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Influenza A viruses are human pathogens with limited therapeutic options. Therefore, it is crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. Here we show that gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway and a binding partner of the virulence factor NS1 protein, inhibits M mRNA nuclear export without altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that preferentially inhibits nuclear export of a subset of viral and cellular mRNAs without altering bulk cellular mRNA export. These findings underscore specific nuclear export requirements for viral mRNAs and phenocopy down-regulation of the mRNA export factor UAP56. This RNA export inhibitor impaired replication of diverse influenza A virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus., Author summary Influenza virus remains a major public health threat killing ~250,000–500,000 people yearly. In pandemic years, influenza infection can lead to even higher mortality rates, as in 1918 when at least 20 million deaths occurred worldwide. Available treatments include vaccines and a few antiviral drugs, but both are limited by mutability of the virus and the development of resistance. Here we leveraged on knowledge of our previous work that uncovered a unique influenza virus pathway inside the nucleus of the host cell, which have the potential to be targeted by a novel therapeutic strategy. We designed and performed a new screen to identify chemical compounds that inhibited viral mRNA production and transport from the nucleus to the cytoplasm of the host cell. Among these compounds, we highlight a novel nuclear export inhibitor of a subset of viral and cellular mRNAs that does not substantially alter bulk cellular RNA export or levels at non-toxic dosages. Consequently, this small molecule inhibits virus replication and therefore can be used as a lead for drug development and serve as an experimental tool for further understanding key steps of the influenza virus mRNA nuclear export pathway.

The RNA helicase LGP2 (Laboratory of Genetics and Physiology 2) is a non-signaling member of the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), whose pivotal role on innate immune responses against RNA viruses is being increasingly uncovered. LGP2 is known to work in synergy with melanoma differentiation-associated gene 5 (MDA5) to promote the antiviral response induced by picornavirus infection. Here, we describe the activity of the foot-and-mouth disease virus (FMDV) Leader protease (Lpro) targeting LGP2 for cleavage. When LGP2 and Lpro were co-expressed, cleavage products were observed in an Lpro dose-dependent manner while co-expression with a catalytically inactive Lpro mutant had no effect on LGP2 levels or pattern. We further show that Lpro localizes and immunoprecipitates with LGP2 in transfected cells supporting their interaction within the cytoplasm. Evidence of LGP2 proteolysis was also detected during FMDV infection. Moreover, the inhibitory effect of LGP2 overexpression on FMDV growth observed was reverted when Lpro was co-expressed, concomitant with lower levels of IFN-β mRNA and antiviral activity in those cells. The Lpro target site in LGP2 was identified as an RGRAR sequence in a conserved helicase motif whose replacement to EGEAE abrogated LGP2 cleavage by Lpro. Taken together, these data suggest that LGP2 cleavage by the Leader protease of aphthoviruses may represent a novel antagonistic mechanism for immune evasion., Spanish Ministry of Economy, Industry and Competitiveness (MINECO) (MS) and P2013/ABI-2906 (co-financed by Autonomous Community of Madrid and EC FEDER funds) (FS)

Influenza A virus usurps host signaling factors to regulate its replication. One example is mTOR, a cellular regulator of protein synthesis, growth and motility. While the role of mTORC1 in viral infection has been studied, the mechanisms that induce mTORC1 activation and the substrates regulated by mTORC1 during influenza virus infection have not been established. In addition, the role of mTORC2 during influenza virus infection remains unknown. Here we show that mTORC2 and PDPK1 differentially phosphorylate AKT upon influenza virus infection. PDPK1-mediated phoshorylation of AKT at a distinct site is required for mTORC1 activation by influenza virus. On the other hand, the viral NS1 protein promotes phosphorylation of AKT at a different site via mTORC2, which is an activity dispensable for mTORC1 stimulation but known to regulate apoptosis. Influenza virus HA protein and down-regulation of the mTORC1 inhibitor REDD1 by the virus M2 protein promote mTORC1 activity. Systematic phosphoproteomics analysis performed in cells lacking the mTORC2 component Rictor in the absence or presence of Torin, an inhibitor of both mTORC1 and mTORC2, revealed mTORC1-dependent substrates regulated during infection. Members of pathways that regulate mTORC1 or are regulated by mTORC1 were identified, including constituents of the translation machinery that once activated can promote translation. mTORC1 activation supports viral protein expression and replication. As mTORC1 activation is optimal midway through the virus life cycle, the observed effects on viral protein expression likely support the late stages of influenza virus replication when infected cells undergo significant stress., Author summary Drug-resistant influenza viruses commonly arise due to frequent genetic changes and current antiviral drugs are not highly efficient. These underscore the need for new antiviral therapies effective against influenza viruses. Understanding how influenza virus uses cellular proteins for infection can potentially identify novel targets for pharmacological intervention. Influenza virus modulates cellular pathways to promote its replication and avoid immune restriction. Here we reveal the interplay between the cellular protein mTOR, which functions in two distinct protein complexes, and influenza virus infection. mTOR complex 1 (mTORC1) is activated during influenza virus infection through a cascade of specific modifications, or phosphorylation events, and by reducing the levels of another cellular protein termed REDD1, which is an mTORC1 inhibitor. Activation of mTORC1 results in additional phosphorylation events that together promote viral protein expression and replication. On the other hand, mTOR complex 2 (mTORC2) phosphorylates AKT at a specific site during infection, which is a process mediated by the viral NS1 protein that is known to regulate viral-mediated cell death. Since these effects occur midway through the virus life cycle in the infected cell, mTORC1 and mTORC2 activation are likely important to regulate the cellular environment in order to facilitate the late stages of viral infection.

