Influenza A virus cap-snatches host RNAs based on their abundance early after infection.

The influenza A virus RNA polymerase cleaves the 5' ends of host RNAs and uses these RNA fragments as primers for viral mRNA synthesis. We performed deep sequencing of the 5' host-derived ends of the eight viral mRNAs of influenza A/Puerto Rico/8/1934 (H1N1) virus in infected A549 cells, and compared the population to those of A/Hong Kong/1/1968 (H3N2) and A/WSN/1933 (H1N1). In the three strains, the viral RNAs target different populations of host RNAs. Host RNAs are cap-snatched based on their abundance, and we found that RNAs encoding proteins involved in metabolism are overrepresented in the cap-snatched populations. Because this overrepresentation could be a reflection of the host response early after infection, and thus of the increased availability of these transcripts, our results suggest that host RNAs are cap-snatched mainly based on their abundance without preferential targeting.

A Conserved Residue, Tyrosine (Y) 84, in H5N1 Influenza A Virus NS1 Regulates IFN Signaling Responses to Enhance Viral Infection.

The non-structural protein, NS1, is a virulence factor encoded by influenza A viruses (IAVs). In this report, we provide evidence that the conserved residue, tyrosine (Y) 84, in a conserved putative SH2-binding domain in A/Duck/Hubei/2004/L-1 [H5N1] NS1 is critical for limiting an interferon (IFN) response to infection. A phenylalanine (F) substitution of this Y84 residue abolishes NS1-mediated downregulation of IFN-inducible STAT phosphorylation, and surface IFNAR1 expression. Recombinant IAV (rIAV) [H1N1] expressing A/Grey Heron/Hong Kong/837/2004 [H5N1] NS1-Y84F (rWSN-GH-NS1-Y84F) replicates to lower titers in human lung epithelial cells and is more susceptible to the antiviral effects of IFN-ß treatment compared with rIAV expressing the intact H5N1 NS1 (rWSN-GH-NS1-wt). Cells infected with rWSN-GH-NS1-Y84F express higher levels of IFN stimulated genes (ISGs) associated with an antiviral response compared with cells infected with rWSN-GH-NS1-wt. In mice, intranasal infection with rWSN-GH-NS1-Y84F resulted in a delay in onset of weight loss, reduced lung pathology, lower lung viral titers and higher ISG expression, compared with mice infected with rWSN-GH-NS1-wt. IFN-ß treatment of mice infected with rWSN-GH-NS1-Y84F reduced lung viral titers and increased lung ISG expression, but did not alter viral titers and ISG expression in mice infected with rWSN-GH-NS1-wt. Viewed altogether, these data suggest that the virulence associated with this conserved Y84 residue in NS1 is, in part, due to its role in regulating the host IFN response.

Influenza Virus Targets Class I MHC-Educated NK Cells for Immunoevasion.

The immune response to influenza virus infection comprises both innate and adaptive defenses. NK cells play an early role in the destruction of tumors and virally-infected cells. NK cells express a variety of inhibitory receptors, including those of the Ly49 family, which are functional homologs of human killer-cell immunoglobulin-like receptors (KIR). Like human KIR, Ly49 receptors inhibit NK cell-mediated lysis by binding to major histocompatibility complex class I (MHC-I) molecules that are expressed on normal cells. During NK cell maturation, the interaction of NK cell inhibitory Ly49 receptors with their MHC-I ligands results in two types of NK cells: licensed ("functional"), or unlicensed ("hypofunctional"). Despite being completely dysfunctional with regard to rejecting MHC-I-deficient cells, unlicensed NK cells represent up to half of the mature NK cell pool in rodents and humans, suggesting an alternative role for these cells in host defense. Here, we demonstrate that after influenza infection, MHC-I expression on lung epithelial cells is upregulated, and mice bearing unlicensed NK cells (Ly49-deficient NKCKD and MHC-I-deficient B2m-/- mice) survive the infection better than WT mice. Importantly, transgenic expression of an inhibitory self-MHC-I-specific Ly49 receptor in NKCKD mice restores WT influenza susceptibility, confirming a direct role for Ly49. Conversely, F(ab')2-mediated blockade of self-MHC-I-specific Ly49 inhibitory receptors protects WT mice from influenza virus infection. Mechanistically, perforin-deficient NKCKD mice succumb to influenza infection rapidly, indicating that direct cytotoxicity is necessary for unlicensed NK cell-mediated protection. Our findings demonstrate that Ly49:MHC-I interactions play a critical role in influenza virus pathogenesis. We suggest a similar role may be conserved in human KIR, and their blockade may be protective in humans.

