Identification and characterization of the helix-destabilizing activity ofrotavirus nonstructural protein NSP2

The rotavirus nonstructural protein NSP2 self-assembles into homomultimers, hinds single-stranded RNA nonspecifically, possesses a Mg2+-dependent nucl eoside triphosphatase (NTPase) activity, and is a component of replication intermediates. Because these properties are characteristics of known viral helicases, we examined the possibility that this was also an activity of NS P2 by using a strand displacement assay and purified bacterially expressed protein, The results revealed that, under saturating concentrations, NSP2 d isrupted both DNA-RNA and RNA-RNA duplexes; hence, the protein possesses he lix-destabilizing activity. However, unlike typical helicases, NSP2 require d neither a divalent cation nor a nucleotide energy source for helix destab ilization. Further characterization showed that NSP2 displayed no polarity in destabilizing a partial duplex, In addition, helix destabilization by NS P2 was found to proceed cooperatively and rapidly. The presence of Mg2+ and other divalent cations inhibited by approximately one-half the activity of NSP2, probably due to the increased stability of the duplex substrate brou ght on by the cations, In contrast, under conditions where NSP2 functions a s an NTPase, its helix-destabilizing activity was less sensitive to the pre sence of Mg2+, suggesting that in the cellular environment the two activiti es associated with the protein, helix destabilization and NTPase, may funct ion together. Although distinct from typical helicases, the helix-destabili zing activity of NSP2 is quite similar to that of the sigma NS protein of r eovirus and to the single-stranded DNA-binding proteins (SSBs) involved in double-stranded DNA replication. The presence of SSB-like nonstructural pro teins in two members of the family Reoviridae suggests a common mechanism o f unwinding viral mRNA prior to packaging and subsequent minus-strand RNA s ynthesis.