The aims of this project are to study the macromolecular regulatory steps during the replication of vesicular stomatitis virus (VSV) and to discover underlying mechanisms by which viruses cause disease, with the hope that novel counter measures will be discovered for the better control of viral diseases. Molecular biochemical techniques will be used throughout. This will be coupled with genetic and biological approaches. The glycoprotein G of VSV will be studied in relation to its natural cleavage products Gs and anchor. Their functions in vitro and as immunogens in mice will be delineated. Virions, containing only anchors, will be tested for immune enhancement using non- neutralizing antibodies and macrophages. The point of cleavage and the overall structure of Gs will be determined by terminal amino acid analysis and, eventually, X-ray crystallography. Also, how efficiently VSV donates its G to HIV during coinfection of H9 cells with RF-HIV will be assessed. Pseudotypes of HIV (VSV) will be characterized by their susceptibility to specific neutralizing antibodies and by their expanded host range. The role of defective interfering (DI) particles in eliciting or modulating immunogenic responses will be studied in mice and in reconstituted immune cell in vitro. Preliminary data show that DI particles differ from standard infectious VSV in their interactions with T lymphocytes. VSV mRNA's will be utilized to detect cytoplasmic regulatory controls. Cloned cDNA's will be engineered to contain mutations, deletions or extra sequences. mRNA made from these cDNA's will be tested in vitro or in vivo for translational efficiency, ribosome binding and dissociation, mRNA stability, and targeting to membrane organelles. Initially, engineered sequences will be made in the 5' and 3' non-coding leader regions Then nonessential coding regions and regions containing other open reading frames will be altered. If sequences in the mRNA are found to respond to cytoplasmic regulation then those sequences will be used in binding assays to detect the proteins or ribonucleoproteins that may be responsible. Also, peptides made from these minor open reading frames will be used to make antibody for the purpose of determining whether these products exist during vsv infections.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
2R37AI020896-08
Application #
3481141
Study Section
Virology Study Section (VR)
Project Start
1979-06-01
Project End
1994-05-31
Budget Start
1989-06-01
Budget End
1990-05-31
Support Year
8
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
Plakhov, I V; Arlund, E E; Aoki, C et al. (1995) The earliest events in vesicular stomatitis virus infection of the murine olfactory neuroepithelium and entry of the central nervous system. Virology 209:257-62
Chang, T L; Reiss, C S; Huang, A S (1994) Inhibition of vesicular stomatitis virus RNA synthesis by protein hyperphosphorylation. J Virol 68:4980-7
Cao, B N; Huneycutt, B S; Gapud, C P et al. (1993) Lymphokine expression profile of resting and stimulated CD4+ CTL clones specific for the glycoprotein of vesicular stomatitis virus. Cell Immunol 146:147-56
Reiss, C S; Gapud, C P; Keil, W (1992) Newly synthesized class II MHC chains are required for VSV G presentation to CTL clones. Cell Immunol 139:229-38
Browning, M J; Huneycutt, B S; Huang, A S et al. (1991) Replication-defective viruses modulate immune responses. J Immunol 147:2685-91
Forger 3rd, J M; Bronson, R T; Huang, A S et al. (1991) Murine infection by vesicular stomatitis virus: initial characterization of the H-2d system. J Virol 65:4950-8
Browning, M J; Huang, A S; Reiss, C S (1990) Cytolytic T lymphocytes from the BALB/c-H-2dm2 mouse recognize the vesicular stomatitis virus glycoprotein and are restricted by class II MHC antigens. J Immunol 145:985-94
Browning, M; Reiss, C S; Huang, A S (1990) The soluble viral glycoprotein of vesicular stomatitis virus efficiently sensitizes target cells for lysis by CD4+ T lymphocytes. J Virol 64:3810-6
Fan, W; Chen, S S; Huang, A S (1989) Multiple thermolabile and temperature-sensitive lesions in mutant tl17 of vesicular stomatitis virus. Intervirology 30:148-55
Chen, S S; Huang, A S (1989) Human cervical carcinoma cell lines contain an antigen identical to the tumor-specific 75 kDa antigen of HeLa cells: detection by viral acquisition. J Virol Methods 24:159-67

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