A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis

Abstract

It is not known how influenza A viruses, important human pathogens, counter PKR activation, a crucial host antiviral response. Here we elucidate this mechanism. We show that the direct binding of PKR to the NS1 protein in vitro that results in inhibition of PKR activation requires the NS1 123-127 amino acid sequence. To establish whether such direct binding of PKR to the NS1 protein is responsible for inhibiting PKR activation in infected cells, we generated recombinant influenza A/Udorn/72 viruses expressing NS1 proteins in which amino acids 123/124 or 126/127 are changed to alanines. In cells infected with these mutant viruses, PKR is activated, eIF-2alpha is phosphorylated and viral protein synthesis is inhibited, indicating that direct binding of PKR to the 123-127 sequence of the NS1 protein is necessary and sufficient to block PKR activation in influenza A virus-infected cells. Unexpectedly, the 123/124 mutant virus is not attenuated because reduced viral protein synthesis is offset by enhanced viral RNA synthesis at very early times of infection. These early viral RNAs include those synthesized predominantly at later times during wild-type virus infection, demonstrating that wild-type temporal regulation of viral RNA synthesis is absent in 123/124 virus-infected cells. Enhanced early viral RNA synthesis after 123/124 virus infection also occurs in mouse PKR-/- cells, demonstrating that PKR activation and deregulation of the time course of viral RNA synthesis are not coupled. These results indicate that the 123/124 site of the NS1A protein most likely functionally interacts with the viral polymerase to mediate temporal regulation of viral RNA synthesis. This interaction would occur in the nucleus, whereas PKR would bind to NS1A proteins in the cytoplasm prior to their import into the nucleus.

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

“…Some of the residues reported to interact with the CPSF30 subunit of the polyadenylation complex are highly conserved, namely Leu144, Gly184, Glu186, Asn188 and the highly surface-exposed Trp187. Residues 123-127 have been shown to interact with a dsRNA-dependent serine/ threonine protein kinase R (PKR) (Min et al, 2007). These residues are surface-exposed and Lys126 is conserved (grade 8), while the other residues are variable.…”

The NS1 gene from bat-derived influenza-like virus H17N10 can be rescued in influenza A PR8 backbone

“…For this, we picked H17N10 NS1, which shares an average of 45 % identity with NS1 molecules of other virus subtypes (Tong et al, 2012). The non-structural protein NS1 acts at multiple levels to inhibit type I IFN synthesis by direct interaction with host factors or through binding to dsRNA (Chen et al, 1999;Donelan et al, 2003;Gack et al, 2009;Li et al, 2006;Min et al, 2006Min et al, , 2007Tan & Katze, 1998), which allows the virus to overcome host defences and replicate efficiently (Hale et al, 2008). The anti-host immune responses of NS1 are attributed to two functional domains of NS1: one is the N-terminal RNA-binding domain (RBD) (Qian et al, 1995) and the other is the C-terminal effector domain (ED) (Li et al, 1998).…”

Differential RNA silencing suppression activity of NS1 proteins from different influenza A virus strains

“…For example, NS1 blocks 29-59-oligoadenylate synthetase-mediated activation of RNase L (Min & Krug, 2006) and limits the induction of IFN-b (Wang et al, 2000;Ludwig et al, 2002). Furthermore, NS1 binds to and inhibits the cytoplasmic dsRNA sensor retinoic acidinducible gene product I (Guo et al, 2007;Mibayashi et al, 2007) and blocks protein kinase R-mediated inhibition of protein synthesis (Bergmann et al, 2000;Min et al, 2007). In addition to its IFN antagonistic activities, NS1 has recently been shown to actively suppress RNA silencing in plant, insect and mammalian cells (Bucher et al, 2004;Delgadillo et al, 2004;Li et al, 2004;Haasnoot et al, 2007).…”