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Influenza A Virus (IAV) is a recurring respiratory virus with limited availability of antiviral therapies. Understanding host proteins essential for IAV infection can identify targets for alternative host-directed therapies (HDTs). Using affinity purification-mass spectrometry and global phosphoproteomic and protein abundance analyses using three IAV strains (pH1N1, H3N2, H5N1) in three human cell types (A549, NHBE, THP-1), we map 332 IAV-human protein-protein interactions and identify 13 IAV-modulated kinases. Whole exome sequencing of patients who experienced severe influenza reveals several genes, including scaffold protein AHNAK, with predicted loss-of-function variants that are also identified in our proteomic analyses. Of our identified host factors, 54 significantly alter IAV infection upon siRNA knockdown, and two factors, AHNAK and coatomer subunit COPB1, are also essential for productive infection by SARS-CoV-2. Finally, 16 compounds targeting our identified host factors suppress IAV replication, with two targeting CDK2 and FLT3 showing pan-antiviral activity across influenza and coronavirus families. This study provides a comprehensive network model of IAV infection in human cells, identifying functional host targets for pan-viral HDT.

A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.

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The human interferon (IFN)-induced MxA protein is a key antiviral host restriction factor exhibiting broad antiviral activity against many RNA viruses, including highly pathogenic avian influenza A viruses (IAV) of the H5N1 and H7N7 subtype. To date the mechanism for how MxA exerts its antiviral activity is unclear, however, additional cellular factors are believed to be essential for this activity. To identify MxA cofactors we performed a genome-wide siRNA-based screen in human airway epithelial cells (A549) constitutively expressing MxA using an H5N1 reporter virus. These data were complemented with a proteomic screen to identify MxA-interacting proteins. The combined data identified SMARCA2, the ATPase subunit of the BAF chromatin remodeling complex, as a crucial factor required for the antiviral activity of MxA against IAV. Intriguingly, our data demonstrate that although SMARCA2 is essential for expression of some IFN-stimulated genes (ISGs), and the establishment of an antiviral state, it is not required for expression of MxA, suggesting an indirect effect on MxA activity. Transcriptome analysis of SMARCA2-depleted A549-MxA cells identified a small set of SMARCA2-regulated factors required for activity of MxA, in particular IFITM2 and IGFBP3. These findings reveal that several virus-inducible factors work in concert to enable MxA restriction of IAV.

How transcription affects genome 3D organization is not well understood. We found that during influenza A (IAV) infection, rampant transcription rapidly reorganizes host cell chromatin interactions. These changes occur at the ends of highly transcribed genes, where global inhibition of transcription termination by IAV NS1 protein causes readthrough transcription for hundreds of kilobases. In these readthrough regions, elongating RNA polymerase II disrupts chromatin interactions by inducing cohesin displacement from CTCF sites, leading to locus decompaction. Readthrough transcription into heterochromatin regions switches them from the inert (B) to the permissive (A) chromatin compartment and enables transcription factor binding. Data from non-viral transcription stimuli show that transcription similarly affects cohesin-mediated chromatin contacts within gene bodies. Conversely, inhibition of transcription elongation allows cohesin to accumulate at previously transcribed intragenic CTCF sites and to mediate chromatin looping and compaction. Our data indicate that transcription elongation by RNA polymerase II remodels genome 3D architecture.

At every step of their replication cycle influenza viruses depend heavily on their host cells. The multifaceted interactions that occur between the virus and its host cell determine the outcome of the infection, including efficiency of progeny virus production, tropism, and pathogenicity. In order to understand viral disease and develop therapies for influenza it is therefore pertinent to study the intricate interplay between influenza viruses and their required host factors. Here, we review the current knowledge on host cell factors required by influenza virus at the different stages of the viral replication cycle. We also discuss the roles of host factors in zoonotic transmission of influenza viruses and their potential for developing novel antivirals.

