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Communicable respiratory viral infections pose both epidemic and pandemic threats and broad-spectrum antiviral strategies could improve preparedness for these events. To discover host antiviral restriction factors that may act as suitable targets for the development of host-directed antiviral therapies, we here conduct a whole-genome CRISPR activation screen with influenza B virus (IBV). A top hit from our screen, beta-1,3-glucuronyltransferase 1 (B3GAT1), effectively blocks IBV infection. Subsequent studies reveal that B3GAT1 activity prevents cell surface sialic acid expression. Due to this mechanism of action, B3GAT1 expression broadly restricts infection with viruses that require sialic acid for entry, including Victoria and Yamagata lineage IBVs, H1N1/H3N2 influenza A viruses (IAVs), and the unrelated enterovirus D68. To understand the potential utility of B3GAT1 induction as an antiviral strategy in vivo, we specifically express B3GAT1 in the murine respiratory epithelium and find that overexpression is not only well-tolerated, but also protects female mice from a lethal viral challenge with multiple influenza viruses, including a pandemic-like H1N1 IAV. Thus, B3GAT1 may represent a host-directed broad-spectrum antiviral target with utility against clinically relevant respiratory viruses. Identification of host antiviral restriction factors could provide targets for antiviral therapy. Here, using a genome-wide CRISPR screen, the authors identify the glycosyltransferase B3GAT1 as a host protein which, when ectopically overexpressed, restricts influenza virus infection in vitro and in mice, as well as other viruses relying on sialic acid for entry. [ABSTRACT FROM AUTHOR]

Influenza A viruses encode their genomes across eight, negative sense RNA segments. The six largest segments produce mRNA transcripts that do not generally splice; however, the two smallest segments are actively spliced to produce the essential viral proteins NEP and M2. Thus, viral utilization of RNA splicing effectively expands the viral coding capacity without increasing the number of genomic segments. As a first step towards understanding why splicing is not more broadly utilized across genomic segments, we designed and inserted an artificial intron into the normally nonsplicing NA segment. This insertion was tolerated and, although viral mRNAs were incompletely spliced, we observed only minor effects on viral fitness. To take advantage of the unspliced viral RNAs, we encoded a reporter luciferase gene in frame with the viral ORF such that when the intron was not removed the reporter protein would be produced. This approach, which we also show can be applied to the NP encoding segment and in different viral genetic backgrounds, led to high levels of reporter protein expression with minimal effects on the kinetics of viral replication or the ability to cause disease in experimentally infected animals. These data together show that the influenza viral genome is more tolerant of splicing than previously appreciated and this knowledge can be leveraged to develop viral genetic platforms with utility for biotechnology applications. Author summary: Unlike most host mRNAs, many viral mRNAs encode multiple discrete, functional proteins. One method influenza A viruses use to increase the protein products from two of their eight RNA genome segments is splicing. Splicing requires host machinery to remove part of the viral mRNA, the intron, to generate a different mRNA product. Although only certain influenza viral segments naturally splice, we were interested in whether additional segments could splice to produce multiple proteins. We inserted artificial introns harboring reporter genes into otherwise nonsplicing genomic segments of an H1N1 influenza A virus and found that this modification was well tolerated by the virus. We further demonstrated that an unrelated H3N2 influenza A virus could similarly support splicing and express a reporter protein from an artificial intron. These findings have implications for our understanding of how viruses expand their coding capacity with a limited genome. Additionally, encoding reporter proteins in spliced intronic sequences also represents a new method of generating reporter viruses requiring limited manipulation of the viral RNA. [ABSTRACT FROM AUTHOR]

