Article Figures & Data
Figures
- Fig 1
Norovirus VP1 and VP2 expression increases when both capsid proteins are present. Western blots of 293 cell lysates transfected with only VP1, VP2, or both VP1 and VP2 were analyzed with rabbit antisera specific for the protein as indicated to the left of each panel. HoV VP2 contained a 3× FLAG epitope and monoclonal anti-FLAG antibody was used for detection. β-Actin served as a loading control.
- Fig 2
Norwalk virus VP1 region encompassing residues 49 to 61 regulates capsid protein expression. (A) Schematic diagram of NV VP1 N-terminal truncation mutants generated. (B) Full-length unmodified (wt) or truncation mutant VP1 proteins (indicated by residue numbers) were coexpressed with VP2 and cell lysates subjected to coimmunoprecipitation. VP1 proteins were immunoprecipitated (IP) and associated VP2 resolved on a Western blot (WB). The presence of each protein in the lysates prior to immunoprecipitation is indicated to the right of each panel (input). NT, N-terminal arm; S, shell domain; P, protruding domain. Numbers denote VP1 amino acid residues.
- Fig 3
Residues IDPWI (underlined) on VP1 are highly conserved among the shell domains of different noroviruses. Amino acid alignment of the N-terminal region of VP1 for which sequences were available from strains GI.1 to GI.14, GII.1 to GII.17, and representative strains from GIII through GVI. Identical residues are shaded gray.
- Fig 4
Isoleucine 52 on the Norwalk virus VP1 interacts with VP2 and maps to the shell domain. (A) Western blot of VP2 coimmunoprecipitated with different full-length VP1 proteins mutated at the indicated residues. (B) Mapping of the surface-exposed residues onto the structure of a VP1 dimer (each monomer shown in blue and green). The centrally located isoleucine 52 is in red. Black residues are buried and hence not visible on the structure.
- Fig 5
Norovirus shell domain with the conserved isoleucine is important for VP2 interaction in NV and HoV. (A) Western blot of VP2 from total cell lysates coexpressed with different NV VP1 proteins (the S domain, the P domain, full-length VP1 [wt], or full-length VP1 mutated at isoleucine 52 [I52A]). Numbers denote band quantitation using ImageJ. β-Actin served as a loading control. (B) Coimmunoprecipitation of HoV full-length VP1 (wt) or I48A mutant with FLAG-tagged VP2 in 293 cells. Coimmunoprecipitated complex was resolved with anti-FLAG antibody. Western blot of the input VP1 uses rabbit polyclonal specific for HoV VP1, and β-actin blot serves as loading control.
- Fig 6
Wild-type NV VP1 and I52 mutant dimerized and assembled into VLPs, but the latter did not incorporate VP2. (A) Electron micrographs and Western blots of VLPs produced from capsid proteins expressed in 293 cells and purified VLPs negatively stained and visualized by EM. Arrows indicate representative VLPs. (B) Purified VLPs were analyzed on Western blot using specific antibodies. Overexposure revealed no additional bands.
- Fig 7
Interaction footprint mapped in the interior of the NV particle. (A) Surface representation of the cross-section of the NV particle highlighting the shell domain (cyan) and the protruding domain (gray). Inset, VP1 dimer with isoleucine 52 in red. (B) The electrostatic potential of the dimeric VP1 shell domain. Negatively (red) and positively (blue) charged areas are contoured at ±57 kT/e. Arrows indicate the positions of isoleucine 52.
