Date of Award
Doctor of Philosophy (PhD)
The purpose of this research was to study in vitro the biochemical mechanisms involved in the regulation of vesicular stomatitis virus mRNA and protein synthesis.
Cell-free extracts from vesicular stomatitis virus (VSV) infected cells synthesized all five VSV specific structural proteins L, G, NS, N and M in vitro. Separation of the free and membrane bound polysomes from these extracts and analysis of the proteins synthesized in vitro showed that only the membrane-bound polysomes could synthesize the viral membrane glycoprotein G. The other four VSV proteins, including the unglycosylated membrane protein M, were synthesized by both free and membrane-bound polysomes. These results suggest that the VS viral proteins are synthesized at different cytoplasmic sites in infected cells.
The viral mRNA species from free and membrane-bound polysomes were isolated and translated in cell-free extracts from wheat embryo and ascites cells. The VSV specific mRNA species contained four classes of polyadenylated mRNA sedimenting at 28S, 19S, 17S and 12S. Partial fractionation and translation of the mRNA species showed that only the mRNA species which was isolated from membrane-bound polysomes and which sedimented in the 16 to 195 region of the sucrose gradient directed the synthesis of the G protein in vitro. The in vitro synthesized G protein, however, had a lower molecular weight (63,000D) than virion G (mw 69,000D).
Ribonucleoprotein particles found in the cytoplasm of VSV-infected cells have been suggested to function in the synthesis of VSV mRNA species in vivo. In order to investigate this possibility, ribonucleoprotein particles were isolated from extracts of VSV-infected L cells and the RNA species synthesized in vitro were characterized. Four classes of RNA sedimenting at 28S, 19S, 17S and 12S were synthesized in vitro. All four classes of RNA were polyadenylated, however, polyadenylation appeared to be less efficient in vitro than in vivo. Methyl groups donated by S-adenosyl methionine were incorporated into all the RNA species synthesized in vitro. The presence of S-adenosyl methionine or of the methylation inhibitor S-adenosyl homocysteine however, did not affect the rate of RNA synthesis nor the nature of the RNA species synthesized in vitro.
The mRNAs synthesized in vitro were translated in the homologous ascites and heterologous wheat embryo cell-free extracts. In both extracts, the products were shown by polyacrylamide gel electrophoresis, and by immunoprecipitation to contain the viral proteins G, NS, N and M and possibly the L protein. Characterization of the G protein synthesized in vitro showed that it contained the same tryptic peptides as virion G. However, as was observed on translation of the polysomal VSV mRNA species, the G protein synthesized in response to in vitro synthesized mRNA had a molecular weight of only 63,000D. These results suggest that the G protein synthesized in vitro may be incompletely glycosylated.
Methylated VSV mRNA was found to be more active in protein synthesis than unmethylated mRNA in both the ascites and wheat embryo systems. S-adenosyl homocysteine further inhibited the translation of unmethylated mRNA but did not affect methylated mRNA translation. Addition of S-adenosyl methionine stimulated the translation of unmethylated mRNA in the wheat embryo extract suggesting the presence of mRNA methylating activity in the protein synthesizing system. The mRNA methylating activity present in the wheat embryo S-30 extract as recovered in the ribosome-free supernatant fraction and was insensitive to the protein synthesis inhibitor pactamycin.
In order to further study the regulation of transcription and translation of viral mRNA species, a system which could both synthesize and translate V5V mRNAs into viral proteins was developed. The system contained purified ribonucleoprotein particles from VSV-infected L cells and ribosomal extracts obtained from unifected HeLa cells. Analysis of the optimal conditions for transcription and translation showed that Mg²⁺, K⁺ and all four ribonucleoside triphosphates were required. The coupled transcription-translation system synthesized four classes of V5V specific mRNAs sedimenting at 28S, 19S, 17S and 12S. Translation of the synthesized mRNA in the coupled system resulted in the synthesis of the viral proteins L, G, NS, N and M.
