Virus-like particles and E1-E4 protein expressed from the human papillomavirus type 11 bicistronic E1-E4-L1 transcript.
Detection of E1-E4 protein in human papillomavirus (HPV 11)-infected tissue is tightly linked to detection of L1 major capsid protein. The only L1-containing transcript identified in HPV 11-infected tissue is the bicistronic E1-E4-L1 mRNA, potentially encoding both the E1-E4 and the L1 proteins. It has not been established that these proteins can be expressed from the E1-E4-L1 transcript. The HPV 11 E1-E4-L1 sequence was cloned by reverse transcriptase polymerase chain reaction into the p1393 vector to produce recombinant baculoviruses. Immunoblots of recombinant baculovirus-infected Sf9 cell lysates demonstrated both the E1-E4 and the L1 proteins. An ELISA was performed on infected Sf9 cells using a monoclonal antibody specific for nondenatured L1, demonstrating that 10 ng of native L1 protein was present per microgram of total nuclear protein. Electron microscopic analysis revealed 50- to 60-nm icosahedral virus-like particles. In vitro transcription/translation was performed using pSPORT constructs containing the E1-E4-L1 sequence or, as controls, monocistronic pSPORT-E1-E4 or L1 constructs. The pSPORT-E1-E4-L1 construct produced the E1-E4 and L1 proteins at a ratio of 17:1. For E1-E4 protein, expression was greater from the pSPORT-E1-E4-L1 construct than from the monocistronic pSPORT-E1-E4 construct. In contrast, more L1 protein was expressed from pSPORT-L1 than from pSPORT-E1-E4-L1. A mutant E1-E4-L1 construct containing no E1-E4 start codon expressed L1 protein in amounts nearly equal to that expressed from the pSPORT-L1 construct. Addition of an antisense oligonucleotide directed at the E1-E4 start codon region to in vitro reactions using pSPORT-E1-E4-L1 was associated with inhibition of E1-E4 protein synthesis and increased translation of L1 protein.
Brown
D R
DR
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202, USA.
Pratt
L
L
Bryan
J T
JT
Fife
K H
KH
Jansen
K
K
D000818:Animals; D036022:Capsid Proteins; D002460:Cell Line; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D016376:Oligonucleotides, Antisense; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics*; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000032:analysis; D018411:Spodoptera / Q000166:cytology; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics*; D014771:Virion
D036022:Capsid Proteins; D004268:DNA-Binding Proteins; C084304:E1 protein, Human papillomavirus type 11; C094776:L1 protein, Human papillomavirus type 11; D016376:Oligonucleotides, Antisense; D009856:Oncogene Proteins, Viral; D012367:RNA, Viral; D014764:Viral Proteins; C067860:oncogene protein E4, type 11, Human papillomavirus