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Journal of Virology, September 2001, p. 8461-8468, Vol. 75, No. 18
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.18.8461-8468.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Examining Human T-Lymphotropic Virus Type 1 Infection and
Replication by Cell-Free Infection with Recombinant Virus
Vectors
David
Derse,1,*
Shawn A.
Hill,1
Patricia A.
Lloyd,2
Hye-kyung
Chung,1 and
Barry A.
Morse1
Basic Research Laboratory, National Cancer Institute,
NCI-Frederick,1 and
SAIC-Frederick,2 Frederick, Maryland
21702
Received 4 May 2001/Accepted 19 June 2001
A sensitive and quantitative cell-free infection assay, utilizing
recombinant human T-cell leukemia virus type 1 (HTLV-1)-based vectors,
was developed in order to analyze early events in the virus replication
cycle. Previous difficulties with the low infectivity and restricted
expression of the virus have prevented a clear understanding of these
events. Virus stocks were generated by transfecting cells with three
plasmids: (i) a packaging plasmid encoding HTLV-1 structural and
regulatory proteins, (ii) an HTLV-1 transfer vector containing either
firefly luciferase or enhanced yellow fluorescent protein genes, and
(iii) an envelope expression plasmid. Single-round infections were
initiated by exposing target cells to filtered supernatants and
quantified by assaying for luciferase activity in cell extracts or by
enumerating transduced cells by flow cytometry. Transduction was
dependent on reverse transcription and integration of the recombinant
virus genome, as shown by the effects of the reverse transcriptase
inhibitor 3'-azido-3'-deoxythymidine (AZT) and by mutation of the
integrase gene in the packaging vector, respectively. The 50%
inhibitory concentration of AZT was determined to be 30 nM in this
HTLV-1 replication system. The stability of HTLV-1 particles,
pseudotyped with either vesicular stomatitis virus G protein or HTLV-1
envelope, was typical of retroviruses, exhibiting a half-life of
approximately 3.5 h at 37°C. The specific infectivity of
recombinant HTLV-1 virions was at least 3 orders of magnitude lower
than that of analogous HIV-1 particles, though both were pseudotyped
with the same envelope. Thus, the low infectivity of HTLV-1 is
determined in large part by properties of the core particle and by the
efficiency of postentry processes.
*
Corresponding author. Mailing address: Basic Research
Laboratory, Bldg. 567, NCI-Frederick, Frederick, MD 21702-1201. Phone: (301) 846-5611. Fax: (301) 846-6863. E-mail:
derse{at}ncifcrf.gov.
Journal of Virology, September 2001, p. 8461-8468, Vol. 75, No. 18
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.18.8461-8468.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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