Construction of a full-length human T cell leukemia virus type I genome from MT-2 cells containing multiple defective proviruses using overlapping polymerase chain reaction.
Human T cell leukemia virus type I (HTLV-I), the etiological agent of adult T cell leukemia, integrates into the host genome as a provirus. Multiple defective copies of the integrated provirus are often present in the host genome. For this reason it is difficult to clone the intact provirus from HTLV-I-infected cells using conventional techniques. Here, we used overlapping polymerase chain reaction (PCR) to construct a full-length provirus of HTLV-I directly from an HTLV-I-transformed cell line, MT-2, which contains multiple defective proviruses. First, four overlapping proviral HTLV-I fragments (1.4-3.9 kb each) were constructed from genomic MT-2 DNA using PCR. Next, the complete HTLV-I proviral DNA (9 kb) was generated from these fragments using asymmetric PCR and cloned into a plasmid vector. 293 T cells transfected with this plasmid produced virus-like particles, and we show that these particles are capable of infecting a human T cell line. We propose that this cloning technique constitutes a powerful tool for constructing infectious molecular clones from cells of patients infected with HTLV-I or other viruses.
Ohsugi
Takeo
T
Division of Microbiology and Genetics, Center for Animal Resources and Development, Institute of Resource Development and Analysis, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan. ohsugi@gpo.kumamoto-u.ac.jp
Kumasaka
Toshio
T
Urano
Toru
T
D002461:Cell Line, Transformed; D016679:Genome, Viral; D023281:Genomics / Q000379:methods*; D015368:Human T-lymphotropic virus 1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008854:Microscopy, Electron; D016133:Polymerase Chain Reaction / Q000379:methods; D011533:Proviruses / Q000235:genetics*; D013601:T-Lymphocytes / Q000378:metabolism / Q000648:ultrastructure / Q000821:virology