Juliet Morrison

MDA-5 Is Cleaved in Poliovirus-Infected Cells

ABSTRACT Infections with RNA viruses are sensed by the innate immune system through membrane-bound Toll-like receptors or the cytoplasmic RNA helicases RIG-I and MDA-5. It is believed that MDA-5 is crucial for sensing infections by picornaviruses, but there have been no studies on the role of this protein during infection with poliovirus, the prototypic picornavirus. Beginning at 4 h postinfection, MDA-5 protein is degraded in poliovirus-infected cells. Levels of MDA-5 declined beginning at 6 h after infection with rhinovirus type 1a or encephalomyocarditis virus, but the protein was stable in cells infected with rhinovirus type 16 or echovirus type 1. Cleavage of MDA-5 is not carried out by either poliovirus proteinase 2Apro or 3Cpro. Instead, degradation of MDA-5 in poliovirus-infected cells occurs in a proteasome- and caspase-dependent manner. Degradation of MDA-5 during poliovirus infection correlates with cleavage of poly(ADP) ribose polymerase (PARP), a hallmark of apoptosis. Induction of apoptosis by puromycin leads to cleavage of both PARP and MDA-5. The MDA-5 cleavage product observed in cells treated with puromycin is ∼90 kDa, similar in size to the putative cleavage product observed in poliovirus-infected cells. Poliovirus-induced cleavage of MDA-5 may be a mechanism to antagonize production of type I interferon in response to viral infection.

Innate Immunity Evasion by Dengue Virus

For viruses to productively infect their hosts, they must evade or inhibit important elements of the innate immune system, namely the type I interferon (IFN) response, which negatively influences the subsequent development of antigen-specific adaptive immunity against those viruses. Dengue virus (DENV) can inhibit both type I IFN production and signaling in susceptible human cells, including dendritic cells (DCs). The NS2B3 protease complex of DENV functions as an antagonist of type I IFN production, and its proteolytic activity is necessary for this function. DENV also encodes proteins that antagonize type I IFN signaling, including NS2A, NS4A, NS4B and NS5 by targeting different components of this signaling pathway, such as STATs. Importantly, the ability of the NS5 protein to bind and degrade STAT2 contributes to the limited host tropism of DENV to humans and non-human primates. In this review, we will evaluate the contribution of innate immunity evasion by DENV to the pathogenesis and host tropism of this virus.

The interferon signaling antagonist function of yellow fever virus NS5 protein is activated by type I interferon.

To successfully establish infection, flaviviruses have to overcome the antiviral state induced by type I interferon (IFN-I). The nonstructural NS5 proteins of several flaviviruses antagonize IFN-I signaling. Here we show that yellow fever virus (YFV) inhibits IFN-I signaling through a unique mechanism that involves binding of YFV NS5 to the IFN-activated transcription factor STAT2 only in cells that have been stimulated with IFN-I. This NS5-STAT2 interaction requires IFN-I-induced tyrosine phosphorylation of STAT1 and the K63-linked polyubiquitination at a lysine in the N-terminal region of YFV NS5. We identified TRIM23 as the E3 ligase that interacts with and polyubiquitinates YFV NS5 to promote its binding to STAT2 and trigger IFN-I signaling inhibition. Our results demonstrate the importance of YFV NS5 in overcoming the antiviral action of IFN-I and offer a unique example of a viral protein that is activated by the same host pathway that it inhibits.

Proteinase 2Apro Is Essential for Enterovirus Replication in Type I Interferon-Treated Cells

ABSTRACT The Picornaviridae family comprises a diverse group of small RNA viruses that cause a variety of human and animal diseases. Some of these viruses are known to induce cleavage of components of the innate immune system and to inhibit steps in the interferon pathway that lead to the production of type I interferon. There has been no study of the effect of picornaviral infection on the events that occur after interferons have been produced. To determine whether members of the Enterovirus genus can antagonize the antiviral activity of interferon-stimulated genes (ISGs), we pretreated cells with alpha interferon (IFN-α) and then infected the cells with poliovirus type 1, 2, or 3; enterovirus type 70; or human rhinovirus type 16. We found that these viruses were able to replicate in IFN-α-pretreated cells but that replication of vesicular stomatitis virus, a Rhabdovirus, and encephalomyocarditis virus (EMCV), a picornavirus of the Cardiovirus genus, was completely inhibited. Although EMCV is sensitive to IFN-α, coinfection of cells with poliovirus and EMCV leads to EMCV replication in IFN-α-pretreated cells. The enteroviral 2A proteinase (2Apro) is essential for replication in cells pretreated with interferon, because amino acid changes in this protein render poliovirus sensitive to IFN-α. The addition of the poliovirus 2Apro gene to the EMCV genome allowed EMCV to replicate in IFN-α-pretreated cells. These results support an inhibitory role for 2Apro in the most downstream event in interferon signaling, the antiviral activities of ISGs.

