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-trans formed 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 (9kb) was generated from these fragments using asymmetric PCR and cloned into a plasmid vector. 293T 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. (C) 2004 Elsevier Inc. All rights reserved.