The role of the rotavirus nonstructural protein, NS35, in the viral replication cycle: Analysis of NS35 interactions with viral RNA and proteins
Date of Award
Doctor of Philosophy (Ph.D.)
Microbiology and Immunology
First Committee Member
John T. Patton, Committee Chair
Based on several lines of evidence, the role of the rotavirus nonstructural protein, NS35, in the viral replication cycle is crucial. The most abundant nonstructural protein in the infected cell, NS35 has been shown to be concentrated in cytoplasmic inclusions referred to as viroplasms. Viroplasms have been proposed to be the site where viral RNA replication and assembly of single-shelled virions take place. The association of NS35 with several viral replication intermediates (RIs) has also been demonstrated. Perhaps the most direct evidence for the significance of NS35 in rotavirus replication and morphogenesis is the severe defect of tsE, a temperature-sensitive mutant rotavirus, in which a lesion has been mapped to the gene encoding NS35. At the nonpermissive temperature, tsE does not undergo RNA replication, establish viroplasms, or produce RNA-containing virions. Two amino acid changes have been detected between the parent virus SA11-R and tsE at positions 152 and 200 (alanine to valine and valine to isoleucine, respectively).To more directly characterize the function(s) of NS35 in rotavirus replication, the interactions of NS35 with viral RNA and proteins were investigated. By gel shift and ultraviolet light cross-linking assays, NS35 was shown to possess nonspecific RNA-binding activity both in vitro and in vivo. No other viral proteins were required for the formation of NS35-RNA complexes in vitro as NS35 translated in rabbit reticulocyte lysates exhibited affinity for poly(U)-sepharose. Attempts to localize the RNA-binding domain in NS35 to a previously described conserved and basic region by deletion mapping yielded no definitive results. All poly(U)-sepharose binding activity was lost when more than four amino acids were deleted from the protein. From these studies, however, a correlation between the affinity of truncated NS35 species for poly(U)-sepharose and their ability to assemble into 12s complexes was apparent.Protein-protein interactions involving NS35 were investigated using the thiol-cleavable cross-linking reagent, dithiobis(succinimidyl propionate), DSP. By this approach, NS35 was found to exist in homomultimers of extensive sizes in the infected cell. Although NS35 was shown to represent the predominant protein component of the viroplasm, detection of vaccinia/T7-expressed NS35 in MA104 cells by immunofluorescence indicated that NS35 was not sufficient for the formation of viroplasms in the absence of other viral proteins.Also detected by protein cross-linking with DSP was the direct interaction of NS35 with the viral polymerase, VP1. The viral guanylyltransferase, VP3, was also found to be associated with the VP1-NS35 (polymerase) complex. No RNA component or RNA-binding activity was found to be associated with the polymerase complex. However, results of pulse-chase experiments indicated that the complex is transient and is, therefore, likely to serve as an intermediate of a more mature complex.
Biology, Molecular; Biology, Microbiology
Kattoura, Maha Dalal, "The role of the rotavirus nonstructural protein, NS35, in the viral replication cycle: Analysis of NS35 interactions with viral RNA and proteins" (1993). Dissertations from ProQuest. 3167.