The K186E Amino Acid Substitution in the Canine Influenza Virus H3N8 NS1 Protein Restores Its Ability To Inhibit Host Gene Expression

Abstract

Canine influenza viruses (CIVs) are the causative agents of canine influenza, a contagious respiratory disease in dogs, and include the equine-origin H3N8 and the avian-origin H3N2 viruses. Influenza A virus (IAV) nonstructural protein 1 (NS1) is a virulence factor essential for counteracting the innate immune response. Here, we evaluated the ability of H3N8 CIV NS1 to inhibit host innate immune responses. We found that H3N8 CIV NS1 was able to efficiently counteract interferon (IFN) responses but was unable to block general gene expression in human or canine cells. Such ability was restored by a single amino acid substitution in position 186 (K186E) that resulted in NS1 binding to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a cellular protein involved in pre-mRNA processing. We also examined the frequency distribution of K186 and E186 among H3N8 CIVs and equine influenza viruses (EIVs), the ancestors of H3N8 CIV, and experimentally determined the impact of amino acid 186 in the ability of different CIV and EIV NS1s to inhibit general gene expression. In all cases, the presence of E186 was responsible for the control of host gene expression. In contrast, the NS1 protein of H3N2 CIV harbors E186 and blocks general gene expression in canine cells. Altogether, our results confirm previous studies on the strain-dependent ability of NS1 to block general gene expression. Moreover, the observed polymorphism on amino acid 186 between H3N8 and H3N2 CIVs might be the result of adaptive changes acquired during long-term circulation of avian-origin IAVs in mammals. Canine influenza is a respiratory disease of dogs caused by two CIV subtypes, the H3N8 and H3N2 viruses, of equine and avian origins, respectively. Influenza NS1 is the main viral factor responsible for the control of host innate immune responses, and changes in NS1 can play an important role in host adaptation. Here we assessed the ability of H3N8 CIV NS1 to inhibit host innate immune responses and gene expression. The H3N8 CIV NS1 did not block host gene expression, but this activity was restored by a single amino acid substitution (K186E), which was responsible for NS1 binding to the host factor CPSF30. In contrast, the H3N2 CIV NS1, which contains E186, blocks general gene expression. Our results suggest that the ability to block host gene expression is not required for influenza virus replication in mammals but might be important in the long-term adaptation of avian-origin influenza viruses to mammals.

Functional Characterization and Direct Comparison of Influenza A, B, C, and D NS1 Proteins in vitro and in vivo

“…PR8 IAV, IBV, ICV, and IDV NS1 proteins were detected by Western blot using an antibody against the HA epitope tag ( Figure 1B), although different levels of expression were noted. We were not able to detect expression of 1918 NS1, mainly because of its ability to inhibit host gene expression, including its own synthesis ( Figure 1B), as previously described (DeDiego et al, 2016;Nogales et al, 2016bNogales et al, , 2017aNogales et al, , 2018a. These results demonstrate that the NS1 proteins from IBV, ICV, and IDV do not inhibit host gene expression, similar to PR8 IAV NS1.…”

“…Gluc expression was evaluated using a luminometer ( Figure 1A) at 24 h post-transfection. As previously described (Steidle et al, 2010;DeDiego et al, 2016;Nogales et al, 2016bNogales et al, , 2017aChauche et al, 2018;Rodriguez et al, 2018), PR8 NS1 did not inhibit Gluc reporter gene expression, whereas 1918 NS1 was able to efficiently inhibit protein expression of the reporter gene. IBV, ICV, and IDV NS1 proteins did not inhibit Gluc expression ( Figure 1A).…”

“…Since the NS1 protein from some IAV strains have been shown to differ in inhibiting host gene expression (Nemeroff et al, 1998;Twu et al, 2006;Hale et al, 2010;Spesock et al, 2011;Dankar et al, 2013;Ayllon et al, 2014;DeDiego et al, 2016;Nogales et al, 2016bNogales et al, , 2017aNogales et al, , 2018aChauche et al, 2018), we selected A/Brevig Mission/1/18 H1N1 (1918) and A/Puerto Rico/8/34 H1N1 (PR8) as representative NS1 proteins with the ability to induce or not, respectively, cellular shutoff. Moreover, the antiviral functions of these two IAV NS1 proteins have been well-described in the literature (Steidle et al, 2010;DeDiego et al, 2016;Nogales et al, 2016bNogales et al, , 2017aNogales et al, , 2018a. To assess if the NS1 proteins from different origins were able to block polymerase II promoter-driven host gene expression (Figure 1), human 293T cells were transiently co-transfected with individual plasmids encoding HA epitope tagged NS1 proteins from IAV (PR8 or 1918), IBV, ICV, or IDV; together with a plasmid expressing the Gaussia luciferase (Gluc) reporter gene ( Figure 1A).…”

“…Studies describing the mechanisms of action of NS1 during viral infection have been primarily focused in IAV (Garcia-Sastre et al, 1998;Nemeroff et al, 1998;Falcon et al, 1999;Burgui et al, 2003;Twu et al, 2006;Guo et al, 2007;Kochs et al, 2007Kochs et al, , 2009Mibayashi et al, 2007;Hale et al, 2008aHale et al, ,b, 2010Steidle et al, 2010;Dankar et al, 2013;Ayllon et al, 2014;DeDiego et al, 2016;Nogales et al, 2016bNogales et al, , 2017aNogales et al, ,d, 2018aChauche et al, 2018;Rodriguez et al, 2018), IBV (Yuan and Krug, 2001;Dauber et al, 2006;Hai et al, 2008;Nogales et al, 2015;Patzina et al, 2017) and, to a less extent, ICV (Nakada et al, 1986;Muraki et al, 2010;Pachler and Vlasak, 2011). Nothing is currently known about the ability of IDV NS1 to counteract the IFN response.…”

Acetylation at K108 of the NS1 protein is important for the replication and virulence of influenza virus

“…The residue 187W in the ED domain is important for the dimerization of the NS1 protein, and the W187R substitution impaired NS1 dimerization and attenuated the virus in vivo [14]. In addition, residues 186E, 189D, and 194V play important roles in the binding of NS1 to cleavage and polyadenylation specificity factor 30 (CPSF30), and mutations in those residues weaken the binding of NS1 to CPSF30 and impair the ability of the NS1 protein to shut off host gene expression [15,16].…”