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Previous Article | Next Article 
Journal of Virology, July 1999, p. 6114-6116, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Retroviral Vectors Pseudotyped with Lymphocytic
Choriomeningitis Virus
Hrvoje
Miletic,1
Michael
Bruns,1
Konstantinos
Tsiakas,1
Birgit
Vogt,1
Roya
Rezai,1
Christopher
Baum,1
Klaus
Kühlke,2
François-Loïc
Cosset,3
Wolfram
Ostertag,1
Heinz
Lother,1 and
Dorothee
von Laer1,*
Heinrich-Pette-Institut für
Experimentelle Virologie und Immunologie an der Universität
Hamburg, 20251 Hamburg,1 and Eufets,
55743 Idar-Oberstein,2 Germany, and
Centre de Génétique Moléculaire et
Cellulaire, Centre National de la Recherche Scientifique, UMR5534,
UCB Lyon-1, 69622 Villeurbanne Cedex,
France3
Received 9 December 1998/Accepted 16 March 1999
 |
ABSTRACT |
Pseudotyping can improve retroviral vector stability and
transduction efficiency. Here, we describe a novel pseudotype of murine
leukemia virus packaged with lymphocytic choriomeningitis virus (LCMV).
This pseudotype was stable during ultracentrifugation and infected
several cell lines from different species. Moreover, LCMV glycoproteins
were not cell toxic.
| |
TEXT |
Low titers limit in vivo
applications of conventional amphotropic retroviral vectors. Virus
particles cannot be concentrated to overcome this problem because of
the instability of the retroviral envelope (22). In
addition, transduction of some cell types, such as hematopoietic
progenitors, has been inefficient (1, 11, 24, 28). Both
vector stability and host range have been improved by packaging murine
leukemia virus (MLV) vectors with the G protein of vesicular stomatitis
virus (VSV) to generate hybrid virions called MLV(VSV) pseudotypes
(13, 28, 29). However, a major drawback of these packaging
systems is that the VSV G protein is cell toxic (7, 9, 17,
31).
Here we describe a retroviral vector pseudotyped with the glycoproteins
of an arenavirus, the lymphocytic choriomeningitis virus (LCMV). LCMV
glycoproteins are synthesized as a 74-kDa precursor protein, GP-C, and
then cleaved into a 35-kDa transmembrane protein, GP-2, and an external
44-kDa protein, GP-1. In contrast to VSV G, LCMV glycoproteins are not
cell toxic (25).
Initially we tested whether LCMV could rescue a retroviral vector. The
env-negative packaging cell line TELCeB was infected with
the LCMV WE strain. TELCeB cells are derived from the human fibroblast
cell line Te671 and contain gag and pol genes as
well as a retroviral lacZ vector (10). These
cells lack viral envelope proteins and do not produce infectious
retrovirus unless an appropriate glycoprotein is provided. After
infection with LCMV, levels of production of wild-type LCMV and rescued
retrovirus were measured daily and expressed as PFU and
LacZ-transferring units (LTU), respectively. The titration assays have
been described previously (16, 18). Additionally, production
of LCMV glycoproteins in infected TELCeB cells was monitored by flow
cytometric analysis with a mouse monoclonal antibody against LCMV GP-1
(6). The results of a representative experiment are shown in
Fig. 1. Retroviral vector was produced
for 6 days, with the maximum level of production of 5 × 104 LTU per ml, together with the highest expression of
LCMV glycoprotein, occurring on day 3. The highest titer for wild-type
LCMV was 3 × 108 per ml on day 2. No cytopathic
effect was observed during replication of LCMV in the packaging cell
line, although high levels of LCMV glycoproteins were expressed.
Supernatants were then incubated with a neutralizing anti-LCMV gp44
monoclonal antibody (in mouse ascites fluid, 1:100) for 1 h
(6). This led to a more than 3-log-unit reduction in vector
titer. As a control, the amphotropic pseudotype of the same retroviral
vector was used and was not neutralized by the anti-LCMV antibody (data
not shown). These data show that the retroviral vector indeed carried
on its surface LCMV glycoproteins that mediate cell entry in the
absence of retroviral envelope proteins.
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FIG. 1.
Rescue of the retroviral vector MFGlnsLacZ by LCMV. The
retroviral env-negative packaging cell line TELCeB was
infected with LCMV at a multiplicity of 0.01. Between days 1 and 7 after infection, supernatants were replaced daily and the titers of
wild-type LCMV and of the LacZ vector were determined by a plaque assay
on L-929 cells and by measurement of lacZ gene transfer to
Sc-1 cells, respectively. Additionally, a portion of the LCMV-infected
TECLeB cells was stained daily with a monoclonal antibody to the LCMV
glycoprotein GP-1 and analyzed by flow cytometry (FACScalibur; Becton
Dickinson, Heidelberg, Germany). The mean fluorescence is shown.  ,
titer of wild-type LCMV in PFU per ml;  , LTU per ml;  , mean
fluorescence of LCMV glycoprotein GP expression.
