Identification of a novel protein in porcine astrovirus that is important for virus replication

Overlapping genes are common in some RNA viruses. It has been proposed that a potential overlapping gene is the ORFX, here termed ORF2b, which overlaps the ORF2 coding sequence in astroviruses. The aim of this study was to determine whether ORF2b is an overlapping gene that encodes a functional protein which is needed for viral replication. Sequence alignment showed that there was an ORF2b in a PAstV type 1 strain of astrovirus, PAstV1-GX1, which was embedded within the larger ORF2. The AUG codon for ORF2b is located 19 nucleotides downstream of the initiation site of ORF2 and contains 369 nucleotides and it codes for a predicted 122-amino-acid protein. A specific polyclonal antibody against the ORF2b protein was raised and used to demonstrate the expression of the new identified gene in virus-infected and pCAGGS-ORF2b-transfected cells. Analysis of purified virions revealed that the ORF2b protein was not incorporated into virus particles. Reverse genetics based on a PAstV type 1 infectious cDNA clone showed that the ORF2b protein was not essential but important for optimal virus infectivity. Knockout of the downstream potential stop codon candidate of ORF2b demonstrated that the C-terminus of the ORF2b protein can be extended by 170 amino acids, suggesting that the C-terminus of the newly identified ORF2b protein may be variable.