Norwalk virus open reading frame 3 encodes a minor structural protein.

Norwalk virus (NV) is a prototype strain of human caliciviruses, a group of viruses that are the major pathogens causing epidemic nonbacterial gastroenteritis (14, 49). The NV genome, a positive-sense, single-stranded RNA molecule approximately 7.7 kb in length, is predicted to contain three open reading frames (ORFs) (29). The first and third ORFs are in reading frame 2 of the cDNA, while ORF 2 is in reading frame 3. The first ORF (ORF 1) is predicted to encode the nonstructural proteins. Sequence analysis has identified similarities to the picornavirus 2C helicase, 3C protease, and 3D RNA-dependent RNA polymerase (29). The second ORF (ORF 2) encodes the capsid protein. Expression of the capsid protein in insect cells infected with baculovirus recombinants results in the self-assembly of empty recombinant virus-like particles (rVLPs) (28, 51). The third ORF (ORF 3) is located at the 3′ end of the genome and codes for a 212-amino-acid protein of unknown function. The predicted molecular weight of the NV ORF 3 protein is 22,479. The ORF 3 protein is a basic protein with a predicted isoelectric point of 10.99, which has led to speculation that it may be involved in nucleic acid binding (13). Purified 38-nm recombinant NV (rNV) VLPs have been characterized antigenically and morphologically (28). Three-dimensional reconstruction studies revealed that these particles fold into T=3 icosahedral structures formed by 180 copies of the capsid protein (40). The finding of virus capsids composed of a single structural protein is a common feature of plant viruses including tomato bushy stunt virus (22) and turnip crinkle virus (25). Caliciviruses and nodaviruses are the only animals viruses described to date with a capsid made of a single structural protein (23, 43). A notable difference between plant virus and NV capsid proteins is that the plant virus capsid proteins have an N-terminal basic domain, which is thought to interact with the RNA during assembly. The NV capsid protein lacks such a basic region, and analysis of the X-ray crystallographic structure of rNV VLPs has revealed that the inner surface of the icosahedral shell is acidic (38). For these reasons, it seems highly possible that the calicivirus ORF 3 protein may aid in RNA encapsidation. The presence of ORF 3 in the genome is conserved throughout all human and animal caliciviruses, suggesting that it plays a role in replication or assembly. ORF 3 or ORF 3-equivalent proteins were detected in feline calicivirus (FCV)-infected cells (24) and in rabbit hemorrhagic disease virus (RHDV)-infected primary hepatocytes (32) as well as RHDV virions (54). The synthesis of the ORF 3 protein in a cell-free translation system has been reported for FCV and Camberwell virus (24, 54). Considerable sequence variability of ORF 3 among members of the Norwalk-like viruses (NLVs) has been reported and is consistent with the idea that ORF 3 might be a structural protein (42, 45). The inability to grow NV and a lack of reagents to detect ORF 3 has hampered studies of the ORF 3 protein. In the present paper, we report the characterization of the NV ORF 3 protein synthesized in a cell-free translation system and expressed in insect cells, purified VLPs, and purified NV virions. These studies were facilitated by generation of ORF 3 peptide antisera which are able to recognize the original peptides by enzyme-linked immunosorbent assay (ELISA) and the ORF 3 protein by immunoprecipitation and Western blot analysis.