An Insect Nidovirus Emerging from a Primary Tropical Rainforest

Zirkel, Florian; Kurth, Andreas; Quan, Phenix-Lan; Briese, Thomas; Ellerbrok, Heinz; Pauli, Georg; Leendertz, Fabian H.; Lipkin, W. Ian; Ziebuhr, John; Drosten, Christian; Junglen, Sandra

Using TMHMM v2.0, three hydrophobic regions comprising putative multiple membrane-spanning domains (TMD1 25 L-L 47 , TMD2 1128 I-Y 1272 , and TMD3 1727 Y-M 1780 ) were identified within the first predicted ORF of the CAVV genome. Three TMDs are also found in ORF1a of CoV and ToV, whereas RoV has four TMDs (18) . However, the position of CAVV TMD1 was similar to that in RoV (18) . Between TMD2 and TMD3, a putative 3CL pro domain was identified. Comparative sequence analysis suggested that the CAVV 3CL pro domain is a cysteine protease with a Cys-His-Asp catalytic triad (see Fig. S1a (Fig. S1c) . Although short regions with low similarity suggest conservation of the ExoN and MT domains, reliable alignments could not be generated and biochemical evidence remains to be obtained to confirm the functionality of these domains. Putative RFS. Expression of the second major ORF of nidoviruses involves a programmed ribosomal frameshift into the Ϫ1 reading frame, occurring just upstream of the ORF1a stop codon (32, 33) . The overlap region of ORF1a and -1b typically contains a slippery heptanucleotide sequence and a downstream RNA pseudoknot structure that together promote ribosomal frameshifting (32, 33) . With minor variations, previously identified nidovirus slippery sequences conform to the XXXYYYZ consensus sequence conserved in many ribosomal slip sites on viral RNAs (for a review, see reference 42). Thus, for example, ArV, CoV, and ToV use 5=-(U/G)UUAAAC and RoV uses 5=-AAAUUUU as a slippery sequence (18, 42) . The short (~35-nt) ORF1a/1b overlap region in CAVV does not contain heptanucleotide sequences related to those of other nidoviruses. The only XXXYYYZ-like sequence identified in this region of the CAVV genome is 7829 CCCUUUG. However, previous systematic mutagenesis studies of ribosomal slip sites (43) revealed that the CCCUUUG heptanucleotide sequence does not promote efficient Ϫ1 ribosomal frameshifting in vitro and, to our knowledge, has not been reported to mediate efficient ribosomal frameshifting in viral or cellular systems. Further analyses revealed 7835 GGAUUUU as a further candidate slip site. The GGAUUUU sequence conforms to the simultaneousslippage model introduced by Jacks et al. (44) . More importantly, data obtained for red clover necrotic mosaic (diantho) virus (RC-NMV) (45, 46) have shown that this sequence mediates efficient Ϫ1 ribosomal frameshifting and thus expression of the downstream p57 polymerase ORF of RCNMV. Furthermore, the CAVV 7835 GGAUUUU sequence is located 5 nt upstream of an energetically favorable RNA secondary structure. Both the length of the spacer element and the presence of a putative stem-loop structure adjacent to the proposed frameshift site support the idea that CAVV ORF1b expression is mediated by Ϫ1 ribosomal frame- shifting at this site. Even though direct experimental evidence would be desirable to further corroborate this prediction, it seems reasonable to suggest that ORF1a/1b-encoded sequences are fused at a 2491 Leu-Asp-Phe-Ser junction site.