Last data update: Apr 22, 2024. (Total: 46599 publications since 2009)
Records 1-18 (of 18 Records) |
Query Trace: Suryaprakash S [original query] |
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A liposome-displayed hemagglutinin vaccine platform protects mice and ferrets from heterologous influenza virus challenge
Sia ZR , He X , Zhang A , Ang JC , Shao S , Seffouh A , Huang WC , D'Agostino MR , Teimouri Dereshgi A , Suryaprakash S , Ortega J , Andersen H , Miller MS , Davidson BA , Lovell JF . Proc Natl Acad Sci U S A 2021 118 (22) Recombinant influenza virus vaccines based on hemagglutinin (HA) hold the potential to accelerate production timelines and improve efficacy relative to traditional egg-based platforms. Here, we assess a vaccine adjuvant system comprised of immunogenic liposomes that spontaneously convert soluble antigens into a particle format, displayed on the bilayer surface. When trimeric H3 HA was presented on liposomes, antigen delivery to macrophages was improved in vitro, and strong functional antibody responses were induced following intramuscular immunization of mice. Protection was conferred against challenge with a heterologous strain of H3N2 virus, and naive mice were also protected following passive serum transfer. When admixed with the particle-forming liposomes, immunization reduced viral infection severity at vaccine doses as low as 2 ng HA, highlighting dose-sparing potential. In ferrets, immunization induced neutralizing antibodies that reduced the upper respiratory viral load upon challenge with a more modern, heterologous H3N2 viral strain. To demonstrate the flexibility and modular nature of the liposome system, 10 recombinant surface antigens representing distinct influenza virus strains were bound simultaneously to generate a highly multivalent protein particle that with 5 ng individual antigen dosing induced antibodies in mice that specifically recognized the constituent immunogens and conferred protection against heterologous H5N1 influenza virus challenge. Taken together, these results show that stable presentation of recombinant HA on immunogenic liposome surfaces in an arrayed fashion enhances functional immune responses and warrants further attention for the development of broadly protective influenza virus vaccines. |
Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes.
Voss WN , Hou YJ , Johnson NV , Delidakis G , Kim JE , Javanmardi K , Horton AP , Bartzoka F , Paresi CJ , Tanno Y , Chou CW , Abbasi SA , Pickens W , George K , Boutz DR , Towers DM , McDaniel JR , Billick D , Goike J , Rowe L , Batra D , Pohl J , Lee J , Gangappa S , Sambhara S , Gadush M , Wang N , Person MD , Iverson BL , Gollihar JD , Dye J , Herbert A , Finkelstein IJ , Baric RS , McLellan JS , Georgiou G , Lavinder JJ , Ippolito GC . Science 2021 372 (6546) 1108-1112 The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following SARS-CoV-2 infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor-binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an N-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multi-donor class of "public" antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that "public" NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape. |
SARS-CoV-2 RBD Neutralizing Antibody Induction is Enhanced by Particulate Vaccination.
Huang WC , Zhou S , He X , Chiem K , Mabrouk MT , Nissly RH , Bird IM , Strauss M , Sambhara S , Ortega J , Wohlfert EA , Martinez-Sobrido L , Kuchipudi SV , Davidson BA , Lovell JF . Adv Mater 2020 32 (50) e2005637 The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a candidate vaccine antigen that binds angiotensin-converting enzyme 2 (ACE2), leading to virus entry. Here, it is shown that rapid conversion of recombinant RBD into particulate form via admixing with liposomes containing cobalt-porphyrin-phospholipid (CoPoP) potently enhances the functional antibody response. Antigen binding via His-tag insertion into the CoPoP bilayer results in a serum-stable and conformationally intact display of the RBD on the liposome surface. Compared to other vaccine formulations, immunization using CoPoP liposomes admixed with recombinant RBD induces multiple orders of magnitude higher levels of antibody titers in mice that neutralize pseudovirus cell entry, block RBD interaction with ACE2, and inhibit live virus replication. Enhanced immunogenicity can be accounted for by greater RBD uptake into antigen-presenting cells in particulate form and improved immune cell infiltration in draining lymph nodes. QS-21 inclusion in the liposomes results in an enhanced antigen-specific polyfunctional T cell response. In mice, high dose immunization results in minimal local reactogenicity, is well-tolerated, and does not elevate serum cobalt levels. Taken together, these results confirm that particulate presentation strategies for the RBD immunogen should be considered for inducing strongly neutralizing antibody responses against SARS-CoV-2. |
Conserved Oligomeric Golgi (COG) Complex Proteins Facilitate Orthopoxvirus Entry, Fusion and Spread.
