Rotavirus Nonstructural Protein NSP2 Self-assembles into Octamers That Undergo Ligand-induced Conformational Changes

Abstract

The nonstructural protein NSP2 is a component of the rotavirus replication machinery and binds singlestranded RNA cooperatively, with high affinity, and independent of sequence. Recently, NSP2 has been shown to form multimers and to possess an NTPase activity, but its precise function remains unclear. In the present study, we have characterized the solution structure of recombinant NSP2 by velocity and equilibrium ultracentrifugation, dynamic light scattering, and circular dichroism spectroscopy. We found that NSP2 exists as an octamer, which is functional in the binding of RNA and ADP. In the presence of magnesium, a partial dissociation of the octamer into smaller oligomers was observed. This was reversed by binding of ADP and RNA. We observed an increased sedimentation rate in the presence of ADP and a nonhydrolyzable ATP analogue, which suggests a change toward a significantly more compact octameric conformation. The secondary structure of NSP2 showed a high fraction of ␤-sheet, Rotavirus is a significant cause of disease in humans and animals. It is a member of the Reoviridae, and its genome consists of 11 segments of double-stranded RNA, which codes for six structural and six nonstructural proteins. The structural proteins include VP2, VP6, and VP7, which form the triple layered icosahedral virion capsid, the spike protein VP4, the RNA polymerase VP1, and the multifunctional capping enzyme VP3 (1) (for a review, see Ref.2). Many studies have addressed the properties of the structural proteins, for example their spatial configuration in the virus particle using cryo-EM (3, 4), their antigenicity, and their role in viral entry, replication, or morphogenesis. Unfortunately, much less is known about the nonstructural proteins that are expressed and left behind in the infected cells.Although it has been shown that these nonstructural proteins are not essential for replicase activity in vitro (5), they are important in several aspects of the replication cycle of the virus in vivo. Some of the more intensively studied nonstructural proteins include, for example, NSP4, which has a membranedestabilizing activity and assists in the budding of newly synthesized inner capsid particles into the lumen of the endoplasmic reticulum (6, 7), where they acquire the outer coat protein VP7. NSP3 binds to the 3Ј-end of the viral mRNA and interacts with eukaryotic translation initiation factor eIF4G to enhance efficiency of translation (8). NSP2 and NSP5 interact to form viroplasms, large inclusions in the cytoplasm where core-like replication intermediates (core RIs) 1 are assembled and RNA replication takes place (2, 9, 10). As was shown in a study of a temperature-sensitive mutant, NSP2 is required for the formation of the viroplasm and is also essential in the synthesis of double-stranded RNA in vivo (11). However, little is known about the mechanism underlying this observation and the function of NSP2 on a molecular level.NSP2 is a 35-kDa protein that forms homomultimers and interacts with the RNA polymerase VP1 (12, 17). F...

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