Impact of Protein Nitration on Influenza Virus Infectivity and Immunogenicity

ABSTRACT Influenza viruses are deadly respiratory pathogens of special importance due to their long history of global pandemics. During influenza virus infections, the host responds by producing interferons, which activate interferon-stimulated genes (ISGs) inside target cells. One of these ISGs is inducible nitric oxide synthase (iNOS). iNOS produces nitric oxide (NO) from arginine and molecular oxygen inside the cell. NO can react with superoxide radicals to form reactive nitrogen species, principally peroxynitrite. While much work has been done studying the many roles of nitric oxide in influenza virus infections, the direct effect of peroxynitrite on influenza virus proteins has not been determined. Manipulations of NO, either by knocking out iNOS or chemically inhibiting NO, produced no change in virus titers in mouse models of influenza infection. However, peroxynitrite has a known antimicrobial effect on various bacteria and parasites, and the reason for its lack of antimicrobial effect on influenza virus titers in vivo remains unclear. Therefore, we wished to test the direct effect of nitration of influenza virus proteins. We examined the impact of nitration on virus infectivity, replication, and immunogenicity. We observed that the nitration of influenza A virus proteins decreased virus infectivity and replication ex vivo. We also determined that the nitration of influenza virus hemagglutinin protein can reduce antibody responses to native virus protein. However, our study also suggests that nitration of influenza virus proteins in vivo is likely not extensive enough to inhibit virus functions substantially. These findings will help clarify the role of peroxynitrite during influenza virus infections. IMPORTANCE Nitric oxide and peroxynitrite produced during microbial infections have diverse and seemingly paradoxical functions. While nitration of lung tissue during influenza virus infection has been observed in both mice and humans, the direct effect of protein nitration on influenza viruses has remained elusive. We addressed the impact of nitration of influenza virus proteins on virus infectivity, replication, and immunogenicity. We observed that ex vivo nitration of influenza virus proteins reduced virus infectivity and immunogenicity. However, we did not detect nitration of influenza virus hemagglutinin protein in vivo. These results contribute to our understanding of the roles of nitric oxide and peroxynitrite in influenza virus infections.