Realegeno S , Priyamvada L , Kumar A , Blackburn JB , Hartloge C , Puschnik AS , Sambhara S , Olson VA , Carette JE , Lupashin V , Satheshkumar PS . Viruses 2020 12 (7) Although orthopoxviruses (OPXV) are known to encode a majority of the genes required for replication in host cells, genome-wide genetic screens have revealed that several host pathways are indispensable for OPXV infection. Through a haploid genetic screen, we previously identified several host genes required for monkeypox virus (MPXV) infection, including the individual genes that form the conserved oligomeric Golgi (COG) complex. The COG complex is an eight-protein (COG1-COG8) vesicle tethering complex important for regulating membrane trafficking, glycosylation enzymes, and maintaining Golgi structure. In this study, we investigated the role of the COG complex in OPXV infection using cell lines with individual COG gene knockout (KO) mutations. COG KO cells infected with MPXV and vaccinia virus (VACV) produced small plaques and a lower virus yield compared to wild type (WT) cells. In cells where the KO phenotype was reversed using a rescue plasmid, the size of virus plaques increased demonstrating a direct link between the decrease in viral spread and the KO of COG genes. KO cells infected with VACV displayed lower levels of viral fusion and entry compared to WT suggesting that the COG complex is important for early events in OPXV infection. Additionally, fewer actin tails were observed in VACV-infected KO cells compared to WT. Since COG complex proteins are required for cellular trafficking of glycosylated membrane proteins, the disruption of this process due to lack of individual COG complex proteins may potentially impair the virus-cell interactions required for viral entry and egress. These data validate that the COG complex previously identified in our genetic screens plays a role in OPXV infection. |
Influenza virus NS1- C/EBPβ gene regulatory complex inhibits RIG-I transcription.
Kumari R , Guo Z , Kumar A , Wiens M , Gangappa S , Katz JM , Cox NJ , Lal RB , Sarkar D , Fisher PB , Garcia-Sastre A , Fujita T , Kumar V , Sambhara S , Ranjan P , Lal SK . Antiviral Res 2020 176 104747 Influenza virus non-structural protein 1 (NS1) counteracts host antiviral innate immune responses by inhibiting Retinoic acid inducible gene-I (RIG-I) activation. However, whether NS1 also specifically regulates RIG-I transcription is unknown. Here, we identify a CCAAT/Enhancer Binding Protein beta (C/EBPbeta) binding site in the RIG-I promoter as a repressor element, and show that NS1 promotes C/EBPbeta phosphorylation and its recruitment to the RIG-I promoter as a C/EBPbeta/NS1 complex. C/EBPbeta overexpression and siRNA knockdown in human lung epithelial cells resulted in suppression and activation of RIG-I expression respectively, implying a negative regulatory role of C/EBPbeta. Further, C/EBPbeta phosphorylation, its interaction with NS1 and occupancy at the RIG-I promoter was associated with RIG-I transcriptional inhibition. These findings provide an important insight into the molecular mechanism by which influenza NS1 commandeers RIG-I transcriptional regulation and suppresses host antiviral responses. |
Adenovirus vector-based multi-epitope vaccine provides partial protection against H5, H7, and H9 avian influenza viruses.
