Characterization of human papillomavirus type 16 E2 protein and subdomains expressed in insect cells.
The E2 open reading frame of human papillomavirus type 16 (HPV-16) encodes a DNA-binding protein which modulates papillomavirus transcription and replication. To investigate the biological and biochemical properties of the HPV-16 E2 protein, we have constructed recombinant baculoviruses which express the full-length molecule and individual N- and C-terminal domains in Sf21 insect cells. In this system the full-length E2 protein was phosphorylated and targeted to the insect cell nucleus. A 93 amino acid C-terminal fragment encompassing the DNA binding and dimerization functions of E2 was also translocated to the nucleus but was not modified by phosphorylation. The E2 N-terminal protein accumulated in the insect cell cytoplasm but was not efficiently phosphorylated. The formation of heterodimers between full-length and N-terminally truncated E2 species was observed when Sf21 cells were co-infected with recombinant viruses and when homodimers were mixed in vitro, suggesting that the dimer interface is not sufficiently stable to prevent subunit exchange in vivo. Both homo- and heterodimeric E2 species were able to bind specifically and in any combination to tandem E2 binding sites from the HPV-16 regulatory region. Furthermore, the HPV-16 E2 protein bound to DNA exhibited a distinct susceptibility profile to pronase digestion, potentially contrasting with that reported for BPV-1 E2. These observations suggest that significant structural and functional differences may exist between the BPV/HPV E2 proteins and have implications for understanding E2-dependent regulation of transcription and replication.
Sanders
C M
CM
Department of Biology, University of York, Heslington, United Kingdom.
Stern
P L
PL
Maitland
N J
NJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics; D009856:Oncogene Proteins, Viral / Q000235:genetics* / Q000378:metabolism; D027383:Papillomaviridae / Q000378:metabolism*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
D017931:DNA Primers; D004268:DNA-Binding Proteins; C054855:E2 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D011994:Recombinant Proteins; D010450:Endopeptidases