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 F1^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 Si9 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. (C) 1996 Academic Press, Inc.