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Journal of Virology, January 2001, p. 1061-1064, Vol. 75, No. 2
Heinrich-Pette-Institut für
Experimentelle Virologie und Immunologie an der Universität
Hamburg, D-20251 Hamburg, Germany
Received 8 August 2000/Accepted 25 October 2000
Cytoplasmic vector systems are generally used for expression of
lymphocytic choriomeningitis virus (LCMV) proteins. However, we
achieved high levels of cell surface glycoproteins using a standard
nuclear expression plasmid. Expression was independent of other LCMV
proteins but was blocked by a missense mutation within the original
LCMV(WE) glycoprotein cDNA.
The lymphocytic choriomeningitis
virus (LCMV) is the prototype arenavirus and a widely used experimental
model for the study of viral persistence and pathogenesis. The envelope
glycoproteins of LCMV (LCMV GP) are initially expressed as a precursor
polypeptide, GP-C, which is posttranslationally processed by a cellular
protease into GP-1 and GP-2. GP-1 interacts with the cellular receptor for LCMV, which has been identified as alpha-dystroglycan. GP-2 contains the fusion peptide and the transmembrane domain
(2-5).
It was previously shown that retroviral vectors can be pseudotyped with
LCMV glycoproteins (11). These pseudotypes, like pseudotypes with the G protein of vesicular stomatitis virus, are
stable during ultracentrifugation, but unlike the vesicular stomatitis
virus G protein, the LCMV glycoproteins are not cytotoxic. A
prerequisite for the generation of safe, helper-free vector particles
for this novel pseudotype is the efficient expression of recombinant
LCMV GP on the surface of retroviral packaging cells. LCMV has a
cytoplasmic life cycle. Therefore, vaccinia virus has been used
frequently to express LCMV GP in order to prevent aberrant splicing of
GP transcripts in the nucleus. In addition, the originally cloned
glycoprotein cDNA of LCMV strain WE [LCMV(WE)] has been expressed by
baculovirus and retroviral expression systems as well as in transgenic
mice (7, 9, 10, 14). These systems were widely used to
study the immune response in mice (6, 8, 12, 13). However,
efficient cell surface expression of recombinant LCMV GP was never
shown. The aim of this study was to express LCMV(WE) glycoproteins on the cell surface by a nuclear expression system that could be used for
the generation of helper virus-free retroviral pseudotypes.
(This article is based on a doctoral study by W.B. in the Faculty of
Biology, University of Hamburg.)
Recombinant LCMV(WE) glycoproteins are not transported to the cell
surface.
The expression plasmid pHCMV-GP(WE) was derived from the
widely used original cDNA of LCMV(WE) S RNA and the pHCMV expression vector (15, 19). pHCMV contains the human cytomegalovirus immediate early promoter, the rabbit beta-globin intron B, and the
rabbit beta-globin polyadenylation sequences. The GP cDNA was amplified
by PCR from the plasmid pY-1-A and inserted between the
BamHI sites of pHCMV by using the restriction sites
introduced by PCR primers (5' CTGGATCCGC CATGGGTCAG
ATTGTGACAA TG 3' and 5' CGGGATCCTT ATCAGCGTCT
TTTCCAGATA G 3') (10). Sequence analysis showed that the
pY-1-A clone and the cDNA in the newly generated pHCMV-GP(WE) encoded
L455 and K457, as also found in other LCMV GP
sequences, and not F455 and R457 as found in
the original publications (15-18). After calcium
phosphate transfection of pHCMV-GP(WE) into 293T cells, no cell surface
expression of recombinant LCMV GP was detectable by flow cytometry with
the monoclonal antibody KL25 (Fig. 1B,
WE) or other monoclonal GP-1-specific and polyclonal anti-LCMV
antibodies (1; data not shown). In contrast, 3 days after infection with LCMV(WE) viral glycoproteins were expressed on the
cell surface as shown by flow cytometry with the KL25 antibody (Fig.
1C).
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.2.1061-1064.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Recombinant Expression of Lymphocytic Choriomeningitis Virus
Strain WE Glycoproteins: a Single Amino Acid Makes the
Difference
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FIG. 1.
