Rotavirus RNA Replication: Single-stranded RNA Extends from the Replicase Particle

Abstract

The rotavirus genome consists of 11 segments of dsRNA that are replicated asymmetrically with plus strand RNA serving as the template for minus strand RNA synthesis. In this study, we have used nondenaturing gel electrophoresis to examine subviral particles that synthesize dsRNA (replicase particles), for possible changes in structure during RNA replication. Analysis of SVPs purified from simian rotavirus SAil-infected MA104 cells and resolved on 0-6% agarose gels containing 50 mM-Tris-glycine pH 8.8 showed that the overall size of particles able to synthesize dsRNA in a cell-free system was 100 nm or more. Electrophoretic analysis of the size of replicase particles as a function of length of incubation in the cell-free system demonstrated that replicase particles decreased in size with increasing length of incubation. However, after 60 to 90 min of incubation, replicase particles no longer changed in size but were similar in size to the rotavirus single-shelled (75 nm), core (60 nm) and precore (45 nm) replicative intermediates which have been described previously. As the size of replicase particles decreased with increasing length of incubation, the number of newly made genome-length dsRNAs in the particles increased. Analysis of the RNA products detected in replicase particles showed that RNA replication is regulated such that the synthesis of full-length dsRNAs in the replicase particle proceeds from the smallest to the largest genome segments. Treatment of replicase particles with single-strand-specific RNase reduced their size to that of replicative intermediates and interfered with their ability to synthesize dsRNA, thus indicating that the plus strand RNA template for replication extends from the replicase particle. This study showed that replicase particles undergo a continuous change in size during RNA replication due apparently to plus strand RNA templates moving into the replicase particle during the synthesis of dsRNA.

Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation

“…Within 2-3 h post-infection, discrete cytoplasmic structures called viroplasms are formed, which contain the non-structural proteins NSP2 and NSP5, together with other viral proteins, including the polymerase, VP1, the guanyltransferase methylase, VP3, and the main inner-core protein, VP2 (Gallegos & Patton, 1989;Zeng et al, 1998). It is in the viroplasm where the initial steps of viral morphogenesis occur, in a process involving the concerted packaging and replication of the 11 positive-polarity ssRNAs to generate the viral dsRNA genomic segments (Patton & Gallegos, 1990;Wentz et al, 1996;Patton et al, 1997). …”

RNA-Binding Activity of the Rotavirus Phosphoprotein NSP5 Includes Affinity for Double-Stranded RNA

“…Characterization of subviral particles from infected cells has indicated that corelike RIs (core RIs) are the simplest complexes that catalyze the synthesis of the dsRNA genome (14,32). Core RIs, which are composed of VP1, VP2, and VP3 and nonstructural proteins NSP2 and NSP5, undergo maturation into double-layered RIs as they synthesize dsRNA.…”

“…32 P-labeled gene 8 mRNA and v40 RNA were prepared by transcribing SacII-linearized vectors pSP65g8(ϩ) and pSP72v40 with T7 RNA polymerase using Ambion MEGAscript and MEGAshortscript kits, respectively (30,35). Reaction mixtures contained 100 Ci of [␣-…”

Function of Rotavirus VP3 Polypeptide in Viral Morphogenesis

“…3aS-labelled subviral particles were obtained as previously described from confluent monolayers of MA-104 cells infected with 5 p.f.u. (Patton & Gallegos, 1990). The pellets containing the subviral particles were suspended in 25 ~1 of buffer HGD (10 mM-HEPES pH 7"6, 10% glycerol, 2mM-dithioerythritol) and subjected to electrophoresis on a 0'6% Tris-glycine agarose gel, then dried and analysed by autoradiography.…”