Universitą degli studi di Pavia
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Bavagnoli research activity
NS1 is a non-structural protein of the influenza virus: it is not a structural component of the virion, but it is expressed at very high levels in infected cells. NS1 inhibits host immune response limiting both interferon (IFN) production and the antiviral effects of IFN-induced proteins, by seizing the CSPF factor, responsible for the polyadenylation of mRNA and subtracting the PAB II protein (poly (A)-binding protein) required for host cell mRNA processing. It was observed that deletions in the carboxy-terminal (C-terminal) region of NS1 can occurr during seasonal epidemics in the circulting influenza A strains, but their biological significance is not as yet clear. Literature data have shown that the last six aminoacids (225-230) of NS1, in strains of avian origin, constitute a PDZ-binding domain (PL), which allows interaction with a wide range of cellular proteins. PDZ domains are protein-protein interaction modules that recognize specific C-terminal sequences to assemble protein complexes. We have identified in the last 12 amino acids of the C-terminal region of H7N1 NS1 a previously unreported rearrangement constituted by two consecutive PL motifs (TPL, Tandem PDZ-Ligand). The PDZ domain is present in a wide range of cellular human proteins: experiments carried out in order to analyze the binding properties of the new TPL domain of NS1, using a protein array, have allowed us to identify the cellular protein RIL (Reversion Induced-LIM) as the primary ligand protein of NS1. Literature data have shown that RIL interacts with the active form of tyrosine kinase Src, playing a leading role in its regulation. It is known that avian NS1 proteins also contain two Src-homology type 3 binding domain (SHB): SHB1 has been shown to mediate the interaction of NS1 with p85β subunit of PI3K kinase, while SHB2 is thought to be able to mediate the interaction with adapter proteins Crk/CrkL.
Since avian influenza virus NS1 is able to physically interact with RIL and since RIL is a known intractor of Src, also consideraing that NS1 possesses two SHB domains, we tested the ability of the viral NS1 protein to physically and functionally interact with Src, both in the presence or absence of RIL, through enzymatic assays and pull down assays. The results shows that NS1 is able to physically interact with Src, and to stimulate its enzymatic activity. However, the exact role of Src activation in avian influenza virus life cycle is still unknown.
Due to its potential binding to multiple partners, NS1 can interfere with different cellular pathways. Our findings are aimed at defining a therapeutic strategy that can reduced the emergence of viral resistance towards antiviral drugs is the selective targeting of host factors required for viral replication.
Since avian influenza virus NS1 is able to physically interact with RIL and since RIL is a known intractor of Src, also consideraing that NS1 possesses two SHB domains, we tested the ability of the viral NS1 protein to physically and functionally interact with Src, both in the presence or absence of RIL, through enzymatic assays and pull down assays. The results shows that NS1 is able to physically interact with Src, and to stimulate its enzymatic activity. However, the exact role of Src activation in avian influenza virus life cycle is still unknown.
Due to its potential binding to multiple partners, NS1 can interfere with different cellular pathways. Our findings are aimed at defining a therapeutic strategy that can reduced the emergence of viral resistance towards antiviral drugs is the selective targeting of host factors required for viral replication.
