BIOLOGICAL-ACTIVITIES OF A PUTATIVE TRUNCATED HEPATITIS-B VIRUS-X GENE-PRODUCT FUSED TO A POLYLYSIN STRETCH

Truncated transcripts terminating within the HBx frame have been recog nized previously in tumor and liver tissue of HCC patients. In this st udy biological activities of a predicted truncated HBx fused to a poly sin stretch (HBtx-polylysin) and of full length HBx were compared in N IH3T3 cells transfected with respective cDNA plasmids. Transactivation of a co-transfecting reporter gene and influence on neo' DNA mediated transformation to G418 resistance were determined. In comparison to f ull length HBx the data indicate for HBtx-polylysin a lower transactiv ating capacity and as judged by the yield of colonies on a solid surfa ce, a lower capacity to stimulate neo(r) DNA mediated transformation. In soft agar the outgrowth into G418 resistant colonies was dependent on co-transfecting HBx cDNA. In providing this condition HBtx-polylysi n had a much higher relative activity than full length HBx. Large coin tegrants consisting of the plasmids carrying truncated HBx cDNA and ne o(r) DNA respectively were identified by chromosomal in situ hybridiza tion. Based on Southern blot analyses extended concatemeres of the HBx cDNA plasmid constituted a main part of the cointegrants. Expression of truncated HBx cDNA was followed on the RNA and the protein level. T he presence of this cDNA could be correlated to a compact spindle like cell appearance, its loss after prolonged passaging in the absence of G418 to a concomitant reversion to the phenotype of the NIH3T3 cell. Interspersed selection for G418 resistance stabilized the morphologica lly transformed phenotype. These results provide a basis to manipulate expression of truncated HBx and to recognize thereby processes leadin g to transformation.