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at both 5' and 3' ends. This double-stranded DNA is integrated into the host
chromosome, catalyzed by the viral integrase. Once DNA is integrated, new
viral RNA synthesis is carried out by the host-cell RNA polymerase, produc-
ing a large number of viral RNA molecule. These RNAs are translated to pro-
duce the capsid, envelope, and reverse transcriptase proteins. Initiation of viral
assembly begins by binding of capsid proteins to the RNA packaging signal
(
sequence) of the viral RNA. Mature viral particles bud out from the host
cells containing two copies of RNA genome, capsid, envelope, and reverse
transcriptase proteins.
The single most important advance in the development of retroviruses as
gene-transfer vectors was achieved by complementation of two systems, pack-
aging cells and retroviral vectors
(2)
. A specialized cell line (termed “packag-
ing cells”) was generated in order to permit the production of high titers of
replication-defective recombinant virus, free of wild-type virus (
Fig. 1
). For
this purpose, the structural genes necessary for particle formation and replica-
tion,
gag
,
pol
, and
env
, are integrated into cell lines without the RNA packag-
ing signal
ψ
sequence. Thus, viral RNA in packaging cells provide proteins
necessary for particle formation but cannot be packaged into a viral particle.
The other component is a retroviral vector in which most structural genes have
been deleted for insertion of the gene of interest. The retroviral vector contains
the
ψ
sequence for RNA packaging and two LTRs that provide transcription,
polyadenylation of viral RNA, and integration of double-stranded viral genome
(
Fig. 2
). When the retroviral vector is transfected into the packaging cells, the
viral RNA containing the gene of interest is transcribed and packaged into
viral particle by gag, pol, and env proteins provided by the packaging cells.
This recombinant virus is utilized to infect the target cells, delivering a gene of
interest at high efficiency. In principle, because no genetic information for virus
production is transferred from the packaging cells, transduced cells are unable
to perpetuate an infection and spread virus to other cells.
Many different packaging cells are available currently:
NIH3T3
based pack-
aging cells, (
PA317
and
PT67
), and 293 based packaging cells, (
BOSC23
and
Φ
ψ
NX
(2-5)
. The viral env protein expressed by the packaging cell line deter-
mines the cellular host range of the packaged virus and allows infection of
different cell types through recognition of specific cellular receptors. The
PT67
packaging cells, a
NIH3T3
based line expressing the
10A1
viral envelope, can
enter cells via two different surface molecules, the amphotropic retrovirus and
the gibbon ape leukemia virus receptors
(3)
. Thus, they exhibit a broader host
range than other packaging cells. Among many packaging cells, we found
NX
cells to be most convenient for a small-scale generation of a high-titer recom-
binant virus. These cells can be obtained from ATCC (Rockville, MD) upon
permission from Dr. Nolan (http://www/leland.stanford.edu/group/nolan/
Φ
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