MOLECULAR CLONING AND CHARACTERIZATION OF AN AMERICAN ISOLATE OF HUMAN T-CELL LEUKEMIA VIRUS TYPE 1

Abstract

All the studies that have been done to date on the regulation of gene expression for HTLV-I have been based on observations drawn from subgenomic clones of the virus. In an attempt to reproduce the events in the course of a natural infection, an intact HTLV-I provirus was molecularly cloned. The provirus originated from an HTLV-I-infected human T-cell line, CS-1. The CS-1 DNA was cloned in bacteriophage lambda and the clone was shown to contain the intact virus as well as 5kb and 4kb of 5' and 3' cellular flanking sequences, respectively. The CS-1 restriction map was generated and compared to maps of other isolates (showing similarity to the prototype Japanese isolate). The HTLV-I LTR was subcloned into a plasmid, sequenced and showed a variation of 1-3% in the DNA sequence compared to other HTLV-I isolates. The promoter sequences in the LTR, however, showed no variation. A cloning strategy was employed to subclone the intact provirus into a plasmid vector such that no cellular sequences would be present. Restriction analysis showed no gross alterations of the virus. Functionality of the cloned intact provirus was verified by its ability to produce viral mRNAs and proteins. HeLa cells transfected with the provirus clone were shown to be transcriptionally active by producing all the three viral mRNAs (genomic, the singly-spliced and the doubly-spliced messages), the levels of which were increased by addition of PMA to the transfected cells. Serum from an HTLV-I-infected ATL patient detected the viral structural proteins being produced by the provirus clone. Future studies with the intact HTLV-I clone will contribute to the establishment of a precise phylogenetic relationship of the viruses of this group and will clarify the pathways used by viral regulatory proteins and their involvement in virus infectivity.