Hassan AO , Amen O , Sayedahmed EE , Vemula SV , Amoah S , York I , Gangappa S , Sambhara S , Mittal SK . PLoS One 2017 12 (10) e0186244 The emergence of H5, H7, and H9 avian influenza virus subtypes in humans reveals their pandemic potential. Although human-to-human transmission has been limited, the genetic reassortment of the avian and human/porcine influenza viruses or mutations in some of the genes resulting in virus replication in the upper respiratory tract of humans could generate novel pandemic influenza viruses. Current vaccines do not provide cross protection against antigenically distinct strains of the H5, H7, and H9 influenza viruses. Therefore, newer vaccine approaches are needed to overcome these potential threats. We developed an egg-independent, adenovirus vector-based, multi-epitope (ME) vaccine approach using the relatively conserved immunogenic domains of the H5N1 influenza virus [M2 ectodomain (M2e), hemagglutinin (HA) fusion domain (HFD), T-cell epitope of nucleoprotein (TNP). and HA alpha-helix domain (HalphaD)]. Our ME vaccine induced humoral and cell-mediated immune responses and caused a significant reduction in the viral loads in the lungs of vaccinated mice that were challenged with antigenically distinct H5, H7, or H9 avian influenza viruses. These results suggest that our ME vaccine approach provided broad protection against the avian influenza viruses. Further improvement of this vaccine will lead to a pre-pandemic vaccine that may lower morbidity, hinder transmission, and prevent mortality in a pandemic situation before a strain-matched vaccine becomes available. |
Critical role of RIG-I and MDA5 in early and late stages of Tulane virus infection.
Chhabra P , Ranjan P , Cromeans T , Sambhara S , Vinje J . J Gen Virol 2017 98 (5) 1016-1026 Human noroviruses are a major cause of acute gastroenteritis worldwide, but the lack of a robust cell culture system or small animal model have hampered a better understanding of innate immunity against these viruses. Tulane virus (TV) is the prototype virus of a tentative new genus, Recovirus, in the family Caliciviridae. Its epidemiology and biological properties most closely resemble human norovirus. The host innate immune response to RNA virus infection primarily involves pathogen-sensing toll-like receptors (TLRs) TLR3 and TLR7 and retinoic acid-inducible gene I-like receptor RIG-I and melanoma differentiation associated gene 5 (MDA5). In this study, by using siRNA knockdown, we report that TV infection in LLC-MK2 cells results in an early [3 h post infection (h p.i.), P<0.05] RIG-I-dependent and type I interferon-mediated antiviral response, whereas an MDA5-mediated antiviral effect was observed at later (12 h p.i.; P<0.05) stages of TV replication. Induction of RIG-I and MDA5 was critical for inhibition of TV replication. Furthermore, pre-activation of the RIG-I/MDA5 pathway prevented TV replication (>900-fold decrease; P<0.05), suggesting that RIG-I and MDA5 ligands could be used to develop novel preventive and therapeutic measures against norovirus. |
Monkeypox virus host factor screen in haploid cells identifies essential role of GARP complex in extracellular virus formation.
Realegeno S , Puschnik AS , Kumar A , Goldsmith C , Burgado J , Sambhara S , Olson VA , Carroll D , Damon I , Hirata T , Kinoshita T , Carette JE , Satheshkumar PS . J Virol 2017 91 (11) Monkeypox virus (MPXV) is a human pathogen that is a member of the Orthopoxvirus genus, which includes Vaccinia virus and Variola virus (the causative agent of smallpox). Human monkeypox is considered an emerging zoonotic infectious disease. To identify host factors required for MPXV infection, we performed a genome-wide insertional mutagenesis screen in human haploid cells. The screen revealed several candidate genes, including those involved in Golgi trafficking, glycosaminoglycan biosynthesis and glycosylphosphatidylinositol (GPI) - anchor biosynthesis. We validated the role of a set of vacuolar protein sorting (VPS) genes during infection, VPS51-54, which comprise the Golgi-associated retrograde protein (GARP) complex. The GARP complex is a tethering complex involved in retrograde transport of endosomes to the trans-Golgi apparatus. Our data demonstrate that VPS52 and VPS54 were dispensable for mature virus (MV) production but were required for extracellular virus (EV) formation. For comparison, a known antiviral compound, ST-246, was used in our