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A recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) lacking the envelope (E) protein is attenuated in vivo. Here we report that E protein PDZ-binding motif (PBM), a domain involved in protein-protein interactions, is a major determinant of virulence. Elimination of SARS-CoV E protein PBM by using reverse genetics caused a reduction in the deleterious exacerbation of the immune response triggered during infection with the parental virus and virus attenuation. Cellular protein syntenin was identified to bind the E protein PBM during SARS-CoV infection by using three complementary strategies, yeast two-hybrid, reciprocal coimmunoprecipitation and confocal microscopy assays. Syntenin redistributed from the nucleus to the cell cytoplasm during infection with viruses containing the E protein PBM, activating p38 MAPK and leading to the overexpression of inflammatory cytokines. Silencing of syntenin using siRNAs led to a decrease in p38 MAPK activation in SARS-CoV infected cells, further reinforcing their functional relationship. Active p38 MAPK was reduced in lungs of mice infected with SARS-CoVs lacking E protein PBM as compared with the parental virus, leading to a decreased expression of inflammatory cytokines and to virus attenuation. Interestingly, administration of a p38 MAPK inhibitor led to an increase in mice survival after infection with SARS-CoV, confirming the relevance of this pathway in SARS-CoV virulence. Therefore, the E protein PBM is a virulence domain that activates immunopathology most likely by using syntenin as a mediator of p38 MAPK induced inflammation.

Innate immune responses mediated by IFN and inflammatory cytokines are critical for controlling virus replication. Nevertheless, exacerbated innate immune responses could be detrimental for the host and feedback mechanisms are needed to maintain the cellular homeostasis. In this work, we describe a completely novel function for IFI44 in negatively modulating the innate immune responses induced after viral infections. We show that decreasing IFI44 expression by using small interfering RNAs (siRNAs) or by generating knockout (KO) cells impairs virus production and increases the levels of IFN responses. Moreover, we report a novel interaction of IFI44 with the cellular protein FKBP5, which in turn interacts with kinases essential for type I and III IFN induction and signaling, such as the inhibitor of nuclear factor kappa B (IκB) kinases IKKα, IKKβ, and IKKε. Our data indicate that binding of IFI44 to FKBP5 decreased the phosphorylation of IRF-3 and IκBα mediated by IKKε and IKKβ, respectively, providing a likely explanation for the function of IFI44 in negatively modulating IFN responses. These results provide new insights into the induction of innate immune responses and suggest that IFI44 is a new potential antiviral target for reducing virus replication. Using multiple viral systems, and performing silencing approaches, overexpression approaches, and experiments in knockout cells, we report, for the first time, that interferon (IFN)-induced protein 44 (IFI44) positively affects virus production and negatively modulates innate immune responses induced after viral infections. Moreover, IFI44 is able to rescue poly(I·C)- and IFN-mediated inhibition of virus growth. Furthermore, we report a novel interaction of IFI44 with the cellular factor FK506-binding protein 5 (FKBP5), which binds to cellular kinases such as the inhibitor of nuclear factor kappa B (IκB) kinases (IKKα, IKKβ, and IKKε). Importantly, in the presence of FKBP5, IFI44 decreases the ability of IKKβ to phosphorylate IκBα and the ability of IKKε to phosphorylate interferon regulatory factor 3 (IRF-3), providing a novel mechanism for the function of IFI44 in negatively modulating IFN responses. Remarkably, these new IFI44 functions may have implications for diseases associated with excessive immune signaling and for controlling virus infections mediated by IFN responses. IMPORTANCE Innate immune responses mediated by IFN and inflammatory cytokines are critical for controlling virus replication. Nevertheless, exacerbated innate immune responses could be detrimental for the host and feedback mechanisms are needed to maintain the cellular homeostasis. In this work, we describe a completely novel function for IFI44 in negatively modulating the innate immune responses induced after viral infections. We show that decreasing IFI44 expression by using small interfering RNAs (siRNAs) or by generating knockout (KO) cells impairs virus production and increases the levels of IFN responses. Moreover, we report a novel interaction of IFI44 with the cellular protein FKBP5, which in turn interacts with kinases essential for type I and III IFN induction and signaling, such as the inhibitor of nuclear factor kappa B (IκB) kinases IKKα, IKKβ, and IKKε. Our data indicate that binding of IFI44 to FKBP5 decreased the phosphorylation of IRF-3 and IκBα mediated by IKKε and IKKβ, respectively, providing a likely explanation for the function of IFI44 in negatively modulating IFN responses. These results provide new insights into the induction of innate immune responses and suggest that IFI44 is a new potential antiviral target for reducing virus replication.

