Structure-based identification of functional residues in the nucleoside-2'-O-methylase domain of Bluetongue virus VP4 capping enzyme

Stewart, Meredith E.; Roy, Polly

Eukaryotic cellular and most viral messenger RNAs (mRNAs) are modified at their 5 0 termini with methylated cap structures, which promote stability and efficient translation [6, 9] . Recent studies on selected viral methylated caps have revealed that cap structures may also have a function in cloaking viral messenger from sensors of the host cell innate immune response [5, 12, 16, 17, 34, 35] . Most viral transcripts are characterized by a cap1 structure [ m7 GpppN 2m ] consisting of a 7-methylguanosine ( m7 G) linked by a 5 0 -5 0 triphosphate bond to the 5 0 end of the mRNA and the methylation of the 2 0 -OH group of the ribose of the first nucleotide. The cap structure is formed by an orchestrated series of reactions including the RNA triphosphatase, guanylyltransferase and the two methylase activities, viz., guanine-N7-methyltransferase (N7MTase) and nucleoside-2 0 -O-methyltransferase (2 0 -O MTase). Members of the Reoviridae, such as Bluetongue virus (BTV), encode specific viral proteins that are responsible for synthesizing the cap1 structures at the 5 0 termini of their RNA transcripts, similar to most eukaryotic and viral transcripts [23, 36] . BTV VP4, a minor structural protein, alone catalyses the formation of cap1 structure and therefore possesses multiple catalytic activities [18, 23, 24] . VP4 forms a complex with the viral polymerase (VP1) and RNA helicase (VP6) proteins, this polymerase complex (PC) is closely associated with 10 segments of dsRNA genome [10, 21, 28] within the doublecapsid virion particle. The PC is located at the 5-fold axis of the inner capsid layer formed by a single protein, VP3, which in turn is enclosed by a second layer of VP7 protein. This double-layered capsid or ''core'' is further enclosed by an outer capsid, composed of two proteins, VP2 and VP5, which are involved in virus attachment and entry into the host cells [27] . After cellular entry, the outer capsid is lost, releasing the core into the cytoplasm [11] where an active polymerase complex, which remains within the http N7MTase, guanine-N7-methyltransferase; m7G, 7-methylguanosine; m7, methyl group associated with m7G; PC, polymerase complex; BSR4, BHK-21 sub-clone expressing VP4; AdoMet, S-adenosyl methionine; VSV, vesicular stomatitis virus; WNV, West Nile virus; JEV, Japanese encephalitis virus unassembled core, synthesizes and extrudes new transcripts through pores situated at the core 5-fold axis [29] . The 2.5 Ã… X-ray crystallographic structure of VP4 showed that the protein has a unique structural organisation whereby each functional domain is arranged in sequential fashion to facilitate each catalytic activity required to form the cap structure of the newly synthesized transcripts [33] . Thus, VP4 offers an ideal model for characterizing each functional domain and determining their relative contributions to the virus replication cycle.