An E1M∧E2C Fusion Protein Encoded by Human Papillomavirus Type 11 Is a Sequence-Specific Transcription Repressor

Cheng Ming Chiang, Thomas R. Broker, Louise T. Chow

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

We have isolated a putative, spliced E5 cDNA of human papillomavirus type 11 (HPV-11) by polymerase chain reaction amplification of cDNAs from an experimental condyloma. Using retrovirus-mediated gene transfer, we isolated two novel HPV-11 cDNAs, one of which had a splice linking nucleotides 1272 and 3377. This transcript also existed in experimental condylomata and in cervical carcinoma cells transfected with cloned genomic HPV-11 DNAs. The 5′ end of the transcript in transfected cells originated upstream of the initiation codon of the E1 open reading frame (ORF). It could conceptually encode a fusion protein consisting of the amino-terminal 23% of the E1 ORF and the carboxy-terminal 40% of the E2 ORF. This E1M∧E2C fusion protein contained both the DNA replication modulator domain E1M, as defined in the bovine papillomavirus system, and the DNA binding domain of the E2 protein, which regulates viral transcriptional activities. Indirect immunofluorescence with polyclonal antibodies raised against the bacterially expressed TrpE-HPV-11 E2 protein demonstrated nuclear localization of the E1M∧E2C protein in cells transiently transfected with an expression plasmid. Immunoprecipitation revealed a specific protein with an apparent molecular weight of 42,000 in transfected cells. The chloramphenicol acetyltransferase assay established that the putative E1M∧E2C protein was a potent transcriptional repressor of both E2-dependent and E2-independent HPV-11 enhancer/ promoter activities. Northern (RNA) blot hybridization indicated the repression was on the transcriptional level. Mutational analysis suggested that the E1M∧E2C protein is an E2-binding site-specific repressor. The fusion protein also repressed bovine papillomavirus type 1 (BPV-1) E2 protein-dependent BPV-1 enhancer activity. When constitutively expressed in mouse C127 cells, the E1M∧E2C protein inhibited BPV-1 transformation and episomal DNA replication, consistent with a role in the modulation of replication.

Original languageEnglish (US)
Pages (from-to)3317-3329
Number of pages13
JournalJournal of Virology
Volume65
Issue number6
StatePublished - Jun 1991

Fingerprint

Human papillomavirus 11
Papillomaviridae
transcription (genetics)
Bovine papillomavirus 1
Proteins
proteins
Open Reading Frames
Complementary DNA
open reading frames
DNA Replication
DNA replication
cells
Retroviridae
Chloramphenicol O-Acetyltransferase
Bovine papillomavirus
Initiator Codon
DNA
chloramphenicol acetyltransferase
Indirect Fluorescent Antibody Technique
start codon

ASJC Scopus subject areas

  • Immunology

Cite this

An E1M∧E2C Fusion Protein Encoded by Human Papillomavirus Type 11 Is a Sequence-Specific Transcription Repressor. / Chiang, Cheng Ming; Broker, Thomas R.; Chow, Louise T.

In: Journal of Virology, Vol. 65, No. 6, 06.1991, p. 3317-3329.

Research output: Contribution to journalArticle

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abstract = "We have isolated a putative, spliced E5 cDNA of human papillomavirus type 11 (HPV-11) by polymerase chain reaction amplification of cDNAs from an experimental condyloma. Using retrovirus-mediated gene transfer, we isolated two novel HPV-11 cDNAs, one of which had a splice linking nucleotides 1272 and 3377. This transcript also existed in experimental condylomata and in cervical carcinoma cells transfected with cloned genomic HPV-11 DNAs. The 5′ end of the transcript in transfected cells originated upstream of the initiation codon of the E1 open reading frame (ORF). It could conceptually encode a fusion protein consisting of the amino-terminal 23{\%} of the E1 ORF and the carboxy-terminal 40{\%} of the E2 ORF. This E1M∧E2C fusion protein contained both the DNA replication modulator domain E1M, as defined in the bovine papillomavirus system, and the DNA binding domain of the E2 protein, which regulates viral transcriptional activities. Indirect immunofluorescence with polyclonal antibodies raised against the bacterially expressed TrpE-HPV-11 E2 protein demonstrated nuclear localization of the E1M∧E2C protein in cells transiently transfected with an expression plasmid. Immunoprecipitation revealed a specific protein with an apparent molecular weight of 42,000 in transfected cells. The chloramphenicol acetyltransferase assay established that the putative E1M∧E2C protein was a potent transcriptional repressor of both E2-dependent and E2-independent HPV-11 enhancer/ promoter activities. Northern (RNA) blot hybridization indicated the repression was on the transcriptional level. Mutational analysis suggested that the E1M∧E2C protein is an E2-binding site-specific repressor. The fusion protein also repressed bovine papillomavirus type 1 (BPV-1) E2 protein-dependent BPV-1 enhancer activity. When constitutively expressed in mouse C127 cells, the E1M∧E2C protein inhibited BPV-1 transformation and episomal DNA replication, consistent with a role in the modulation of replication.",
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