The interferon-induced transmembrane (IFITM) protein family is a group of antiviral restriction factors that impair flexibility and inhibit membrane fusion at the plasma or the endosomal membrane, restricting viral progression at entry. While IFITMs are widely known to inhibit several single-stranded RNA viruses, there are limited reports available regarding their effect in double-stranded DNA viruses. In this work, we have analyzed a possible antiviral function of IFITMs against a double stranded DNA virus, the African swine fever virus (ASFV). Infection with cell-adapted ASFV isolate Ba71V is IFN sensitive and it induces IFITMs expression. Interestingly, high levels of IFITMs caused a collapse of the endosomal pathway to the perinuclear area. Given that ASFV entry is strongly dependent on endocytosis, we investigated whether IFITM expression could impair viral infection. Expression of IFITM1, 2 and 3 reduced virus infectivity in Vero cells, with IFITM2 and IFITM3 having an impact on viral entry/uncoating. The role of IFITM2 in the inhibition of ASFV in Vero cells could be related to impaired endocytosis-mediated viral entry and alterations in the cholesterol efflux, suggesting that IFITM2 is acting at the late endosome, preventing the decapsidation stage of ASFV. Copyright © 2016 Muñoz-Moreno et al.

A novel H1N1 influenza A virus caused the first pandemic of the 21st century in 2009. Hospitals had an increased demand of health consultations, that made it difficult to estimate the incidence of infection in hospital personnel due to asymptomatic presentations and the under notification of cases. To estimate and compare the rate of exposure of high versus low risk health personnel to 2009 pandemic H1N1 (H1N1pdm2009) influenza A virus in a University Hospital in Chile, we performed a comparative and prospective study. Serum samples were obtained from 117 individuals that worked in the emergency room (ER) and the operating room (OR) during the peak of the pandemic. Antibody titers were determined by the hemagglutination inhibition (HI) assay. Of the samples analyzed, 65% were workers at the ER and 35% at the OR. Of the total number of the subjects tested, 29.1% were seropositive. One out of 3 (36.8%) workers at the ER had positive HI titers, meanwhile only 1 out of 7 (14.6%) workers from the OR was seropositive to the virus. The possibility of being infected in the ER as compared to the OR was 3.4 times greater (OR 3.4; CI 95%, 1.27–9.1), and the individuals of the ER had almost twice as much antibody titers against H1N1pdm2009 than the personnel in the OR, suggesting the potential of more than one exposure to the virus. Of the 34 seropositive subjects, 12 (35.3%) did not develop influenza like illness, including 2 non-clinical personnel involved in direct contact with patients at the ER. Considering the estimated population attack rate in Chile of 13%, both groups presented a higher exposure and seropositive rate than the general population, with ER personnel showing greater risk of infection and a significantly higher level of antibodies. This data provide a strong rationale to design improved control measures aimed at all the hospital personnel, including those coming into contact with the patients prior to triage, to prevent the propagation and transmission of respiratory viruses, particularly during a pandemic outbreak.

