Evidence for the Involvement of Influenza A (Fowl Plague Rostock) Virus Protein P2 in ApG and mRNA Primed in vitro RNA Synthesis

Abstract

SUMMARYEleven temperature-sensitive (ts) mutants of influenza A (fowl plague, Rostock) virus were analysed for in vitro RNA transcriptase activity in reactions primed by ApG or globin rnRNA at 31 °C or at 40.5 °C, the restrictive temperature for ts mutant growth. Only those ts mutants studied which were defective in RNA segment 1, coding for the virion P2 protein, were defective in RNA transcriptase activity when compared to wild-type virus. Mutants having a defect in the P2 protein had no significant RNA transcriptase activity in reactions at 40.5 °C primed by globin mRNA. However, one mutant showed RNA transcriptase activity similar to wild-type virus at 40.5 °C when ApG (0.3 raM) was used as primer. The results suggest that influenza (fowl plague, Rostock) P2 protein is directly involved in the mRNA priming reaction, as well as in the RNA transcription reaction in vitro.The eight distinct negative-strand RNA segments of fowl plague virus, an avian influenza A virus, can be transcribed in vitro by the virion-bound RNA-dependent RNA polymerase (for reviews, see Barry & Mahy, 1979~ Hay & Skehel, 1979, Under optimal conditions the products of the influenza virion RNA-dependent RNA polymerase reaction have many of the properties of virus mRNA synthesized in infected cells, in that they are approx, full-length, polyadenylated transcripts of each genome segment (Plotch & Krug, 1977, 1978: Hay et al., 1977. However, the structures of in vivo and in vitro synthesized mRNA do differ in that the in vitro mRNAs are shorter and lack a 5' methylated cap structure (Hay et al., 1977. Furthermore, primary transcription in vivo is sensitive to the drugs actinomycin D and alpha-amanitin (inhibitors of host cell DNA-dependent RNA polymerase ll), whereas transcription in vitro is insensitive to these drugs (Scholtissek & Rott, 1970;Penhoet et aL, 1971;Mahy et aL, 1972;Lamb & Choppin, 1977;Spooner & Barry, 1977). In addition, the influenza virion transcriptase was shown to possess another unusual property in that a number of guanosine related compounds, particularly the dinucleoside adenyl (3' to 5') guanosine (ApG), stimulate RNA synthesis in vitro by acting as primers for RNA synthesis (McGeoch & Kitron, 1975;Plotch & Krug, 1977).Recently, an explanation for these findings became apparent when it was reported that various eukaryotic mRNAs stimulate the in vitro transcription reaction (Bouloy et al., 1978) and that the 5'-terminal cap structure plus 9 to 15 nucleotides is transferred to the 5' end of the transcript in vitro (Plotch et al., 1979;Bouloy et al., 1980. The authors suggested that capped host cell RNA molecules are used as 'primers' for synthesis of influenza mRNAs in vivo and that the continued synthesis of these primer molecules is the alpha-amanitin-sensitive step in influenza virus replication (Kruget aL, 1979). In an attempt to determine which virion proteins participate in this priming reaction, we have studied the ApG and globin mRNA-primed in vitro transcriptase activities of a number of genetically characteriz...

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

“…The PB2 protein is an essential constituent of the transcriptase complex and has been shown to interact with the type 1 cap structure of host cell heterogeneous nuclear RNA, utilizing it as a primer for mRNA synthesis (Ulmanen et al, 1981Braam et al, 1983). With the PB2 mutants tested, A-pG-primed transcription, in which cap recognition by RNA polymerase is not required, was affected to the same degree as reovirus mRNA-primed transcription, suggesting that the PB2 protein is also involved in the transcription process at some stage other than the cap interaction, as reported previously (Scholtissek & Bowles, 1975;Krug et al, 1975;Sugiura et al, 1975;Nichol et al, 1981;Ghendon et al, 1982). One of the PB2 mutants, ICRC27, exhibited multiple abnormal phenotypes and, therefore, was subjected to further investigation.…”

Identification of the Cap-binding Protein of Two Strains of Influenza A/FPV

“…Thus the virus-specific cap-binding protein may function within the cell nucleus, consistent with its role in the priming of virus-specific mRNA synthesis. These results are also consistent with the results we have obtained with temperature-sensitive mutants of A/FPV/Rostock which have shown that a functional P2 protein is required for utilization of the host cell mRNA primer for virus-specific mRNA synthesis Nichol et al, 1981). Horisberger (1980) has shown by non-equilibrium isoelectrophoresis that the three P proteins of influenza virus comprise two basic and one acidic protein.…”

Extragenic Suppression of a ts Phenotype During Recombination Between ts Mutants of Two Fowl Plague Virus Strains with a ts Mutation in Gene 1

“…a complex of proteins involved in the transcription process. The products of segments 1 and 2 of fowl plague virus both migrate into the nucleus after synthesis (Briedis et al, 1981) where they probably function in messenger RNA synthesis (Mahy et al, 1981;Nichol et al, 1981). A recent report described another example of suppressor recombination with influenza virus involving segments 1 and 8 (Scholtissek & Spring, 1981); one of the segment 8 products, the NS1 protein, also migrates into the cell nucleus after synthesis and may interact there with the transcription complex.…”