A hidden gene in astroviruses encodes a cell-permeabilizing protein involved in virus release

Valeria Lulla; Andrew E. Firth

ORFX overlaps ORF2, which encodes the structural polyprotein, and therefore the expression of XP is likely required in late stages of the virus replication cycle. To rule out the possibility of XP directly influencing viral RNA replication, we developed a replicon system comprising an intact astrovirus genome up to the end of ORFX, followed by a 2A-RLuc cassette fused in either the ORF2 or ORFX reading frame, followed by the last 624 nt of the virus genome and a poly-A tail (Fig. 4A) . These replicons provide a direct measurement of translated product associated with activity of the subgenomic promoter. Two ORFX knock-out mutations were copied in both versions of the replicon to evaluate the significance of XP for RNA replication (Fig. 4B ). Experiments were performed in BSR and Huh7.5.1 cell lines, both of which have been reported to support astrovirus replication 16, 17 . Replication was confirmed by an 1200-2100 fold difference in relative luciferase activity between wt and an RdRp knockout mutant (GDD → GNN) at 9 and 12 hours post transfection (Fig. 4C, Fig. S12A ). The mutations introduced to knock out ORFX had minor effect on luciferase activity when 2A-RLuc was fused in the ORF2 reading frame (Fig. 4C, Fig. S12A ). Consistent with our Ribo-Seq results (Fig. 2C ), luciferase activity for the ORFX reading frame was 14% (BSR cells; Fig. 4C ) and 21% (Huh7.5.1 cells; Fig. S12A ) of luciferase activity for the ORF2 reading frame. Thus, ORFX is efficiently translated but has only a minor (if any) direct effect on viral RNA replication.