In bluetongue virus (BTV)-infected cells, large cytoplasmic aggregates are formed, termed viral inclusion bodies (VIBs), which are believed to be the sites of viral replication and morphogenesis. The BTV nonstructural protein NS2 is the major component of VIBs. NS2 undergoes intracellular phosphorylation and possesses a strong single-stranded RNA binding activity. By changing phosphorylated amino acids to alanines and aspartates, we have mapped the phosphorylated sites of NS2 to two serine residues at positions 249 and 259. Since both of these serines are within the context of protein kinase CK2 recognition signals, we have further examined if CK2 is involved in NS2 phosphorylation by both intracellular colocalization and an in vitro phosphorylation assay. In addition, we have utilized the NS2 mutants to determine the role of phosphorylation on NS2 activities. The data obtained demonstrate that NS2 phosphorylation is not necessary either for its RNA binding properties or for its ability to interact with the viral polymerase VP1. However, phosphorylated NS2 exhibited VIB formation while unmodified NS2 failed to assemble as VIBs although smaller oligomeric forms of NS2 were readily formed. Our data reveal that NS2 phosphorylation controls VIBs formation consistent with a model in which NS2 provides the matrix for viral assembly.Protein phosphorylation is a ubiquitous protein modification that controls a number of intracellular processes. In eukaryotic systems, phosphorylation occurs almost exclusively on serine, threonine, or tyrosine residues (26). Also for RNA viruses, including vesicular stomatitis virus, ebola virus, human immunodeficiency virus type 1 (HIV-1), and rubella virus, protein phosphorylation has been shown to regulate vital processes such as virus transcription and replication, RNA binding activity, and virus assembly (9,22,28,34). The nonstructural protein 2 (NS2) is the only phosphorylated protein of the 10 viral proteins synthesized during a bluetongue virus (BTV) infection (25).BTV is the prototype of the Orbivirus genus in the Reoviridae family and hence characteristically possesses a doublestranded RNA genome enclosed by three consecutive capsid layers of multiple proteins. The BTV genome consists of 10 segments, each encoding one protein. There are seven structural proteins (viral polymerase 1 [VP1] through VP7), of which two are outer capsid proteins (VP2 and VP5) and the remaining five are associated with the BTV core. The viral core consists of a double-layered shell composed of VP3 and VP7, and within the core, there are three virus-encoded proteins (VP1, VP4, and VP6), all of which are enzymatically involved in viral transcription and replication. VP4 is the mRNA-capping enzyme, exhibiting guanyltransferase and methyltransferase activities, VP6 is a double-stranded RNA helicase, and the largest protein, VP1, is the viral RNA-dependent RNA polymerase (5,32,36,41). Core particles are transcriptionally active, producing and releasing mRNA. The remaining three BTV proteins are nonstructural...

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...

The Reoviridae have double-stranded RNA genomes of 10-12 segments, each in a single copy in the mature virion. The basis of genome segment sorting during virus assembly that ensures each virus particle contains the complete viral genome is unresolved. Bluetongue virus (BTV) NS2 is a single-stranded RNA-binding protein that forms inclusion bodies in infected cells. Here, we demonstrate that the specific interaction between NS2 and a stem-loop structure present in BTV S10 RNA, and phylogenetically conserved in other BTV serotypes, is abolished by mutations predicted to disrupt the structure. Subsequently, we mapped RNA regions in three other genomic segments of BTV that are bound preferentially by NS2. However, structure probing of these RNAs did not reveal secondary structure motifs that obviously resembled the stem-loop implicated in the NS2-S10 interaction. In addition, the specific binding by NS2 to two different viral RNAs was found to occur independently. Together, these data support the hypothesis that the recognition by NS2 of different RNA structures may be the basis for discrimination between viral RNAs during virus assembly.