Citation: Ma, W. J., Garcia-Sastre, A., & Schwemmle, M. (2015). Expected and Unexpected Features of the Newly Discovered Bat Influenza A-like Viruses. Plos Pathogens, 11(6), 6. doi:10.1371/journal.ppat.1004819

Upon viral infection, the production of type I interferon (IFN) and the subsequent upregulation of IFN stimulated genes (ISGs) generate an antiviral state with an important role in the activation of innate and adaptive host immune responses. The ubiquitin-like protein (UBL) ISG15 is a critical IFN-induced antiviral molecule that protects against several viral infections, but the mechanism by which ISG15 exerts its antiviral function is not completely understood. Here, we report that ISG15 plays an important role in the regulation of macrophage responses. ISG15-/- macrophages display reduced activation, phagocytic capacity and programmed cell death activation in response to vaccinia virus (VACV) infection. Moreover, peritoneal macrophages from mice lacking ISG15 are neither able to phagocyte infected cells nor to block viral infection in co-culture experiments with VACV-infected murine embryonic fibroblast (MEFs). This phenotype is independent of cytokine production and secretion, but clearly correlates with impaired activation of the protein kinase AKT in ISG15 knock-out (KO) macrophages. Altogether, these results indicate an essential role of ISG15 in the cellular immune antiviral response and point out that a better understanding of the antiviral responses triggered by ISG15 may lead to the development of therapies against important human pathogens.

Work in St. Andrews was supported by the Medical Research Council, UK (RER and RJR), and the Scottish Funding Council (RJR). Influenza A virus NS1 protein is a multifunctional virulence factor consisting of an RNA binding domain (RBD), a short linker, an effector domain (ED), and a C-terminal 'tail'. Although poorly understood, NS1 multimerization may autoregulate its actions. While RBD dimerization seems functionally conserved, two possible apo ED dimers have been proposed (helix-helix and strand-strand). Here, we analyze all available RBD, ED, and full-length NS1 structures, including four novel crystal structures obtained using EDs from divergent human and avian viruses, as well as two forms of a monomeric ED mutant. The data reveal the helix-helix interface as the only strictly conserved ED homodimeric contact. Furthermore, a mutant NS1 unable to form the helix-helix dimer is compromised in its ability to bind dsRNA efficiently, implying that ED multimerization influences RBD activity. Our bioinformatical work also suggests that the helix-helix interface is variable and transient, thereby allowing two ED monomers to twist relative to one another and possibly separate. In this regard, we found a mAb that recognizes NS1 via a residue completely buried within the ED helix-helix interface, and which may help highlight potential different conformational populations of NS1 (putatively termed 'helix-closed' and 'helix-open') in virus-infected cells. 'Helix-closed' conformations appear to enhance dsRNA binding, and 'helix-open' conformations allow otherwise inaccessible interactions with host factors. Our data support a new model of NS1 regulation in which the RBD remains dimeric throughout infection, while the ED switches between several quaternary states in order to expand its functional space. Such a concept may be applicable to other small multifunctional proteins. Publisher PDF

The influenza virus PB1-F2 is an 87-amino acid mitochondrial protein that previously has been shown to induce cell death, although the mechanism of apoptosis induction has remained unclear. In the process of characterizing its mechanism of action we found that the viral PB1-F2 protein sensitizes cells to apoptotic stimuli such as tumor necrosis factor alpha, as demonstrated by increased cleavage of caspase 3 substrates in PB1-F2-expressing cells. Moreover, treatment of purified mouse liver mitochondria with recombinant PB1-F2 protein resulted in cytochrome c release, loss of the mitochondrial membrane potential, and enhancement of tBid-induced mitochondrial permeabilization, suggesting a possible mechanism for the observed cellular sensitization to apoptosis. Using glutathione-S-transferase pulldowns with subsequent mass spectrometric analysis, we identified the mitochondrial interactors of the PB1-F2 protein and showed that the viral protein uniquely interacts with the inner mitochondrial membrane adenine nucleotide translocator 3 and the outer mitochondrial membrane voltage-dependent anion channel 1, both of which are implicated in the mitochondrial permeability transition during apoptosis. Consistent with this interaction, blockers of the permeability transition pore complex (PTPC) inhibited PB1-F2-induced mitochondrial permeabilization. Based on our findings, we propose a model whereby the proapoptotic PB1-F2 protein acts through the mitochondrial PTPC and may play a role in the down-regulation of the host immune response to infection., Synopsis PB1-F2 is a short polypeptide encoded by influenza viruses. While the role of this viral protein is not completely understood, it is known to localize in the mitochodria of the infected cell and to promote cell death. The authors found that PB1-F2 sensitizes cells to death through interactions with two mitochondrial proteins, ANT3 and VDAC1. These interactions promote the permeabilization of the mitochodria and facilitate the release of mitochondrial products that trigger cell death (apoptosis). PB1-F2-mediated cell death through the mitochondria is likely to contribute to the pathogenicity of the influenza virus.

