Journal of Virology, November 1998, p. 8873-8883, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Molecular and Medical Genetics Section,
Received 2 April 1998/Accepted 14 July 1998
Previously we designed novel pseudotyped high-titer
replication defective human immunodeficiency virus type 1 (HIV-1)
vectors to deliver genes into nondividing cells (J. Reiser, G. Harmison, S. Kluepfel-Stahl, R. O. Brady, S. Karlsson, and M. Schubert, Proc. Natl. Acad. Sci. USA 93:15266-15271, 1996). Since then
we have made several improvements with respect to the safety,
flexibility, and efficiency of the vector system. A three-plasmid
expression system is used to generate pseudotyped HIV-1 particles by
transient transfection of human embryonic kidney 293T cells with a
defective packaging construct, a plasmid coding for a heterologous
envelope (Env) protein, and a vector construct harboring a reporter
gene such as neo, ShlacZ (encoding a phleomycin
resistance/
-galactosidase fusion protein), HSA (encoding
mouse heat-stable antigen), or EGFP (encoding enhanced
green fluorescent protein). The packaging constructs lack functional
Vif, Vpr, and Vpu proteins and/or a large portion of the Env coding
region as well as the 5' and 3' long terminal repeats, the Nef
function, and the presumed packaging signal. Using G418 selection, we
routinely obtained vector particles pseudotyped with the vesicular
stomatitis virus G glycoprotein (VSV-G) with titers of up to 8 × 107 CFU/µg of p24, provided that a functional Tat coding
region was present in the vector. Vector constructs lacking a
functional Tat protein yielded titers of around 4 × 106 to 8 × 106 CFU/µg of p24. Packaging
constructs with a mutation within the integrase (IN) core domain
profoundly affected colony formation and expression of the reporter
genes, indicating that a functional IN protein is required for
efficient transduction. We explored the abilities of other Env proteins
to allow formation of pseudotyped HIV-1 particles. The rabies virus and
Mokola virus G proteins yielded high-titer infectious pseudotypes,
while the human foamy virus Env protein did not. Using the improved
vector system, we successfully transduced contact-inhibited primary
human skin fibroblasts and postmitotic rat cerebellar neurons and
cardiac myocytes, a process not affected by the lack of the accessory
proteins.
*
Corresponding author. National Institutes of Health,
Building 10, Room 3D04, 10 Center Dr., MSC 1260, Bethesda, MD
20892-1260. Phone: (301) 594-3129. Fax: (301) 496-9480. E-mail:
jreiser{at}helix.nih.gov.
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