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SummaryEnterohaemorrhagic 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.

SummaryNewcastle 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.Newcastle disease virus (NDV) is a member of the Paramyxoviridae family of enveloped negative-stranded RNA viruses, which also includes Sendai virus, the various parainfluenza viruses, measles virus, respiratory syncytial virus and the henipaviruses. The NDV genome contains six genes, which encode six major proteins: nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN) and the large (L) polymerase (Lamb and Parks, 2007). NDV also produces the V and W proteins by RNA editing during P gene transcription. The P gene mRNA is edited by insertion of one or two additional G residues into a run of G's within the conserved editing site, thus generating the V-and W-encoding mRNAs, respectively (Steward et al., 1993 Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. , 1997). NDV is also being used as a vaccine vector (Huang et al., 2003a;Dinapoli et al., 2009) and oncolytic agent due to its ability to kill tumor cells (Elankumaran et al., 2006;Freeman et al., 2006). NIH Public AccessCleavage of the F protein precursor (F 0 ) produces the active fusion protein (Scheid and Choppin, 1974) and is the primary determinant of virulence as determined by the number of basic residues in the cleavage site (Glickman et al., 1988;Nagai et al., 1976;Toyoda et al., 1989). However, other viral proteins contribute to virulence (Panda et al., 2004;Peeters et al., 1999). Recombinant viruses lacking V have impaired growth in cell culture and embryonated chicken eggs and are highly attenuated in young chickens (Huang et al., 2003b;Mebatsion et al., 2001). These mutant viruses also exhibit increased sensitivity to exogenous interferon (IFN) (Elankumaran et al., 2006;Huang et al., 2003b). Us...

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Newcastle disease virus (NDV) is a member of the genus Avulavirus of the Paramyxoviridae family of enveloped negative-stranded RNA viruses. The envelope of NDV virions contains two types of glycoprotein spikes, the hemagglutinin-neuraminidase (HN) and fusion (F) proteins. HN is responsible for attachment of the virus to sialic acid-containing cell surface receptors. It also possesses neuraminidase (NA) activity that cleaves sialic acid from progeny virus particles to prevent viral self-aggregation. HN also promotes the fusion activity of the F protein responsible for virus-cell and cell-cell fusion (18).NDV causes respiratory, neurological, or enteric disease in many species of birds, resulting in significant losses to the poultry industry worldwide. Strains of the virus are classified into three pathotypes based on the severity of disease in chickens. Avirulent strains that produce mild or asymptomatic infections are termed lentogenic, whereas virulent strains that cause acute infections with high mortality are termed velogenic. Strains of intermediate virulence are termed mesogenic (1). Velogenic strains are further categorized as either neurotropic or viscerotropic.It is widely accepted that cleavage of the fusion protein precursor (F 0 ) is the primary determinant of NDV virulence. F 0 is cleaved at a basic amino acid-rich region, resulting in the formation of the active fusion protein consisting of disulfidelinked F 1 and F 2 polypeptides (18). Virulent strains have four basic residues in the cleavage site, whereas avirulent strains have only two (3,20). The F 0 of virulent NDV strains is cleaved by host proteases found in a wide range of tissues, whereas that of avirulent strains is cleaved only by trypsin-like proteases secreted by a limited number of tissues in the respiratory and intestinal tracts (14).However, the susceptibility to cleavage of the F protein is not the sole determinant of NDV virulence. Modification of a lentogenic F cleavage site to a velogenic one increased virulence, but not to the level of velogenic strains (15,16). This indicates that other viral proteins in addition to F also contribute to virulence. Huang et al. (4) recently showed that the HN protein plays a role in viral tropism and virulence. The HN gene of the Beaudette C (BC) mesogenic recombinant strain rBeaudette C was exchanged with that of lentogenic recombinant strain rLaSota, creating a BC virus having the HN of LaSota and a LaSota virus having the HN of BC. Pathogenicity studies showed that the BC virus having the HN of LaSota decreased in virulence and the LaSota virus having the HN of BC increased in virulence, indicating that HN plays a role in this process.We previously characterized a panel of monoclonal antibodies (MAbs) raised against the HN glycoprotein of the velogenic Australia-Victoria/32 (AV) strain of NDV. These MAbs were used in competition antibody binding assays and additive neutralization assays to delineate seven antigenic sites that form a continuum on HN (5, 6, 10). Escape mutants were selected wit...