The pattern recognition receptor RIG-I is critical for Type-I interferon production. However, the global regulation of RIG-I signaling is only partially understood. Using a human genome-wide RNAi-screen, we identified 226 novel regulatory proteins of RIG-I mediated interferon-β production. Furthermore, the screen identified a metabolic pathway that synthesizes the inositol pyrophosphate 1-IP7 as a previously unrecognized positive regulator of interferon production. Detailed genetic and biochemical experiments demonstrated that the kinase activities of IPPK, PPIP5K1 and PPIP5K2 (which convert IP5 to1-IP7) were critical for both interferon induction, and the control of cellular infection by Sendai and influenza A viruses. Conversely, ectopically expressed inositol pyrophosphate-hydrolases DIPPs attenuated interferon transcription. Mechanistic experiments in intact cells revealed that the expression of IPPK, PPIP5K1 and PPIP5K2 was needed for the phosphorylation and activation of IRF3, a transcription factor for interferon. The addition of purified individual inositol pyrophosphates to a cell free reconstituted RIG-I signaling assay further identified 1-IP7 as an essential component required for IRF3 activation. The inositol pyrophosphate may act by β-phosphoryl transfer, since its action was not recapitulated by a synthetic phosphonoacetate analogue of 1-IP7. This study thus identified several novel regulators of RIG-I, and a new role for inositol pyrophosphates in augmenting innate immune responses to viral infection that may have therapeutic applications., Author Summary The innate immune system is critical for viral infection control by host organisms. The type I interferons are a family of major antiviral cytokines produced upon the activation of innate immune pattern recognition receptors (PRRs) by viruses. The RIG-I is a major PRR that uniquely detects RNA viruses within the cytoplasm. In this study, we aimed to discover cellular genes and pathways that play regulatory roles in the transcriptional induction of type I interferon-β (IFNβ). Using a human genome wide RNA interference (RNAi) screening, we identified 226 genes whose expression is important for proper IFNβ production. Through bioinformatics-based mining of the RNAi screen results, we identified that the cellular pathway synthesizing inositol pyrophosphates, a class of inositol phosphates with high-energy diphosphates, is a key positive regulator of RIG-I mediated IFNβ production. The kinases IPPK, PPIP5K1 and PPIP5K2, that synthesize inositol pyrophosphate 1-IP7, regulated IFNβ response in a catalytically dependent manner. Mechanistic studies identified that 1-IP7 synthesis pathway was needed for efficient phosphorylation of IRF3. The DIPP family of inositol pyrophosphate hydrolases negatively regulated the IFNβ response, upon ectopic expression. In summary, this study generated a global view of the regulation of RIG-I signaling, and identified inositol pyrophosphates as important regulators of antiviral response.

This study analyzed a heterologous prime-boost vaccine approach against HIV-1 using three different antigenically unrelated negative-stranded viruses (NSV) expressing HIV-1 Gag as vaccine vectors: rabies virus (RABV), vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV). We hypothesized that this approach would result in more robust cellular immune responses than those achieved with the use of any of the vaccines alone in a homologous prime-boost regimen. To this end, we primed BALB/c mice with each of the NSV-based vectors. Primed mice were rested for thirty-five days after which we administered a second immunization with the same or heterologous NSV-Gag viruses. The magnitude and quality of the Gag-specific CD8(+) T cells in response to these vectors post boost were measured. In addition, we performed challenge experiments using vaccinia virus expressing HIV-1 Gag (VV-Gag) thirty-three days after the boost inoculation. Our results showed that the choice of the vaccine used for priming was important for the detected Gag-specific CD8(+) T cell recall responses post boost and that NDV-Gag appeared to result in a more robust recall of CD8(+) T cell responses independent of the prime vaccine used. However, the different prime-boost strategies were not distinct for the parameters studied in the challenge experiments using VV-Gag but did indicate some benefits compared to single immunizations. Taken together, our data show that NSV vectors can individually stimulate HIV-Gag specific CD8(+) T cells that are effectively recalled by other NSV vectors in a heterologous prime-boost approach. These results provide evidence that RABV, VSV and NDV can be used in combination to develop vaccines needing prime-boost regimens to stimulate effective immune responses.

