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The characterization and processing of the nonstructural proteins of Sindbis virus

Citation

Hardy, Winters Reef (1990) The characterization and processing of the nonstructural proteins of Sindbis virus. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-06082007-084319

Abstract

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SHORT ABSTRACT:

The nonstructural proteins of Sindbis virus, the type alphavirus of the family Togaviridae, are produced by proteolytic cleavage of two polyprotein precursors. One precursor (P123) is 1,896 amino acids in length and contains the sequences of nsP1, nsP2, and nsP3, while the second (P1234) of 2,513 amino acids is produced by readthrough of an opal termination codon and contains a fourth nonstructural protein, nsP4.

In order to study the kinetics of processing of these polyproteins, monospecific antisera were produced in rabbits to fusion protein antigens containing the N-terminal two-thirds of the trpE protein of E. coli fused to a large part of the sequence within each of the nonstructural proteins of Sindbis virus. Using these antisera the following details of processing were elucidated: i) most nonstructural proteins arise from the processing of the completed precursor P123 and its cleavage product, P12; ii) in the P123 precursor, the primary cleavage occurs in trans, between nsP2 and nsP3 to generate P12 and nsP3 with a half-life of [...]19 min. in vivo, followed by processing of P12 to produce nsP1 and nsP2, either intramolecularly or in trans, at a rate which exceeds that of the first cleavage; iii) processing of the 3-4 site is complex; and iv) nsP3 was found to be phosphorylated during posttranslational modification.

In vitro, processing could be inhibited with antibodies to nsP2, but not with antisera to the other three nonstructural proteins, suggesting that the nonstructural proteinase is in nsP2. Deletion-mapping studies were performed which further localized the proteinase to a region of approximately 334 amino acids at the C terminus of nsP2. From a comparison of the deduced sequences of several alphaviruses in this part of nsP2 with the sequences of cellular proteinases, a hypothesis was presented that the proteinase is a thiol protease related to papain. Finally, the examination of several temperature-sensitive mutants of Sindbis virus has confirmed the importance of this region as only mutants that possessed a mutation in the C-terminal domain of nsP2 produced aberrant processing patterns at the nonpermissive temperature.

Item Type:Thesis (Dissertation (Ph.D.))
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Richards, John H.
Thesis Committee:
  • Campbell, Judith L. (chair)
  • Baldeschwieler, John D.
  • Emr, Scott D.
Defense Date:19 September 1989
Record Number:CaltechETD:etd-06082007-084319
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-06082007-084319
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:2519
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:26 Jun 2007
Last Modified:26 Dec 2012 02:52

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