Protein kinase R (PKR) expression is induced by interferons. This protein is activated by double-stranded (ds) RNAs or RNAs containing duplex regions, produced after different stimuli, such as after viral infections, leading to the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α), and subsequently inhibiting cellular and viral protein translation. This function may lead to different effects such as to impairing the replication of RNA viruses by inhibiting viral protein translation, and to modulating the innate immune responses after viral infections by affecting the translation of effector proteins. In this work, we identify, for the first time, an interaction of IFN alpha inducible protein 27 (IFI27) with PKR-activating protein (PACT or PRKRA) and with PKR, showing that the interaction of IFI27 with PACT is likely mediated by dsRNAs or RNAs containing duplex regions, and that the interaction of IFI27 with PKR is PACT-dependent. Interestingly, using IFI27 knocked-down, knocked-out and overexpressing tumour-derived, established cells, we show that these interactions trigger a potentiation of the activity of PKR and, therefore, a decrease in protein translation. Moreover, we find that IFI27 increases PKR function in cells infected with different RNA viruses such as Severe Acute Respiratory virus 2 (SARS-CoV-2), and Vesicular Stomatitis virus (VSV), and in cells transfected with the dsRNA analog poly(I:C), suggesting a broad effect of IFI27 on PKR activation. Moreover, we show that IFI27 expression increases the formation of stress granules (SGs) at the cell cytoplasm, correlating with the increased PKR activation mediated by IFI27, as it has been shown that the translational arrest induced by activated PKR leads to the formation of SGs. Mechanistically, we describe that this ability of IFI27 to activate PKR is dependent on its interaction with PACT. Further understanding of the regulation of PKR activity will allow us to develop new antiviral drugs to modulate this signalling axis, which is crucial in RNA virus infections.

The tripartite motif-containing (TRIM) 34 protein is an interferon (IFN)-induced protein whose expression is upregulated after influenza A virus (IAV) infection. Here, we identify a previously unknown function for TRIM34 as a negative regulator of innate immune responses following IFN treatment and IAV infection, even in mice, suggesting that this effect is broad and conserved. Complementary overexpression and silencing experiments in cultured cells show that TRIM34 expression positively correlates with IAV titers, indicating that TRIM34 promotes viral replication, likely by counteracting antiviral responses. Moreover, we show novel interactions of TRIM34 with cellular proteins involved in the nucleocytoplasmic transport of host mRNAs and proteins, even in mock-infected cells. Remarkably, these TRIM34 interactions seem independent of TRIM34 E3 ubiquitin ligase activity and affect the nuclear import of IFN-regulatory factor 3 (IRF3) and the nuclear export of host mRNAs encoding antiviral functions, without affecting the nuclear export of viral mRNAs. These results provide a likely mechanism by which TRIM34 dampens host innate immune responses. These TRIM34-mediated effects could be further exploited to develop new antiviral drugs against IAV, and potentially other viral infections.

Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) envelope (E) gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC) activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS) leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na+/K+ ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1β were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1β was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS-CoV virulence.

Influenza A viruses (IAV) can infect a broad range of animal hosts, including humans. In humans, IAV causes seasonal annual epidemics and occasional pandemics, representing a serious public health and economic problem, which is most effectively prevented through vaccination. The defense mechanisms that the host innate immune system provides restrict IAV replication and infection. Consequently, to successfully replicate in interferon (IFN)-competent systems, IAV has to counteract host antiviral activities, mainly the production of IFN and the activities of IFN-induced host proteins that inhibit virus replication. The IAV multifunctional proteins PA-X and NS1 are virulence factors that modulate the innate immune response and virus pathogenicity. Notably, these two viral proteins have synergistic effects in the inhibition of host protein synthesis in infected cells, although using different mechanisms of action. Moreover, the control of innate immune responses by the IAV NS1 and PA-X proteins is subject to a balance that can determine virus pathogenesis and fitness, and recent evidence shows co-evolution of these proteins in seasonal viruses, indicating that they should be monitored for enhanced virulence. Importantly, inhibition of host gene expression by the influenza NS1 and/or PA-X proteins could be explored to develop improved live-attenuated influenza vaccines (LAIV) by modulating the ability of the virus to counteract antiviral host responses. Likewise, both viral proteins represent a reasonable target for the development of new antivirals for the control of IAV infections. In this review, we summarize the role of IAV NS1 and PA-X in controlling the antiviral response during viral infection.

A large number of human genes associated with viral infections contain single nucleotide polymorphisms (SNPs), which represent a genetic variation caused by the change of a single nucleotide in the DNA sequence. SNPs are located in coding or non-coding genomic regions and can affect gene expression or protein function by different mechanisms. Furthermore, they have been linked to multiple human diseases, highlighting their medical relevance. Therefore, the identification and analysis of this kind of polymorphisms in the human genome has gained high importance in the research community, and an increasing number of studies have been published during the last years. As a consequence of this exhaustive exploration, an association between the presence of some specific SNPs and the susceptibility or severity of many infectious diseases in some risk population groups has been found. In this review, we discuss the relevance of SNPs that are important to understand the pathology derived from influenza A virus (IAV) infections in humans and the susceptibility of some individuals to suffer more severe symptoms. We also discuss the importance of SNPs for IAV vaccine effectiveness.

