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 been reported. The long wait for such provirus clones reflects the difficulties inherent in propagating HTLV-I in vitro, and thus a rigorous demonstration of infectivity has awaited improved cell culture systems and sensitive detection techniques for HTLV-I. An intact HTLV-I provirus, originating from an American ATL patient, was subcloned into a plasmid vector and was designated pCS-HTLV. Transient transfections of mammalian cells with pCS-HTLV resulted in the synthesis of viral proteins and mRNAs which were assembled into virions that had physical and morphological characteristics typical of HTLV-I particles. The ability of these virus particles to infect cells, replicate, and produce infectious progeny was demonstrated initially in short term, cell-free infection assays by monitoring the expression of specific viral mRNAs. These studies have been extended in cell culture systems that support continuous virus production. Primary T-lymphocytes have been infected either 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. Furthermore, fetal rhesus lung cells (FRhL) were shown to be permissive for HTLV-I replication and sustained virus expression after infection with pCS-HTLV. Continuous FRhL cell lines now have been established that express various HTLV-I proviruses and mutants. These provirus clones and cell lines provide us with the means to address long-standing questions dealing with the biology of HTLV-I.
Derse
D
D
Laboratory of Leukocyte Biology, National Cancer Institute, Frederick, Maryland 21702-1201, USA.
Mikovits
J
J
Waters
D
D
Brining
S
S
Ruscetti
F
F
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018920:Coculture Techniques; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D015368:Human T-lymphotropic virus 1 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008168:Lung / Q000166:cytology / Q000821:virology; D008253:Macaca mulatta; D008967:Molecular Biology; D011533:Proviruses / Q000235:genetics / Q000502:physiology; D013601:T-Lymphocytes / Q000166:cytology / Q000821:virology; D014779:Virus Replication / Q000502:physiology