Krister Melén

Molecular pathogenesis of influenza A virus infection and virus-induced regulation of cytokine gene expression

Despite vaccines and antiviral substances influenza still causes significant morbidity and mortality world wide. Better understanding of the molecular mechanisms of influenza virus replication, pathogenesis and host immune responses is required for the development of more efficient means of prevention and treatment of influenza. Influenza A virus, which replicates in epithelial cells and leukocytes, regulates host cell transcriptional and translational systems and activates, as well as downregulates apoptotic pathways. Influenza A virus infection results in the production of chemotactic (RANTES, MIP-1 alpha, MCP-1, MCP-3, and IP-10), pro-inflammatory (IL-1 beta, IL-6, IL-18, and TNF-alpha), and antiviral (IFN-alpha/beta) cytokines. Cytokine gene expression is associated with the activation of NF-kappa B, AP-1, STAT and IRF signal transducing molecules in influenza A virus-infected cells. In addition of upregulating cytokine gene expression, influenza A virus infection activates caspase-1 enzyme, which is involved in the proteolytic processing of proIL-1 beta and proIL-18 into their biologically active forms. Influenza A virus-induced IFN-alpha/beta is essential in hosts antiviral defence by activating the expression of antiviral Mx, PKR and oligoadenylate synthetase genes. IFN-alpha/beta also prolongs T cell survival, upregulates IL-12 and IL-18 receptor gene expression and together with IL-18 stimulates NK and T cell IFN-gamma production and the development of Th1-type immune response.

Inflammatory responses in influenza A virus infection.

Influenza A virus causes respiratory tract infections, which are occasionally complicated by secondary bacterial infections. Influenza A virus replicates in epithelial cells and leukocytes resulting in the production of chemokines and cytokines, which favor the extravasation of blood mononuclear cells and the development of antiviral and Th1-type immune response. Influenza A virus-infected respiratory epithelial cells produce limited amounts of chemokines (RANTES, MCP-1, IL-8) and IFN-alpha/beta, whereas monocytes/macrophages readily produce chemokines such as RANTES, MIP-1alpha, MCP-1, MCP-3, IP-10 and cytokines TNF-alpha, IL-1beta, IL-6, IL-18 and IFN-alpha/beta. The role of influenza A virus-induced inflammatory response in relation to otitis media is being discussed.

NF-κB Is Transported into the Nucleus by Importin α3 and Importin α4

NF-κB transcription factors are retained in the cytoplasm in an inactive form until they are activated and rapidly imported into the nucleus. We identified importin α3 and importin α4 as the main importin α isoforms mediating TNF-α-stimulated NF-κB p50/p65 heterodimer translocation into the nucleus. Importin α3 and α4 are close relatives in the human importin α family. We show that importin α3 isoform also mediates nuclear import of NF-κB p50 homodimer in nonstimulated cells. Importin α3 is shown to directly bind to previously characterized nuclear localization signals (NLSs) of NF-κB p50 and p65 proteins. Importin α molecules are known to have armadillo repeats that constitute the N-terminal and C-terminal NLS binding sites. We demonstrate by site-directed mutagenesis that NF-κB p50 binds to the N-terminal and p65 to the C-terminal NLS binding site of importin α3. In vitro competition experiments and analysis of cellular NF-κB suggest that NF-κB binds to importin α only when it is free of IκBα. The present study demonstrates that the nuclear import of NF-κB is a highly regulated process mediated by a subset of importin α molecules.

Arginine/Lysine-rich Nuclear Localization Signals Mediate Interactions between Dimeric STATs and Importin α5

Interferon stimulation results in tyrosine phosphorylation, dimerization, and nuclear import of STATs (signal transducers and activators of transcription). Proteins to be targeted into the nucleus usually contain nuclear localization signals (NLSs), which interact with importin α. Importin α binds to importin β, which docks the protein complex to nuclear pores, and the complex translocates into the nucleus. Here we show that baculovirus-produced and -activated STAT1 homodimers and STAT1-STAT2 heterodimers directly interacted with importin α5 (NPI-1). This interaction was very stable and was dependent on lysines 410 and 413 of STAT1. Only STAT dimers that had two intact NLS elements, one in each monomer, were able to bind to importin α5. STAT-importin α5 complexes apparently consisted of two STAT and two importin α molecules. STAT NLS-dependent colocalization of importin α5 with STAT1 or STAT2 was seen in the nucleus of transfected cells. γ-Activated sequence DNA elements efficiently inhibited STAT binding to importin α5 suggesting that the DNA and importin α binding sites are close to each other in STAT dimers. Our results demonstrate that specific NLSs in STATs mediate direct interactions of STAT dimers with importin α, which activates the nuclear import process.

