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

Area of Science:

• Virology
• Molecular Biology
• Structural Biology

Background:

• Norwalk virus (NV) causes epidemic nonbacterial gastroenteritis.
• Cultivation of NV is challenging, necessitating molecular approaches for studying its genome and proteins.
• The function of the NV open reading frame 3 (ORF 3) protein remained uncharacterized.

Purpose of the Study:

• To characterize the Norwalk virus ORF 3 protein.
• To investigate the association of the ORF 3 protein with virus-like particles (VLPs) and NV virions.

Main Methods:

• Expression of NV ORF 3 protein in a cell-free translation system (rabbit reticulocyte lysates).
• Expression of NV ORF 3 protein in insect cells using baculovirus recombinants (ORFs 2+3 and ORF 3 alone).
• Western blot analysis and indirect immunofluorescence studies.
• Analysis of purified NV from infected volunteers.

Main Results:

• Cell-free expression yielded a 23K ORF 3 protein, not N-linked glycosylated.
• Expression in insect cells with ORFs 2+3 produced a single 23K protein, while ORF 3 alone yielded multiple forms (23K-35K).
• The ORF 3 protein localized to the cytoplasm of infected insect cells.
• The 23K ORF 3 protein associated with recombinant VLPs.
• Purified NV virions contained a 35K ORF 3 protein and higher molecular weight forms.

Conclusions:

• The Norwalk virus ORF 3 protein is a minor structural component of the virion.
• The ORF 3 protein exists in multiple forms, with a 35K form being significant in NV virions.