The N-terminal half of the influenza virus NS1 protein is sufficient for nuclear retention of mRNA and enhancement of viral mRNA translation

Abstract

A collection of C-terminal deletion mutants of the influenza A virus NS1 gene has been used to define the regions of the NS1 protein involved in its functionality. Immunofluorescence analyses showed that the NS1 protein sequences downstream from position 81 are not required for nuclear transport. The capacity of these mutants to bind RNA was studied by in vitro binding tests using a model vRNA probe. These experiments showed that the N-terminal 81 amino acids of NS1 protein are sufficient for RNA binding activity. The collection of mutants also served to map the NS1 sequences required for nuclear retention of mRNA and for stimulation of viral mRNA translation, using the NP gene as reporter. The results obtained indicated that the N-terminal 113 amino acids of NS1 protein are sufficient for nuclear retention of mRNA and stimulation of viral mRNA translation. The possibility that this region of the protein may be sufficient for virus viability is discussed in relation to the sequences of NS1 genes of field isolates and to the phenotype of known viral mutants affected in the NS1 gene.

Large-scale analysis of influenza A virus sequences reveals potential drug target sites of non-structural proteins

“…The major role of NS1 is to antagonize the antiviral response of the host by preventing the activation of NF-kB and induction of alpha/betainterferon (IFN-a/b) (Wang et al, 2000). However, NS1 is a multifunctional protein that is additionally involved in several processes: (i) inhibiting the pre-mRNA 39-end processing (Fortes et al, 1994) by binding to two 39-end processing factors, namely cleavage and polyadenylation specificity factor and poly(A)-binding protein II (Chen et al, 1999;Nemeroff et al, 1998); (ii) blocking the post-transcriptional processing and nuclear export of cellular mRNA (Fortes et al, 1994); (iii) stimulating the translation of matrix (M1) proteins (Enami et al, 1994;Marió n et al, 1997); (iv) inhibiting the activation of a protein kinase that phosphorylates the elf-2 translation initiation factor by binding to doublestranded (ds)RNA (Lu et al, 1995;Aragó n et al, 2000); (v) induction of the phosphatidylinositol-3-kinase (PI3K)/Akt signalling pathway in order to support virus replication (Ehrhardt et al, 2006(Ehrhardt et al, , 2007. The NS1 protein is 230-237 aa, depending on the strain, and has a molecular mass of approximately 26 kDa (reviewed by Hale et al, 2008b).…”

A Human Sequence Homologue of Staufen Is an RNA-Binding Protein That Is Associated with Polysomes and Localizes to the Rough Endoplasmic Reticulum

“…dmStaufen protein is the product of a maternal mRNA of D. melanogaster that is involved in the accumulation of bicoid and oskar mRNAs at the anterior pole of the embryo and the posterior pole of the oocyte, respectively (48). It contains double-stranded RNA (dsRNA)-binding domains that associate with bicoid mRNA through a precise secondary structure in its 3Ј UTR (18).Our group has been studying the influenza virus nonstructural protein NS1, an RNA-binding protein (26,33,42) that may be involved in several regulatory processes during viral infection (reviewed in reference 37). These include the modulation of pre-mRNA splicing (20,21,32), the retention of poly(A)-containing RNA in the nucleus (20,42), and the stimulation of viral mRNA translation (10,14).…”

“…Our group has been studying the influenza virus nonstructural protein NS1, an RNA-binding protein (26,33,42) that may be involved in several regulatory processes during viral infection (reviewed in reference 37). These include the modulation of pre-mRNA splicing (20,21,32), the retention of poly(A)-containing RNA in the nucleus (20,42), and the stimulation of viral mRNA translation (10,14).…”

“…The thSTL DNA fragment of clone C was transferred in frame to pRSET vector to generate plasmid pRSTL, in which the thSTL protein was fused to a His tag and the T7 antigenic tag. The recombinant pRSTL plasmid was transformed into Escherichia coli BL21(DE3)(pLysS), the expression of His-thSTL was induced by isopropyl-␤-D-thiogalactopyranoside, and the recombinant protein was purified by chromatography on Ni-nitrilotriacetic acid (NTA)-agarose as described previously (33). In addition, the hStaufen-like cDNA sequence encoding the dsRNA-binding domains was also fused in frame to pRSET vector to generate plasmid pRHST.…”

“…Transfection of HeLa cells with expression plasmids driven by polymerase II was done with cationic liposomes (44) as described elsewhere (33). When expression vectors driven by the T7 promoter were used COS-1 cells were previously infected with vaccinia virus vTF7-3 and then transfected as indicated above.…”