that can interfere with host cell signaling and antiviral response. Previously, serine protease NS3/4A was shown to block host cell interferon (IFN) production by proteolytic cleavage of MAVS and TRIF, the adaptor molecules of the RIG‐I and TLR3 signaling pathways, respectively. This study shows that another HCV protease, NS2 can interfere efficiently with cytokine gene expression. NS2 and its proteolytically inactive mutant forms were able to inhibit type I and type III IFN, CCL5 and CXCL10 gene promoters activated by Sendai virus infection. However, the CXCL8 gene promoter was not inhibited by NS2. In addition, constitutively active RIG‐I (ΔRIG‐I), MAVS, TRIF, IKKε, and TBK1‐induced activation of IFN‐β promoter was inhibited by NS2. Cotransfection experiments with IKKε or TBK1 together with interferon regulatory factor 3 (IRF3) and HCV expression constructs revealed that NS2 in a dose‐dependent manner inhibited IKKε and especially TBK1‐induced IRF3 phosphorylation. GST pull‐down experiments with GST‐NS2 and in vitro‐translated and cell‐expressed IKKε and TBK1 demonstrated direct physical interactions of the kinases with NS2. Further evidence that the IKKε/TBK1 kinase complex is the target for NS2 was obtained from the observation that the constitutively active form of IRF3 (IRF3‐5D) activated readily IFN‐β promoter in the presence of NS2. The present study identified HCV NS2 as a potent interferon antagonist, and describes an explanation of how NS2 downregulates the major signaling pathways involved in the development of host innate antiviral responses. J. Med. Virol. 85:71–82, 2012.

Hepatitis C virus core, NS3, NS4B and NS5A are the major immunogenic proteins in humoral immunity in chronic HCV infection

BackgroundThe viral genome of hepatitis C virus constitutes a 9.6-kb single-stranded positive-sense RNA which encodes altogether 11 viral proteins. In order to study the humoral immune responses against different HCV proteins in patients suffering from chronic HCV infection, we produced three structural (core, E1 and E2) and six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) in Sf 9 insect cells by using the baculovirus expression system.ResultsThe recombinant HCV core, E1, E2, NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins were purified and used in Western blot analysis to determine antibody responses against individual HCV protein in 68 HCV RNA and antibody positive human sera that were obtained from patients suffering from genotype 1, 2, 3 or 4 infection. These sera were also analysed with INNO-LIA Score test for HCV antibodies against core, NS3, NS4AB and NS5A, and the results were similar to the ones obtained by Western blot method. Based on our Western blot analyses we found that the major immunogenic HCV antigens were the core, NS4B, NS3 and NS5A proteins which were recognized in 97%, 86%, 68% and 53% of patient sera, respectively. There were no major genotype specific differences in antibody responses to individual HCV proteins. A common feature within the studied sera was that all except two sera recognized the core protein in high titers, whereas none of the sera recognized NS2 protein and only three sera (from genotype 3) recognised NS5B.ConclusionThe data shows significant variation in the specificity in humoral immunity in chronic HCV patients.

Hepatitis C virus proteins interfere with the activation of chemokine gene promoters and downregulate chemokine gene expression

The hepatitis C virus (HCV) non-structural (NS) 3/4A protein complex inhibits the retinoic acid inducible gene I (RIG-I) pathway by proteolytically cleaving mitochondria-associated CARD-containing adaptor protein Cardif, and this leads to reduced production of beta interferon (IFN-beta). This study examined the expression of CCL5 (regulated upon activation, normal T-cell expressed and secreted, or RANTES), CXCL8 (interleukin 8) and CXCL10 (IFN-gamma-activated protein 10, or IP-10) chemokine genes in osteosarcoma cell lines that inducibly expressed NS3/4A, NS4B, core-E1-E2-p7 and the entire HCV polyprotein. Sendai virus (SeV)-induced production of IFN-beta, CCL5, CXCL8 and CXCL10 was downregulated by the NS3/4A protein complex and by the full-length HCV polyprotein. Expression of NS3/4A and the HCV polyprotein reduced the binding of interferon regulatory factors (IRFs) 1 and 3 and, to a lesser extent, nuclear factor (NF)-kappaB (p65/p50) to their respective binding elements on the CXCL10 promoter during SeV infection. Furthermore, binding of IRF1 and IRF3 to the interferon-stimulated response element-like element, and of c-Jun and phosphorylated c-Jun to the activator protein 1 element of the CXCL8 promoter, was reduced when NS3/4A and the HCV polyprotein were expressed. In cell lines expressing NS3/4A and the HCV polyprotein, the subcellular localization of mitochondria was changed, and this was kinetically associated with the partial degradation of endogenous Cardif. These results indicate that NS3/4A alone or as part of the HCV polyprotein disturbs the expression of IRF1- and IRF3-regulated genes, as well as affecting mitogen-activated protein kinase kinase- and NF-kappaB-regulated genes.

