Characterization of a temperature-sensitive mutant in the RNA polymerase PB2 subunit gene of influenza A/WSN/33 virus

Abstract

The temperature-sensitive mutant ts-1 of influenza virus A/WSN/33 carries mutations in the gene encoding RNA polymerase PB2 subunit. Effect of temperature on various steps of viral RNA synthesis was examined using disrupted virions of ts-1 mutant. The initiation of RNA synthesis with dinucleotide ApG primer was not affected by elevated temperature, whereas that with primer RNA containing 5'-terminal cap-1 structure was temperature-sensitive. The result supports the previous notion deduced from the UV-crosslinking experiments, that PB2 is involved in the cap-1 dependent initiation of RNA synthesis. In addition, the ts-1 mutant showed a defect in RNA chain elongation. Nucleotide sequence analysis of RNA segment 1 of ts-1 mutant revealed that the amino acid number 417 is essential for the recognition of cap-1 structures and/or the interaction with catalytic unit of the RNA polymerase.

Two separate sequences of PB2 subunit constitute the RNA cap‐binding site of influenza virus RNA polymerase

“…The PB1binding site is located at the N-terminal proximal region , while the nuclear localization signals (NLS) are mapped in two regions (Mukaigawa & Nayak 1991). The PB2 mutations affecting the function of PB2 or virus growth are shown along the PB2 polypeptide (Yamanaka et al 1990;Mukaigawa & Nayak 1991;Lawson et al 1992;Subbarao et al 1993Subbarao et al , 1995Perales et al 1996). 1983) and capped RNA-primed transcription initiation (Yamanaka et al 1990); (ii) capped RNA can be crosslinked to PB2 protein Blaas et al 1982;Penn et al 1982; this study) and (iii) the isolated PB2 alone is able to bind capped RNA (Shi et al 1996). In agreement with these ®ndings, short segments with low levels of sequence similarity to cellular cap-binding proteins exist in the PB2 protein (de la Luna et al 1989) (Fig.…”

Genetically engineered live attenuated influenza A virus vaccine candidates

“…We chose the PB2 gene as a target for mutagenesis for several reasons: mutations in PB2 have been associated with ts and attenuated (att) phenotypes (29,43,52), several ts mutations in PB2 have been molecularly characterized (6,20,23), and a helper virus for rescue of PB2 into infectious influenza viruses has been described (45). We chose to generate the following mutations: (i) mutations associated with ts phenotypes, for which it was possible to introduce new codons that would require more than one nucleotide change to reencode the wild-type amino acid (E65G, P112S, N265S, or I310T); (i) mutations which are associated with marked attenuation in other ts viruses of particular interest (Y658H, found in the PB2 gene of the ts mutants ts1A2 and ts1E) (23); or (iii) mutations which have potential functional importance by virtue of their location within a region of partial sequence similarity with the cellular cap-binding protein, eIF-4E (7).…”

Molecular Assembly of the Influenza Virus RNA Polymerase: Determination of the Subunit-Subunit Contact Sites

“…(ll) Nuclear localization signals (Jones et oL, 1986;IVlukaigawa & Nayak, 1991 ;Nieto et o/., 1994) ; ([~) cap-binding sequences of PB2 (a) (de la Luna et cd., 1989). The sites of mutations so far determined are indicated by arrowheads (a) (Subbarao eta/., 1993 ;Yamanaka et aL, 1990). The RNA polymerase consensus motifs of PB1 are indicated as A, B, C and D (b) (Delarue et oL, 1990;Poch et o/., 1989).…”