Two forms of the G protein were synthesized in the coupled system, G₁ (mw 63,000D) and G₂ (mw 67,000D). Analysis of the tryptic peptides showed that G₁, G2 and virion G had identical peptide sequences. The synthesis of G₂ required the presence of membranes and only G₁ was synthesized in the absence of membranes. Affinity chromatography on concanavalin A sepharose columns showed that G₂ but not G₁ was a glycoprotein. Removal of sialic acid residues from G by neuraminidase resulted in a product with an electrophoretic mobility identical to that of G₂. Digestion of G₂ or G with a mixture of neuraminidase, β-galactosidase and β-N-acetylglucosaminidase however, produced a protein of molecular weight 65,000D. These data suggest that G₂ is the desialated G and is formed by the glycosylation of polypeptide G₁ in the presence of membranes.
Addition of stripped crude microsomal membranes to a translation system containing free ribosomes resulted in the formation of membrane-bound polysomes and in the conversion of G₁ to the glycosylated protein G₂. The G₂ protein synthesized by the reconstructed membrane-bound polysomes was segregated into the microsomal vesicles and was, therefore, protected against proteolytic digestion. Stripped membranes were required at an early stage of protein synthesis for the synthesized protein to be inserted into membranes and glycosylated. The G₂ protein, however, was not completely protected from proteolytic digestion showing that a portion of the peptide chain presumably the carboxyterminal was present on the cytoplasmic side of the membrane vesicle. These results suggest that membrane glycoprotein may be synthesized and transported across membranes in a manner analogous to secretory proteins except that the membrane glycoproteins are not completely discharged across the membrane.
In conclusion, the results obtained have confirmed and extended previous studies on the nature and biosynthesis of the VSV specific mRNA species. In addition, the studies of in vitro protein synthesis have provided new insights into the mechanisms involved in the biosynthesis, glycosylation and insertion into membranes of membrane glycoproteins. This research has led to the following publications:
1. Ghosh, H.P., Toneguzzo, F and Wells, S. (1973) Synthesis in vitro of Vesicular Stomatitis Virus proteins in cytoplasmic extracts of L cells. Biochem. Biophys. Res. Comm., 54, 228.
2. Toneguzzo, F and Ghosh, H.P. (1975) Cell-free synthesis of Vesicular Stomatitis Virus proteins: Translation of membrane-bound polyribosomal mRNAs. FEBS Letters, 50, 369.
3. Ghosh, H.P. and Toneguzzo, F. (1975) Synthesis in vitro of mRNA by RNA polymerase induced in VSV-infected L cells and translation of the mRNA species into viral proteins. pp.259-269 in A.L. Haenni and G. Beaud (ed.) INSERM Colloque; In vitro transcription and translation of viral genomes. Institut National de la Sante et de la Recherche Medicale, Paris.
4. Toneguzzo, F. and Ghosh, H.P. (1976) Characterization and Translation of Methylated and Unmethylated Vesicular Stomatitis Virus mRNA synthesized in vitro by ribonucleoprotein particles from Vesicular Stomatitis Virus infected L cells. J. Virol., 17, 477.
5. Toneguzzo, F. and Ghosh, H.P. (1977) Synthesis and Glycosylation in vitro of the Vesicular Stomatitis Virus glycoprotein G in a coupled transcription-translation system. Proc. Natl. Acad. Sci. (U.S.A.) 74, 1516.
6. Toneguzzo, F. and Ghosh, H.P. Insertion into membranes and glycosylation of the Vesicular Stomatitis Virus membrane glycoprotein G. (1977) Proc. Natl. Acad. Sci. U.S.A. (in press).
Toneguzzo, Frances, "Synthesis in vitro of Vesicular Stomatitis Virus Messenger RNA and Proteins" (1977). Open Access Dissertations and Theses. Paper 3168.