Influenza A virus NS1 protein activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway by direct interaction with the p85 subunit of PI3K

Abstract

Influenza A virus infection activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but the mechanism is not clear. Here, it is reported that influenza A virus NS1 protein is responsible for PI3K/Akt pathway activation. It was demonstrated that the NS1 protein interacts with the p85 regulatory subunit of PI3K via direct binding to the SH3 and C-terminal SH2 domains of p85. Consensus binding motifs for SH3 and SH2 domains were found in influenza A virus NS1, namely an SH2-binding motif (YXXXM) at aa 89, SH3-binding motif 1 (PXXP) around aa 164 and SH3-binding motif 2 around aa 212. Mutant virus encoding NS1 protein with mutations in the SH-binding motifs failed to interact with SH domains of p85 and did not activate the PI3K/Akt pathway. The mutant virus is attenuated in Madin-Darby canine kidney cells. Our study has established a novel function of NS1: by interacting with p85 via the SH-binding motifs, NS1 can activate the PI3K/Akt pathway.RNA segment 8 of Influenza A virus encodes two proteins, NS1 and NS2/NEP (Lamb, 1989). NS1 is a multifunctional protein that is translated from unspliced mRNA (Lamb & Choppin, 1979). Two functional domains have been identified in the NS1 protein: an RNA-binding domain near the N terminus and an effector domain in the C terminus . The RNA-binding activity of the NS1 protein correlates to its ability to inhibit cellular pre-mRNA splicing (Lu et al., 1994;Qiu et al., 1995). It is also linked to the ability to counteract cellular alpha/beta interferon functions efficiently (Garcia-Sastre et al., 1998) by inhibiting the activation of the protein kinase PKR (Lu et al., 1995;Hatada et al., 1999) and transcription factors NF-kB, IRF-3 and IRF-7 (Talon et al., 2000;Wang et al., 2000). Within the effector domain, two binding sites of cellular proteins were identified. The binding site for CPSF is positioned around aa 186 and the PABII-binding site is located between aa 223 and 237 (Li et al., 2001). These binding sites are required for the inhibition of 39-end processing of cellular pre-mRNAs; thus, they are important for influenza virus replication (Noah et al., 2003).Phosphatidylinositol 3-kinases (PI3Ks) are a family of cellular, heterodimeric enzymes that consist of a regulatory subunit (p85) and a catalytic subunit (p110). PI3K is activated by binding of the SH domain in the p85 subunit to autophosphorylated tyrosine kinase receptors, or to non-receptor tyrosine kinases or some viral proteins in the cytoplasm (Carpenter et al., 1993;Street et al., 2004). After activation, the p110 subunit of PI3K phosphorylates the lipid substrate phosphatidylinositol-4,5-bisphosphate to produce phosphatidylinositol-3,4,5-trisphosphate (Toker & Cantley, 1997). This molecule serves as a lipid second messenger and is able to regulate phosphorylation of a number of kinases, including Akt. Akt is activated via phosphorylation at Thr308 and Ser473 (Alessi et al., 1996). Phosphorylated Akt plays a central role in modulating diverse downstream signalling pathways associated with cell survival, pro...

A new influenza virus virulence determinant: The NS1 protein four C-terminal residues modulate pathogenicity

“…The NS1 protein has been reported to have multiple functions including controlling the temporal synthesis of viral-specific mRNA and viral genomic RNAs (12,13) and interacting with the cellular protein phosphatidylinositol-3-kinase (PI3-kinase) (14)(15)(16), which may cause a delay in virus-induced apoptosis (17). However, the majority of previous work has focused on the ability of NS1 to circumvent the host cell antiviral responses.…”

Structural basis for suppression of a host antiviral response by influenza A virus

“…By identifying the replication defect of a recombinant influenza A/Udorn/72 (Ud) virus that encodes an NS1A protein lacking dsRNA-binding activity, it was established that the primary role of NS1A dsRNA-binding activity is the inhibition of the IFN-␣/␤-induced oligo A synthetase/RNase L pathway, and that NS1A dsRNA-binding activity has no detectable role in inhibiting the production of IFN-␤ mRNA or inhibiting the activation of protein kinase R (PKR) (4,5). The rest of the NS1A protein, which is referred to as the effector domain, has binding sites for several cellular proteins, including: the cellular 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a cellular factor required for the 3Ј end processing of cellular pre-mRNAs, thereby inhibiting the production of all cellular mRNAs, including IFN-␤ mRNA (6-10); p85␤, resulting in the activation of phosphatidylinositol-3-kinase signaling (11)(12)(13)(14); and PKR, resulting in the inhibition of PKR activation (15).…”

Deep sequencing of the mouse lung transcriptome reveals distinct long non-coding RNAs expression associated with the high virulence of H5N1 avian influenza virus in mice

“…Most importantly, a number of studies have shown that influenza virus can effectively activate the MAPK pathways and the activation of MAPK family members plays an important role in viral replication, proinflammatory and apoptotic response in various cells during influenza virus infection [69–74]. Moreover, the PI3K/Akt/mTOR pathway also activated during influenza virus infection and function as supporting viral effective replication [75–80]. …”