Clewley, Jonathan P., Jonathan P. (1976) Purification of Semliki Forest virus RNA - dependent RNA polymerase. PhD thesis, University of Warwick.

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Abstract

Replicase activity was found in the cytoplasm of the infected cell and, after removal of nuclei, the replication complex was characterized by isopycnic sucrose gradient centrifugation. A membranous band at 35% (w/w) sucrose contained replicase activity and was the site of RNA synthesis. At
1 3/4 hr post-infection the replicase was associated with input virion RNA but not input virion proteins.

Replicase present in the post-nuclear supernatant was competent to synthesize all forms of virus-specific RNA found in the infected cell. However, on further purification there was a loss of the production of discrete single- stranded RNA in vitro. This loss was probably caused by endogenous ribonuclease activity and disruption of the replication complex.

Recovery of the replicase rich band from isopycnic gradients allowed it to be analysed on linear sucrose gradients. The replication complex had an S-value of 250 which, on nonionic detergent treatment, was converted to a 25S structure.

The replicase activity of this 25S complex was specifically bound to an oligo(dT)- cellulose column bearing attached 42S virion RNA. Analysis of the polypeptides eluted off in the fractions having replicase activity showed that there were only 3 labelled bands visible on polyacrylamide gels. Two of these comigrated with two non-structural virus polypeptides identified in infected cells. The third polypeptide of the purified replicase preparation (MW 40,000 daltons) is probably a host protein. Material from mock- infected cells purified exactly as the replicase contained only the 40,000 dalton protein.

It is therefore concluded that SFV replicase contains 2 virus-specific polypeptides which have MWs of 90,000 and 63,000 daltons.

Item Type:	Thesis (PhD)
Subjects:	Q Science > Q Science (General) Q Science > QP Physiology
Library of Congress Subject Headings (LCSH):	Semliki Forest virus -- Purification, Semliki Forest virus, RNA polymerases, RNA -- Analysis
Official Date:	January 1976
Dates:	Date Event January 1976 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Kennedy, Ian
Sponsors:	Science Research Council (Great Britain)
Extent:	viii, 146 leaves
Language:	eng

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