Increased MDSC Accumulation and Th2 Biased Response to Influenza A Virus Infection in the Absence of TLR7 in Mice

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

5'PPP-RNA induced RIG-I activation inhibits drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza virus replication

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>in vivo </it>in mice.</p> <p>Conclusions</p> <p>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.</p

The conserved N-terminal basic residues and zinc-finger motifs of HIV-1 nucleocapsid restrict the viral cDNA synthesis during virus formation and maturation

Reverse transcription of the genomic RNA by reverse transcriptase occurs soon after HIV-1 infection of target cells. The viral nucleocapsid (NC) protein chaperones this process via its nucleic acid annealing activities and its interactions with the reverse transcriptase enzyme. To function, NC needs its two conserved zinc fingers and flanking basic residues. We recently reported a new role for NC, whereby it negatively controls reverse transcription in the course of virus formation. Indeed, deleting its zinc fingers causes reverse transcription activation in virus producer cells. To investigate this new NC function, we used viruses with subtle mutations in the conserved zinc fingers and its flanking domains. We monitored by quantitative PCR the HIV-1 DNA content in producer cells and in produced virions. Results showed that the two intact zinc-finger structures are required for the temporal control of reverse transcription by NC throughout the virus replication cycle. The N-terminal basic residues also contributed to this new role of NC, while Pro-31 residue between the zinc fingers and Lys-59 in the C-terminal region did not. These findings further highlight the importance of NC as a major target for anti-HIV-1 drugs

The 3′ Untranslated Regions of Influenza Genomic Sequences Are 5′PPP-Independent Ligands for RIG-I

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 ∼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-β (IFN-β) 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-β 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

NLRX1 Protein Attenuates Inflammatory Responses to Infection by Interfering with the RIG-I-MAVS and TRAF6-NF-κB Signaling Pathways

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

Analysis of the Initiating Events in HIV-1 Particle Assembly and Genome Packaging

HIV-1 Gag drives a number of events during the genesis of virions and is the only viral protein required for the assembly of virus-like particles in vitro and in cells. Although a reasonable understanding of the processes that accompany the later stages of HIV-1 assembly has accrued, events that occur at the initiation of assembly are less well defined. In this regard, important uncertainties include where in the cell Gag first multimerizes and interacts with the viral RNA, and whether Gag-RNA interaction requires or induces Gag multimerization in a living cell. To address these questions, we developed assays in which protein crosslinking and RNA/protein co-immunoprecipitation were coupled with membrane flotation analyses in transfected or infected cells. We found that interaction between Gag and viral RNA occurred in the cytoplasm and was independent of the ability of Gag to localize to the plasma membrane. However, Gag:RNA binding was stabilized by the C-terminal domain (CTD) of capsid (CA), which participates in Gag-Gag interactions. We also found that Gag was present as monomers and low-order multimers (e.g. dimers) but did not form higher-order multimers in the cytoplasm. Rather, high-order multimers formed only at the plasma membrane and required the presence of a membrane-binding signal, but not a Gag domain (the CA-CTD) that is essential for complete particle assembly. Finally, sequential RNA-immunoprecipitation assays indicated that at least a fraction of Gag molecules can form multimers on viral genomes in the cytoplasm. Taken together, our results suggest that HIV-1 particle assembly is initiated by the interaction between Gag and viral RNA in the cytoplasm and that this initial Gag-RNA encounter involves Gag monomers or low order multimers. These interactions per se do not induce or require high-order Gag multimerization in the cytoplasm. Instead, membrane interactions are necessary for higher order Gag multimerization and subsequent particle assembly in cells

Mutations of the Mouse ELMO Domain Containing 1 Gene (Elmod1) Link Small GTPase Signaling to Actin Cytoskeleton Dynamics in Hair Cell Stereocilia

Stereocilia, the modified microvilli projecting from the apical surfaces of the sensory hair cells of the inner ear, are essential to the mechanoelectrical transduction process underlying hearing and balance. The actin-filled stereocilia on each hair cell are tethered together by fibrous links to form a highly patterned hair bundle. Although many structural components of hair bundles have been identified, little is known about the signaling mechanisms that regulate their development, morphology, and maintenance. Here, we describe two naturally occurring, allelic mutations that result in hearing and balance deficits in mice, named roundabout (rda) and roundabout-2J (rda2J). Positional cloning identified both as mutations of the mouse ELMO domain containing 1 gene (Elmod1), a poorly characterized gene with no previously reported mutant phenotypes. The rda mutation is a 138 kb deletion that includes exons 1–5 of Elmod1, and rda2J is an intragenic duplication of exons 3–8 of Elmod1. The deafness associated with these mutations is caused by cochlear hair cell dysfunction, as indicated by conspicuous elongations and fusions of inner hair cell stereocilia and progressive degeneration of outer hair cell stereocilia. Mammalian ELMO-family proteins are known to be involved in complexes that activate small GTPases to regulate the actin cytoskeleton during phagocytosis and cell migration. ELMOD1 and ELMOD2 recently were shown to function as GTPase-activating proteins (GAPs) for the Arf family of small G proteins. Our finding connecting ELMOD1 deficiencies with stereocilia dysmorphologies thus establishes a link between the Ras superfamily of small regulatory GTPases and the actin cytoskeleton dynamics of hair cell stereocilia

Structure and immunogenicity of alternative forms of the simian immunodeficiency virus gag protein expressed using Venezuelan equine encephalitis virus replicon particles

Venezuelan equine encephalitis virus replicon particles (VRP) were engineered to express different forms of SIV Gag to compare expression in vitro, formation of intra- and extracellular structures and induction of humoral and cellular immunity in mice. The three forms examined were full-length myristylated SIV Gag (Gagmyr+), full-length Gag lacking the myristylation signal (Gagmyr-), or a truncated form of Gagmyr- comprising only the matrix and capsid domains (MA/CA). Comparison of VRP-infected primary mouse embryo fibroblasts, mouse L929 cells and primate Vero cells showed comparable expression levels for each protein, as well as extracellular virus-like particles (VRP-Gagmyr+), and distinctive cytoplasmic aggregates (VRP-Gagmyr-) with each cell type. VPR were used to immunize BALB/c mice, and immune responses were compared using an interferon (IFN)-γ ELISPOT assay and a serum antibody ELISA. Although all three VRP generated similar levels of IFN-γ-producing cells at 1 week post-boost, at 10 weeks post-boost the MA/CA-VRP-induced response was maintained at a significantly higher level relative to that induced by Gagmyr+-VRP. Antibody responses to MA/CA-VRP and Gagmyr+-VRP were not significantly different