H7N9 and Other Pathogenic Avian Influenza Viruses Elicit a Three-Pronged Transcriptomic Signature That Is Reminiscent of 1918 Influenza Virus and Is Associated with Lethal Outcome in Mice

ABSTRACT Modulating the host response is a promising approach to treating influenza, caused by a virus whose pathogenesis is determined in part by the reaction it elicits within the host. Though the pathogenicity of emerging H7N9 influenza virus in several animal models has been reported, these studies have not included a detailed characterization of the host response following infection. Therefore, we characterized the transcriptomic response of BALB/c mice infected with H7N9 (A/Anhui/01/2013) virus and compared it to the responses induced by H5N1 (A/Vietnam/1203/2004), H7N7 (A/Netherlands/219/2003), and pandemic 2009 H1N1 (A/Mexico/4482/2009) influenza viruses. We found that responses to the H7 subtype viruses were intermediate to those elicited by H5N1 and pdm09H1N1 early in infection but that they evolved to resemble the H5N1 response as infection progressed. H5N1, H7N7, and H7N9 viruses were pathogenic in mice, and this pathogenicity correlated with increased transcription of cytokine response genes and decreased transcription of lipid metabolism and coagulation signaling genes. This three-pronged transcriptomic signature was observed in mice infected with pathogenic H1N1 strains such as the 1918 virus, indicating that it may be predictive of pathogenicity across multiple influenza virus strains. Finally, we used host transcriptomic profiling to computationally predict drugs that reverse the host response to H7N9 infection, and we identified six FDA-approved drugs that could potentially be repurposed to treat H7N9 and other pathogenic influenza viruses. IMPORTANCE Emerging avian influenza viruses are of global concern because the human population is immunologically naive to them. Current influenza drugs target viral molecules, but the high mutation rate of influenza viruses eventually leads to the development of antiviral resistance. As the host evolves far more slowly than the virus, and influenza pathogenesis is determined in part by the host response, targeting the host response is a promising approach to treating influenza. Here we characterize the host transcriptomic response to emerging H7N9 influenza virus and compare it with the responses to H7N7, H5N1, and pdm09H1N1. All three avian viruses were pathogenic in mice and elicited a transcriptomic signature that also occurs in response to the legendary 1918 influenza virus. Our work identifies host responses that could be targeted to treat severe H7N9 influenza and identifies six FDA-approved drugs that could potentially be repurposed as H7N9 influenza therapeutics.

Integrated Omics Analysis of Pathogenic Host Responses during Pandemic H1N1 Influenza Virus Infection: The Crucial Role of Lipid Metabolism

Pandemic influenza viruses modulate proinflammatory responses that can lead to immunopathogenesis. We present an extensive and systematic profiling of lipids, metabolites, and proteins in respiratory compartments of ferrets infected with either 1918 or 2009 human pandemic H1N1 influenza viruses. Integrative analysis of high-throughput omics data with virologic and histopathologic data uncovered relationships between host responses and phenotypic outcomes of viral infection. Proinflammatory lipid precursors in the trachea following 1918 infection correlated with severe tracheal lesions. Using an algorithm to infer cell quantity changes from gene expression data, we found enrichment of distinct T cell subpopulations in the trachea. There was also a predicted increase in inflammatory monocytes in the lung of 1918 virus-infected animals that was sustained throughout infection. This study presents a unique resource to the influenza research community and demonstrates the utility of an integrative systems approach for characterization of lipid metabolism alterations underlying respiratory responses to viruses.

Genomic Profiling of Collaborative Cross Founder Mice Infected with Respiratory Viruses Reveals Novel Transcripts and Infection-Related Strain-Specific Gene and Isoform Expression

Genetic variation between diverse mouse species is well-characterized, yet existing knowledge of the mouse transcriptome comes largely from one mouse strain (C57BL/6J). As such, it is unlikely to reflect the transcriptional complexity of the mouse species. Gene transcription is dynamic and condition-specific; therefore, to better understand the mouse transcriptional response to respiratory virus infection, we infected the eight founder strains of the Collaborative Cross with either influenza A virus or severe acute respiratory syndrome coronavirus and sequenced lung RNA samples at 2 and 4 days after infection. We found numerous instances of transcripts that were not present in the C57BL/6J reference annotation, indicating that a nontrivial proportion of the mouse genome is transcribed but poorly annotated. Of these novel transcripts, 2150 could be aligned to human or rat genomes, but not to existing mouse genomes, suggesting functionally conserved sequences not yet recorded in mouse genomes. We also found that respiratory virus infection induced differential expression of 4287 splicing junctions, resulting in strain-specific isoform expression. Of these, 59 were influenced by strain-specific mutations within 2 base pairs of key intron–exon boundaries, suggesting cis-regulated expression. Our results reveal the complexity of the transcriptional response to viral infection, previously undocumented genomic elements, and extensive diversity in the response across mouse strains. These findings identify hitherto unexplored transcriptional patterns and undocumented transcripts in genetically diverse mice. Host genetic variation drives the complexity and diversity of the host response by eliciting starkly different transcriptional profiles in response to a viral infection.

