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Role of ATP and protein kinases in vesicular stomatitis virus RNA synthesis
54 ROLE OF ATP AND PROTEIN KINASES IN VESICULAR STOMATITIS VIRUS RNA SYNTHESIS William B. Helfman, J. David Beckes, Lisa C. Childers and Jacaues Department of Biology and Molecular Biology Institute San Diego State University, San Diego, California 92182, U. S. A.
Perrault.
A mutational alteration in the template-associated N protein of the VSV polR mutants is responsible for the replication-like synthesis carried out by purified virions in vitro (Cell 35, 175-185, 1983). We report here that a single substitution, is responsible for this phenotype. arg to his at position 179 of the N protein, Furthermore, the ability of the polR mutants to bypass the requirement for cleavage of the 8,a bond of ATP for transcription initiation (synthesis in the presence of AMPPNP), as well as the change in synthesis kinetics with varying ATP concentrations, are both due to the same single site mutation in the N protein. In light of these results, we have explored the role of virion-associated protein kinases in the regulation of viral RNA synthesis. Using a number of biochemical parameters, including protein and nucleotide substrate preferences, we have identified at least three distinct protein kinase activities. One of these appears to be altered in the polR mutants. We suggest that specific phosphorylation of virus proteins regulates the switch from transcription to replication.
55 TRANSLATIONAL CONTROLOF THE SENDAI VIRUS P/C GENE JOSEPH CURRAN, SILVIA VIDAL, DANIEL KOLAKOFSKY, Dept. of Microbiology, CMU University of Geneva Medical School, 1211 Geneva 4, Switzerland
Specific antisera detect a 10 kDa1 protein (X) in infected cells, representing the last 95 residues of P (568 aa). X does not appear to be derived from a precursor P in vivo, and in vitro X can be made under conditions in which P synthesis has been arrested by hybridized DNA fragments or specific cleavage of the mRNA. X initiation is nevertheless cap dependant. Ribosomes which initiate X appear to pass directly from the cap to the initiation codon. To unambiguously determine where P, C C’ and Y proteins were initiated, the first three ATG’s in the P/C mRNA were each changed to GCG. P was found to start on the first, C on the second and Y on the third ATG. Using deletion analysis and the introduction of in frame amber codons, an upstream ACG was identified as the most likely initiation site for C’. When changed to an ATC, C’ was overexpressed. N-terminal sequencing of the C’ protein also indicated that it initiated on the ACG codon. In vivo, C is by far the most highly expressed of these proteins, although it starts from the third initiation codon. Experiments suggest that all the ribosomes which initiate at C’ and P are scanning from the cap and this is also true for the majority of ribosomes which initiate at C. However, 10-30X of ribosomal initiations at C appear to be capindependant and scanning independant.
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Perrault.
A mutational alteration in the template-associated N protein of the VSV polR mutants is responsible for the replication-like synthesis carried out by purified virions in vitro (Cell 35, 175-185, 1983). We report here that a single substitution, is responsible for this phenotype. arg to his at position 179 of the N protein, Furthermore, the ability of the polR mutants to bypass the requirement for cleavage of the 8,a bond of ATP for transcription initiation (synthesis in the presence of AMPPNP), as well as the change in synthesis kinetics with varying ATP concentrations, are both due to the same single site mutation in the N protein. In light of these results, we have explored the role of virion-associated protein kinases in the regulation of viral RNA synthesis. Using a number of biochemical parameters, including protein and nucleotide substrate preferences, we have identified at least three distinct protein kinase activities. One of these appears to be altered in the polR mutants. We suggest that specific phosphorylation of virus proteins regulates the switch from transcription to replication.
55 TRANSLATIONAL CONTROLOF THE SENDAI VIRUS P/C GENE JOSEPH CURRAN, SILVIA VIDAL, DANIEL KOLAKOFSKY, Dept. of Microbiology, CMU University of Geneva Medical School, 1211 Geneva 4, Switzerland
Specific antisera detect a 10 kDa1 protein (X) in infected cells, representing the last 95 residues of P (568 aa). X does not appear to be derived from a precursor P in vivo, and in vitro X can be made under conditions in which P synthesis has been arrested by hybridized DNA fragments or specific cleavage of the mRNA. X initiation is nevertheless cap dependant. Ribosomes which initiate X appear to pass directly from the cap to the initiation codon. To unambiguously determine where P, C C’ and Y proteins were initiated, the first three ATG’s in the P/C mRNA were each changed to GCG. P was found to start on the first, C on the second and Y on the third ATG. Using deletion analysis and the introduction of in frame amber codons, an upstream ACG was identified as the most likely initiation site for C’. When changed to an ATC, C’ was overexpressed. N-terminal sequencing of the C’ protein also indicated that it initiated on the ACG codon. In vivo, C is by far the most highly expressed of these proteins, although it starts from the third initiation codon. Experiments suggest that all the ribosomes which initiate at C’ and P are scanning from the cap and this is also true for the majority of ribosomes which initiate at C. However, 10-30X of ribosomal initiations at C appear to be capindependant and scanning independant.
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