Controlling the antiviral interferon response: Type I interferons initiate the changes in gene expression that are critical for fighting viral infections. However, restraining the type I interferon response is equally important for avoiding pathology resulting from inflammation. Froggatt et al. identified an interferon-stimulated gene (ISG) that encodes the transcription factor ETV7, which suppressed the expression of other ISGs, including those critical for controlling influenza viruses in lung epithelial cells. These results suggest that regulatory factors such as ETV7 may regulate the balance between control of viral infection and the excessive inflammation associated with severe viral disease. The type I interferon (IFN) response is an important component of the innate immune response to viral infection. Precise control of IFN responses is critical because insufficient expression of IFN-stimulated genes (ISGs) can lead to a failure to restrict viral spread, whereas excessive ISG activation can result in IFN-related pathologies. Although both positive and negative regulatory factors control the magnitude and duration of IFN signaling, it is also appreciated that several ISGs regulate aspects of the IFN response themselves. In this study, we performed a CRISPR activation screen to identify previously unknown regulators of the type I IFN response. We identified the strongly induced ISG encoding ETS variant transcription factor 7 (ETV7) as a negative regulator of the type I IFN response. However, ETV7 did not uniformly suppress ISG transcription. Instead, ETV7 preferentially targeted a subset of antiviral ISGs that were particularly important for IFN-mediated control of influenza viruses. Together, our data assign a function for ETV7 as an IFN response regulator and also identify ETV7 as a potential therapeutic target to increase innate antiviral responses and enhance IFN-based antiviral therapies. [ABSTRACT FROM AUTHOR]

Antiviral therapeutics are a front-line defense against virally induced diseases. Because viruses frequently mutate to escape direct inhibition of viral proteins, there is interest in targeting the host proteins that the virus must co-opt to complete its replication cycle. However, a detailed understanding of the interactions between the virus and the host cell is necessary in order to facilitate development of host-directed therapeutics. As a first step, we performed a genome-wide loss of function screen using the alphacoronavirus HCoV-229E to better define the interactions between coronaviruses and host-factors. We report the identification and validation of an ER-resident host protein, TMEM41B, as an essential host factor for not only HCoV-229E but also genetically distinct coronaviruses including the pandemic beta-coronavirus SARS-CoV-2. We show that the protein is required at an early, but post-receptor engagement, stage of the viral lifecycle. Further, mechanistic studies revealed that although the protein was not enriched at replication complexes, it likely contributes to viral replication complex formation via mobilization of cholesterol and other lipids to facilitate host membrane expansion and curvature. Continued study of TMEM41B and the development of approaches to prevent its function may lead to broad spectrum anti-coronavirus therapeutics. Author summary: The COVID-19 pandemic has highlighted the burden that emerging coronaviruses can place on global health systems. Due to difficulty in predicting the characteristics of future pandemic viruses, pan-coronaviral therapeutics are highly desired. While many antivirals directly target viral proteins to inhibit their functions, targeting broadly required host-dependency factors represents an orthogonal approach that may have the added benefit of lower susceptibly to the emergence of viral escape mutants. One limitation of this approach, however, is that essential host factors for viral replication must be identified and characterized prior to development of novel therapeutics. In this work, we performed a genome-wide host-dependency factor screen with the endemic seasonal human coronavirus HCoV-229E. We identified TMEM41B, an ER-resident protein, as a critical host-dependency factor for not only HCoV-229E but also distantly related coronaviruses as well. Mechanistic studies revealed that TMEM41B is likely required to facilitate appropriate membrane composition for the establishment of intracellular viral replication complexes. Thus, the conserved requirement for TMEM41B is likely due to the fact that all coronaviruses establish similar structures for RNA replication. Future studies understanding how to pharmacologically target TMEM41B and its associated lipid mobilization pathways may lead to the development of broad-spectrum anti-coronavirus therapeutics. [ABSTRACT FROM AUTHOR]

The cell-cell signaling process called quorum sensing allows bacteria to control behaviors in response to changes in population density. In Vibrio harveyi, the master quorum-sensing transcription factor LuxR is a member of the TetR family of transcription factors that both activates and represses genes to coordinate group behaviors, including bioluminescence. Here, we show that integration host factor (IHF) is a key coactivator of the luxCDABE bioluminescence genes that is required together with LuxR for precise timing and expression levels of bioluminescence during quorum sensing. IHF binds to multiple sites in the luxCDABE promoter and bends the DNA in vitro. IHF and LuxR synergistically bind luxCDABE promoter DNA at overlapping, essential binding sites that are required for maximal gene expression in vivo. RNA-seq analysis demonstrated that IHF regulates 300 genes in V. harveyi, and among these are a core set of 19 genes that are also directly bound and regulated by LuxR. We validated these global analyses by demonstrating that both IHF and LuxR are required for transcriptional activation of the osmotic stress response genes betIBA-proXWV. These data suggest that IHF plays an integral role in one mechanism of transcriptional activation by the LuxR-type family of quorum-sensing regulators in vibrios. [ABSTRACT FROM AUTHOR]