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 members with long open reading frames (ORF) for retroviral proteins. The existence of a biologically active provirus with replicative capacities has not yet been demonstrated. To confirm the assumption that HERV-K codes for the previously observed retrovirus-like particles (human teratocarcinoma-derived virus, HTDV) in human teratocarcinoma cells, we have constructed recombinant full-length HERV-K cDNA-based baculoviruses with gag, pro, pol, and env ORFs. Two viral constructs were used for infections of insect cells, one bearing 67 bp of the 5' untranslated region upstream of the 5' splice donor (SD) site and of the retroviral genes, the second omitting the SD sequence. For both recombinant viruses, indirect immunofluorescence and laser scan analyses revealed expression of HERV-K Gag protein. Electron microscopy studies demonstrated efficient production of virus-like particles (VLPs) at the cytoplasmic cell membranes. These VLPs are morphologically identical with the HTDV phenotype. In immunoelectron microscopy of ultrathin frozen sections, anti-HERV-K Gag antibodies specifically reacted with HERV-K VLPs. In Western blots, in addition to the 76-kDa precursor protein, the putative major core protein with an apparent molecular mass of 32 kDa exhibited predominant immunoreactivity with anti-Gag antiserum. In contrast, neither HERV-K Env nor cORF proteins could be detected due to inefficient mRNA splicing. Purified particles from insect cell culture supernatants tested in an ultrasensitive reverse transcriptase assay revealed weak polymerase activity. The data demonstrate that HERV-K codes for retroviral particles of the HTDV phenotype.
Tönjes
R R
RR
Paul-Ehrlich-Institut, Langen, Germany. Toenjes@em.uni-frankfurt.de
Boller
K
K
Limbach
C
C
Lugert
R
R
Kurth
R
R
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D011995:Recombination, Genetic; D012190:Retroviridae / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D018411:Spodoptera / Q000166:cytology; D014407:Tumor Cells, Cultured; D014771:Virion / Q000648:ultrastructure; D019065:Virus Assembly
D004279:DNA, Viral; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase