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-β, 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-κB activation. Thus, NLRX1 functions as a checkpoint of overzealous inflammation.
► NLRX1 attenuates IFN induction by preventing the interaction between RIG-I and MAVS ► NLRX1 functions as a negative regulator of IFN-I and IL-6 during influenza infection ► NLRX1 attenuates inflammation by intersecting the TRAF6 pathway to affect NF-κB
The emergence and spread of Zika virus (ZIKV) presented a challenge to the diagnosis of ZIKV infections in areas with transmission of dengue (DENV) and chikungunya (CHIKV) viruses. To facilitate ...detection of ZIKV infections, and differentiate these infections from DENV and CHIKV, we developed the Trioplex real-time RT-PCR assay (Trioplex assay). Here, we describe the optimization of multiplex and singleplex formats of the assay for a variety of chemistries and instruments to facilitate global standardization and implementation. We evaluated the analytical performance of all Trioplex modalities for detection of these three pathogens in serum and whole blood, and for ZIKV in urine. The limit of detection for the three viruses and in different RNA-extraction modalities is near 10
genome copy equivalents per milliliter (GCE/mL). Simultaneous testing of more than one specimen type from each patient provides a 6.4% additional diagnostic sensitivity. Overall, the high sensitivity of the Trioplex assay demonstrates the utility of this assay ascertaining Zika cases.
Influenza A virus (IAV), similar to other viruses, exploits the machinery of human host cells for its survival and replication. We identified α‐actinin‐4, a host cytoskeletal protein, as an ...interacting partner of IAV nucleoprotein (NP). We confirmed this interaction using co‐immunoprecipitation studies, first in a coupled in vitro transcription‐translation assay and then in cells either transiently co‐expressing the two proteins or infected with whole IAV. Importantly, the NP–actinin‐4 interaction was observed in several IAV subtypes, including the 2009 H1N1 pandemic virus. Moreover, immunofluorescence studies revealed that both NP and actinin‐4 co‐localized largely around the nucleus and also in the cytoplasmic region of virus‐infected A549 cells. Silencing of actinin‐4 expression resulted in not only a significant decrease in NP, M2 and NS1 viral protein expression, but also a reduction of both NP mRNA and viral RNA levels, as well as viral titers, 24 h post‐infection with IAV, suggesting that actinin‐4 was critical for viral replication. Furthermore, actinin‐4 depletion reduced the amount of NP localized in the nucleus. Treatment of infected cells with wortmannin, a known inhibitor of actinin‐4, led to a decrease in NP mRNA levels and also caused the nuclear retention of NP, further strengthening our previous observations. Taken together, the results of the present study indicate that actinin‐4, a novel interacting partner of IAV NP, plays a crucial role in viral replication and this interaction may participate in nuclear localization of NP and/or viral ribonucleoproteins.
Structured digital
•NP physically interacts with actinin-4 by anti bait coimmunoprecipitation (1, 2)•NP and actnin-4 colocalize by fluorescence microscopy (View interaction)•NP physically interacts with actinin-4 by anti bait coimmunoprecipitation (View interaction)•NP binds to actinin-4 by anti tag coimmunoprecipitation (1, 2)•NP physically interacts with actinin-4 by anti bait coimmunoprecipitation (View interaction)•NP physically interacts with actinin-4 by anti tag coimmunoprecipitation (View interaction)•NP physically interacts with actinin-4 by anti bait coimmunoprecipitation (View interaction) •NP physically interacts with actinin-4 by anti bait coimmunoprecipitation (View interaction) •NP physically interacts with actinin-4 by two hybrid (1, 2) •NP physically interacts with actinin-4 by anti bait coimmunoprecipitation (View interaction)
Human host proteins aiding the influenza A viral nucleoprotein in replication and transcription of virus and its intracellular shuttling are not well studied. The study presented here reports the dynamics of interaction between NP and actinin‐4 and the pivotal role of cytoskeletal protein in not only trafficking of NP, but also boosting its mRNA and vRNA levels, ultimately elevating viral titers.
Emergence of drug-resistant strains of influenza viruses, including avian H5N1 with pandemic potential, 1918 and 2009 A/H1N1 pandemic viruses to currently used antiviral agents, neuraminidase ...inhibitors and M2 Ion channel blockers, underscores the importance of developing novel antiviral strategies. Activation of innate immune pathogen sensor Retinoic Acid Inducible Gene-I (RIG-I) has recently been shown to induce antiviral state.
In the present investigation, using real time RT-PCR, immunofluorescence, immunoblot, and plaque assay we show that 5'PPP-containing single stranded RNA (5'PPP-RNA), a ligand for the intracytoplasmic RNA sensor, RIG-I can be used as a prophylactic agent against known drug-resistant avian H5N1 and pandemic influenza viruses. 5'PPP-RNA treatment of human lung epithelial cells inhibited replication of drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza viruses in a RIG-I and type 1 interferon dependant manner. Additionally, 5'PPP-RNA treatment also inhibited 2009 H1N1 viral replication in vivo in mice.
