Polypeptides specified by the influenza virus genome 2. Assignment of protein coding functions to individual genome segments by in vitro translation

Cytoplasmic RNA extracted from chick embryo fibroblasts infected with influenza A (fowl plague) virus (FPV) was translated in a wheat germ cell-free protein-synthesizing system. Polypeptides which comigrated during SDS-polyacrylamide gel electrophoresis with marker virus-specific polypeptides P₁, P₂, P3, NP, M, and NS were synthesized in vitro. The NP, M, and NS polypeptides were positively identified by tryptic peptide mapping. The polypeptide component of the virus glycoprotein HA was also synthesized in vitro, and was identified by tryptic peptide mapping. RNA extracted from purified FPV (vRNA) did not direct the synthesis of any recognizable virus-specific polypeptides in vitro, either as a total preparation, or as individual RNA genome segments. The protein coding functions of the vRNA segments were identified by hybridization of individual segments to a preparation of infected cell cytoplasmic RNA. On subsequent translation of the RNA in vitro, synthesis of the virus-specific polypeptide corresponding to the hybridized vRNA segment was specifically reduced. We conclude that, for FPV, virion RNA segments 1–3 code for the three P polypeptides and segments 4, 5, 6, 7, and 8 code for polypeptides HA, NP, NA, M, and NS, respectively.     

Purification and enzymatic properties of an RNA polymerase-RNA complex from influenza virus

An RNA polymerase-viral RNA complex was purified from influenza A/PR/8 virions by combination of cesium trifluoroacetate centrifugation and phosphocellulose column chromatography. Surface proteins were removed from the detergent-treated virions by the centrifugation. Starting from the M protein-free ribonucleoprotein (RNP) fraction, an RNA polymerase-RNA complex lacking NP protein was isolated by repeated chromatography on phosphocellulose columns. The isolated RNA polymerase-RNA complex, which is composed of PB1, PB2, PA and vRNA, cleaved capped poly(A) endonucleolytically at 10-12 nucleotides from the 5' end and incorporated GMP into the 3' end of the resulting capped fragments. In the presence of all four ribonucleotide triphosphate substrates, the cleaved fragments were elongated to polynucleotides in the absence of exogenous vRNA. The RNA synthesis was primed not only by capped polynucleotides but also dinucleotide ApG. These results indicate that the purified RNA polymerase-RNA complex is as active in viral mRNAs synthesis as native RNP and that NP protein is not required for the catalytic function.     

Replication in vitro of the influenza virus genome: selective dissociation of RNA replicase from virus-infected cell ribonucleoprotein complexes

Summary Replication of the influenza virus genome involves two discrete step reactions: vRNA-directed primer-independent (unprimed) synthesis of cRNA; and cRNA-directed unprimed synthesis of vRNA. Nuclear extracts from both MDCK and HeLa cells infected with influenza virus A/PR8/34 exhibited unprimed synthesis of both cRNA and vRNA strands (a parameter of RNA replication). Ribonucleoprotein (RNP) complexes with the replication activity were isolated from these nuclear extracts by glycerol gradient centrifugation in the presence of 0.1 M KCl. At 0.5 M KCl, however, these complexes were dissociated into stripped RNP and soluble protein fractions. The soluble fraction contained the activity of exogenous template-dependent unprimed RNA synthesis, indicating that the RNA replicase is dissociated from RNP upon exposure to high salt concentrations. On the other hand, the high salt-treated RNP catalyzed only primer-dependent RNA synthesis, but regained a low level activity of exogenous template-dependent unprimed RNA synthesis by adding nuclear extracts from uninfected cells, suggesting that host factor(s) is involved in the functional interconversion of influenza virus RNA polymerase.     

Investigation on the biological activity of fowl plague virus ribonucleoprotein.

Fowl plague virus (FPV) ribonucleoprotein (RNP) bands in sucrose density gradient in a heterogeneous peak with sedimentation coefficients from 45 to 70 S, whereas in cesium chloride gradient it has a homogeneous density of 1.33-1.34 g/cm3. FPV RNP contains 7.4-8% RNA. Upon inoculation of chick embryo cell cultures. FPV RNP shows no infectivity, does not induce virus-specific protein synthesis and does not participate in complementation or recombination interactions with ts mutants of FPV. The biological activity of FPV RNP demonstrable under certain experimental conditions is due to admixture of undestroyed virions and is completely eliminated by treatment of the preparation with gamma-globulin fraction of antiserum to FPV haemagglutinin, but not with antiserum to RNP proteins.     

Nucleotide sequences of the PA and PB1 genes of B/Ann Arbor/1/66 virus: comparison with genes of B/Lee/40 and type A influenza viruses

The complete sequences of the PA and PB1 genome RNA segments of B/Ann Arbor/1/66 virus have been determined. The PA vRNA is 2308 bases long. Its complementary RNA has a single open reading frame of 2187 bases, capable of encoding a PA protein of 726 amino acids with a molecular weight of 83,175 Da. The predicted PA polypeptide has an overall net charge of -7.5 at pH 7.0. The PB1 vRNA is 2369 bases long. Its complementary RNA has a single open reading frame of 2277 bases, capable of encoding a PB1 protein of 752 amino acids with a molecular weight of 84,332 Da. The predicted PB1 polypeptide has an overall net charge of +18.5 at pH 7.0. Sequence homology comparisons of the PA and PB1 polypeptides from B/Ann Arbor/1/66 virus to the PA and PB1 polypeptides of type A influenza virus reveal respective homologies of approximately 38 and 60%. This high cross-type homology (61%) was previously reported for the PB1 protein of B/Lee/40 virus (Kemdirim et al., 1986). The cross-type homology for the PA protein is similar to that of other non-polymerase proteins, but is substantially lower than that seen for the PB1 protein. Thus, the high cross-type homology that exists for the PB1 gene does not appear to be a characteristic of all polymerase genes.     

The synthesis of influenza virus negative-strand RNA takes place in insoluble complexes present in the nuclear matrix fraction

The replication of influenza virus RNA in vitro has been studied by cell fractionation of MDCK-infected cells and characterization of in vitro synthesized RNA. Analysis of the RNA product polarity by liquid hybridization to excess single-stranded DNA probes shows that only the RNP complexes present in the nuclear matrix fraction are able to synthesize negative-polarity RNA. This RNA product has been characterized as authentic vRNA by size analysis, RNase-protection by unlabelled, positive-polarity riboprobes and T1-fingerprinting. Priming the in vitro reaction with ApG stimulates preferentially the synthesis of positive-polarity RNA, while ApGpU stimulates both positive and negative-polarity RNA synthesis.