The purpose of this research is to study the mechanism of replication of the negative-stranded RNA viruses using vesicular stomatitis virus (VSV) as a prototype. The unifying aim of this proposal is to identify factors relevant to the control of expression of information in the genome of the negative-strand virus. In particular the questions we propose to ask are designed to evaluate the role of several factors in controlling whether the VSV negative strand template will be expressed by transcription to yield discrete mRNAs or by replication to produce the genome-sized virion complementary (VC) RNA.
Our specific aims are as follows: 1. Evaluation of the relation of the discrete secondary structures which we have identified in the VSV genome-size RNAs, but not the mRNAs, to possible biological function. To do this the location of these structures on the genome will be mapped and then the size, number, sequence relatedness and primary sequence in the structures will be determined. This information will be related to the location and known sequence of the virion RNA termini, intercistronic and leader RNA junctions. 2. Analysis of the virion RNA and RNP in situ for interacting structures. This will test whether or not the secondary structures observed in VSV RNAs are due to interactions of contiguous sequences or distance sequences in the genome. 3. Analysis of the architecture of VSV RNA synthetic structures by electron microscopy. 4. Study of the relation of the VSV NS protein structure, conformation and levels of phosphorylation to RNA transcription and replication. 5. Characterization of an in vitro translation assisted RNA synthesis system. 6. Characterization of RNAs synthesized by respiratory syncytial (RS) virus in vivo and in vitro. Identification of polyadenylated mRNA species and assignment of mRNA coding capacity by in vitro translation of separated messages.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI012464-10
Application #
3125192
Study Section
Virology Study Section (VR)
Project Start
1978-04-01
Project End
1986-03-31
Budget Start
1985-04-01
Budget End
1986-03-31
Support Year
10
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Mondal, Arindam; Victor, Ken G; Pudupakam, R S et al. (2014) Newly identified phosphorylation site in the vesicular stomatitis virus P protein is required for viral RNA synthesis. J Virol 88:1461-72
Harouaka, Djamila; Wertz, Gail W (2012) Second-site mutations selected in transcriptional regulatory sequences compensate for engineered mutations in the vesicular stomatitis virus nucleocapsid protein. J Virol 86:11266-75
Rainsford, Edward W; Harouaka, Djamila; Wertz, Gail W (2010) Importance of hydrogen bond contacts between the N protein and RNA genome of vesicular stomatitis virus in encapsidation and RNA synthesis. J Virol 84:1741-51
Harouaka, Djamila; Wertz, Gail W (2009) Mutations in the C-terminal loop of the nucleocapsid protein affect vesicular stomatitis virus RNA replication and transcription differentially. J Virol 83:11429-39
Galloway, Summer E; Wertz, Gail W (2009) A temperature sensitive VSV identifies L protein residues that affect transcription but not replication. Virology 388:286-93
Galloway, Summer E; Wertz, Gail W (2008) S-adenosyl homocysteine-induced hyperpolyadenylation of vesicular stomatitis virus mRNA requires the methyltransferase activity of L protein. J Virol 82:12280-90
Barr, John N (2007) Bunyavirus mRNA synthesis is coupled to translation to prevent premature transcription termination. RNA 13:731-6
Flanagan, E Brian; Schoeb, Trenton R; Wertz, Gail W (2003) Vesicular stomatitis viruses with rearranged genomes have altered invasiveness and neuropathogenesis in mice. J Virol 77:5740-8
Hardy, R W; Wertz, G W (2000) The Cys(3)-His(1) motif of the respiratory syncytial virus M2-1 protein is essential for protein function. J Virol 74:5880-5
Whelan, S P; Barr, J N; Wertz, G W (2000) Identification of a minimal size requirement for termination of vesicular stomatitis virus mRNA: implications for the mechanism of transcription. J Virol 74:8268-76

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