Minimum Requirements for Bluetongue Virus Primary Replication In Vivo

Abstract

The replication mechanism of bluetongue virus (BTV) has been studied by an in vivo reverse genetics (RG) system identifying the importance of certain BTV proteins for primary replication of the virus. However, a unique in vitro cell-free virus assembly system was subsequently developed, showing that it did not require the same set of viral components, which is indicative of differences in these two systems. Here, we studied the in vivo primary replicase complex more in-depth to determine the minimum components of the complex. We showed that while NS2 is an essential component of the primary replication stage during BTV infection, NS1 is not an essential component but may play a role in enhancing BTV protein synthesis. Furthermore, we demonstrated that VP7, a major structural protein of the inner core, is not required for primary replication but appears to stabilize the replicase complex. In contrast, VP3, the other major structural core protein, is an essential component of the complex, together with the three minor enzymatic proteins (VP1, VP4, and VP6) of the core. In addition, our data have demonstrated that the smallest minor protein, VP6, which is known to possess an RNA-dependent helicase activity, may also act as an RNA translocator during assembly of the primary replicase complex.

Rapid Generation of Replication-Deficient Monovalent and Multivalent Vaccines for Bluetongue Virus: Protection against Virulent Virus Challenge in Cattle and Sheep

“…5). The NA titers ranged between 16 and 128 for all serotypes tested, and there was no apparent interference with respect to the ability of the animal to respond to each of the vaccine strains present in the cocktail (Fig. 5).…”

“…Reassortant viruses were rescued from confluent monolayers of complementing cells (BSR9) transfected with six plasmids that drive the expression of BTV proteins VP1, VP3, VP4, VP6, NS1, and NS2 as described previously (16) prior to transfection with seven T7-derived single-stranded RNAs (ssRNAs) from BTV-1, one of the modified S9 (S9⌬ or S9⌬-EGFP) ssRNAs, and a combination of S2 and S6 ssRNAs from a particular serotype.…”

Trafficking of Bluetongue Virus Visualized by Recovery of Tetracysteine-Tagged Virion Particles

“…Thus, live-cell imaging of cells infected with fluorescently labeled poliovirus has revealed that after internalization through a clathrin-, caveolin-, and flotillin-independent, but actin-and tyrosine kinase-dependent, pathway, the virus releases its RNA rapidly from vesicles located very close to the plasma membrane and does not require endocytic acidification or microtubule-dependent transport (14). In this study, however, for the first time, we have successfully identified exposed loop regions in the BTV outer capsid protein VP2 and have generated replication-competent tagged viruses via reverse genetics (28,33). Since, to date, investigation of the functional role of VP2 in BTV entry has been limited to studies based mainly on recombinant protein expression (9), the tagging of VP2 and its successful incorporation into BTV particles have provided valuable insights into its role during virus entry and trafficking.…”

“…3B). As described previously (33), uncapped S2 T7 transcripts (BTV1-S2) were generated for all constructs (BTV1-S2 94 , BTV1-S2 352 , and BTV1-S2 420 ) to recover TC-tagged mutant viruses. The reverse genetics system used transfection of BSR cells with 9 T7-derived RNA transcripts (S1 and S3 to S10) together with either wild-type (WT) BTV1-S2 or each of the tagged S2 transcripts.…”

“…The T7 BTV capped (BTV1 S1, BTV1 S3 to S10) and uncapped (BTV1 S2, BTV1 S2 with a TC tag) transcripts were generated as described previously (28). The mutant BTV particles were recovered by following a method described previously (33). Genomic dsRNA from cells infected with control or mutant BTV was analyzed as described previously (28).…”