Type I interferon response against viral and non-viral gene transfer in human tumor and primary cell lines

Type I interferon (IFN‐α/β) response is one of the major host defence mechanisms against viruses. Some recent reports suggest that IFNs may interfere with the efficacy of both non‐viral and virus‐vector‐mediated therapeutic gene transfer.

Human kinome analysis reveals novel kinases contributing to virus infection and retinoic-acid inducible gene I-induced type I and type III IFN gene expression

Activation of host innate antiviral responses are mediated by retinoic-acid inducible gene I (RIG-I)-like receptors, RIG-I and melanoma differentiation-associated gene 5, and TLRs 3, 7, 8 and 9, recognising different types of viral nucleic acids. The major components of the RIG-I- and TLR pathways have putatively been identified, but previously unrecognised kinases may contribute to virus infection-induced activation of the IFN response. Here, we screened a human kinase cDNA library, termed the kinome, using an IFN-λ1 promoter-driven luciferase reporter assay in HEK293 cells during Sendai virus infection. Of the 568 kinases analysed, nearly 50 enhanced IFN-λ1 gene expression at least twofold in response to Sendai virus infection. The best activators were FYN (FYN oncogene related to SRC, FGR, YES), serine/threonine kinase 24, activin A receptor type 1 and SRPK1 (SFRS protein kinase 1). These kinases enhanced RIG-I-dependent IFN-λ1 promoter activation via IFN-stimulated response and NF-κB elements, as confirmed using mutant IFN-λ1 promoter constructs. FYN and SRPK1 enhanced IFN-λ1 and CXCL10 protein production via the RIG-I pathway, and stimulated RIG-I and MyD88-dependent phosphorylation of IRF3 and IRF7 transcription factors, respectively. We conclude that several previously unrecognised kinases, particularly FYN and SRPK1, positively regulate IFN-λ1 and similarly regulated cytokine and chemokine genes during viral infection.

P4: Epidemiology of hepatitis C virus infections in Finland between the years 1995 and 2011

PURPOSE OF THE STUDY: Recent studies have identified a seroprevalence of hepatitis C of 1.6% in the United States and 0.7% in Germany. A significant variation has, however, been detected between rural and metropolitan areas. Identification of hepatitis C virus (HCV)-positive persons for appropriate counseling and management is the major focus of national prevention programs. Currently, routine testing is recommended for persons most likely to have HCV infection. Our aim was to assess the prevalence of hepatitis C in the German Ruhr region, which is one of the largest and most densely populated metropolitan areas in Europe. METHODS: Between 06/2009 and 07/2010 8435 consecutive patients (46.4% male, 53.6% female) who were admitted to the emergency unit of a tertiary care center (St. Josef Hospital, Ruhr-University Bochum, Germany) were investigated. Tests included analysis of antibodies to HCV (anti-HCV), ASTand ALT-levels and HCV RNA, if applicable. Thirty patients with known replicative hepatitis C infection served as internal control. SUMMARY OF RESULTS: The seroprevalence of anti-HCV was 3.45% (291/8435 patients). 194/291 (66.67%) seropositive individuals were amendable for further evaluation. Viral replication was detected in 70.62% of these patients (137/194) equating to a prevalence of replicative and chronic hepatitis C of 1.62% in this population. Age and sex distribution did not differ between anti-HCV positive patients and negative controls. Following analysis of liver transaminases, significant differences in AST levels between anti-HCV positive patients and negative controls (AST antiHCV positive = 60.8 4.2 U/L; AST controls = 37.7 4.5 U/L; p < 0.01) were detected. No differences were shown between ALT levels between these groups (ALT anti-HCV positive = 51.5 4.1 U/L; ALT controls = 49.5 13.9 U/L; p > 0.05). CONCLUSIONS: The prevalence of hepatitis C in densely populated metropolitan areas seems to be higher than previously expected (3.45% versus 0.4–0.7%). Revisiting the risk stratification and a more extensive screening might contribute to more effective prevention programs. The mere evaluation of transaminases is not suitable for predicting either seroprevalence of anti-HCV antibodies or chronic hepatitis C. Furthermore, the pre-clinical identification of HCV-positive individuals might significantly reduce the risk of accidental hepatitis C transmission among healthcare workers and would lead to an increase of the early hepatitis C diagnosis targeting to an early lead-in of the antiviral therapy avoiding late complications.