Deep sequencing reveals the eight facets of the influenza A/HongKong/1/1968 (H3N2) virus cap-snatching process.

The influenza A virus RNA polymerase cleaves the 5' end of host pre-mRNAs and uses the capped RNA fragments as primers for viral mRNA synthesis. We performed deep sequencing of the 5' ends of viral mRNAs from all genome segments transcribed in both human (A549) and mouse (M-1) cells infected with the influenza A/HongKong/1/1968 (H3N2) virus. In addition to information on RNA motifs present, our results indicate that the host primers are divergent between the viral transcripts. We observed differences in length distributions, nucleotide motifs and the identity of the host primers between the viral mRNAs. Mapping the reads to known transcription start sites indicates that the virus targets the most abundant host mRNAs, which is likely caused by the higher expression of these genes. Our findings suggest negligible competition amongst RdRp:vRNA complexes for individual host mRNA templates during cap-snatching and provide a better understanding of the molecular mechanism governing the first step of transcription of this influenza strain.

Influenza A/Hong Kong/156/1997(H5N1) virus NS1 gene mutations F103L and M106I both increase IFN antagonism, virulence and cytoplasmic localization but differ in binding to RIG-I and CPSF30.

BACKGROUND: The genetic basis for avian to mammalian host switching in influenza A virus is largely unknown. The human A/HK/156/1997 (H5N1) virus that transmitted from poultry possesses NS1 gene mutations F103L + M106I that are virulence determinants in the mouse model of pneumonia; however their individual roles have not been determined. The emergent A/Shanghai/patient1/2013(H7N9)-like viruses also possess these mutations which may contribute to their virulence and ability to switch species. METHODS: NS1 mutant viruses were constructed by reverse genetics and site directed mutagenesis on human and mouse-adapted backbones. Mouse infections assessed virulence, virus yield, tissue infection, and IFN induction. NS1 protein properties were assessed for subcellular distribution, IFN antagonism (mouse and human), CPSF30 and RIG-I domain binding, host transcription (microarray); and the natural prevalence of 103L and 106I mutants was assessed. RESULTS: Each of the F103L and M106I mutations contributes additively to virulence to reduce the lethal dose by >800 and >3,200 fold respectively by mediating alveolar tissue infection with >100 fold increased infectious yields. The 106I NS1 mutant lost CPSF binding but the 103L mutant maintained binding that correlated with an increased general decrease in host gene expression in human but not mouse cells. Each mutation positively modulated the inhibition of IFN induction in mouse cells and activation of the IFN-ß promoter in human cells but not in combination in human cells indicating negative epistasis. Each of the F103L and M106I mutations restored a defect in cytoplasmic localization of H5N1 NS1 in mouse cells. Human H1N1 and H3N2 NS1 proteins bound to the CARD, helicase and RD RIG-I domains, whereas the H5N1 NS1 with the same consensus 103F and 106M mutations did not bind these domains, which was totally or partially restored by the M106I or F103L mutations respectively. CONCLUSIONS: The F103L and M106I mutations in the H5N1 NS1 protein each increased IFN antagonism and mediated interstitial pneumonia in mice that was associated with increased cytoplasmic localization and altered host factor binding. These mutations may contribute to the ability of previous HPAI H5N1 and recent LPAI H7N9 and H6N1 (NS1-103L+106M) viruses to switch hosts and cause disease in humans.

