Bluetongue virus VP6 acts early in the replication cycle and can form the basis of chimeric virus formation.
A minor core protein, VP6, of bluetongue virus (BTV) possesses nucleoside triphosphatase, RNA binding, and helicase activities. Although the enzymatic functions of VP6 have been documented in vitro using purified protein, its definitive role in BTV replication remains unclear. In this study, using a recently developed T7 transcript-based reverse genetics system for BTV, we examined the importance of VP6 in virus replication. We show that VP6 is active early in replication, consistent with a role as part of the transcriptase or packaging complex, and that its action can be provided in trans by a newly developed complementary cell line. Furthermore, the genomic segment encoding VP6 was mutated to reveal the cis-acting sequences required for replication or packaging, which subsequently enabled the construction of a chimeric BTV expressing enhanced green fluorescent protein. These data confirm that one of the 10 genome segments of BTV can be replaced with a chimeric RNA containing the essential packaging and replication signals of BTV and the coding sequence of a foreign gene.
Matsuo
Eiko
E
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom.
Roy
Polly
P
D000818:Animals; D001820:Bluetongue virus / Q000502:physiology*; D002460:Cell Line; D006224:Cricetinae; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D014758:Viral Core Proteins / Q000502:physiology*; D010948:Viral Plaque Assay; D019065:Virus Assembly; D014779:Virus Replication
D014758:Viral Core Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins