Brzeski, Henry (1977) The synthesis and function of the nonstructural polypeptides of Sindbis virus. PhD thesis, University of Warwick.

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Abstract

Sindbis virus an alphavirus, has a single stranded RNA genome with a molecular weight of 4.2 million (42S RNA). Inside infected cells two RNA species are found. One, 42S RNA, is assumed to code for the RNA polymerase(s) and is also destined to enter mature virus, the second, 26S RNA, has a molecular weight of 1.8 million and is a subgenomic fragment representing the 3’ end of 42S RNA. The 26S RNA functions as a messenger for the structural proteins of the virus. The aim of this work was to discover the mechanism by which Sindbis virus, in particular, regulated the rate at which it synthesised the two RNA species such that the rate of structural protein and virion RNA synthesis were co-ordinated for efficient virus assembly.

It was considered that the regulatory ability would probably lie with the nonstructural polypeptides i.e. those comprising the RNA polymerase. For this reason it was first necessary to characterise the nonstructural polypeptides synthesised by Sindbis virus as no such polypeptides had been previously described. Pulse and pulse-chase experiments conducted early in the infectious cycle indicated the existence of three new stable polypeptides nsp60, nsp82 and nsp89 along with three other polypeptides p215, pl50 and p76 which were unstable under chase conditions. Inhibition of proteolytic enzymes resulted in the accumulation of p215 and a larger polypeptide p230 along with a concomitant reduction in the amount of smaller polypeptides synthesised, suggesting that p230 was the entire translation product of the nonstructural region of 42S RNA. This precursor-product relationship was confirmed by tryptic peptide mapping. The gene order was established as S' -nsp60-nsp89-nsp82-3' and the order of cleavage of the precursor was also determined.

Having characterised the nonstructural polypeptides it became necessary to determine the mechanism by which 26S RNA was transcribed from a 42S negative strand RNA. This was achieved by determining the relative sensitivities of the synthesis of each RNA species to UV inactivation.

The rate of inactivation of 26S RNA was consistent with its transcription from a template with a molecular weight of 1.8 million (which is the size of 26S RNA) and so, since there is no negative strand of the same size as 26S RNA, it was concluded that 26S RNA was synthesised by internal initiation of transcription on the 42S negative strand RNA. Further analysis of the UV inactivation data Indicated that, on average, a single template RNA supported the synthesis of one molecule of 42S and three molecules of 26S RNA.

In an attempt to characterise the polymerase(s) responsible for the synthesis of 26S and 42S RNA, 15 RNA -ve temperature-sensitive mutants were examined with respect to the rate at which they synthesised the two RNA species. These mutants are unable to synthesise RNA when Incubated continuously at the restrictive temperature and hence are assumed to have a lesion in the gene(s) coding for the polymerase(s). Under shift-up conditions no mutant suffered a loss of 428 RNA synthetic ability but all mutants showed a relative decrease in the rate of 26S RNA synthesis and in the case of three mutants this was marked. Examinations of the polypeptide phenotypes of these mutants suggested that there was a basic polymerase activity responsible for the transcription of both 42S and 26S RNA but that a virus specified polypeptide was required for the Initiation of 26S RNA synthesis. Experimental evidence was consistent with this regulatory polypeptide being nsp89.

Finally, a number of experiments performed in this laboratory suggested that one of the structural polypeptides could also regulate the 42S to 26S RNA ratio. These results are summarised and further results are presented which Indicate that this ability resides in the core protein. The data are assembled into a model which suggests a mechanism whereby the rate of synthesis of virion (42S) RNA and the structural proteins can be closely monitored and regulated such that they are produced in optimal amounts for the assembly of mature virus.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QP Physiology Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Togaviruses, Polypeptides, RNA -- Synthesis
Official Date:	September 1977
Dates:	Date Event September 1977 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Kennedy, S. I. T.
Extent:	[12], 128, [11] leaves
Language:	eng

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