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

Abstract

Orbiviruses are insect-transmitted, non-enveloped viruses with a ten-segmented dsRNA genome of which the bluetongue virus (BTV) is the prototype. Viral non-structural protein NS3/NS3a is encoded by genome segment 10 (Seg-10), and is involved in different virus release mechanisms. This protein induces specific release via membrane disruptions and budding in both insect and mammalian cells, but also the cytopathogenic release that is only seen in mammalian cells. NS3/NS3a is not essential for virus replication in vitro with BTV Seg-10 containing RNA elements essential for virus replication, even if protein is not expressed. Recently, new BTV serotypes with distinct NS3/NS3a sequence and cell tropism have been identified. Multiple studies have hinted at the importance of Seg-10 in orbivirus replication, but the exact prerequisites are still unknown. Here, more insight is obtained with regard to the needs for orbivirus Seg-10 and the balance between protein expression and RNA elements. Multiple silent mutations in the BTV NS3a ORF destabilized Seg-10, resulting in deletions and sequences originating from other viral segments being inserted, indicating strong selection at the level of RNA during replication in mammalian cells in vitro. The NS3a ORFs of other orbiviruses were successfully exchanged in BTV1 Seg-10, resulting in viable chimeric viruses. NS3/NS3a proteins in these chimeric viruses were generally functional in mammalian cells, but not in insect cells. NS3/NS3a of the novel BTV serotypes 25 and 26 affected virus release from Culicoides cells, which might be one of the reasons for their distinct cell tropism.

Bluetongue virus serotype 27: Experimental infection of goats, sheep and cattle with three BTV-27 variants reveal atypical characteristics and likely direct contact transmission BTV-27 between goats

“…In addition, differences in tissue culture susceptibility observed between the three variants (and in comparison with BTV-25 and 26) raise questions pertaining to the nature of the viral genetic control(s) affecting transmission and susceptibility. Moreover, recent studies demonstrated that interactions between BT viral proteins are crucial in terms of tissue tropism and particularly in insect cells (Feenstra, Drolet, Boonstra, & van Rijn, 2015;Feenstra, van Gennip, Schreuder, & van Rijn, 2016;Nunes et al, 2014 andPullinger et al, 2016). Reverse genetics using mono-reassortants of BTV-26 genes in a BTV-1 background has been able to provide answers for some of these (Pullinger et al, 2016).…”

Requirements and comparative analysis of reverse genetics for bluetongue virus (BTV) and African horse sickness virus (AHSV)

“…Previously, RG for BTV using exclusively in vitro synthesized capped run-off RNA transcripts, have resulted in reassortants, mutants and variants with spontaneously introduced changes during rescue [ 27 , 40 ]; [ 11 , 33 ]. On the other hand, several modifications appeared not viable [ 32 , 35 , 40 ]. Here, expression plasmids with optimized ORFs of VP1, 3, 4, 6, and NS1 and 2 of BTV6/net08 were successfully used (Table 3 ).…”

“…In order to increase the success rate for rescue of mutants and reassortants, the most optimal system is the combination of all seven expression plasmids with optimized ORFs and capped RNA transcripts for the second transfection. BTV1 and BTV8 variants were generated using BTV6 based ORF-optimized expression plasmids [ 35 ], and virulent AHSV5 was similarly recovered with AHSV4LP based expression plasmids (published elsewhere). We propose that one set of expression plasmids of the respective orbivirus species in combination with a set of ten even uncapped run-off RNA transcripts will be sufficient to quickly recover new orbivirus variants.…”

“…Recovery of reassortants showed that the constellation of the BTV genome is flexible [ 23 , 26 – 31 ]. RG has also been used to study viral proteins [ 11 , 14 ], as well as the role of RNA sequences in virus replication [ 32 – 35 ].…”