Mutation in NS2, a nonstructural protein of influenza A virus, extragenically causes aberrant replication and expression of the PA gene and leads to generation of defective interfering particles.

Abstract

Several consecutive undiluted passages of infectious virus are usually required to obtain defective interfering particles of influenza virus. In contrast, a reassortant (Wa-182) of influenza A/WSN, which we isolated, whose NS gene was replaced with the NS gene of A/Aichi, was readily converted to defective interfering form after only a single high-multiplicity infection. The defective interfering particles of Wa-182 were devoid of the PA gene (RNA segment 3) but possessed several species of subgenomic RNAs of the PA gene origin. Such aberrant replication of the PA gene was shown to be caused by an extragenic effect of the NS gene of Wa-182, because, when the NS gene of Wa-182 was singly transferred to the wild-type A/Ann Arbor/6/60 virus, the recipient showed exactly the same features. Analysis of nucleotide sequence demonstrated that the NS gene ofWa-182 contained three point mutations relative to the wild-type NS gene that resulted in two amino acid substitutions in the nonstructural protein NS2, suggesting that the mutation in NS2 protein affected the normal replication of the PA gene of Wa-182. The results also suggest that the NS2 protein plays an important role in the synthesis of intact genome RNAs.Defective interfering (DI) particles of influenza virus are produced by serial undiluted passages of a standard infectious virus (1). DI particles have a reduced amount of virion RNA (vRNA) segments accompanied by the acquisition of smaller RNA segments (DI RNAs), the amount of which correlates with the interfering ability of the particles (2-4). The majority of DI RNAs arise through simple internal deletion, retaining their original 3'-and 5'-terminal regions, and derived predominantly from polymerase genes (PA, PBI, or PB2) (5-7). Although the generation of DI RNAs may occur during the process of vRNA replication (8), its precise mechanism is not understood, probably because primary DI RNA products from their progenitor genes are unavailable.The first step in replication on influenza virus RNA is the production of full-length transcripts of the vRNAs that serve as templates for synthesis of additional vRNAs. The second step is copying template RNAs into vRNAs. These steps are initiated without a primer and are not terminated at the poly(A) site used during viral mRNA synthesis (9, 10). The synthesis of template RNA-and vRNA requires the synthesis of nucleocapsid protein not associated with nucleocapsids (10). However, the transition from template RNA to vRNA synthesis may require the synthesis of other virus-specific proteins, such as nonstructural protein (NS protein). In fact, some temperature-sensitive mutants of the NS gene of influenza A virus failed to produce vRNAs at the nonpermissive temperature, whereas they normally synthesized both the mRNAs and template RNAs at that temperature (11).By crossing influenza viruses A/WSN and A/Aichi, we have isolated a reassortant (Wa-182) that is readily converted to DI particles lacking the PA gene in a one-step growth process after a high-multiplicity inf...

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

“…We have, however, isolated an influenza A virus mutant, Wa-182, with a unique phenotype. We have demonstrated that after a single cycle of replication of Wa-182 at a high m.o.i., DI particles lacking the PA gene were predominantly produced (Odagiri & Tobita, 1990). In contrast, when the mutant was grown at low m.o.i., infectious progeny virus that retained the PA gene was produced.…”

Genome packaging in influenza A virus

“…Sequencing of DI RNAs showed that they were derived from genomic segments by internal deletion, and retained the terminal regions of the segment (Davis et al, 1980;Davis & Nayak, 1979;Duhaut & Dimmock, 1998, 2000Duhaut & McCauley, 1996;Hughes et al, 2000;Jennings et al, 1983;Moss & Brownlee, 1981;Nakajima et al, 1979;Nayak & Sivasubramanian, 1983;Nayak et al, 1982;Noble & Dimmock, 1995). 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).…”

“…These are truncated forms of genome segments which retain their ability to replicate and be packaged and which, due to their smaller size, out-compete full-length segments. Viruses containing DI RNAs arise in influenza A during high-multiplicity passage (von Magnus, 1954), particularly in certain genetic backgrounds (Nakajima et al, 1979;Odagiri & Tobita, 1990;Ueda et al, 1980). Sequencing of DI RNAs showed that they were derived from genomic segments by internal deletion, and retained the terminal regions of the segment (Davis et al, 1980;Davis & Nayak, 1979;Duhaut & Dimmock, 1998, 2000Duhaut & McCauley, 1996;Hughes et al, 2000;Jennings et al, 1983;Moss & Brownlee, 1981;Nakajima et al, 1979;Nayak & Sivasubramanian, 1983;Nayak et al, 1982;Noble & Dimmock, 1995).…”

NS2/NEP protein regulates transcription and replication of the influenza virus RNA genome

“…In addition to the role of NS2/NEP in the export of vRNPs, it has been shown that a point mutation at position 32 in the NS2/NEP protein of the A/WSN/33 influenza virus Wa-182 results in the production of defective interfering (DI) particles lacking an intact PA gene after a single highmultiplicity cycle of infection (Odagiri & Tobita, 1990;Odagiri et al, 1994). Furthermore, a study in which an influenza A chloramphenicol acetyltransferase (CAT) reporter gene was used to assess RNA synthesis showed that the NS2/NEP protein inhibited RNA synthesis by reducing the levels of vRNA, cRNA and mRNA (Bullido et al, 2001).…”