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|
We then analyzed whether the LCMV helper function required retroviral
gag and pol. 293 cells and 293gp2 cells, the
latter containing MLV gag and pol, were
transfected with an MLV-based retroviral vector containing the
neo gene (MP1N) and were selected with G418 (12,
28). The resulting cell lines, 293MP1N and 293gp2MP1N, were then
infected with LCMV. 293gpMP1N produced infectious retroviral progeny
(3 × 103 G418-resistant transfer units [GTU] per
ml; for the method of titration, see reference 28),
but no vector could be detected in the supernatant of LCMV-infected
293MP1N cells, which did not contain retroviral gag or
pol (detection limit, 1 GTU per ml). As a control, the cells
were also infected with a replication-competent amphotropic helper
(23). An infectious vector that transferred neo
resistance was recovered from both cells lines at titers of 1 × 104 and 6 × 104 per ml for 293MP1N and
293gp2MP1N, respectively. Thus, as expected for a classical retroviral
pseudotype, retroviral genomic RNA was not packaged by LCMV into
infectious virions in the absence of gag and pol
gene products.
To verify that MLV(LCMV) pseudotypes indeed mediated stable
transduction with integration of the transgene into the target cell
genome, DNA of 12 G418-resistant clones was subjected to Southern blot
analysis after restriction with HindIII, a single cutter, and a neo probe (26). In 10 clones, one
copy of the integrated retroviral vector genome per cell was detected,
and in the remaining 2 clones, two copies were detected (data not shown). Transduction with the MLV(LCMV) pseudotype thus led to stable
integration of the transgene, which also supports the conclusion that
the MLV(LCMV) pseudotype indeed contained a functional retroviral core.
However, the hybrid vector particles were produced by LCMV infection of
env-negative packaging cell lines, in which all LCMV proteins were expressed. Therefore, we could not completely exclude the
possibility that, in addition to the glycoproteins, further LCMV
proteins are required for efficient vector production. This issue is
important for the generation of helper-free packaging systems and is
currently addressed by expressing isolated LCMV genes in
env-negative packaging cells.
Amphotropic retroviruses lose infectivity upon ultracentrifugation,
most likely because of the lability of the retroviral envelope
glycoproteins (22). We tested whether the MLV(LCMV) pseudotypes are more stable. TELCeB cells were infected with LCMV or with amphotropic helper virus. Equal amounts of both virus progeny
were pelleted and purified by ultracentrifugation through a 0 to 40%
Urografin gradient as described previously (19). Titers of a
representative gradient (in LTU per ml) are shown in Fig.
2. LCMV pseudotypes were recovered
without loss of infectivity, in contrast to a >3-log-unit loss of
infectious amphotropic virus. The reverse transcriptase activities in
the virion peaks showed that the amounts of virus particles harvested
from the gradient were similar for both pseudotypes (data not shown).
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FIG. 2.
MLV(LCMV) pseudotypes retain infectivity upon
ultracentrifugation. TELCeB cells were infected with LCMV or
amphotropic helper virus. Supernatants were harvested and frozen. The
MLV(LCMV) and amphotropic pseudotype titers were determined. Equal
quantities of the infectious vector were pelleted by
ultracentrifugation and then subjected to purification on a 0 to 40%
Urografin gradient. Vector titers and densities in each fraction were
determined.  , amphotropic pseudotype;  , MLV(LCMV) pseudotype.
|
|
LCMV pseudotypes were also stable when they were stored at 4°C.
Within the observation period of 3 days, the loss of titer was less
than twofold. One cycle of freezing (
80°C) and thawing led to a
twofold reduction in pseudotype titer.
The tropism of MLV(LCMV) pseudotypes was analyzed. Vector titers on
different cell lines relative to the titer on the mouse fibroblast line
Sc-1 are shown in Table 1. The human
hematopoietic progenitor cell lines K562 and TF-1, the human hepatoma
cell line HUH-7, and the human glioma cell line nce-G112 could be
efficiently infected (14, 30). All these lines are derived
from cell types that are relevant targets in gene therapy. Moreover,
canine thymus cells (Cf2Th) and hamster cells, the latter of which are
normally resistant to transduction with MLV-derived vectors, were also highly susceptible to the MLV(LCMV) pseudotypes.
Wild-type LCMV has been shown to infect several cell types from
different tissues and species (2, 3, 5, 15, 20, 27).
Alpha-dystroglycan, which is widely expressed in different tissues, was
recently found to be a receptor for LCMV (4, 8). It was,
therefore, not surprising that in our analysis MLV(LCMV) pseudotypes
also infected several different cell lines, including hamster cells,
which are resistant to amphotropic retroviral vectors (21).
An interesting property of LCMV is that single amino acid changes in
the glycoprotein can alter cell tropism, indicating that the LCMV
glycoproteins may use different closely related receptors (or one
receptor with different posttranslational modifications) on different
cell types (20, 27). This flexibility of the glycoprotein
may be of considerable advantage for the design of vectors with
preferential tropism for specific tissues and cell types.
In conclusion, although several questions remain open, especially
regarding the feasibility of a helper-free packaging system, MLV(LCMV)
pseudotypes are a promising alternative to current retroviral pseudotypes and may allow stable production of broad-host-range retroviral vectors which can be concentrated by ultracentrifugation.
| |
ACKNOWLEDGMENTS |
We thank Carol Stocking for critical reading of the manuscript.
This work was supported by Deutsche Forschungsgemeinschaft grant LA
1135/3-1 and by Bundesministerium für Bildung und Forschung grant
01KV9811.4.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Heinrich-Pette-Institut, Martinistr. 52, D-20251 Hamburg, Germany.
Phone: 49-40-48051274. Fax: 49-40-48051187. E-mail:
laer{at}hpi.uni-hamburg.de.
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Journal of Virology, July 1999, p. 6114-6116, Vol. 73, No. 7
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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