Human host factors required for influenza virus replication.

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.

The interferon antagonistic activities of the V proteins from two strains of Newcastle disease virus correlate with their known virulence properties.

Newcastle disease virus (NDV) is an avian paramyxovirus that exists as hundreds of strains with widely different virulence properties. The NDV V protein exhibits interferon (IFN) antagonistic activity, which contributes to the virulence of the virus. The IFN-antagonistic activities of the V proteins from the avirulent strain La Sota and the moderately virulent strain Beaudette C (BC) were compared in an assay for the rescue of a recombinant NDV expressing the green fluorescent protein (NDV-GFP). Consistent with the virulence properties of the two viruses, the BC V protein exhibits a 4-fold greater ability to rescue replication of NDV-GFP than the La Sota V protein. Four amino acid differences in the C-terminal region of V, as well as the N-terminal region, contribute to the difference in IFN-antagonistic activity between the two V proteins.

Enterohaemorrhagic Escherichia coli Tir requires a C-terminal 12-residue peptide to initiate EspF-mediated actin assembly and harbours N-terminal sequences that influence pedestal length.

Enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) both utilize type III secretion systems that translocate the effector protein Tir into the plasma membrane of mammalian cells in order to stimulate localized actin assembly into 'pedestals'. The Tir molecule that EPEC delivers is phosphorylated within its C-terminus on tyrosine-474, and a clustered 12-residue phosphopeptide encompassing this residue initiates an efficient signalling cascade that triggers actin polymerization. In addition to Y474, tyrosine-454 of EPEC Tir is phosphorylated, although inefficiently, and promotes actin polymerization at low levels. In contrast to EPEC Tir, EHEC Tir lacks Y474 and triggers pedestal formation in a phosphotyrosine-independent manner by interacting with an additional effector protein, EspF(U). To identify EHEC Tir sequences that regulate localized actin assembly, we circumvented the strict requirements for type III translocation and directly expressed Tir derivatives in mammalian cells by transfection. Infection of Tir-expressing cells with a Tir-deficient EHEC strain demonstrated that ectopically expressed Tir localizes to the plasma membrane, is modified by mammalian serine-threonine kinases and is fully functional for actin pedestal formation. Removal of portions of the cytoplasmic N-terminus of Tir resulted in the generation of abnormally long pedestals, indicating that this region of EHEC Tir influences pedestal length. In the presence of the entire N-terminal domain, a 12-residue peptide from the C-terminus of EHEC Tir is both necessary and sufficient to recruit EspF(U) and initiate actin pedestal formation. This peptide encompasses the portion of EHEC Tir analogous to the EPEC Tir-Y454 region and is present within the Tir molecules of all pedestal-forming bacteria, suggesting that this sequence harbours a conserved signalling function.

Monoclonal antibody routinely used to identify avirulent strains of Newcastle disease virus binds to an epitope at the carboxy terminus of the hemagglutinin-neuraminidase protein and recognizes individual mesogenic and velogenic strains.

Newcastle disease virus (NDV) strains are classified as having high (velogenic), intermediate (mesogenic), or low (lentogenic) pathogenesis and virulence in chickens. Recent studies have established that the hemagglutinin-neuraminidase (HN) protein plays an important role in viral tropism and virulence. A monoclonal antibody (AVS-I) has previously been shown to be specific for lentogenic strains of NDV (Srinivasappa et al., Avian Dis. 30:562-567, 1986) and is routinely used to identify these strains. We have used competition antibody binding assays with a previously characterized panel of monoclonal antibodies, binding to chimeric HN proteins, and the characterization of an escape mutant to localize the binding site of AVS-I to the extreme carboxy terminus of the protein. In addition, we have shown that AVS-I does recognize at least one mesogenic strain and one velogenic strain of the virus, calling into question the potential of this antibody as a diagnostic reagent for avirulent NDV strains.