An estimated 50 million dengue virus (DENV) infections occur annually and more than forty percent of the human population is currently at risk of developing dengue fever (DF) or dengue hemorrhagic fever (DHF). Despite the prevalence and potential severity of DF and DHF, there are no approved vaccines or antiviral therapeutics available. An improved understanding of DENV immune evasion is pivotal for the rational development of anti-DENV therapeutics. Antagonism of type I interferon (IFN-I) signaling is a crucial mechanism of DENV immune evasion. DENV NS5 protein inhibits IFN-I signaling by mediating proteasome-dependent STAT2 degradation. Only proteolytically-processed NS5 can efficiently mediate STAT2 degradation, though both unprocessed and processed NS5 bind STAT2. Here we identify UBR4, a 600-kDa member of the N-recognin family, as an interacting partner of DENV NS5 that preferentially binds to processed NS5. Our results also demonstrate that DENV NS5 bridges STAT2 and UBR4. Furthermore, we show that UBR4 promotes DENV-mediated STAT2 degradation, and most importantly, that UBR4 is necessary for efficient viral replication in IFN-I competent cells. Our data underscore the importance of NS5-mediated STAT2 degradation in DENV replication and identify UBR4 as a host protein that is specifically exploited by DENV to inhibit IFN-I signaling via STAT2 degradation., Author Summary Dengue virus (DENV) is the leading cause of mosquito-borne viral illness and death in humans. At present, there are no vaccines and no specific antiviral therapeutics to prevent or treat DENV infections. We previously described that the NS5 protein of DENV inhibits type I interferon signaling in virus-infected cells by mediating STAT2 degradation. This property allows DENV to overcome the antiviral effects of type I interferon, contributing to viral replication in the host. We have now obtained new insight into the mechanism by which DENV NS5 induces STAT2 degradation. NS5 bridges STAT2 with the cellular protein UBR4, a member of a family of predicted E3 ligases, resulting in UBR4-mediated STAT2 degradation. Elimination of UBR4 or mutations in NS5 that prevent its binding to UBR4 prevent NS5 from inducing STAT2 degradation. Importantly, UBR4 is required for optimal DENV replication in the presence of a competent type I interferon system. Our data demonstrate the requirement of a host factor, UBR4, for DENV to overcome the antiviral interferon response. This information might be important for the design of specific DENV inhibitors that prevent dengue virus from evading innate immunity.

Influenza A viruses can adapt to new host species, leading to the emergence of novel pathogenic strains. There is evidence that highly pathogenic viruses encode for non-structural 1 (NS1) proteins that are more efficient in suppressing the host immune response. The NS1 protein inhibits type-I interferon (IFN) production partly by blocking the TRIM25 ubiquitin E3 ligase-mediated Lys63-linked ubiquitination of the viral RNA sensor RIG-I, required for its optimal downstream signaling. In order to understand possible mechanisms of viral adaptation and host tropism, we examined the ability of NS1 encoded by human (Cal04), avian (HK156), swine (SwTx98) and mouse-adapted (PR8) influenza viruses to interact with TRIM25 orthologues from mammalian and avian species. Using co-immunoprecipitation assays we show that human TRIM25 binds to all tested NS1 proteins, whereas the chicken TRIM25 ortholog binds preferentially to the NS1 from the avian virus. Strikingly, none of the NS1 proteins were able to bind mouse TRIM25. Since NS1 can inhibit IFN production in mouse, we tested the impact of TRIM25 and NS1 on RIG-I ubiquitination in mouse cells. While NS1 efficiently suppressed human TRIM25-dependent ubiquitination of RIG-I 2CARD, NS1 inhibited the ubiquitination of full-length mouse RIG-I in a mouse TRIM25-independent manner. Therefore, we tested if the ubiquitin E3 ligase Riplet, which has also been shown to ubiquitinate RIG-I, interacts with NS1. We found that NS1 binds mouse Riplet and inhibits its activity to induce IFN-β in murine cells. Furthermore, NS1 proteins of human but not swine or avian viruses were able to interact with human Riplet, thereby suppressing RIG-I ubiquitination. In conclusion, our results indicate that influenza NS1 protein targets TRIM25 and Riplet ubiquitin E3 ligases in a species-specific manner for the inhibition of RIG-I ubiquitination and antiviral IFN production., Author Summary Influenza viruses cause annual epidemics and occasionally, major global pandemics. To establish productive infection these viruses have mechanisms to evade host immune responses, including the type-I interferon (IFN) response. An important component of the IFN system is the helicase RIG-I that recognizes viral RNA, and is subsequently ubiquitinated by TRIM25 ubiquitin E3 ligase to induce downstream signaling resulting in IFN-α/β production. The NS1 protein of influenza A viruses binds to human TRIM25 and inhibits TRIM25-dependent RIG-I ubiquitination and downstream RIG-I signaling. An important unresolved question is how viruses can inhibit the RIG-I pathway when infecting new hosts. Here we show that while human TRIM25 is able to bind to different NS1 proteins, chicken TRIM25 binds preferentially to the NS1 from an avian virus. Strikingly, mouse TRIM25 was unable to bind NS1. We found that NS1 blocks RIG-I signaling in mouse and human cells by different mechanisms. While NS1 inhibits human TRIM25-mediated RIG-I ubiquitination, in mouse cells NS1 suppresses RIG-I signaling by binding to and inhibiting the ubiquitin E3 ligase Riplet. These results help understand the immune evasion strategies used by influenza virus in different species, and may partly explain the ability of this virus to adapt to different host species.

