Human papillomavirus type 18 E6*, E6, and E7 protein synthesis in cell-free translation systems and comparison of E6 and E7 in vitro translation products to proteins immunoprecipitated from human epithelial cells.
Expression of the E6 and E7 transforming genes of human papillomavirus type 18 (HPV18) occurs via structurally bicistronic mRNAs in which the downstream open reading frame (ORF) E7 is preceded either by the full-length ORF E6 or by a spliced ORF, E6*. We have used in vitro transcription and translation of HPV18 cDNAs in order to analyze the synthesis of E6*, E6, and E7 proteins and to compare the E6 and E7 in vitro translation products with the authentic proteins immunoprecipitated from cervical cancer cells. In wheat germ extract, in vitro translation resulted in the production of all three proteins, E6*, E6, and E7. In rabbit reticulocyte lysate, however, only the E6 and E7 proteins were produced. The lack of E6* protein was due neither to template RNA degradation nor to an inhibitory influence of the RNA 5' leader sequences, thus indicating the possibility of either inhibition of synthesis or degradation of E6* protein in reticulocyte lysate. The E7 protein was synthesized from both E6*-E7 and E6-E7 RNAs. In vitro-synthesized and authentic HPV18 E7 proteins revealed identical electrophoretic mobilities in two-dimensional gel electrophoresis, thus indicating similar modifications. By using a monoclonal antibody against the N terminus of HPV18 E6* and E6, an 18-kDa protein was detected not only in HPV18-positive but also in HPV18-negative epithelial cells. The 18-kDa proteins and the in vitro-synthesized HPV18 E6 protein exhibited comparable electrophoretic characteristics in two-dimensional gels. These results suggest the possible existence of a cellular protein related to HPV18 E6.
Roggenbuck
B
B
Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Larsen
P M
PM
Fey
S J
SJ
Bartsch
D
D
Gissmann
L
L
Schwarz
E
E
D002474:Cell-Free System; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D015180:Electrophoresis, Gel, Two-Dimensional; D004848:Epithelium / Q000382:microbiology; D006801:Humans; D066298:In Vitro Techniques; D007526:Isoelectric Point; D008970:Molecular Weight; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D027383:Papillomaviridae / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D012326:RNA Splicing; D011994:Recombinant Proteins
D004268:DNA-Binding Proteins; C052603:E6 protein, Human papillomavirus type 18; C052633:E7 protein, Human papillomavirus type 18; D009856:Oncogene Proteins, Viral; D011994:Recombinant Proteins