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Examining the molecular genetics of HTLV-I with an infectious molecular clone of the virus and permissive cell culture systems.

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1
Laboratory of Leukocyte Biology, National Cancer Institute, Frederick, Maryland 21702-1201, USA.

Abstract

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.

[Indexed for MEDLINE]

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