Influenza A virus (IAV) is a leading cause of respiratory tract disease worldwide. Anti-viral CD8(+) T lymphocytes responding to IAV infection are believed to eliminate virally infected cells by direct cytolysis but may also contribute to pulmonary inflammation and tissue damage via the release of pro-inflammatory mediators following recognition of viral antigen displaying cells. We have previously demonstrated that IAV antigen expressing inflammatory cells of hematopoietic origin within the infected lung interstitium serve as antigen presenting cells (APC) for infiltrating effector CD8(+) T lymphocytes; however, the spectrum of inflammatory cell types capable of serving as APC was not determined. Here, we demonstrate that viral antigen displaying neutrophils infiltrating the IAV infected lungs are an important cell type capable of acting as APC for effector CD8(+) T lymphocytes in the infected lungs and that neutrophils expressing viral antigen as a result of direct infection by IAV exhibit the most potent APC activity. Our findings suggest that in addition to their suggested role in induction of the innate immune responses to IAV, virus clearance, and the development of pulmonary injury, neutrophils can serve as APCs to anti-viral effector CD8(+) T cells within the infected lung interstitium.

Influenza A virus infects 5-20% of the population annually, resulting in ~35,000 deaths and significant morbidity. Current treatments include vaccines and drugs that target viral proteins. However, both of these approaches have limitations, as vaccines require yearly development and the rapid evolution of viral proteins gives rise to drug resistance. In consequence additional intervention strategies, that target host factors required for the viral life cycle, are under investigation. Here we employed arrayed whole-genome siRNA screening strategies to identify cell-autonomous molecular components that are subverted to support H1N1 influenza A virus infection of human bronchial epithelial cells. Integration across relevant public data sets exposed druggable gene products required for epithelial cell infection or required for viral proteins to deflect host cell suicide checkpoint activation. Pharmacological inhibition of representative targets, RGGT and CHEK1, resulted in significant protection against infection of human epithelial cells by the A/WS/33 virus. In addition, chemical inhibition of RGGT partially protected against H5N1 and the 2009 H1N1 pandemic strain. The observations reported here thus contribute to an expanding body of studies directed at decoding vulnerabilities in the command and control networks specified by influenza virulence factors.

