Cloning, expression, and immunogenicity of the assembly protein of varicella-zoster virus and detection of the products of open reading frame 33.
Herpesviruses produce assembly proteins (AP) that act as scaffolding proteins for the assembly of the viral capsids. The products of the assemblin gene, which encodes both maturational protease and AP, have been established for herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (CMV). We cloned an inframe ORF (encoding amino acids 304-605), found within the ORF 33 assemblin gene of VZV, into a yeast expression vector. The 34-kDa AP was expressed as a fusion protein with the particle-forming Ty p1 protein, resulting in high-level production of hybrid AP-virus-like particles (AP-VLPs). When AP-VLPs were injected into mice and rabbits, antibodies were produced that reacted with, but that did not neutralise, native VZV. Three of four inbred strains of mice immunised with AP-VLPs produced a VZV-specific T-cell response. The mouse and rabbit sera reacted with six bands on native VZV by Western blot analysis. The dominant bands were found at 34 and 38 kDa. Bands were also seen at 66, 63, 41, and 31 kDa. The 38-kDa protein may represent the mature AP derived from the 41-kDa precursor AP, itself the release product from the full-length 66-kDa assemblin. The 34-kDa protein probably represents the product of the inframe co-translational gene within ORF 33 encoding amino acids 304-605. The genetic organisation and proteolytic maturation of VZV assemblin are, therefore, analogous to those of other herpesviruses.
Garcia-Valcarcel
M
M
British Biotech Pharmaceuticals Ltd, Oxford, England.
Fowler
W J
WJ
Harper
D R
DR
Jeffries
D J
DJ
Layton
G T
GT
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000032:analysis / Q000235:genetics*; D005260:Female; D014645:Herpesvirus 3, Human / Q000235:genetics* / Q000276:immunology / Q000502:physiology*; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016366:Open Reading Frames; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000276:immunology; D012697:Serine Endopeptidases; D014764:Viral Proteins / Q000235:genetics* / Q000276:immunology*; D019065:Virus Assembly
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; C071910:assemblin