Analysis of influenza A virus temperature-sensitive mutants with mutations in RNA segment 8

Abstract

Temperature-sensitive (ts) mutants of influenza virus strain A/Udorn/72 (H3N2 subtype) with lesions in RNA segment 8 exhibited intrasegmental complementation, and were divided in two complementation groups (HI and H2) on MDCK cells. The nucleotide sequence of segment 8 was determined for three of these mutants. The H1 strains, ICR1629 and SPC45, have a single amino acid substitution in the coding region of the non-structural protein NS 1, whereas the H2 strain, ICR516, has a substitution in the NS2-coding region. With both NS1 ts mutants, the synthesis of two late proteins, the matrix protein (M1) and haemagglutinin (HA), was greatly reduced and NS1 synthesis also decreased at 40 °C (non-permissive temperature) compared to that at 34 °C (permissive temperature). The synthesis of each virus-specific RNA was analysed using a quantitative hybridization method. However, at 40 °C, the levels of individual mRNAs including those for the late proteins, were almost the same as those at 34 °C, and attained the wild-type levels later in the infection (5 h postinfection) when the synthesis of the late proteins and the NS1 protein was severely reduced. The observations suggest that the NS 1 protein, which is a nuclear protein, is involved in some post-transcriptional processes in the synthesis of the late proteins and the NS 1 protein.

Genetic Analysis of Influenza Virus NS1 Gene: a Temperature-Sensitive Mutant Shows Defective Formation of Virus Particles

“…Although it is not essential for virus viability (23), mutation in NS1 protein leads to defective viruses, some of which show temperature-sensitive phenotypes (12,14,17,27,28,40,60,(63)(64)(65). In addition, NS1 mutants are less virulent than wt viruses in experimental infections in animals (2,15,23,68).…”

“…It is translated from the colinear transcript of segment 8, which also encodes NS2 protein from a spliced mRNA (31, 34). NS1 accumulates in the nucleus early in the infection (4, 32) and when it is expressed from cDNA (26,35,56), but at late times in the infection it is also found in the cytoplasm (49), in association with polysomes (8,16,32).Although NS1 protein is apparently nonessential, as a recombinant virus has been generated that lacks the gene (23), several virus mutants have been isolated or generated which contain mutations in the NS1 protein and are severely hampered for replication (12,14,17,27,28,40,60,(63)(64)(65). The phenotypes of these mutant viruses indicate that NS1 protein may be involved in several steps in the virus infectious cycle, including transcription and/or replication of virus RNA, late virus protein synthesis, and interference with the cellular gene expression, and that it eventually modulates the virulence of virus infections in vivo (2, 23, 68).…”

“…Although NS1 protein is apparently nonessential, as a recombinant virus has been generated that lacks the gene (23), several virus mutants have been isolated or generated which contain mutations in the NS1 protein and are severely hampered for replication (12,14,17,27,28,40,60,(63)(64)(65). The phenotypes of these mutant viruses indicate that NS1 protein may be involved in several steps in the virus infectious cycle, including transcription and/or replication of virus RNA, late virus protein synthesis, and interference with the cellular gene expression, and that it eventually modulates the virulence of virus infections in vivo (2, 23, 68).…”

Involvement of the influenza A virus PB2 protein in the regulation of viral gene expression

“…To study further the mechanism of viral gene expression, we have analysed temperature-sensitive (ts) mutants of influenza A/Udorn/72 (H3N2) virus (Shimizu et al, 1982a, b;Hatada et al, 1990). Using six ts mutants of the PB2 gene, we studied the synthesis of viral RNAs and proteins in infected MDCK cells to identify the functional roles of the PB2 protein in viral RNA synthesis and its regulation.…”

Binding of influenza A virus NS1 protein to dsRNA in vitro

“…The protein is encoded by RNA segment 8 together with another non-structural protein, NS2 (Inglis et al, 1980;Lamb & Lai, 1980). The function(s) of NS 1 remains to be determined despite many studies including analysis of temperature-sensitive mutants of the protein (Lamb, 1989;Hatada et al, 1990;Alonso-Caplen et al, 1992), having been performed.…”