experiments demonstrating that EV titers in VPS52 and VPS54 knockout (KO) cells were comparable to levels exhibited by ST-246 treated wildtype cells. Confocal microscopy was used to examine actin tail formation, one of the viral egress mechanisms for cell-to-cell dissemination, and revealed an absence of actin tails in VPS52KO or VPS54KO infected cells. Further evaluation of these cells by electron microscopy demonstrated a decrease in wrapped viruses (WV) compared to wild type control. Collectively, our data demonstrate the role of GARP complex genes in double-membrane wrapping of MV necessary for EV formation, implicating the host endosomal trafficking pathway in orthopoxvirus infection.IMPORTANCE Human monkeypox is an emerging zoonotic infectious disease caused by Monkeypox virus (MPXV). Of the two MPXV clades, the Congo Basin strain is associated with severe disease, higher mortality, and increased human-to-human transmission relative to the West African strain. Monkeypox is endemic to regions of western and central Africa but was introduced into the United States in 2003 from the importation of infected animals. The threat of MPXV and other orthopoxviruses is increasing due to the absence of routine smallpox vaccination leading to a higher proportion of naive populations. In this study, we have identified and validated candidate genes that are required for MPXV infection, specifically the Golgi-associated retrograde protein (GARP) complex. Identifying host targets required for infection that prevents extracellular virus formation such as the GARP complex or the retrograde pathway can provide a potential target for anti-viral therapy. |
Influenza virus exploits tunneling nanotubes for cell-to-cell spread.
Kumar A , Kim JH , Ranjan P , Metcalfe MG , Cao W , Mishina M , Gangappa S , Guo Z , Boyden ES , Zaki S , York I , Garcia-Sastre A , Shaw M , Sambhara S . Sci Rep 2017 7 40360 Tunneling nanotubes (TNTs) represent a novel route of intercellular communication. While previous work has shown that TNTs facilitate the exchange of viral or prion proteins from infected to naive cells, it is not clear whether the viral genome is also transferred via this mechanism and further, whether transfer via this route can result in productive replication of the infectious agents in the recipient cell. Here we present evidence that lung epithelial cells are connected by TNTs, and in spite of the presence of neutralizing antibodies and an antiviral agent, Oseltamivir, influenza virus can exploit these networks to transfer viral proteins and genome from the infected to naive cell, resulting in productive viral replication in the naive cells. These observations indicate that influenza viruses can spread using these intercellular networks that connect epithelial cells, evading immune and antiviral defenses and provide an explanation for the incidence of influenza infections even in influenza-immune individuals and vaccine failures. |
Human Heat shock protein 40 (Hsp40/DnaJB1) promotes influenza A virus replication by assisting nuclear import of viral ribonucleoproteins.
Batra J , Tripathi S , Kumar A , Katz JM , Cox NJ , Lal RB , Sambhara S , Lal SK . Sci Rep 2016 6 19063 A unique feature of influenza A virus (IAV) life cycle is replication of the viral genome in the host cell nucleus. The nuclear import of IAV genome is an indispensable step in establishing virus infection. IAV nucleoprotein (NP) is known to mediate the nuclear import of viral genome via its nuclear localization signals. Here, we demonstrate that cellular heat shock protein 40 (Hsp40/DnaJB1) facilitates the nuclear import of incoming IAV viral ribonucleoproteins (vRNPs) and is important for efficient IAV replication. Hsp40 was found to interact with NP component of IAV RNPs during early stages of infection. This interaction is mediated by the J domain of Hsp40 and N-terminal region of NP. Drug or RNAi mediated inhibition of Hsp40 resulted in reduced nuclear import of IAV RNPs, diminished viral polymerase function and attenuates overall viral replication. Hsp40 was also found to be required for efficient association between NP and importin alpha, which is crucial for IAV RNP nuclear translocation. These studies demonstrate an important role for cellular chaperone Hsp40/DnaJB1 in influenza A virus life cycle by assisting nuclear trafficking of viral ribonucleoproteins. |
NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection.