PB1-F2 is a 90 amino acid protein that is expressed from the +1 open reading frame in the PB1 gene of some influenza A viruses and has been shown to contribute to viral pathogenicity. Notably, a serine at position 66 (66S) in PB1-F2 is known to increase virulence compared to an isogenic virus with an asparagine (66N) at this position. Recently, we found that an influenza virus expressing PB1-F2 N66S suppresses interferon (IFN)-stimulated genes in mice. To characterize this phenomenon, we employed several in vitro assays. Overexpression of the A/Puerto Rico/8/1934 (PR8) PB1-F2 protein in 293T cells decreased RIG-I mediated activation of an IFN-β reporter and secretion of IFN as determined by bioassay. Of note, the PB1-F2 N66S protein showed enhanced IFN antagonism activity compared to PB1-F2 wildtype. Similar observations were found in the context of viral infection with a PR8 PB1-F2 N66S virus. To understand the relationship between NS1, a previously described influenza virus protein involved in suppression of IFN synthesis, and PB1-F2, we investigated the induction of IFN when NS1 and PB1-F2 were co-expressed in an in vitro transfection system. In this assay we found that PB1-F2 N66S further reduced IFN induction in the presence of NS1. By inducing the IFN-β reporter at different levels in the signaling cascade, we found that PB1-F2 inhibited IFN production at the level of the mitochondrial antiviral signaling protein (MAVS). Furthermore, immunofluorescence studies revealed that PB1-F2 co-localizes with MAVS. In summary, we have characterized the anti-interferon function of PB1-F2 and we suggest that this activity contributes to the enhanced pathogenicity seen with PB1-F2 N66S- expressing influenza viruses., Author Summary Influenza viruses can cause global pandemics and are thus a major health concern. The novel H1N1 pandemic virus infected a large number of people, but resulted in relatively mild symptoms in the majority of cases. In contrast, the avian H5N1 viruses are associated with a high mortality rate, but are not transmitted from human to human. Understanding the viral and host factors that play a role in causing disease is crucial in developing effective vaccines and therapeutics. Furthermore, finding viral markers for high virulence may help predict the impact of newly emerging pandemic influenza viruses. We have previously established that a single amino acid substitution (N66S) in the viral PB1-F2 protein causes increased virulence in an H5N1 and the 1918 pandemic virus. Here we show that PB1-F2 N66S reduces the induction of interferon (IFN), a potent antiviral molecule secreted by cells in response to infection. Furthermore, we demonstrate that the inhibition of IFN by PB1-F2 N66S occurs at the level of the mitochondrial antiviral signaling protein (MAVS), a key player in the IFN production pathway. Our work here characterizes a new function for the PB1-F2 protein and how this function can lead to increased disease severity.

The recent 2009 pandemic H1N1 virus infection in humans has resulted in nearly 5,000 deaths worldwide. Early epidemiological findings indicated a low level of infection in the older population (>65 years) with the pandemic virus, and a greater susceptibility in people younger than 35 years of age, a phenomenon correlated with the presence of cross-reactive immunity in the older population. It is unclear what virus(es) might be responsible for this apparent cross-protection against the 2009 pandemic H1N1 virus. We describe a mouse lethal challenge model for the 2009 pandemic H1N1 strain, used together with a panel of inactivated H1N1 virus vaccines and hemagglutinin (HA) monoclonal antibodies to dissect the possible humoral antigenic determinants of pre-existing immunity against this virus in the human population. By hemagglutinination inhibition (HI) assays and vaccination/challenge studies, we demonstrate that the 2009 pandemic H1N1 virus is antigenically similar to human H1N1 viruses that circulated from 1918–1943 and to classical swine H1N1 viruses. Antibodies elicited against 1918-like or classical swine H1N1 vaccines completely protect C57B/6 mice from lethal challenge with the influenza A/Netherlands/602/2009 virus isolate. In contrast, contemporary H1N1 vaccines afforded only partial protection. Passive immunization with cross-reactive monoclonal antibodies (mAbs) raised against either 1918 or A/California/04/2009 HA proteins offered full protection from death. Analysis of mAb antibody escape mutants, generated by selection of 2009 H1N1 virus with these mAbs, indicate that antigenic site Sa is one of the conserved cross-protective epitopes. Our findings in mice agree with serological data showing high prevalence of 2009 H1N1 cross-reactive antibodies only in the older population, indicating that prior infection with 1918-like viruses or vaccination against the 1976 swine H1N1 virus in the USA are likely to provide protection against the 2009 pandemic H1N1 virus. This data provides a mechanistic basis for the protection seen in the older population, and emphasizes a rationale for including vaccination of the younger, naïve population. Our results also support the notion that pigs can act as an animal reservoir where influenza virus HAs become antigenically frozen for long periods of time, facilitating the generation of human pandemic viruses., Author Summary Influenza A viruses generally infect individuals of all ages and cause severe respiratory disease in very young children and elderly people (>65 years). However, the 2009 pandemic H1N1 virus infection is predominantly seen in children and adults (

