The rotavirus RNA-binding protein NS35 (NSP2) forms 10S multimers and interacts with the viral RNA polymerase

Abstract

The gene 8 product of SA11 rotavirus, NS35 (NSP2), is a nonspecific RNA-binding protein that accumulates in cytoplasmic inclusions (viroplasms) and is required for genome replication. To gain additional information on the role of NS35 in virus replication, lysates of simian rotavirus SA11-infected cells were treated with the thio-cleavable crosslinking agent, dithiobis(succinimidyl propionate) (DSP). Gel electrophoresis of NS35-specific immunoprecipitates recovered from the crosslinked lysates indicated that infected cells contained NS35 multimers, the largest consisting of four or more molecules of the protein. Sedimentation analysis of NS35 expressed in rabbit reticulocyte lysates by cell-free translation and in vTF7-3-infected cells by transfection with a gene 8-containing transcription vector showed that NS35 assembles into multimers of approximately 10S and that the formation of the multimers does not require other viral proteins. The 10S multimers were also detected in rotavirus-infected cells, providing evidence that they function in virus replication. The lack of RNase sensitivity indicates that the 10S multimers probably lack an RNA component. However, by an NS35-specific RNA capture assay, the multimers were shown to possess the RNA-binding activity previously demonstrated for NS35. Despite its ability to multimerize and bind RNA, indirect immunofluorescence assays showed that when transiently expressed in cells, NS35 alone is not sufficient to induce the formation of viroplasms. DSP-crosslinking of infected cell lysates and immunoprecipitation also revealed that NS35 interacts with the putative viral RNA polymerase VP1. Analysis of cytoplasmic extracts resolved by sedimentation on glycerol gradients suggested that the VP1-NS35 complexes are soluble and RNA-free. Complexes formed from NS35 multimers, VP1, and viral messenger RNA may function to coordinate RNA packaging and the assembly of viral cores.