The Function and Replication of Sindbis Virus-Specific RNA's in Infected Cells

Citation

Simmons, Daniel Tawil (1974) The Function and Replication of Sindbis Virus-Specific RNA's in Infected Cells. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/grvs-e385. https://resolver.caltech.edu/CaltechTHESIS:04132021-203938881

Abstract

During infection with Sindbis virus, two species of Sindbis-specific single-stranded RNA are synthesized. One of them, 49S RNA is the RNA of the vinis and has a molecular weight of 4.3 ± 0.3 x 10⁶ daltons. This molecular weight was estimated by a variety of methods, including polyacrylamide gel electrophoresis, sedimentation after reaction with fonnaldehyde and analysis of the molecular weight of its double-stranded form. The other species of single-stranded RNA, 26S RNA, was found only in infected cells and has a molecular weight of 1.6 x 10⁶ daltons, determined by sedimentation in dimethylsulfoxide. Hybridization-competition experiments showed that 26S RNA is a specific one-third of the 49S RNA genome.

In infected cells, 26S RNA was primarily associated with ribosomes, and was found to be the predominant species of viral messenger RNA. A small amount (10 % by weight) of the messenger RNA in the cells was Sindbis 49S RNA. No other unique and separate species of Sindbis messenger RNA could be detected in infected cells.

The two species of Sindbis single-stranded RNA were translated in lysates of rabbit reticulocytes. Sindbis 26S RNA was translated primarily into the nucleocapsid protein and into a protein shown by others to be the precursor of the two glycoproteins of the virus. These results indicated that Sindbis 26S RNA codes solely for the structural proteins of the virus.

Sindbis 49S RNA was translated in vitro into 8 or 9 polypeptides ranging in molecular weight from 60,000 to 180,000 daltons. None of these polypeptides coincided with any known Sindbis proteins.

The replication of Sindbis-specific RNA was studied by analyzing the forms of double-stranded RNA (or replicative forms) in infected cells. When RNA from infected cells was treated with ribonuclease, three species of Sindbis-specific double-stranded RNA (RF's I, II, and III) were isolated. Their molecular weights were determined to be 8.8 x 10⁶ daltons for RFI, 5.6 x 10⁶ daltons for RFII, and 2.9 x 10⁶ daltons for RFIII.

By hybridization-competition experiments, it was shown that RFI is the double-stranded form of 49S RNA, RFIII, the double-stranded form of 26S RNA, and RFII, the double-stranded form of a species of RNA with molecular weight of 2.8 x 10⁶ daltons and identical to two-thirds of the genome.

The size and structure of Sindbis replicative intermediates (RI's) were studied and found to consist of a double-stranded region the size of RFI and of various lengths of single-stranded tails. Our model for the replication of Sindbis-spccific RNA predicts that Sindbis RI's exist in two classes, called RIa and RIb. RIa is the template for the synthesis of 49S RNA and is reduced to RFI when treated with ribonuclease. When Rib is treated with ribonuclease, it is reduced to RF's II and III due to a single-stranded gap in the "plus" strand of the RI (the virus RNA is "plus"-stranded). The portion of RIb corresponding to RFIII is the template for the synthesis of 26S RNA, and the portion corresponding to RFII is the template for synthesis of a species of RNA of 2.8 x 10⁶ daltons, which has not been detected in its single-stranded form. We hypothesize that there must be two different regions on RIb where chain synthesis is initiated since 26S RNA is synthesized at a much faster rate than the product of RFII.

Thesis Files