Mapping of clinical and expression quantitative trait loci in a sex-dependent effect of host susceptibility to mouse-adapted influenza H3N2/HK/1/68.

Seasonal influenza outbreaks and recurrent influenza pandemics present major challenges to public health. By studying immunological responses to influenza in different host species, it may be possible to discover common mechanisms of susceptibility in response to various influenza strains. This could lead to novel therapeutic targets with wide clinical application. Using a mouse-adapted strain of influenza (A/HK/1/68-MA20 [H3N2]), we produced a mouse model of severe influenza that reproduces the hallmark high viral load and overexpression of cytokines associated with susceptibility to severe influenza in humans. We mapped genetic determinants of the host response using a panel of 29 closely related mouse strains (AcB/BcA panel of recombinant congenic strains) created from influenza-susceptible A/J and influenza-resistant C57BL/6J (B6) mice. Combined clinical quantitative trait loci (QTL) and lung expression QTL mapping identified candidate genes for two sex-specific QTL on chromosomes 2 and 17. The former includes the previously described Hc gene, a deficit of which is associated with the susceptibility phenotype in females. The latter includes the phospholipase gene Pla2g7 and Tnfrsf21, a member of the TNFR superfamily. Confirmation of the gene underlying the chromosome 17 QTL may reveal new strategies for influenza treatment.

Identification of RNA helicase A as a cellular factor that interacts with influenza A virus NS1 protein and its role in the virus life cycle.

Influenza A virus NS1 protein has multiple functions in the infected cell during the virus life cycle. Identification of novel cellular factors that interact with NS1 and understanding their functions in virus infection are of great interest. Recombinant viruses carrying a tagged NS1 are valuable for investigation of interactions between NS1 and cellular factors in the context of virus infection. Here, we report the generation of replication-competent recombinant influenza A viruses bearing a Strep tag in the NS1 protein. Purification of a protein complex associated with Strep-tagged NS1 from virus-infected cells followed by mass spectrometry revealed a number of attractive host factors. Among them, we focused our study on RNA helicase A (RHA) in this report. Through biomedical and functional analyses, we demonstrated that RHA interacts with NS1 in an RNA-dependent manner. Knockdown of RHA resulted in a significant reduction on virus yield and polymerase activity in a minigenome assay. Our cell-free viral genome replication assay showed that viral RNA replication and transcription can be enhanced by addition of RHA, and the enhanced effect of RHA required its ATP-dependent helicase activity. In summary, we established a system to identify cellular factors that interact with NS1 protein during virus infection and furthermore demonstrated that RHA interacts with NS1 and enhances viral replication and transcription.

Negative regulation of lung inflammation and immunopathology by TNF-α during acute influenza infection.

Lung immunopathology is the main cause of influenza-mediated morbidity and death, and much of its molecular mechanisms remain unclear. Whereas tumor necrosis factor-α (TNF-α) is traditionally considered a proinflammatory cytokine, its role in influenza immunopathology is unresolved. We have investigated this issue by using a model of acute H1N1 influenza infection established in wild-type and TNF-α-deficient mice and evaluated lung viral clearance, inflammatory responses, and immunopathology. Whereas TNF-α was up-regulated in the lung after influenza infection, it was not required for normal influenza viral clearance. However, TNF-α deficiency led not only to a greater extent of illness but also to heightened lung immunopathology and tissue remodeling. The severe lung immunopathology was associated with increased inflammatory cell infiltration, anti-influenza adaptive immune responses, and expression of cytokines such as monocyte chemoattractant protein-1 (MCP-1) and fibrotic growth factor, TGF-ß1. Thus, in vivo neutralization of MCP-1 markedly attenuated lung immunopathology and blunted TGF-ß1 production following influenza infection in these hosts. On the other hand, in vivo transgenic expression of MCP-1 worsened lung immunopathology following influenza infection in wild-type hosts. Thus, TNF-α is dispensable for influenza clearance; however, different from the traditional belief, this cytokine is critically required for negatively regulating the extent of lung immunopathology during acute influenza infection.

