Sequence Specificity in the Interaction of Bluetongue Virus Non-structural Protein 2 (NS2) with Viral RNA

Abstract

The non-structural protein NS2 of Bluetongue virus (BTV) is synthesized abundantly in virus-infected cells and has been suggested to be involved in virus replication. The protein, with a high content of charged residues, possesses a strong affinity for single-stranded RNA species but, to date, all studies have failed to identify any specificity in the NS2-RNA interaction. In this report, we have examined, through RNA binding assays using highly purified NS2, the specificity of interaction with different single-stranded RNA (ssRNA) species in the presence of appropriate competitors. The data obtained show that NS2 indeed has a preference for BTV ssRNA over nonspecific RNA species and that NS2 recognizes a specific region within the BTV10 segment S10. The secondary structure of this region was determined and found to be a hairpin-loop with substructures within the loop. Modification-inhibition experiments highlighted two regions within this structure that were protected from ribonuclease cleavage in the presence of NS2. Overall, these data imply that a function of NS2 may be to recruit virus messenger RNAs (that also act as templates for synthesis of genomic RNAs) selectively from other RNA species within the infected cytosol of the cell during virus replication.Viruses that have a segmented RNA genome face a challenging task in the recruitment and assortment of specific viralcoded RNA species in the infected cells. It is commonly believed that each of the viral segments must possess some specific sequence or structures, which is recognized by one or more virally encoded proteins to facilitate these processes. Generally, viral RNA-binding proteins are distinguished by being in one of two categories. The first includes proteins that are associated with the nucleocapsid (nucleoproteins) (1) and are involved in the replication process (transcription and packaging) of the viral genome (2, 3). The second includes proteins that play an essential role in recruiting, transporting (4), modifying (5), and translating (6) viral RNA. These proteins can also interact with cellular RNA to suppress the expression of regulatory genes (7), so protecting the viral RNA from cellular recognition mechanisms to use the cellular machinery for virus propagation (6). Members of the Reoviridae have segmented double-stranded RNA genomes enclosed within the double capsids of the virions. During virus entry into the host cells the outer capsid proteins are lost, allowing the viral core to initiate the transcription of genomic RNAs. The newly synthesized single-stranded RNA species are subsequently released into the cytosol and in turn serve both as templates for viral doublestranded RNA genome synthesis and also act as messengers for the synthesis of viral proteins within the cytoplasm (see review, see Ref. 8). However, to date it is not known how the 10 -12 RNA segments are specifically recruited and transported to the virus replication and assembly sites within the cytoplasm and if any specific sequence is involved. Bluetongue virus is an o...

Phosphorylation of Bluetongue Virus Nonstructural Protein 2 Is Essential for Formation of Viral Inclusion Bodies

“…NS2 binds ssRNA molecules in general, although it exhibits a higher RNA affinity for BTV ssRNA than for the other nonspecific RNA molecules (30,31). It was possible that the phosphorylation status of NS2 may influence its selective RNA binding ability.…”

“…RNA binding assays were essentially carried out as described by Lymperopoulos et al (30). In a typical binding assay, each type of NS2 at the concentration of 3.7 M (75 pmol) was incubated with 8 nM of 32 P-labeled ssRNA probe (1,125 nucleotides [nt]) that was derived from BTV segment 8 (S8).…”

“…This has also been shown for proteins of other RNA viruses (13,18,19). For NS2, which exhibits a preferential RNA binding activity for BTV plus-strand RNAs over non-BTV RNAs, a correlation of its phosphorylated state and RNA binding properties has been assumed (30). This was based on the fact that NS2 used in experiments that failed to identify an RNA binding specificity was expressed in Escherichia coli and consequently not phos- Regarding protein-protein interactions, we showed that NS2 colocalizes intracellularly with the BTV core proteins that are components of the polymerase complex.…”

“…It has the enzymatic ability of hydrolyzing nucleotide triphosphates to nucleotide monophosphates (23,42). The protein also binds single-stranded RNA (ssRNA) very efficiently, and BTV RNAs are preferentially bound over nonspecific RNAs (30,31). An important structural feature of NS2 is its ability to form multimers (45).…”

Minimum Requirements for Bluetongue Virus Primary Replication In Vivo

“…NS2 is the main component of viral inclusion bodies (VIBs) in virus-infected cells where assembly of core particle takes place (18,19,37,40,41). or BSR (bottom) infected cell lysates were spun down over a 30% (wt/vol) sucrose cushion and subjected to CsCl equilibrium centrifugation.…”