Research Article: Pathogenicity of the H1N1 influenza virus enhanced by functional synergy between the NPV100I and NAD248N pair

Date Published: May 31, 2019

Publisher: Public Library of Science

Author(s): Woo-Jong Kim, Kye-Yeon Hur, Han Wook Park, Seung-Woo Lee, Joo-Yeon Yoo, Balaji Manicassamy.


By comparing and measuring covariations of viral protein sequences from isolates of the 2009 pH1N1 influenza A virus (IAV), specific substitutions that co-occur in the NP-NA pair were identified. To investigate the effect of these co-occurring substitution pairs, the V100I substitution in NP and the D248N substitution in NA were introduced into laboratory-adapted WSN IAVs. The recombinant WSN with the covarying NPV100I-NAD248N pair exhibited enhanced pathogenicity, as characterized by increased viral production, increased death and inflammation of host cells, and high mortality in infected mice. Although direct interactions between the NPV100I and NAD248N proteins were not detected, the RNA-binding ability of NPV100I was increased, which was further strengthened by NAD248N, in expression-plasmid-transfected cells. Additionally, the NAD248N protein was frequently recruited within lipid rafts, indirectly affecting the RNA-binding ability of NP as well as viral release. Altogether, our data indicate that the covarying NPV100I-NAD248N pair obtained from 2009 pH1N1 IAV sequence information function together to synergistically augment viral assembly and release, which may explain the observed enhanced viral pathogenicity.

Partial Text

The genome of influenza A virus (IAV), a member of the Orthomyxoviridae family, consists of a segmented negative-sense single-strand RNA, and changes in the influenza viral genome are frequent events due to the accumulation of mutations and reassortment of RNA segments [1]. In addition to seasonal flu, IAV pandemic infections occasionally occur, with severe impacts on public health and society. There have been extensive efforts using various approaches to understand the nature of influenza pandemics. Among them, sequence comparison and reverse genetics using recombinant virus in murine or primate systems have been useful for identifying key RNA segments or sequence substitutions that contribute to influenza viral pathogenicity.

In this report, we demonstrate that functional synergy between the covarying NPV100I and NAD248N mutation pairs of the 2009 pH1N1 IAV explains the enhanced pathogenicity of this influenza virus. A single-amino acid substitution at the 100th residue of the NP protein increased its vRNA binding potential, which was significantly enhanced when the NAD248N protein was present. The ability of the NA protein to be recruited to the lipid raft compartment, which directly influences viral particle release, was also augmented with a single-amino acid change at the 248th residue of the NA protein. Therefore, our data indicate that the covarying NPV100I and NAD248N pair functions to boost viral component assembly and virus particle release, which may explain the increased production of influenza virus (Fig 6).