Treating viral exacerbations of chronic obstructive pulmonary disease: insights from a mouse model of cigarette smoke and H1N1 influenza infection.

BACKGROUND: Chronic obstructive pulmonary disease is a progressive lung disease that is punctuated by periods of exacerbations (worsening of symptoms) that are attributable to viral infections. While rhinoviruses are most commonly isolated viruses during episodes of exacerbation, influenza viruses have the potential to become even more problematic with the increased likelihood of an epidemic. METHODOLOGY AND PRINCIPAL FINDINGS: This study examined the impact of current and potential pharmacological targets namely the systemic corticosteroid dexamethasone and the peroxisome proliferator-activated receptor-gamma agonist pioglitazone on the outcome of infection in smoke-exposed mice. C57BL/6 mice were exposed to room air or cigarette smoke for 4 days and subsequently inoculated with an H1N1 influenza A virus. Interventions were delivered daily during the course of infection. We show that smoke-exposed mice have an exacerbated inflammatory response following infection. While smoke exposure did not compromise viral clearance, precision cut lung slices from smoke-exposed mice showed greater expression of CC (MCP-1, -3), and CXC (KC, MIP-2, GCP-2) chemokines compared to controls when stimulated with a viral mimic or influenza A virus. While dexamethasone treatment partially attenuated the inflammatory response in the broncho-alveolar lavage of smoke-exposed, virally-infected animals, viral-induced neutrophilia was steroid insensitive. In contrast to controls, dexamethasone-treated smoke-exposed influenza-infected mice had a worsened health status. Pioglitazone treatment of virally-infected smoke-exposed mice proved more efficacious than the steroid intervention. Further mechanistic evaluation revealed that a deficiency in CCR2 did not improve the inflammatory outcome in smoke-exposed, virally-infected animals. CONCLUSIONS AND SIGNIFICANCE: This animal model of cigarette smoke and H1N1 influenza infection demonstrates that smoke-exposed animals are differentially primed to respond to viral insult. While providing a platform to test pharmacological interventions, this model demonstrates that treating viral exacerbations with alternative anti-inflammatory drugs, such as PPAR-gamma agonists should be further explored since they showed greater efficacy than systemic corticosteroids.

Cigarette smoke impacts immune inflammatory responses to influenza in mice.

RATIONALE: Studies have shown that cigarette smoke impacts respiratory host defense mechanisms; however, it is poorly understood how these smoke-induced changes impact the overall ability of the host to deal with pathogenic agents. OBJECTIVE: The objective of this study was to investigate the impact of mainstream cigarette smoke exposure on immune inflammatory responses and viral burden after respiratory infection with influenza A. METHODS: C57BL/6 mice were sham- or smoke-exposed for 3 to 5 mo and infected with either 2.5 x 10(3) pfu (low dose) or 2.5 x 10(5) pfu (high dose) influenza virus. MEASUREMENTS AND MAIN RESULTS: Although smoke exposure attenuated the airway's inflammatory response to low-dose infection, we observed increased inflammation in smoke-exposed compared with sham-exposed mice after infection with high-dose influenza, despite a similar rate of viral clearance. The heightened inflammatory response was associated with increased expression of tumor necrosis factor-alpha, interleukin-6, and type 1 IFN in the airway, and increased mortality. Importantly, smoke exposure did not interfere with the development of influenza-specific memory responses; sham- and smoke-exposed animals were equally protected upon viral rechallenge. CONCLUSION: Our study suggests that, in mice, cigarette smoke affects primary antiviral immune-inflammatory responses, whereas secondary immune protection remains intact.

Influenza virus receptor specificity and cell tropism in mouse and human airway epithelial cells.

