Multimers formed by the rotavirus nonstructural protein NSP2 bind to RNA and have nucleoside triphosphatase activity

The nonstructural protein NSP2 is a component of rotavirus replication inte rmediates and accumulates in cytoplasmic inclusions (viroplasms), sites of genome RNA replication and the assembly of subviral particles. To better un derstand the structure and function of the protein, C-terminally His-tagged NSP2 was expressed in bacteria and purified to homogeneity. In its purifie d form, the protein did not exist as a monomer but rather was present as an 8S-10S homomultimer consisting of 6 +/- 2 subunits of recombinant NSP2 (rN SP2), As shown by gel mobility shift assays, the rNSP2 multimers hound to R NA in discrete cooperative steps to form higher-order RNA-protein complexes . The RNA-binding activity of the rNSP2 multimers was determined to be nons pecific and to have a strong preference for single-stranded RNA over double -stranded RNA for which it displayed little affinity. Enzymatic analysis re vealed that rNSP2 possessed an associated nucleoside triphosphatase (NTPase ) activity in vitro, which in the presence of Mg2+ catalyzed the hydrolysis of each of the four NTPs to NDPs with equal efficiency. Evidence indicatin g that the hydrolysis of NTP resulted in the covalent linkage of the gamma- phosphate to rNSP2 was obtained. Additional experiments showed that NSP2 ex pressed transiently in MA014 cells is phosphorylated. We propose that NSP2 functions as a molecular motor, catalyzing the packaging of viral mRNA into core-like replication intermediates through the energy derived from its NT Pase activity.