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Functional analysis of viral nonstructural and structural proteins

Citation

Hahn, Young Shin Lim (1989) Functional analysis of viral nonstructural and structural proteins. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-06072007-075259

Abstract

The genera Alphavirus and Flavivirus contain enveloped RNA viruses which are similar in size, morphology, and RNA content, and were once considered to belong to the same family, the Togaviridae. Recently the flaviviruses were reclassified as a separate family, the Flaviviridae, because they differ markedly from alphaviruses in replication strategy and mode of assembly as well as genome organization. In this thesis, representatives of both groups have been studied in order to understand the functions of the various virus-encoded proteins in replication and pathogenesis. Part I describes the mapping of temperature-sensitive (ts) RNA- mutants of the alphavirus Sindbis virus, to elucidate the function of each nonstructural protein during RNA replication. Part II includes the determination of the complete nucleotide sequence of the flavivirus dengue 2, comparative analysis of conserved elements in the 3' untranslated region of various flaviviruses, and expression of dengue 2 structural proteins in a recombinant vaccinia virus. During alphavirus replication, the parental 49S plus-strand RNA is transcribed into a complementary minus-strand RNA which serves as the template for the synthesis of both plus-strand 49S genomic RNA and 26S subgenomic RNA. The nonstructural proteins, which are involved in viral RNA replication, are translated from the genomic 49S RNA as two polyprotein precursors that are processed by cotranslational or posttranslational cleavage into four final polypeptide products. ts RNA- mutants of Sindbis virus have been isolated previously and grouped by complementation into four groups (A, B, F, G); these mutants fail to make RNA after infection at a nonpermissive temperature and were presumed to contain ts lesions in the nonstructural proteins. Over a number of years, work in our own and other laboratories has established the details of the RNA- phenotypes of these mutants, which include defects in RNA chain elongation, in initiation of genomic and 26S RNA synthesis, in regulation of minus-strand template synthesis, and in processing of polyprotein precursors. However, it is only with the mapping described here that it has been possible to unambiguously assign these functions to particular nonstructural polypeptides. The mutations responsible for the is phenotype of Sindbis complementation group F mutants ts6, tsl10, and tsl18, have been determined. ts6 and ts110 have a single base substitution in nsP4 resulting in a replacement of Gly by Glu at position 153 or position 324, respectively. It is of interest that nsP4 contains the Gly-Asp-Asp motif characteristic of a number of viral replicases, and this together with the fact that all RNA synthesis in ts6-infected cells is shut off upon shift-up from the permissive to the nonpermissive temperature suggests that nsP4 is the viral RNA polymerase or elongation enzyme. tsl18 is a double mutant where one mutation is in nsP2 (the Val at residue 425 is changed to Ala). This mutation alone causes the formation of minute plaques at the nonpermissive condition without a reduction in the plaque number. The second change (Gln-93 of nsP4 changed to Arg) has little apparent phenotype on its own, but in combination with the change in nsP2 it leads to a temperature-sensitive phenotype. This suggests that nsP2 and nsP4 interact with one another in a complex. We also have mapped representatives of RNA- complementation groups A, B, and G. Mutants belonging to groups A and G have been found to map in nsP2, suggesting that this protein is required for initiation of 26S RNA synthesis, proteolytic processing of the nonstructural precursor, and shut off of minus-strand synthesis. tsl1, the only member of group B, has a mutation in nsP1, indicating that this protein is responsible for initiation of minus-strand synthesis. To understand the role of viral structural proteins in the pathogenesis of flaviviruses, we studied one of the dengue viruses, which constitute a worldwide health problem of increasing dimensions, and determined the complete nucleotide sequence of the PRl59-S1 strain of dengue 2 virus except for l5 nucleotides at the 5' end. There is one long open reading frame which is translated to give the structural proteins, capsid (C), membrane-like protein (M), and envelope protein (E), followed by nonstructural proteins, NS1, NS2, NS3, NS4, and NS5. The individual proteins appear to be produced by posttranslational cleavage of a precursor polyprotein. There are nucleotide sequences in the 5' terminal region (in the coding region for the capsid protein) and in the 3' terminal region (in the 3' untranslated sequence) that are invariant among flaviviruses examined to date and that may be involved in cyclization of the RNA. These sequences are presumed to be important for viral replication. In addition, the 3' terminal 79 nucleotides are capable of forming a hairpin structure. We have expressed the structural proteins of dengue 2 using a recombinant vaccinia virus to study the role of these proteins in the immunological response. The vaccinia recombinant containing a cDNA copy of the 5' region of the dengue genome virus expressed dengue structural proteins which are correctly cleaved and modified. This suggests that the sequences encoding the structural proteins specify all the necessary catalytic activities or recognition signals required to ensure the proper synthesis and maturation of the polypeptides. And also, this recombinant can generate dengue-specific antibodies in mice which neutralize viral infectivity.

Item Type:Thesis (Dissertation (Ph.D.))
Degree Grantor:California Institute of Technology
Division:Biology
Major Option:Biology
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Strauss, James H.
Thesis Committee:
  • Strauss, James H. (chair)
  • Attardi, Giuseppe
  • Rothenberg, Ellen V.
  • Emr, Scott D.
  • Sternberg, Paul W.
Defense Date:8 February 1989
Record Number:CaltechETD:etd-06072007-075259
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-06072007-075259
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:2497
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:14 Jun 2007
Last Modified:26 Dec 2012 02:52

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