Antigenic drift of the hemagglutinin (HA) and neuraminidase (NA) influenza virus proteins contributes to reduced vaccine efficacy. To analyze antigenic drift in human seasonal H1N1 viruses derived from the 2009 pandemic H1N1 virus (pH1N1-like viruses) accounts for the limited effectiveness (around 40%) of vaccination against pH1N1-like viruses during the 2015-2016 season, nasal washes/swabs collected from adult subjects in the Rochester, NY area, were used to sequence and isolate the circulating viruses. The HA and NA proteins from viruses circulating during the 2015-2016 season encoded eighteen and fourteen amino acid differences, respectively, when compared to A/California/04/2009, a strain circulating at the origin of the 2009 pandemic. The circulating strains belonged to subclade 6B.1, defined by HA amino acid substitutions S101N, S179N, and I233T. Hemagglutination-inhibiting (HAI) and HA-specific neutralizing serum antibody (Ab) titers from around 50% of pH1N1-like virus-infected subjects and immune ferrets were 2-4 fold lower for the 2015-2016 circulating strains compared to the vaccine strain. In addition, using a luminex-based mPlex HA assay, the binding of human sera from subjects infected with pH1N1-like viruses to the HA proteins from circulating and vaccine strains was not identical, strongly suggesting antigenic differences in the HA protein. Additionally, NA inhibition (NAI) Ab titers in human sera from pH1N1-like virus-infected subjects increased after the infection and there were measurable antigenic differences between the NA protein of circulating strains and the vaccine strain using both ferret and human antisera. Despite having been vaccinated, infected subjects exhibited low HAI Ab titers against the vaccine and circulating strains. This suggests that poor responses to the H1N1 component of the vaccine as well as antigenic differences in the HA and NA proteins of currently circulating pH1N1-like viruses could be contributing to risk of infection even after vaccination.

Viroporins are viral proteins with ion channel (IC) activity that play an important role in several processes, including virus replication and pathogenesis. While many coronaviruses (CoVs) encode two viroporins, severe acute respiratory syndrome CoV (SARS-CoV) encodes three: proteins 3a, E, and 8a. Additionally, proteins 3a and E have a PDZ-binding motif (PBM), which can potentially bind over 400 cellular proteins which contain a PDZ domain, making them potentially important for the control of cell function. In the present work, a comparative study of the functional motifs included within the SARS-CoV viroporins was performed, mostly focusing on the roles of the IC and PBM of E and 3a proteins. Our results showed that the full-length E and 3a proteins were required for maximal SARS-CoV replication and virulence, whereas viroporin 8a had only a minor impact on these activities. A virus missing both the E and 3a proteins was not viable, whereas the presence of either protein with a functional PBM restored virus viability. E protein IC activity and the presence of its PBM were necessary for virulence in mice. In contrast, the presence or absence of the homologous motifs in protein 3a did not influence virus pathogenicity. Therefore, dominance of the IC and PBM of protein E over those of protein 3a was demonstrated in the induction of pathogenesis in mice. IMPORTANCE Collectively, these results demonstrate key roles for the ion channel and PBM domains in optimal virus replication and pathogenesis and suggest that the viral viroporins and PBMs are suitable targets for antiviral therapy and for mutation in attenuated SARS-CoV vaccines. Collectively, these results demonstrate key roles for the ion channel and PBM domains in optimal virus replication and pathogenesis and suggest that the viral viroporins and PBMs are suitable targets for antiviral therapy and for mutation in attenuated SARS-CoV vaccines.

Interferons (IFNs), IFN-stimulated genes (ISGs), and inflammatory cytokines mediate innate immune responses, and are essential to establish an antiviral response. Within the innate immune responses, retinoic acid-inducible gene I (RIG-I) is a key sensor of virus infections, mediating the transcriptional induction of IFNs and inflammatory proteins. Nevertheless, since excessive responses could be detrimental to the host, these responses need to be tightly regulated. In this work, we describe, for the first time, how knocking-down or knocking-out the expression of IFN alpha-inducible protein 6 (IFI6) increases IFN, ISG, and pro-inflammatory cytokine expression after the infections with Influenza A Virus (IAV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and Sendai Virus (SeV), or poly(I:C) transfection. We also show how overexpression of IFI6 produces the opposite effect, in vitro and in vivo , indicating that IFI6 negatively modulates the induction of innate immune responses. Knocking-out or knocking-down the expression of IFI6 diminishes the production of infectious IAV and SARS-CoV-2, most likely because of its effect on antiviral responses. Importantly, we report a novel interaction of IFI6 with RIG-I, most likely mediated through binding to RNA, that affects RIG-I activation, providing a molecular mechanism for the effect of IFI6 on negatively regulating innate immunity. Remarkably, these new functions of IFI6 could be targeted to treat diseases associated with an exacerbated induction of innate immune responses and to combat viral infections, such as IAV and SARS-CoV-2.