Identification of the defective genes in three mutant groups of influenza virus

Abstract

Seven complementation-recombination groups of temperature-sensitive (ts) influenza WSN virus mutants have been previously isolated. Recently two of these groups (IV and VI) were shown to possess defects in the neuraminidase and the hemagglutinin gene, respectively, and two groups (I and III) were reported to have defects in the P3 and P1 proteins which are required for complementary RNA synthesis. In this communication we report on the defects in the remaining three mutant groups. Wild-type (ts+) recombinants derived from ts mutants and different non-ts influenza viruses were analyzed on RNA polyacrylamide gels. This technique permitted the identification of the P2 protein, the nucleoprotein, and the M protein as the defective gene products in mutant groups II, V, and VII, respectively. Based on the physiological behavior of mutants in groups II and V, it appears that P2 protein and nucleoprotein are required for virion RNA synthesis during influenza virus replication.

De novo replication of the influenza virus RNA genome is regulated by DNA replicative helicase, MCM

“…Since the influenza virus genome consists of eight-segmented and singlestranded RNAs, we detected eight segments of newly synthesized RNAs. The electrophoretic mobility of these RNAs synthesized in the presence of IREF-1 was similar to that of the virus genome RNAs prepared from purified virions (data not shown) and that of RNAs synthesized in the presence of the ApG dinucleotide primer complementary to the nucleotide positions 1 and 2 of the 3 0 -terminus of vRNA (lane 5, and see Ritchey and Palese, 1977;Honda et al, 1986). To determine the polarity of newly synthesized RNA, the RNA possibly synthesized from segment 8 was excised from the gel, and the isolated RNA was then digested with RNase T1.…”

“…MCM interacted with the viral RNA polymerase through its contact with PA ( Figure 4). Previous genetic analyses suggested that PA participates in the replication process, although its precise role had not been well established (Sugiura et al, 1975;Ritchey and Palese, 1977;Kawaguchi et al, 2005). Recently, it has been reported that the influenza virus vRNP interacts with nucleosomes (Garcia-Robles et al, 2005).…”

“…In contrast, the genome replication is primer-independent and generates full-length vRNA through cRNA synthesis. Genetic analyses suggest that PA participates in the replication process, although the precise function of PA is not well established (Sugiura et al, 1975;Ritchey and Palese, 1977). Recently, we found that PA is involved in the assembly of a functional polymerase (Kawaguchi et al, 2005).…”

Hyperphosphorylation of mutant influenza virus matrix protein, M1, causes its retention in the nucleus

“…MDBK cells were infected with WSN or ts51 at 32ЊC (lanes 3, 4, 7, and 8) or at 39.5ЊC (lanes 1, 2, 5, and 6). Cells were labeled with32 P i or [ 35 S]methionine at early times (2 to 5 h [lanes 1, 3, 5, and 7]) or at late times (6 to 8 h [lanes 2, 4, 6, and 8]) and analyzed on an SDS-PAGE gel containing 12% acrylamide. M1 and NP are indicated.…”

Influence of membrane (M) protein on influenza A virus virion transcriptase activity in vitro and its susceptibility to rimantadine

“…Membrane (M) protein, which constitutes about 40% of the total amount of virion proteins, forms the internal membrane of a virion. The exact function of M protein in the reproduction of orthomyxoviruses is not yet clear, although results of studies on temperature-sensitive mutants confirm that this protein participates in the late stages of virion morphogenesis (14). Carroll and Wagner (3) recently showed for a vesicular stomatitis virus model that when M protein isolated from virions was added to a system containing vesicular stomatitis virus ribonucleoprotein (RNP), the transcriptase activity was decreased by approximately 40%.…”