Analysis of the L1 gene product of human papillomavirus type 16 by expression in a vaccinia virus recombinant.
The L1 open reading frame of human papillomavirus type 16 (HPV16) has been expressed in vaccinia virus under the control of both the 7.5K early and late promoter, and the 4b major late promoter. Antibodies to a beta-galactosidase fusion protein containing a C-terminal portion of the HPV16 L1 gene product were used to compare the levels of L1 expression in the two recombinants, and showed that greater levels of expression were obtained when the gene was placed under the control of the 4b late promoter. Immunofluorescence studies revealed a nuclear location of the L1 gene product when expressed in vaccinia virus. Antibodies to the beta-galactosidase fusion protein detected a major polypeptide species of 57K and a minor species of 64K in Western blots of recombinant-infected cell lysates. The 64K species was not detected when cells were infected in the presence of tunicamycin, indicating that the primary translation product of the HPV16 L1 open reading frame is modified by N-linked glycosylation when expressed in vaccinia virus. Whereas antibodies to HPV16 L1 fusion proteins and to a peptide containing amino acids from the C terminus of HPV16 L1 reacted well in Western blots with the HPV16 L1 target expressed in vaccinia virus, no reactivity was observed with antibodies to bovine papillomavirus type 1 particles or to a HPV6b fusion protein.
Browne
H M
HM
Department of Pathology, University of Cambridge, U.K.
Churcher
M J
MJ
Stanley
M A
MA
Smith
G L
GL
Minson
A C
AC
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D006801:Humans; D007118:Immunoassay; D009693:Nucleic Acid Hybridization; D027383:Papillomaviridae / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D014616:Vaccinia virus / Q000235:genetics; D014764:Viral Proteins / Q000032:analysis / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D001616:beta-Galactosidase