Our findings suggest that 5'PPP-RNA mediated activation of RIG-I can suppress replication of influenza viruses irrespective of their genetic make-up, pathogenicity, and drug-sensitivity status.
Toll-like receptors (TLRs) play an important role in the induction of innate and adaptive immune response against influenza A virus (IAV) infection; however, the role of Toll-like receptor 7 (TLR7) ...during the innate immune response to IAV infection and the cell types affected by the absence of TLR7 are not clearly understood. In this study, we show that myeloid derived suppressor cells (MDSC) accumulate in the lungs of TLR7 deficient mice more so than in wild-type C57Bl/6 mice, and display increased cytokine expression. Furthermore, there is an increase in production of Th2 cytokines by TLR7(-/-) compared with wildtype CD4+ T-cells in vivo, leading to a Th2 polarized humoral response. Our findings indicate that TLR7 modulates the accumulation of MDSCs during an IAV infection in mice, and that lack of TLR7 signaling leads to a Th2-biased response.
Influenza A virus (IAV), similar to other viruses, exploits the machinery of human host cells for its survival and replication. We identified alpha-actinin-4, a host cytoskeletal protein, as an ...interacting partner of IAV nucleoprotein (NP). We confirmed this interaction using co-immunoprecipitation studies, first in a coupled in vitro transcription-translation assay and then in cells either transiently co-expressing the two proteins or infected with whole IAV. Importantly, the NP-actinin-4 interaction was observed in several IAV subtypes, including the 2009 H1N1 pandemic virus. Moreover, immunofluorescence studies revealed that both NP and actinin-4 co-localized largely around the nucleus and also in the cytoplasmic region of virus-infected A549 cells. Silencing of actinin-4 expression resulted in not only a significant decrease in NP, M2 and NS1 viral protein expression, but also a reduction of both NP mRNA and viral RNA levels, as well as viral titers, 24 h post-infection with IAV, suggesting that actinin-4 was critical for viral replication. Furthermore, actinin-4 depletion reduced the amount of NP localized in the nucleus. Treatment of infected cells with wortmannin, a known inhibitor of actinin-4, led to a decrease in NP mRNA levels and also caused the nuclear retention of NP, further strengthening our previous observations. Taken together, the results of the present study indicate that actinin-4, a novel interacting partner of IAV NP, plays a crucial role in viral replication and this interaction may participate in nuclear localization of NP and/or viral ribonucleoproteins. Structured digital abstract *NP physically interacts with actinin-4 by anti bait coimmunoprecipitation (1, 2)*NP and actnin-4 colocalize by fluorescence microscopy (View interaction)*NP physically interacts with actinin-4 by anti bait coimmunoprecipitation (View interaction)*NP binds to actinin-4 by anti tag coimmunoprecipitation (1, 2)*NP physically interacts with actinin-4 by anti bait coimmunoprecipitation (View interaction)*NP physically interacts with actinin-4 by anti tag coimmunoprecipitation (View interaction)*NP physically interacts with actinin-4 by anti bait coimmunoprecipitation (View interaction) *NP physically interacts with actinin-4 by anti bait coimmunoprecipitation (View interaction) *NP physically interacts with actinin-4 by two hybrid (1, 2) *NP physically interacts with actinin-4 by anti bait coimmunoprecipitation (View interaction) PUBLICATION ABSTRACT
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
Nlrx1
−/−
mice exhibited increased expression of antiviral signaling molecules IFN-β, 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-κB activation. Thus, NLRX1 functions as a checkpoint of overzealous inflammation.
All retroviruses contain three genes that encode the Gag, Pol, and Env polyproteins. Each of these precursors is in turn cleaved to form the multiple protein products found within the mature ...infectious virion. The Env glycoproteins are inserted in the lipid bilayer at the surface of the virion and interact with host-encoded receptors to mediate entry into the target cell. The cleaved products of the Pol protein, reverse transcriptase and integrase, are responsible for converting the diploid single-stranded viral RNA genome into a double-stranded DNA copy and for integrating this copy into the host genome. The Gag polyprotein functions at two distinct stages in the viral replication cycle. After expression from the integrated provirus, Gag directs the formation and budding of nascent viral particles as well as the concomitant incorporation of the enzymes of the virus via Gag-Pol fusion proteins. Late during or immediately following budding, proteolytic cleavage of Gag liberates its main constituents, matrix, capsid (CA), and nucleocapsid. Each of these proteins is thought to function early during entry, although the exact role of each is not yet clear. The experiments in this thesis focus on the role of the Gag precursor in the budding process as well as on the role of CA in post-budding events. Specifically, we were able to demonstrate that the domains of the Rous sarcoma virus (RSV) Gag protein responsible for the production of dense particles and for the final release of nascent particles were functionally replaceable by regions from other Gag proteins. We also showed that the replacement of the amino terminus of RSV Gag with that of a heterologous membrane-binding domain could target the chimeric protein to the Golgi apparatus instead of the plasma membrane. Finally, we identified suppressor mutations that restored infectivity to viruses containing lesions in a highly conserved region of CA. These compensatory mutations were found in regions of CA both near and far from the original mutation as well as in sequences not contained in the mature CA protein.
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