Rotavirus morphogenesis: domains in the major inner capsid protein essential for binding to single-shelled particles and for trimerization.

A cell-free system containing rotavirus subviral particles (SVPs), rabbit reticulocyte lysate, and [35S]methionine was programmed to synthesize viral protein by the addition of messenger RNA (mRNA). Electrophoretic analysis of single-shelled particles recovered from the system by CsCl centrifugation showed that newly made VP6 assembled into the particles in vitro. Electrophoretic analysis also showed that the newly made VP6 which bound to single-shelled particles in vitro was arranged in trimeric units. To identify the domain within VP6 essential for assembly into single-shelled particles, amino- and carboxyl-truncated species of VP6 were assayed for the ability to associate with single-shelled particles in the cell-free system. The truncated proteins were introduced into the system by adding VP6 mRNAs containing 5'- and 3'-terminal deletions. The terminally deleted mRNAs were prepared using SP6 RNA polymerase to transcribe portions of cDNAs of the rotavirus SA11 gene for VP6 (gene 6). Analysis of the ability of truncated VP6 to associate with single-shelled particles showed that a domain essential for assembly resides at the carboxyl-end of VP6 located between amino acid residues 251 and 397. To contrast the domain for assembly with that for trimerization, amino- and carboxyl-truncated species of VP6 were also examined by electrophoretic assay for the ability to trimerize in vitro. The results showed that the domain for trimerization resides near the center of VP6 located between amino acid residues 105 and 328. Comparison of the domains for assembly and trimerization showed that they are unique but may overlap. The fact that some truncated species of VP6, although able to bind to single-shelled particles were unable to form trimers in vitro, suggests that trimerization of VP6 is not prerequisite for the assembly of single-shelled particles.