Characterization of the roles of the major inner capsid proteins, VP2 and VP6, in rotavirus replication using SA11 temperature-sensitive mutants F and G

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Microbiology and Immunology

First Committee Member

John T. Patton, Committee Chair


Temperature-sensitive mutants of rotavirus SA11 were previously developed and organized into 10 of a possible 11 recombination groups. Two of these mutants, tsF and tsG map to genes encoding VP2 (segment 2) and VP6 (segment 6), respectively. Subviral particles (SVPs) isolated from infected cells were analyzed for associated replicase activity (ssRNA $\to$ dsRNA) in vitro. Replicase activity of tsF SVPs from cells maintained at nonpermissive temperature was 4-5-fold less than that of SVPs from cells maintained at permissive temperature). In contrast, no significant difference was detected between the levels of replicase activity associated with tsG SVPs from cells maintained at either permissive or nonpermissive temperatures. SVPs with replicase activity recovered from tsG-infected cells maintained at nonpermissive temperature co-migrated with virion-derived cores on gels and contained VP2, but not VP6. Taken together, these data indicated that VP2, but not VP6 was necessary for rotavirus replicase activity.As viral assembly is dramatically reduced in tsF and tsG-infected cells maintained at nonpermissive temperature, the nucleotide sequences of segment 2 and segment 6 of tsF and tsG, respectively, were determined in order to identify domains that may be important for rotavirus morphogenesis. While only a single mutation was noted in the predicted sequence of tsF VP2, two mutations were identified at the N-terminus of tsG VP6. Analysis of SVPs recovered from tsF-infected cells maintained at nonpermissive temperature suggested that tsF VP2 may not assemble into core structures at the elevated temperature. Thus, the location of this mutation may define sequences in VP2 that are important for viral assembly. In the case of tsG VP6, the mutations lie outside of domains shown previously to be important for trimer formation and in vitro assembly into single-shelled particles. Immunofluorescence studies indicated that in tsG-infected cells maintained at permissive temperature, VP6 was closely associated with viroplasmic inclusions, the putative sites of single-shelled particle assembly. However, in tsG-infected cells maintained at nonpermissive temperature, VP6 was distributed throughout the cytoplasm. These results suggested that the N-terminus of VP6, while not required for binding to core particles, is important for the targeting to viroplasmic inclusions.


Biology, Molecular; Biology, Microbiology

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