Ranjan P , Singh N , Kumar A , Neerincx A , Kremmer E , Cao W , Davis WG , Katz JM , Gangappa S , Lin R , Kufer TA , Sambhara S . Eur J Immunol 2014 45 (3) 758-72 The NLR protein, NLRC5, is an important regulator of MHC I gene expression, however, the role of NLRC5 in other innate immune responses is less well defined. In the present study, we report that NLRC5 binds RIG-I and that this interaction is critical for robust antiviral responses against influenza virus. Overexpression of NLRC5 in the human lung epithelial cell line, A549, and normal human bronchial epithelial (NHBE) cells resulted in impaired replication of influenza virus A/Puerto Rico/8/34 virus (PR8) and enhanced IFN-beta expression. Influenza virus leads to induction of IFN-beta that drives RIG-I and NLRC5 expression in host cells. Our results suggest that NLRC5 extends and stabilizes influenza virus-induced RIG-I expression and delays expression of the viral inhibitor protein NS1. We show that NS1 binds to NLRC5 to suppress its function. Interaction domain mapping revealed that NLRC5 interacts with RIG-I via its N-terminal death domain and that NLRC5 enhanced antiviral activity in a LRR-domain-independent manner. Taken together, our findings identify a novel role for NLRC5 in RIG-I-mediated antiviral host responses against influenza virus infection, distinguished from the role of NLRC5 in MHC class I gene regulation. |
Activation of the RIG-I pathway during influenza vaccination enhances the germinal center reaction, promotes T follicular helper cell induction, and provides a dose-sparing effect and protective immunity.
Kulkarni RR , Rasheed MA , Bhaumik SK , Ranjan P , Cao W , Davis C , Marisetti K , Thomas S , Gangappa S , Sambhara S , Kaja MK . J Virol 2014 88 (24) 13990-4001 Pattern Recognition Receptors (PRR) sense certain molecular patterns uniquely expressed by pathogens. Retinoic-acid-inducible gene I (RIG-I) is a cytosolic PRR that senses viral nucleic acids and induces innate immune activation and secretion of type-I IFNs. Here, using influenza vaccine antigens, we investigated the consequences of activating the RIG-I pathway on antigen-specific adaptive immune responses. We found that mice immunized with influenza vaccine antigens co-administered with 5' ppp-dsRNA, a RIG-I ligand, developed robust levels of hemagglutination inhibiting antibodies, enhanced germinal center reaction and T follicular helper cell responses. In addition, RIG-I activation enhanced antibody affinity maturation and plasma cell responses in draining lymph nodes, spleen, and bone marrow and conferred protective immunity against virus challenge. Importantly, activation of RIG-I pathway was able to reduce the antigen requirement by 10-100 folds in inducing optimal influenza-specific cellular and humoral responses including protective immunity. The effects induced by 5' ppp-dsRNA were significantly dependent on type-I IFN and IPS-1 (adapter protein downstream of RIG-I pathway) signaling, but were independent of MyD88 or TLR3 mediated pathways. Our results show that activation of RIG-I-like receptor pathway programs the innate immunity to achieve qualitatively and quantitatively enhanced protective cellular adaptive immune responses even at low antigen doses and thus, indicate the potential utility of RIG-I ligands as molecular adjuvants for the viral vaccines. STUDY IMPORTANCE STATEMENT: The recently discovered RNA helicase family of RIG-I-like receptors (RLRs) is a critical component of host defense mechanisms responsible for detecting viruses and triggering innate anti-viral cytokines that help control viral replication and dissemination. In this study we show that the RLR-pathway can be effectively exploited for enhancing adaptive immunity and protective immune memory against viral infection. Our results show that activation of RIG-I pathway along with influenza vaccination programs the innate immunity to induce qualitatively and quantitatively superior protective adaptive immunity against pandemic influenza viruses. More importantly, the RIG-I activation at the time of vaccination allows induction of robust adaptive responses even at sparing vaccine antigen doses. These results highlight the potential utility of exploiting RIG-I pathway for enhancing viral vaccine specific immunity and have broader implications for designing better vaccines in general. |
RIG-I goes beyond naked recognition.
