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

Initial predictions on structural protein genes. Sequence analyses of proteins predicted to be expressed from the 5 major ORFs in the 3=-proximal region of the CAVV genome revealed little (if any) similarity with other viral (and cellular) proteins, confirming that CAVV diverged profoundly from other nidoviruses and complicating functional assignments of these proteins. As summarized in Table 2 , two proteins are expected to be expressed from sgRNA 2. ORF2a is predicted to encode a type I glycoprotein featuring a C-terminal membrane-spanning domain and multiple glycosylation sites. Based on these predictions, the protein likely represents a functional equivalent of the S protein of other nidoviruses, which remains to be confirmed in further studies. The predicted translation start codon of ORF2b is the second AUG on the subgenomic mRNA2, located just downstream of the ORF2a start codon, suggesting that ORF2b may be translated by a leaky scanning mechanism. ORF2b encodes a highly basic protein (pK a , 10.8) with a molecular mass of 24 kDa. Both its size and its charge suggest that this protein may be the viral nucleocapsid protein. Among nidoviruses, this upstream position of the (presumed) N protein gene in the CAVV genome is unusual but has its precedent in members of the family Roniviridae (52). ORF3a and -3b encode proteins with predicted molecular masses of 18 and 14 kDa, respectively. Database searches failed to reveal close homologs of these proteins. Protein analysis software predicts the presence of membrane-spanning domains in both proteins (residues 95 to 117 in ORF3a and residues 73 to 95 in ORF3b), suggesting that both proteins are integral membrane proteins. The ORF3a protein likely contains a signal peptidase cleavage site, 15 Ala-Met-Ser|Ala-Glu, and is predicted to be glycosylated, further supporting a role as a membrane-spanning structural protein of the virus. The specific mechanism used to express the ORF3b gene product is unclear but may involve internal ribosomal entry, as shown previously for several downstream ORFs expressed from coronavirus subgenomic mRNAs (53) (54) (55) (56) . Further studies are required to establish if ORF3a and ORF3b proteins have functions related to those of the membrane-spanning M and E proteins of other nidoviruses. ORF4 encodes a small protein of 50 amino acid residues with unknown functions.