Nuclear and Nucleolar Targeting of Influenza A Virus NS1 Protein: Striking Differences between Different Virus Subtypes

ABSTRACT Influenza A virus nonstructural protein 1 (NS1A protein) is a virulence factor which is targeted into the nucleus. It is a multifunctional protein that inhibits host cell pre-mRNA processing and counteracts host cell antiviral responses. We show that the NS1A protein can interact with all six human importin α isoforms, indicating that the nuclear translocation of NS1A protein is mediated by the classical importin α/β pathway. The NS1A protein of the H1N1 (WSN/33) virus has only one N-terminal arginine- or lysine-rich nuclear localization signal (NLS1), whereas the NS1A protein of the H3N2 subtype (Udorn/72) virus also has a second C-terminal NLS (NLS2). NLS1 is mapped to residues 35 to 41, which also function in the double-stranded RNA-binding activity of the NS1A protein. NLS2 was created by a 7-amino-acid C-terminal extension (residues 231 to 237) that became prevalent among human influenza A virus types isolated between the years 1950 to 1987. NLS2 includes basic amino acids at positions 219, 220, 224, 229, 231, and 232. Surprisingly, NLS2 also forms a functional nucleolar localization signal NoLS, a function that was retained in H3N2 type virus NS1A proteins even without the C-terminal extension. It is likely that the evolutionarily well-conserved nucleolar targeting function of NS1A protein plays a role in the pathogenesis of influenza A virus.

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.

NF-κB p52, RelB and c-Rel are transported into the nucleus via a subset of importin α molecules

In resting cells NF-kappaB transcription factors are retained in the cytoplasm as latent inactive complexes, until they are activated and rapidly transported into the nucleus. We show that all NF-kappaB proteins are imported into the nucleus via a subset of importin alpha isoforms. Our data indicate that the NF-kappaB components of the classical and alternative pathways have somewhat different specifities to importin alpha molecules. Based on the results from binding experiments of in vitro-translated and Sendai virus infection-induced or TNF-alpha-stimulated endogenous NF-kappaB proteins, it can be predicted that the specifity of NF-kappaB proteins to importin alpha molecules is different and changes upon the composition of the imported dimer. p52 protein binds directly to importin alpha3, alpha4, alpha5 and alpha6 and c-Rel binds to importin alpha5, alpha6 and alpha7 via a previously described monopartite nuclear localization signals (NLSs). Here we show that RelB, instead, has a bipartite arginine/lysine-rich NLS that mediates the binding of RelB to importin alpha5 and alpha6 and subsequent nuclear translocation of the protein. Moreover, we show that the nuclear import of p52/RelB heterodimers is mediated exclusively by the NLS of RelB. In addition, we found that the NLS of p52 mediates the nuclear import of p52/p65 heterodimers.

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.

Interferon-induced gene expression and signaling in human hepatoma cell lines

BACKGROUND/AIM Interferon(IFN)-alpha alone or combined with other antiviral substances has been extensively used for the treatment of viral infections of the liver. Since the molecular mechanisms of IFN action in liver cells are relatively poorly characterized, we studied IFN-induced gene expression and signaling in human hepatoma, HepG2 and HuH7 cell lines. METHODS/RESULTS IFN binding to its specific cell surface receptor leads to activation of the Janus family tyrosine kinase (JAK) - signal transducer and activator of transcription (STAT) pathway. We observed that in HepG2 and HuH7 cells IFN-inducible genes were upregulated by IFNs, but relatively high concentrations of IFN-alpha were needed to turn on MxA (an antiviral gene) and MxB gene expression. The basal expression of IFN-alpha receptor (IFNAR1 and IF-NAR2) JAK1 and TYK2 mRNAs was readily detectable, and their expression was not significantly altered by treatment with either IFN-alpha or IFN-gamma. Hepatoma cells possessed relatively low basal expression levels of IFN signaling molecules STAT1, STAT2 and p48, but their expression was strongly upregulated by both types of IFNs. Pretreatment of HepG2 or HuH7 with low IFN-gamma doses, followed by stimulation with IFN-alpha, resulted in a marked enhancement of the formation of IFN-alpha-specific signaling complex ISGF3. CONCLUSION The results indicate positive feedback mechanisms in the IFN signaling system in hepatoma cells.

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.