The 1918 Influenza Virus PB2 Protein Enhances Virulence through the Disruption of Inflammatory and Wnt-Mediated Signaling in Mice

ABSTRACT The 1918-1919 influenza pandemic remains the single greatest infectious disease outbreak in the past century. Mouse and nonhuman primate infection models have shown that the 1918 virus induces overly aggressive innate and proinflammatory responses. To understand the response to viral infection and the role of individual 1918 genes on the host response to the 1918 virus, we examined reassortant avian viruses nearly identical to the pandemic 1918 virus (1918-like avian virus) carrying either the 1918 hemagglutinin (HA) or PB2 gene. In mice, both genes enhanced 1918-like avian virus replication, but only the mammalian host adaptation of the 1918-like avian virus through reassortment of the 1918 PB2 led to increased lethality. Through the combination of viral genetics and host transcriptional profiling, we provide a multidimensional view of the molecular mechanisms by which the 1918 PB2 gene drives viral pathogenicity. We demonstrate that 1918 PB2 enhances immune and inflammatory responses concomitant with increased cellular infiltration in the lung. We also show for the first time, that 1918 PB2 expression results in the repression of both canonical and noncanonical Wnt signaling pathways, which are crucial for inflammation-mediated lung regeneration and repair. Finally, we utilize regulatory enrichment and network analysis to define the molecular regulators of inflammation, epithelial regeneration, and lung immunopathology that are dysregulated during influenza virus infection. Taken together, our data suggest that while both HA and PB2 are important for viral replication, only 1918 PB2 exacerbates lung damage in mice infected with a reassortant 1918-like avian virus. IMPORTANCE As viral pathogenesis is determined in part by the host response, understanding the key host molecular driver(s) of virus-mediated disease, in relation to individual viral genes, is a promising approach to host-oriented drug efforts in preventing disease. Previous studies have demonstrated the importance of host adaptive genes, HA and PB2, in mediating disease although the mechanisms by which they do so are still poorly understood. Here, we combine viral genetics and host transcriptional profiling to show that although both 1918 HA and 1918 PB2 are important mediators of efficient viral replication, only 1918 PB2 impacts the pathogenicity of an avian influenza virus sharing high homology to the 1918 pandemic influenza virus. We demonstrate that 1918 PB2 enhances deleterious inflammatory responses and the inhibition of regeneration and repair functions coordinated by Wnt signaling in the lungs of infected mice, thereby promoting virus-associated disease.

Evaluation of the innate immune responses to influenza and live-attenuated influenza vaccine infection in primary differentiated human nasal epithelial cells

The host innate immune response to influenza virus is a key determinant of pathogenic outcomes and long-term protective immune responses against subsequent exposures. Here, we present a direct contrast of the host responses in primary differentiated human nasal epithelial cell (hNEC) cultures following infection with either a seasonal H3N2 influenza virus (WT) or the antigenically-matched live-attenuated vaccine (LAIV) strain. Comparison of the transcriptional profiles obtained 24 and 36h post-infection showed that the magnitude of gene expression was greater in LAIV infected relative to that observed in WT infected hNEC cultures. Functional enrichment analysis revealed that the antiviral and inflammatory responses were largely driven by type III IFN induction in both WT and LAIV infected cells. However, the enrichment of biological pathways involved in the recruitment of mononuclear leukocytes, antigen-presenting cells, and T lymphocytes was uniquely observed in LAIV infected cells. These observations were reflective of the host innate immune responses observed in individuals acutely infected with influenza viruses. These findings indicate that cell-intrinsic type III IFN-mediated innate immune responses in the nasal epithelium are not only crucial for viral clearance and attenuation, but may also play an important role in the induction of protective immune responses with live-attenuated vaccines.

STAT2 signaling and dengue virus infection.

Dengue virus (DENV) is an important human pathogen whose byzantine relationship with the immune response is poorly understood. DENV causes dengue fever and dengue hemorrhagic fever/dengue shock syndrome, diseases for which palliative care is the only treatment. DENV immunopathogenesis studies are complicated by the lack of an immunocompetent small-animal model, and this has hindered anti-DENV drug and vaccine development. This review describes strategies that DENV uses to evade the type I interferon response and focuses on how data gained from the study of DENV NS5-mediated STAT2 degradation may be used to create immunocompetent DENV mouse models and design anti-DENV therapeutics.