Our laboratory will study the mechanism(s) involved in the regulation of the functions of the virus-specific, virion-associated RNA polymerases of the two minus-stranded RNA animal viruses, vesicular stomatitis virus (VSV) and influenza A. Specifically we have designed experiments to examine the modification(s) of these two enzymes which allows them to switch from transcription of their genomes into virus-specific mRNAs which are capped, methylated and polyadenylated and are incomplete copies of the genomic sequence to a replicative mode which leads to synthesis of full length (+) and (-) copies of the genome that are assembled during synthesis with nucleocapsid proteins to form RNP complexes. We are presently cloning the small subunit of the VSV polymerase (NS) for expression in E. coli and in animal cells to obtain sufficient amounts of NS with which to carry out analyses of the phosphorylation sites on NS. We will also sequence the cDNA to determine the amino acid sequence of this protein. If we can obtain reasonable levels of expression for NS we hope to use the NS protein for in vitro reconstitution studies with purified RNPs and L protein. Using monospecific antisera versus both VSV enzyme subunits (L1, NS1) we are studying the interaction of this enzyme with its RNP template by immunoelectron microscopic techniques. Since we can inhibit influenza transcription (due to its reliance on host cell mRNA for caps and primers) with actino D, we will establish an in vitro replication system from cell nuclear extracts for flu by coupling transcription and translation to drive flu replication in a manner similar to our in vitro VSV replication system. Using specific antibodies to the flu RNA polymerase (PB1, PB2 PA) we will establish whether all three subunits are involved in flu replication as well as transcription. In a similar manner we will study the role, if any, of flu nonstructural protein (NS1) in vitro replication system in which we can selectively inhibit transcription (actino D) or replication (cycloheximide) to study the enzyme modification(s) involved and the role of cellular and/or virus factors in it.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI012316-12
Application #
3125160
Study Section
Virology Study Section (VR)
Project Start
1975-05-01
Project End
1990-11-30
Budget Start
1986-12-01
Budget End
1987-11-30
Support Year
12
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Utah
Department
Type
Schools of Medicine
DUNS #
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Szewczyk, B; Pilat, Z; Bienkowska-Szewczyk, K et al. (1998) Elution of glycoproteins from replicas of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Electrophoresis 19:220-3
Peng, Q; Galarza, J M; Shi, L et al. (1996) Influenza A virus RNA-dependent RNA polymerase cleaves influenza mRNA in vitro. Virus Res 42:149-58
Shi, L; Galarza, J M; Summers, D F (1996) Recombinant-baculovirus-expressed PB2 subunit of the influenza A virus RNA polymerase binds cap groups as an isolated subunit. Virus Res 42:1-9
Shi, L; Summers, D F; Peng, Q et al. (1995) Influenza A virus RNA polymerase subunit PB2 is the endonuclease which cleaves host cell mRNA and functions only as the trimeric enzyme. Virology 208:38-47
Neufeld, K L; Galarza, J M; Richards, O C et al. (1994) Identification of terminal adenylyl transferase activity of the poliovirus polymerase 3Dpol. J Virol 68:5811-8
Galarza, J M; Sowa, A; Hill, V M et al. (1992) Influenza A virus NP protein expressed in insect cells by a recombinant baculovirus is associated with a protein kinase activity and possesses single-stranded RNA binding activity. Virus Res 24:91-106
Hill, V M; Summers, D F (1990) A minor microtubule-associated protein is responsible for the stimulation of vesicular stomatitis virus transcription in vitro. J Gen Virol 71 ( Pt 2):289-98
Szewczyk, B; Laver, W G; Summers, D F (1988) Purification, thioredoxin renaturation, and reconstituted activity of the three subunits of the influenza A virus RNA polymerase. Proc Natl Acad Sci U S A 85:7907-11
Szewczyk, B; Summers, D F (1987) Fluorescent staining of proteins transferred to nitrocellulose allowing for subsequent probing with antisera. Anal Biochem 164:303-6
Hill, V M; Harmon, S A; Summers, D F (1986) Stimulation of vesicular stomatitis virus in vitro RNA synthesis by microtubule-associated proteins. Proc Natl Acad Sci U S A 83:5410-3

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