Norwalk virus open reading frame 3 encodes a minor structural protein

Norwalk virus (NV) is a causative agent of acute epidemic nonbacterial gast roenteritis in humans. The inability to cultivate NV has required the use o f molecular techniques to examine the genome organization and functions of the viral proteins. The function of the NV protein encoded by open reading frame 3 (ORF 3) has been unknown. In this paper, we report the characteriza tion of the NV ORF 3 protein expressed in a cell-free translation system an d in insect cells and show its association with recombinant virus-like part icles (VLPs) and NV virions. Expression of the ORF 3 coding region in rabbi t reticulocyte lysates resulted in the production of a single protein with an apparent molecular weight of 23,000 (23K protein), which is not modified by N-linked glycosylation. The ORF 3 protein was expressed in insect cells by using two different baculovirus recombinants; one recombinant contained the entire 3' end of the genome beginning with the ORF 2 coding sequences (ORFs 2+3), and the second recombinant contained ORF 3 alone. Expression fr om the construct containing both ORF 2 and ORF 3 resulted in the expression of a single protein (23K protein) detected by Western blot analysis with O RF 3-specific peptide antisera. However, expression from a construct contai ning only the ORF 3 coding sequences resulted in the production of multiple forms of the ORF 3 protein ranging in size from 23,000 to 35,000. Indirect -immunofluorescence studies using an ORF 3 peptide antiserum showed that th e ORF 3 protein is localized to the cytoplasm of infected insect cells. The 23K ORF 3 protein was consistently associated with recombinant VLPs purifi ed from the media of insect cells infected with a baculovirus recombinant c ontaining the entire 3' end of the NV genome. Western blot analysis of NV p urified from the stools of NV-infected volunteers revealed the presence of a 35K protein as well as multiple higher-molecular-weight bands specificall y recognized by an ORF 3 peptide antiserum. These results indicate that the ORF 3 protein is a minor structural protein of the virion.