Recent human infections caused by the highly pathogenic avian influenza virus H5N1 strains emphasize an urgent need for assessment of factors that allow viral transmission, replication, and intra-airway spread. Important determinants for virus infection are epithelial cell receptors identified as glycans terminated by an alpha2,3-linked sialic acid (SA) that preferentially bind avian strains and glycans terminated by an alpha2,6-linked SA that bind human strains. The mouse is often used as a model for study of influenza viruses, including recent avian strains; however, the selectivity for infection of specific respiratory cell populations is not well described, and any relationship between receptors in the mouse and human lungs is incompletely understood. Here, using in vitro human and mouse airway epithelial cell models and in vivo mouse infection, we found that the alpha2,3-linked SA receptor was expressed in ciliated airway and type II alveolar epithelial cells and was targeted for cell-specific infection in both species. The alpha2,6-linked SA receptor was not expressed in the mouse, a factor that may contribute to the inability of some human strains to efficiently infect the mouse lung. In human airway epithelial cells, alpha2,6-linked SA was expressed and functional in both ciliated and goblet cells, providing expanded cellular tropism. Differences in receptor and cell-specific expression in these species suggest that differentiated human airway epithelial cell cultures may be superior for evaluation of some human strains, while the mouse can provide a model for studying avian strains that preferentially bind only the alpha2,3-linked SA receptor.

Immunoglobulin mimicry by Hepatitis C Virus envelope protein E2.

Hepatitis C virus (HCV) establishes persistent infection in the majority of infected individuals. The currently accepted hypothesis of immune evasion by antigenic variation in hypervariable region 1 (HVR1) of glycoprotein E2 does not however, explain the lack of subsequent immune recognition. Here, we show that the N-terminal region of E2 is antigenically and structurally similar to human immunoglobulin (Ig) variable domains. E2 is recognized by anti-human IgG antibodies and also possesses common amino acid (aa) sequence features of the conserved v-gene framework regions of human Ig light chains in particular but also heavy chains and T cell receptors. Using a position specific scoring system, the degree of similarity of HVR1 to Ig types correlated with immune escape and persistence in humans and experimentally infected chimpanzees. We propose a unique role for threshold levels of Ig molecular mimicry in HCV biology that not only advances our concept of viral immune escape and persistent infection but also provides insight into host-dependent disease patterns.

VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents.

Ideally, an oncolytic virus will replicate preferentially in malignant cells, have the ability to treat disseminated metastases, and ultimately be cleared by the patient. Here we present evidence that the attenuated vesicular stomatitis strains, AV1 and AV2, embody all of these traits. We uncover the mechanism by which these mutants are selectively attenuated in interferon-responsive cells while remaining highly lytic in 80% of human tumor cell lines tested. AV1 and AV2 were tested in a xenograft model of human ovarian cancer and in an immune competent mouse model of metastatic colon cancer. While highly attenuated for growth in normal mice, both AV1 and AV2 effected complete and durable cures in the majority of treated animals when delivered systemically.

Mammalian reovirus core protein micro 2 initiates at the first start codon and is acetylated.

Mammalian reovirus is an enteric virus that contains a double-stranded RNA genome. The genome consists of ten RNA segments that encode eight structural and three non-structural proteins. The structural proteins form a double-layered structure. The innermost layer, called the core, consists of five proteins (lambda1, lambda2, lambda3, micro 2, and sigma2). Protein lambda3 is the RNA-dependent RNA polymerase (RdRp) and micro 2 is thought to be an RdRp cofactor. Translation of most reovirus proteins is known to commence at the first start codon. However, the translation initiation site of the viral core protein micro 2, encoded by the M1 RNA segment, has been in dispute. Although the theoretical molecular weight of micro 2 is 83 267 Da the actual molecular weight is unknown because micro 2 runs aberrantly in SDS-PAGE and has resisted characterization by Edman degradation, indicating that the amino terminus is post-translationally modified. In this study, we used proteolysis coupled with MALDI-Qq-TOFMS to determine that translation of micro 2 initiates at the first AUG codon, that its actual molecular weight approximates the theoretical value of 83 kDa, that the amino terminal methionine residue is removed, and that the next amino acid (alanine) is post-translationally acetylated.