Activation of the latent kinase PKR is a potent innate defense reaction of vertebrate cells towards viral infections, which is triggered by recognition of viral double-stranded (ds) RNA and results in a translational shutdown. A major gap in our understanding of PKR's antiviral properties concerns the nature of the kinase activating molecules expressed by influenza and other viruses with a negative strand RNA genome, as these pathogens produce little or no detectable amounts of dsRNA. Here we systematically investigated PKR activation by influenza B virus and its impact on viral pathogenicity. Biochemical analysis revealed that PKR is activated by viral ribonucleoprotein (vRNP) complexes known to contain single-stranded RNA with a 5′-triphosphate group. Cell biological examination of recombinant viruses showed that the nucleo-cytoplasmic transport of vRNP late in infection is a strong trigger for PKR activation. In addition, our analysis provides a mechanistic explanation for the previously observed suppression of PKR activation by the influenza B virus NS1 protein, which we show here to rely on complex formation between PKR and NS1's dsRNA binding domain. The high significance of this interaction for pathogenicity was revealed by the finding that attenuated influenza viruses expressing dsRNA binding-deficient NS1 proteins were rescued for high replication and virulence in PKR-deficient cells and mice, respectively. Collectively, our study provides new insights into an important antiviral defense mechanism of vertebrates and leads us to suggest a new model of PKR activation by cytosolic vRNP complexes, a model that may also be applicable to other negative strand RNA viruses., Author Summary Upon viral infection of vertebrate cells, a vigorous innate defense response is initiated via the recognition of viral double-stranded (ds) RNA by the protein kinase PKR, resulting in the cessation of protein synthesis and subsequent blockage of viral propagation. The activation of PKR's potent antiviral response against influenza and other viruses with a negative strand RNA genome has presented a conundrum, however, as previous attempts failed to detect dsRNA in cells infected with these viruses. Here, we identify genomic RNA within the ribonucleoprotein (RNP) of influenza viruses as a non-canonical activator of the latent kinase PKR. Cell biological examinations revealed that the transfer of viral RNP from the nucleus to the cytoplasm provides a strong stimulus for PKR activation. Moreover, we provide insight into mechanisms of pathogenesis by showing PKR and the NS1 protein of influenza B virus forms a complex in infected cells, which inhibits PKR activation. This interaction seems to be crucial for viral pathogenicity, as a strong attenuation of NS1 mutant viruses was largely rescued in PKR-deficient mice and cells. Taken together, these findings suggest a new model for the induction and inhibition of PKR by influenza virus that may also apply to viruses with a similar genome structure.

Fatal human respiratory disease associated with the 1918 pandemic influenza virus and potentially pandemic H5N1 viruses is characterized by severe lung pathology, including pulmonary edema and extensive inflammatory infiltrate. Here, we quantified the cellular immune response to infection in the mouse lung by flow cytometry and demonstrate that mice infected with highly pathogenic (HP) H1N1 and H5N1 influenza viruses exhibit significantly high numbers of macrophages and neutrophils in the lungs compared to mice infected with low pathogenic (LP) viruses. Mice infected with the 1918 pandemic virus and a recent H5N1 human isolate show considerable similarities in overall lung cellularity, lung immune cell sub-population composition and cellular immune temporal dynamics. Interestingly, while these similarities were observed, the HP H5N1 virus consistently elicited significantly higher levels of pro-inflammatory cytokines in whole lungs and primary human macrophages, revealing a potentially critical difference in the pathogenesis of H5N1 infections. These results together show that infection with HP influenza viruses such as H5N1 and the 1918 pandemic virus leads to a rapid cell recruitment of macrophages and neutrophils into the lungs, suggesting that these cells play a role in acute lung inflammation associated with HP influenza virus infection. In addition, primary macrophages and dendritic cells were also susceptible to 1918 and H5N1 influenza virus infection in vitro and in infected mouse lung tissue., Author Summary Patients who succumbed to influenza during the 1918 pandemic had severe lung pathology marked by extensive inflammatory infiltrate, indicating a robust immune response in the lung. Similar findings have been reported from H5N1-infected patients, raising the question as to why people expire in the presence of a strong immune response. We addressed this question by characterizing the immune cell populations in the mouse lung following infection with the 1918 pandemic virus and two H5N1 viruses isolated from fatal cases. Our data shows excessive immune cell infiltration in the lungs contributing to severe consolidation and tissue architecture destruction in mice infected with highly pathogenic (HP) influenza viruses, supporting the histopathological observations of lung tissue from 1918 and H5N1 fatalities. We found that certain cells of the innate immune system, specifically macrophages and neutrophils, increase significantly into the mouse lung shortly following HP virus infection. Interestingly, lung macrophages and dendritic cells were shown to be susceptible to 1918 and H5N1 virus infection in vitro or ex vivo, suggesting a possible mechanism of immunopathogenesis. Identification of the precise inflammatory cells associated with lung inflammation will be important for the development of treatments that could potentially enhance or modulate host innate immune responses.