Expression of human papillomavirus type 6 L1 and L2 isolated in China and self assembly of virus-like particles by the products.
To study variations of genome late region of human papillomavirus type 6 (HPV-6) isolated in China and assembling capabilities of the encoded capsid proteins, HPV-6 L1 and L2 sequences were cloned and used for expression in Bac-to-Bac baculovirus expression systems (Gibco BRL). Based upon L1 and L2 overlapping sequence two sequences (GenBank accession number AY015006, AY015008) of HPV-6 late region (2869 bp long) were assembled and classified into HPV-6b by phylogenetic analysis. Compared with prototype sequence, nine point mutations were found, including four missense mutations. L1, instead of L2, could self-assemble into virus-like particles (VLPs) in Sf9 nucleus. VLPs self-assembled by L1 alone (L1-VLPs) and by L1 plus L2 (L1+L2-VLPs) were purified and further characterized. Both types of VLPs were spherical particles with a diameter of approximately 50 nm. L1+L2 VLPs comprising L1 and L2 in the molar ratio of about 4:1 possessed the HPV-6 L1 VLP surface and conformational epitopes. In co-expression assay with a series of MOI combination of L1 and L2 recombinant baculoviruses (total MOI=10), existence of L2 of certain level enhanced L1 production by 0.8 fold and VLP production by 3 - 4 folds under experimental conditions. In conclusion, variation rate of HPV-6 genome late region is less than 0.28% and the substitutions A to G at position 7081 and G to A at 7099 may represent region characteristics. The cloned HPV-6 L1 and L2 sequences can be expressed efficiently in Sf9 cells, and the expressed products (L1 or L1+L2) can self-assemble into VLPs that resemble naturally occurring virions.
Wang
Miao
M
Institute of Basic Medical Sciences, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100005, China. wangmiao1973@yahoo.com
Wang
Li-Liang
LL
Chen
Lian-Feng
LF
Han
Ye-Hua
YH
Zou
Yu-Hong
YH
Si
Jing-Yi
JY
Song
Guo-Xing
GX
D000818:Animals; D015153:Blotting, Western; D002213:Capsid / Q000378:metabolism; D036022:Capsid Proteins; D002460:Cell Line; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D008854:Microscopy, Electron; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics* / Q000378:metabolism; D027383:Papillomaviridae / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D017422:Sequence Analysis, DNA; D014162:Transfection; D014764:Viral Proteins; D019065:Virus Assembly
D036022:Capsid Proteins; D004279:DNA, Viral; C082746:L2 oncogene viral capsid protein, Human papillomavirus type 6; D009856:Oncogene Proteins, Viral; D014764:Viral Proteins; C091560:L1 protein, Human papillomavirus type 6