RNA Elements in Open Reading Frames of the Bluetongue Virus Genome Are Essential for Virus Replication

Abstract

Members of the Reoviridae family are non-enveloped multi-layered viruses with a double stranded RNA genome consisting of 9 to 12 genome segments. Bluetongue virus is the prototype orbivirus (family Reoviridae, genus Orbivirus), causing disease in ruminants, and is spread by Culicoides biting midges. Obviously, several steps in the Reoviridae family replication cycle require virus specific as well as segment specific recognition by viral proteins, but detailed processes in these interactions are still barely understood. Recently, we have shown that expression of NS3 and NS3a proteins encoded by genome segment 10 of bluetongue virus is not essential for virus replication. This gave us the unique opportunity to investigate the role of RNA sequences in the segment 10 open reading frame in virus replication, independent of its protein products. Reverse genetics was used to generate virus mutants with deletions in the open reading frame of segment 10. Although virus with a deletion between both start codons was not viable, deletions throughout the rest of the open reading frame led to the rescue of replicating virus. However, all bluetongue virus deletion mutants without functional protein expression of segment 10 contained inserts of RNA sequences originating from several viral genome segments. Subsequent studies showed that these RNA inserts act as RNA elements, needed for rescue and replication of virus. Functionality of the inserts is orientation-dependent but is independent from the position in segment 10. This study clearly shows that RNA in the open reading frame of Reoviridae members does not only encode proteins, but is also essential for virus replication.

Balance of RNA sequence requirement and NS3/NS3a expression of segment 10 of orbiviruses

“…Thus, NS3/NS3a is crucial in vivo for virus transmission between the insect vector and the ruminant host. However, NS3/NS3a protein is not essential for virus replication in vitro (van Gennip et al, 2014), whereas RNA elements in the Seg-10 ORF are essential for BTV replication (Feenstra et al, 2014b). Indeed, we observed here a strong selection on the Seg-10 RNA sequence at the expense of NS3/NS3a expression, when studying silent mutations in Seg-10.…”

“…RNA sequences within the NS3 ORF were shown to be essential for virus replication in vitro, independent of protein expression. Deleted RNA sequences were compensated by RNA inserts from other viral segments during virus rescue without restoring NS3/NS3a expression (Feenstra et al, 2014b). Only small out-of-frame deletions in BTV Seg-10 were stable as long as NS3/NS3a-related protein was expressed and mutations disturbing NS3/NS3a expression were restored by point mutations (van Gennip et al, 2014).…”

“…BTV8 without NS3/NS3a expression (BTV8[NS3]min) was generated by incorporation of BTV8 Seg-10 DC containing an out-of-frame deletion from position 102-263 (Feenstra et al, 2014b Analysis of mutants and reassortants by PCR, sequencing and immunostaining. Stability of rescued virus mutants was investigated during six subsequent passages on BSR cells.…”

“…RNA of passaged virus was isolated using the High Pure Viral RNA kit (Roche) according to manufacturer's protocol. Entire Seg-10 was reverse transcribed and amplified using the one-step RT-PCR kit (Qiagen) with Seg-10 primers (F-full-S10* 59-GTTAAAAAGTGTCGCTGCC-39 and Rfull-S10 59-GTAAGTGTGTAGTGTCGCGCAC-39) as described previously (Feenstra et al, 2014b). Purified amplicons were sequenced using the same primers and the BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems) in an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems).…”

“…Likely, Seg-10 is the first recruited segment, followed by the other small segments, which then probably form a complex or complexes with the larger segments that are then packaged as a whole into the subcore particle . Recently, it has been shown that RNA elements in the ORF of Seg-10 are essential for virus rescue, irrespective of NS3-related expression (Feenstra et al, 2014b). However, the mechanism and exact RNA sequences required are still unknown.…”