EXAMINING THE MOLECULAR-GENETICS OF HTLV-I WITH AN INFECTIOUS MOLECULAR CLONE OF THE VIRUS AND PERMISSIVE CELL-CULTURE SYSTEMS

Infectious molecular clones of HTLV-I proviruses have only recently be en reported. The long wait for such provirus clones reflects the diffi culties inherent in propagating HTLV-I in vitro, and thus a rigorous d emonstration of infectivity has awaited improved cell culture systems and sensitive detection techniques for HTLV-I. An intact HTLV-I provir us, originating from an American ATL patient, was subcloned into a pla smid vector and was designated pCS-HTLV. Transient transfections of ma mmalian cells with pCS-HTLV resulted in the synthesis of viral protein s and mRNAs which were assembled into virions that had physical and mo rphological characteristics typical of HTLV-I particles. The ability o f these virus particles to infect cells, replicate, and produce infect ious progeny was demonstrated initially in short term, cell-free infec tion assays by monitoring the expression of specific viral mRNAs. Thes e studies have been extended in cell culture systems that support cont inuous virus production. Primary T-lymphocytes have been infected eith er with cell-free supernatant fluids from, or by coculture with, cells transiently transfected with pCS-HTLV, giving rise to continuous, IL- 2-dependent cell lines that have been in culture for >1 year. Furtherm ore, fetal rhesus lung cells (FRhL) were shown to be permissive for HT LV-I replication and sustained virus expression after infection with p CS-HTLV. Continuous FRhL cell lines now have been established that exp ress various HTLV-I proviruses and mutants. These provirus clones and cell lines provide us with the means to address long-standing question s dealing with the biology of HTLV-I.