Two Nonoverlapping Domains on the Norwalk Virus Open Reading Frame 3 (ORF3) Protein Are Involved in the Formation of the Phosphorylated 35K Protein and in ORF3-Capsid Protein Interactions

Abstract

Expression of the Norwalk virus open reading frame 3 (ORF3) inSpodoptera frugiperda (Sf9) cells yields two major forms, the predicted 23,000-molecular-weight (23K) form and a larger 35K form. The 23K form is able to interact with the ORF2 capsid protein and be incorporated into virus-like particles. In this paper, we provide mass spectrometry evidence that both the 23K and 35K forms are composed only of the ORF3 protein. Norwalk virus (NV) is the prototype strain of the Norovirus genus in the family Caliciviridae (24). The noroviruses are a group of viruses that are the major pathogens causing epidemic nonbacterial gastroenteritis (8, 31). The NV genome is a positive-sense, single-stranded RNA molecule approximately 7.7 kb in length predicted to encode three open reading frames (ORFs) (16,18). The first ORF (ORF1) encodes the nonstructural proteins. The second ORF (ORF2) 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) (17, 32). The third ORF (ORF3) is located at the 3Ј end of the genome and codes for a 212-amino-acid protein that has been characterized as a minor structural protein (9). Expression of the NV ORF3 region in insect cells yields two forms of ORF3 protein, a 23,000-molecular-weight (23K) unphosphorylated form and a 35K phosphorylated form (9). While the ORF3 protein is not essential for the formation of empty VLPs (10,13,17,21,32), the ORF3 23K protein is associated with recombinant NV (rNV) VLPs purified from insect cell cultures infected either with a baculovirus recombinant expressing the entire 3Ј end of the genome or by coinfection with baculovirus recombinants that individually express the ORF2 and ORF3 proteins (9).Protein expression and interaction studies of the human caliciviruses remain limited due to the lack of a tissue culture system for virus propagation. Conservation of ORF3 in all caliciviruses suggests that it performs an important function in the virus life cycle. The ORF3 protein is present in feline calicivirus (FCV) and rabbit hemorrhagic disease virus (RHDV) particles (22,29). However, no studies have examined the potential domains present in the ORF3 protein. In this paper, we report domains of the ORF3 protein responsible for formation of the larger 35K form and for ORF3 proteincapsid protein interaction. In addition, we report further characterization of the 35K form. These studies were facilitated by the generation of baculovirus recombinants that express mutant ORF3 proteins in insect cells and ORF3-specific peptide antisera. The baculovirus recombinants were also coinfected with an ORF2 baculovirus recombinant to examine the ability of the ORF3 mutant proteins to interact with the ORF2 protein allowing incorporation into rNV VLPs. MATERIALS AND METHODSPlasmids and recombinant baculoviruses. For N-terminal HA-tagged ORF3, the hemagglutinin (HA) tag was cloned onto the N terminus of the ORF3 protein by PCR amplification using pri...

Feline Calicivirus VP2 Is Essential for the Production of Infectious Virions

“…Interestingly, this region corresponds to aa 60 to 85 of FCV VP2 and shows 41% amino acid similarity to the VP2 protein of NV. Deletion of this region in the FCV VP2 protein [clones pF3N , pF3N , pF3N , and pF3N ] dramatically affects the production of infectious virus, which is consistent with the proposal by Glass et al (8) that this region might play a critical role in the interaction between VP1 and VP2. However, the presence of this sequence alone is not sufficient for term2], prevented production of infectious virus particles, suggesting that the N-and C-terminal modifications of the VP2 sequence were likely to affect the folding of this protein and its function in particle assembly.…”

“…The domain of the NV VP2 involved in the protein-protein interactions with VP1 was mapped to an internal region located between aa 108 and 152 (8) that has been described as relatively conserved among the VP2 proteins of different caliciviruses (8). Interestingly, this region corresponds to aa 60 to 85 of FCV VP2 and shows 41% amino acid similarity to the VP2 protein of NV.…”

“…It was proposed that due to its basic characteristics, VP2 might interact with RNA and the internal acidic domains of the calicivirus virion, providing a structural basis for encapsidation of the viral RNA into particles (26,30,43). The sequence between aa 108 and 152 of Norwalk virus (NV) VP2 has been mapped as a domain responsible for protein-protein interactions between the NV VP2 and VP1 proteins (8), but no evidence for direct interactions between VP2 and RNA has been reported. Recently, it was demonstrated that the presence of VP2 could protect VP1 from protease degradation and increase the stability of NV recombinant virus-like particles (1).…”

Norwalk Virus Minor Capsid Protein VP2 Associates within the VP1 Shell Domain

“…In contrast, the P domain (residues 226 to 530) is the most exposed and variable region on VP1 forming protrusions from the viral surface and therefore is not expected to interact with VP2. Previously, we showed that the first 20 N-terminal residues on VP1 are not important for VP2 association (33) or for VLP formation (17).…”

A DNA-launched reverse genetics system for rabbit hemorrhagic disease virus reveals that the VP2 protein is not essential for virus infectivity

“…Although the VP2 protein was proposed to be a minor structural protein, the biological function of VP2 remains to be fully elucidated. In 2003, the function of Norwalk virus VP2 was studied by the Estes group (Bertolotti-Ciarlet et al, 2003;Glass et al, 2003); their results showed that the protein could interact with capsid protein and regulate the expression and stability of the viral capsid protein VP1. Two years later, Sosnovtsev et al (2005) showed that FCV VP2 was essential for the production of infectious virions, as deletion of VP2 resulted in complete loss of infectivity.…”