Genome-wide screening, cloning, chromosomal assignment, and expression of full-length human endogenous retrovirus type K

The human genome harbors 25 to 50 proviral copies of the endogenous retrovi rus type K (HERV-K), some of which code for the characteristic retroviral p roteins Gag, Pol, and Env. For a genome-wide cloning approach of full-lengt h and intact HERV-K proviruses, a human P1 gene library was screened with a gag-specific probe. Both HERV-K type 1 and 2 clones were Isolated. Sixteen HERV-K type 2 proviral genomes were characterized by direct coupled in vit ro transcription-in vitro translation assays to analyze the coding potentia l of isolated gag, pal, and env amplicons from individual P1 clones. After determination of long terminal repeat (LTR) sequences and adjacent chromoso mal integration sites by inverse PCR techniques, two HERV-K type 2 provirus es displaying long retroviral open reading frames (ORFs) were assigned to c hromosomes 7 (C7) and 19 (C19) by using a human-rodent monochromosomal cell hybrid mapping panel. HERV-K(C7) shows an altered (YIDD-to-CIDD) motif in the reverse transcriptase domain. HERV-K(C19) is truncated in the 5' LTR an d harbors a defective protease gene due to a point mutation. Direct amplifi cation of proviral structures from single chromosomes by using chromosomal flanking primers was performed by long PCR for HERV-K(C7) and HERV-K(C19) a nd for type 1 proviruses HERV-K10 and HERV-K18 from chromosomes 5 and 1, re spectively. HERV-K18, in contrast to HERV-K10, bears no intact gag ORF and shows close homology to HERV-K/IDDMK(1,2)22. In transfection experiments, H ERV-K(C7) and HERV-K cDNA-based expression vectors yielded the proteins Gag and cORF whereas HERV-K10 vectors yielded Gag alone. The data suggest that the human genome does not contain an entire, intact proviral copy of HERV- K.