Temperature-sensitive mutants of influenza WSN virus defective in virus-specific RNA synthesis

Abstract

Influenza WSN virus temperature-sensitive (ts) mutants were examined for defects in viral complementary RNA (cRNA) synthesis. The synthesis of viral cRNA was determined by hybridizing RNA from infected cells to radiolabeled virion RNA of known specific activity. Mutants in complementation groups I and III synthesized little, or no, cRNA at the nonpermissive temperature (39.5 C). When cells infected by these mutants were incubated for 5 h at the permissive temperature (33 C) and were then shifted to 39.5 C, net synthesis of cRNA ceased. This strongly suggests that mutants in these two complementation groups possess a ts defect in the transciptase complex. Mutants in group II and group V synthesize reduced amounts of cRNA at 39.5 C. In contrast to the group I and group III mutants, cRNA synthesis in cells infected by a group II or a group V mutant continues after a shift-up. This indicated that these mutants do not possess a ts transcriptase complex and that these mutants are most probably defective in some step in the amplification of cRNA synthesis. As will be discussed, the most likely defect in these mutants is in the synthesis of virion-type RNA. These results suggest that there are two influenza viral gene functions required for transcription and most likely two additional gene functions required for RNA replication.

Influenza virus RNA polymerase PA subunit is a novel serine protease with Ser624 at the active site

“…Influenza virus RNA polymerase PA subunit has been implicated in the viral RNA genome replication by analyses of temperature-sensitive (ts) mutations in the PA gene (Krug et al 1975;Scholtissek & Bowles 1975;Scholtissek et al 1976;Mahy et al 1981; Mowshowitz 1996). Because we did not observe the protease-inducing effect which they claimed occurred in our baculovirus or plasmid expression system, we have decided to examine the protease activity using a purified PA protein.…”

“…The most likely role for PA is as an RNA helicase or in the recruitment of RNA helicase, considering that: (i) temperature sensitive mutation of PA impaired the viral replication at nonpermissive temperatures (Krug et al 1975;Scholtissek & Bowles 1975;Scholtissek et al 1976;Mahy et al 1981;Mowshowitz 1981), (ii) cell extracts containing PB1, PB2 and PA carried the replication activity of a short model RNA template but those missing PA lost the replication activity although they retained vRNA 3 cRNA synthesis activity , (iii) addition of PA to cells expressing PB1, PB2 and NP in trans recovered the genome replication activity on a model RNA template (Nakagawa et al 1996). However, there is no direct evidence that PA has a helicase activity.…”

“…However, information about the role of PA in viral replication is limited. Temperature-sensitive (ts) mutations in the PA gene affected only vRNA synthesis, and not mRNA synthesis (Krug et al 1975;Mahy et al 1981;Mowshowitz 1981;Scholtissek & Bowles 1975;Scholtissek et al 1976). Nakagawa et al (1996) demonstrated that PA was essential for cRNA-dependent vRNA synthesis.…”

Influenza Virus Nucleoprotein Interacts with Influenza Virus Polymerase Proteins

“…In the influenza virus infectious cycle, NP plays a critical role in switching the transcription of viral mRNA to the replication of cRNA and vRNA. Studies with ts mutants have shown that different ts NP mutants can affect both cRNA and vRNA synthesis independently (17,19). Biochemical studies have further shown that RNP-free soluble NP is required for switching mRNA to cRNA synthesis in vitro (6,17).…”

Fine mapping of the subunit binding sites of influenza virus RNA polymerase

“…However, the information about the role of PA on viral replication remains limited. Temperature-sensitive (ts) mutations in the PA gene affected only vRNA synthesis, but not mRNA synthesis (25,30,33,45,46). Nakagawa et al demonstrated that PA was essential for cRNA-dependent vRNA synthesis (36).…”