Bowzard JB , Ranjan P , Sambhara S . Cell Host Microbe 2013 13 (3) 247-9 It is currently unclear at which point during viral replication that RNA genomes are first recognized as nonself by the immune system. In this issue of Cell Host & Microbe, Weber et al. show that incoming nucleocapsid-bound genomes are sufficient to bind and activate innate immune sensors. |
Critical role of an antiviral stress granule containing RIG-I and PKR in viral detection and innate immunity.
Onomoto K , Jogi M , Yoo JS , Narita R , Morimoto S , Takemura A , Sambhara S , Kawaguchi A , Osari S , Nagata K , Matsumiya T , Namiki H , Yoneyama M , Fujita T . PLoS One 2012 7 (8) e43031 Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) function as cytoplasmic sensors for viral RNA to initiate antiviral responses including type I interferon (IFN) production. It has been unclear how RIG-I encounters and senses viral RNA. To address this issue, we examined intracellular localization of RIG-I in response to viral infection using newly generated anti-RIG-I antibody. Immunohistochemical analysis revealed that RLRs localized in virus-induced granules containing stress granule (SG) markers together with viral RNA and antiviral proteins. Because of similarity in morphology and components, we termed these aggregates antiviral stress granules (avSGs). Influenza A virus (IAV) deficient in non-structural protein 1 (NS1) efficiently generated avSGs as well as IFN, however IAV encoding NS1 produced little. Inhibition of avSGs formation by removal of either the SG component or double-stranded RNA (dsRNA)-dependent protein kinase (PKR) resulted in diminished IFN production and concomitant enhancement of viral replication. Furthermore, we observed that transfection of dsRNA resulted in IFN production in an avSGs-dependent manner. These results strongly suggest that the avSG is the locus for non-self RNA sensing and the orchestration of multiple proteins is critical in the triggering of antiviral responses. |
The 3' untranslated regions of influenza genomic sequences are 5'PPP-independent ligands for RIG-I.
Davis WG , Bowzard JB , Sharma SD , Wiens ME , Ranjan P , Gangappa S , Stuchlik O , Pohl J , Donis RO , Katz JM , Cameron CE , Fujita T , Sambhara S . PLoS One 2012 7 (3) e32661 Retinoic acid inducible gene-I (RIG-I) is a key regulator of antiviral immunity. RIG-I is generally thought to be activated by ssRNA species containing a 5'-triphosphate (PPP) group or by unphosphorylated dsRNA up to approximately 300 bp in length. However, it is not yet clear how changes in the length, nucleotide sequence, secondary structure, and 5' end modification affect the abilities of these ligands to bind and activate RIG-I. To further investigate these parameters in the context of naturally occurring ligands, we examined RNA sequences derived from the 5' and 3' untranslated regions (UTR) of the influenza virus NS1 gene segment. As expected, RIG-I-dependent interferon-beta (IFN-beta) induction by sequences from the 5' UTR of the influenza cRNA or its complement (26 nt in length) required the presence of a 5'PPP group. In contrast, activation of RIG-I by the 3' UTR cRNA sequence or its complement (172 nt) exhibited only a partial 5'PPP-dependence, as capping the 5' end or treatment with CIP showed a modest reduction in RIG-I activation. Furthermore, induction of IFN-beta by a smaller, U/A-rich region within the 3' UTR was completely 5'PPP-independent. Our findings demonstrated that RNA sequence, length, and secondary structure all contributed to whether or not the 5'PPP moiety is needed for interferon induction by RIG-I. |
Influenza A virus nucleoprotein exploits Hsp40 to inhibit PKR activation.
