An amino acid change in the non-structural NS2 protein of an influenza A virus mutant is responsible for the generation of defective interfering (DI) particles by amplifying DI RNAs and suppressing complementary RNA synthesis

Abstract

The mutated non-structural NS2 protein of an influenza A virus mutant, Wa-182, has been shown to be responsible for the production of defective interfering (DI) particles lacking the PA gene after a single cycle high-multiplicity infection. Using a subclone of Wa-182, A3/e-3, that inherited the Wa-182 phenotype but contained only a marginal amount of DI RNAs derived from the PA gene, we showed that replication of the PA genome RNA was suppressed primarily at the step of complementary RNA (cRNA) synthesis. On the other hand, the small amounts of DI RNA species present in the stock of A3/e-3 were shown to be replicated efficiently. These findings suggested that the suppression of cRNA synthesis of the PA gene was caused by preferential amplification of the DI RNAs. The suppression of PA gene cRNA synthesis subsequently resulted in suppression of both virion RNA synthesis and secondary transcription of the PA gene. Such aberrant replication of the PA gene was found to be attributable to an amino acid change in the NS2 protein at position 32, from isoleucine to threonine. These results suggest that the NS2 protein plays a role in promoting normal replication of the genomic RNAs by preventing the replication of short-length RNA species.

Biological Activities of 'Noninfectious' Influenza A Virus Particles

“…Sequence analysis of DI RNAs shows that the size and location of the deletions vary widely from stock to stock and tend to evolve over repeated passage[23,32]. While the cis -acting factors that influence DI formation remain obscure, single amino acid substitutions in either NEP (also known as NS2) or the acidic polymerase protein (PA) can enhance the production of DI RNAs [38–40]. These findings demonstrate that trans -acting viral genetic determinants of DI formation do exist.…”

Genome packaging in influenza A virus

“…As it became possible to determine the segment from which a DI RNA was derived, it became apparent that in many cases, their presence correlated with reduced amounts of the parent segment in virus particles (Akkina et al, 1984;Nakajima et al, 1979;Odagiri & Tobita, 1990;Ueda et al, 1980). Furthermore, this interference was shown to act at the level of packaging (rather than solely at the point of synthesis in cells) and also to affect the incorporation of the homologous segment in mixed infections of DI-containing and non-defective wild-type virus stocks (Duhaut & Dimmock, 2002;Duhaut & McCauley, 1996;Odagiri & Tashiro, 1997;Odagiri et al, 1994). This segment-specific competition implied that packaging involved selection for distinctive features shared by the segment and its DI RNA.…”

In Vitro Analysis of Virus Particle Subpopulations in Candidate Live-Attenuated Influenza Vaccines Distinguishes Effective from Ineffective Vaccines

“…The DIPs of the D-delNS1 variants constituted from 86 to 98% of the BAP population, making them the predominant BAPs ( Table 1). On that basis, it is tempting to infer a causal relationship between the truncated NS1 proteins common to the D-del variants and the propensity to generate DIPs, perhaps akin to that reported for the NS2/NEP protein (30) and in keeping with the generally recognized role of the NS1 protein in replication.…”