Influenza A virus is an RNA virus that encodes up to 11 proteins and this small coding capacity demands that the virus use the host cellular machinery for many aspects of its life cycle. Knowledge of these host cell requirements not only informs us of the molecular pathways exploited by the virus but also provides further targets that could be pursued for antiviral drug development. Here we use an integrative systems approach, based on genome-wide RNA interference screening, to identify 295 cellular cofactors required for early-stage influenza virus replication. Within this group, those involved in kinase-regulated signalling, ubiquitination and phosphatase activity are the most highly enriched, and 181 factors assemble into a highly significant host-pathogen interaction network. Moreover, 219 of the 295 factors were confirmed to be required for efficient wild-type influenza virus growth, and further analysis of a subset of genes showed 23 factors necessary for viral entry, including members of the vacuolar ATPase (vATPase) and COPI-protein families, fibroblast growth factor receptor (FGFR) proteins, and glycogen synthase kinase 3 (GSK3)-beta. Furthermore, 10 proteins were confirmed to be involved in post-entry steps of influenza virus replication. These include nuclear import components, proteases, and the calcium/calmodulin-dependent protein kinase (CaM kinase) IIbeta (CAMK2B). Notably, growth of swine-origin H1N1 influenza virus is also dependent on the identified host factors, and we show that small molecule inhibitors of several factors, including vATPase and CAMK2B, antagonize influenza virus replication.

There are two issues related to women and pathways to STEM careers. Recruitment to the field is one aspect. The second issue is the retention of women in STEM fields. This paper reports on the structured interviews with 14 women who have taken career breaks sometime after receiving their PhD in order to understand the pressures in place to take such breaks, how women remain connected (or not) to their field during the break and issues related to re-entering the field after a career break. Suggestions based on the interviews include enhancements for women’s preparation for STEM fields and changes to the STEM culture itself. We suggest the inclusion workshops to detail what they might expect in their career and a provision for networking opportunities. We urge changes in the fee structure for society membership, differences in how resumes are reviewed as well as changes in maternity and child care leave., There are two issues related to women and pathways to STEM careers. Recruitment to the field is one aspect. The second issue is the retention of women in STEM fields. This paper reports on the structured interviews with 14 women who have taken career breaks sometime after receiving their PhD in order to understand the pressures in place to take such breaks, how women remain connected (or not) to their field during the break and issues related to re-entering the field after a career break. Suggestions based on the interviews include enhancements for women’s preparation for STEM fields and changes to the STEM culture itself. We suggest the inclusion workshops to detail what they might expect in their career and a provision for networking opportunities. We urge changes in the fee structure for society membership, differences in how resumes are reviewed as well as changes in maternity and child care leave., There are two issues related to women and pathways to STEM careers. Recruitment to the field is one aspect. The second issue is the retention of women in STEM fields. This paper reports on the structured interviews with 14 women who have taken career breaks sometime after receiving their PhD in order to understand the pressures in place to take such breaks, how women remain connected (or not) to their field during the break and issues related to re-entering the field after a career break. Suggestions based on the interviews include enhancements for women’s preparation for STEM fields and changes to the STEM culture itself. We suggest the inclusion workshops to detail what they might expect in their career and a provision for networking opportunities. We urge changes in the fee structure for society membership, differences in how resumes are reviewed as well as changes in maternity and child care leave.

Hepatitis C virus (HCV) can be classified into six genotypes (1-6) which show 30% nucleotide sequence variability throughout the genome. HCV genotypes 1, 2 and 3 have a world-wide distribution but their prevalence differs from one geographical area to another. In Scotland there is an approximate 50/50 split between individuals infected with HCV genotype 1 and genotype 3. There is little evidence that disease progression or severity differs between the genotypes. On the other hand, one difference which has been consistently demonstrated is the better response of patients infected with genotypes 2 and 3 to interferon treatment than those infected with genotype 1. HCV encodes three structural proteins, core, E1 and E2. The two glycoproteins, E1 and E2, are bE1ieved to be the envE1ope proteins. The lack of a cE1l culture system for the production of HCV virions has meant that the proteins within the envE1ope of the HCV virion remain uncharacterised. However, recombinant forms of the E1 and E2 proteins have been shown to localise to the endoplasmic reticulum (ER) when expressed in cultured cE1ls and to interact with one another to form a complex (Ralston et al., 1993). E2 has also recently been shown to bind to the cE1l-surface molecule, CD81, which, as a result, has been proposed to be an HCV receptor (Pileri et al., 1998). These characteristics of the HCV glycoproteins have been described using constructs derived from genotype 1 only. Comparison of the properties of E1 and E2 with those of other genotypes would identify both conserved features, which are possibly essential to the virus life cycle, and genotype-specific features. In this study, the E1 and E2 proteins of genotype 3 were compared with those of genotype 1 with respect to antibody recognition, subcE1lular localisation, complex formation, glycosylation status and CD81-binding. The role of E2 in mediating resistance to interferon (IFN) through a proposed interaction with PKR (Taylor et al., 1999) was also inv