Sharma K , Tripathi S , Ranjan P , Kumar P , Garten R , Deyde V , Katz JM , Cox NJ , Lal RB , Sambhara S , Lal SK . PLoS One 2011 6 (6) e20215 BACKGROUND: Double-stranded RNA dependent protein kinase (PKR) is a key regulator of the anti-viral innate immune response in mammalian cells. PKR activity is regulated by a 58 kilo Dalton cellular inhibitor (P58(IPK)), which is present in inactive state as a complex with Hsp40 under normal conditions. In case of influenza A virus (IAV) infection, P58(IPK) is known to dissociate from Hsp40 and inhibit PKR activation. However the influenza virus component responsible for PKR inhibition through P58(IPK) activation was hitherto unknown. PRINCIPAL FINDINGS: Human heat shock 40 protein (Hsp40) was identified as an interacting partner of Influenza A virus nucleoprotein (IAV NP) using a yeast two-hybrid screen. This interaction was confirmed by co-immunoprecipitation studies from mammalian cells transfected with IAV NP expressing plasmid. Further, the IAV NP-Hsp40 interaction was validated in mammalian cells infected with various seasonal and pandemic strains of influenza viruses. Cellular localization studies showed that NP and Hsp40 co-localize primarily in the nucleus. During IAV infection in mammalian cells, expression of NP coincided with the dissociation of P58(IPK) from Hsp40 and decrease PKR phosphorylation. We observed that, plasmid based expression of NP in mammalian cells leads to decrease in PKR phosphorylation. Furthermore, inhibition of NP expression during influenza virus replication led to PKR activation and concomitant increase in eIF2alpha phosphorylation. Inhibition of NP expression also led to reduced IRF3 phosphorylation, enhanced IFN beta production and concomitant reduction of virus replication. Taken together our data suggest that NP is the viral factor responsible for P58(IPK) activation and subsequent inhibition of PKR-mediated host response during IAV infection. SIGNIFICANCE: Our findings demonstrate a novel role of IAV NP in inhibiting PKR-mediated anti-viral host response and help us understand P58(IPK) mediated inhibition of PKR activity during IAV infection. |
NLRX1 protein attenuates inflammatory responses to infection by interfering with the RIG-I-MAVS and TRAF6-NF-?B signaling pathways.
Allen IC , Moore CB , Schneider M , Lei Y , Davis BK , Scull MA , Gris D , Roney KE , Zimmermann AG , Bowzard JB , Ranjan P , Monroe KM , Pickles RJ , Sambhara S , Ting JP . Immunity 2011 34 (6) 854-65 The nucleotide-binding domain and leucine-rich-repeat-containing (NLR) proteins regulate innate immunity. Although the positive regulatory impact of NLRs is clear, their inhibitory roles are not well defined. We showed that Nlrx1(-/-) mice exhibited increased expression of antiviral signaling molecules IFN-beta, STAT2, OAS1, and IL-6 after influenza virus infection. Consistent with increased inflammation, Nlrx1(-/-) mice exhibited marked morbidity and histopathology. Infection of these mice with an influenza strain that carries a mutated NS-1 protein, which normally prevents IFN induction by interaction with RNA and the intracellular RNA sensor RIG-I, further exacerbated IL-6 and type I IFN signaling. NLRX1 also weakened cytokine responses to the 2009 H1N1 pandemic influenza virus in human cells. Mechanistically, Nlrx1 deletion led to constitutive interaction of MAVS and RIG-I. Additionally, an inhibitory function is identified for NLRX1 during LPS activation of macrophages where the MAVS-RIG-I pathway was not involved. NLRX1 interacts with TRAF6 and inhibits NF-kappaB activation. Thus, NLRX1 functions as a checkpoint of overzealous inflammation. |
PAMPer and tRIGer: ligand-induced activation of RIG-I.
Bowzard JB , Davis WG , Jeisy-Scott V , Ranjan P , Gangappa S , Fujita T , Sambhara S . Trends Biochem Sci 2011 36 (6) 314-9 Retinoic-acid-inducible gene-I (RIG-I) is an important component of the innate immune response to many RNA viruses that limits viral replication until adaptive immunity becomes available to clear the infection. Upon binding to the nucleic acid genomes and replication intermediates of these viruses, RIG-I undergoes a complex activation process that involves post-translational modifications and structural rearrangements. Once activated, RIG-I upregulates well-studied signal transduction pathways that lead to the production of type-I interferons (IFNs) and a large variety of antiviral IFN-stimulated genes. Thus, an effective antiviral response is dependent on the interaction between pathogen-derived ligands and RIG-I. Recent work has begun to clarify the required characteristics of RIG-I activators and is setting the stage for the identification of authentic ligands used during viral infection. |
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