CHARACTERIZATION OF HUMAN ENDOGENOUS RETROVIRUS TYPE-K VIRUS-LIKE PARTICLES GENERATED FROM RECOMBINANT BACULOVIRUSES

The family of human endogenous retrovirus type K (HERV-K) comprises me mbers with long open reading frames (ORF) for retroviral proteins. The existence of a biologically active provirus with replicative capaciti es has not yet been demonstrated. To confirm the assumption that HERV- K codes for the previously observed retrovirus-like particles (human t eratocarcinoma-derived virus, HTDV) in human teratocarcinoma cells, we have constructed recombinant full-length HERV-K cDNA-based baculoviru ses with gag, pro, pol, and env ORFs. Two viral constructs were used f or infections of insect cells, one bearing 67 bp of the 5' untranslate d region upstream of the 5' splice donor (SD) site and of the retrovir al genes, the second omitting the SD sequence. For both recombinant vi ruses, indirect immunofluorescence and laser scan analyses revealed ex pression of HERV-K Gag protein. Electron microscopy studies demonstrat ed efficient production of virus-like particles (VLPs) at the cytoplas mic cell membranes. These VLPs are morphologically identical with the HTDV phenotype. In immunoelectron microscopy of ultrathin frozen secti ons, anti-HERV-K Gag antibodies specifically reacted with HERV-K VLPs. In Western blots, in addition to the 76-kDa precursor protein, the pu tative major core protein with an apparent molecular mass of 32 kDa ex hibited predominant immunoreactivity with anti-Gag antiserum. In contr ast, neither HERV-K Env nor cORF proteins could be detected due to ine fficient mRNA splicing. Purified particles from insect cell culture su pernatants tested in an ultrasensitive reverse transcriptase assay rev ealed weak polymerase activity. The data demonstrate that HERV-K codes for retroviral particles of the HTDV phenotype. (C) 1997 Academic Pre ss.