The proline-to-leucine mutation at amino acid 110 leads
to processing and cell surface expression of LCMV GP. (A) Western blot
analysis of recombinant LCMV GP. 293T cells were transfected with pHCMV
expression plasmids encoding C-terminally HA-tagged LCMV GP variants or
untagged LCMV GP (control). Forty-eight hours after transfection
proteins were extracted, and samples were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, transferred to
nitrocellulose filters, and analyzed by immunostaining with the anti-HA
tag-specific antibody 3F10. (B) Fluorescence-activated cell sorter
analysis of recombinant LCMV GP. 293T cells were mock treated (shaded
lines) or transfected with pHCMV expression plasmids for LCMV GP
variants (open lines). Forty-eight hours after transfection, LCMV GP
was detected by flow cytometry with the monoclonal antibody KL25. (C)
LCMV GP expression after infection of 293T cells with LCMV. Three days
after infection with a multiplicity of infection of 0.01, LCMV GP was
detected by flow cytometry with monoclonal antibody KL25. WE-HPI,
pHCMV-GP(WE-HPI); WE, pHCMV-GP(WE); WEP110L,
pHCMV-GP(WEP110L), which encodes a proline-to-leucine
mutation at amino acid 110; WE-HPIL110P,
pHCMV-GP(WE-HPIL110P), which encodes a leucine-to-proline
mutation at amino acid 110; GP-C, precursor glycoprotein; GP-2,
C-terminal cleavage product of GP-C containing the HA tag. wt, wild
type.
Recombinant LCMV(WE) GP-C is expressed but not correctly processed. To test whether inefficient protein expression was caused by a lack of transcription or aberrant splicing of the LCMV GP message expressed in the nucleus, total RNA was extracted from pHCMV-GP(WE)-transfected 293T cells. In Northern blot analysis, an LCMV GP mRNA of correct length was detected. No aberrant splicing of this transcript was found by reverse transcription-PCR and sequence analysis of cloned PCR products (seven cDNA clones from three independent PCRs). Furthermore, the level of the GP mRNA in transfected cells was higher than the level found in LCMV-infected cells (data not shown).
To analyze translation and processing of LCMV glycoproteins, an expression vector encoding C-terminally hemagglutinin (HA)-tagged LCMV GP was constructed as described above using the PCR primers 5' CTGGATCCGC CAT GGGTCAG ATTGTGACAA TG 3' and 5' CGGGATC CTT ATCAAGCGTA ATCTGGAACA TCGTATGGGT AGCGTCTTTT CCAGATAGT 3'. Two days after transfection of this construct into 293T cells, protein extracts were analyzed by Western blotting with an anti-HA tag-specific antibody (3F10; Roche Diagnostics GmbH, Mannheim, Germany). The precursor glycoprotein GP-C was efficiently expressed but not processed into mature GP-1 and GP-2 (Fig. 1A, WE).LCMV GP cDNA recloned from virus-infected cells encodes a glycoprotein that is efficiently processed and transported. The reason for an inefficient processing of recombinant LCMV GP-C compared to virus-expressed GP-C could be that the sequences of the recombinant and the wild-type GP differ. Therefore, the GP cDNA was recloned by reverse transcription-PCR of total RNA extracted 2 days after infection of 293T cells with a multiplicity of infection of LCMV(WE) of 0.01. The PCR product was sequenced directly or after blunt-end ligation into the EcoRV site of pBluescript. Within the open reading frame, the sequence of the recloned GP cDNA differed by 28 nucleotides (1.9%) from the published LCMV(WE) GP sequence, by 218 nucleotides (14.6%) from the LCMV(Arm53b) GP sequence, and by 213 nucleotides (14.3%) from the LCMV(CHV2) GP sequence (15, 16, 18). The encoded amino acids had 12 differences (2.4%) with LCMV(WE) GP, 23 differences (4.6%) with LCMV(Arm53b) GP, and 28 differences (5.6%) with LCMV(CHV2) GP. This indicates that the recloned LCMV GP was indeed a WE serotype, and it was therefore named LCMV GP(WE-HPI).
The new GP(WE-HPI) sequence was inserted as described above into pHCMV. Two days after transfection of 293T cells, protein was extracted and analyzed by Western blotting. In contrast to the GP(WE) sequence, the GP(WE-HPI) sequence encoded an LCMV glycoprotein, which was processed as shown by the detection of GP-2, the C-terminal cleavage product containing the HA tag (Fig. 1A, WE-HPI). Cell surface expression was analyzed again 2 days after transfection by flow cytometry with the monoclonal antibody KL25. In contrast to the GP(WE) construct, the pHCMV-GP(WE-HPI) plasmid directed efficient cell surface expression of LCMV GP (Fig. 1B, WE-HPI). This shows that processing and cell surface expression of recombinant LCMV GP are independent of other LCMV proteins.A single mutation is responsible for the block in LCMV glycoprotein
processing and transport.
To analyze which amino acids in the
original GP(WE) sequence impeded processing and cell surface expression
of LCMV GP, chimeric glycoprotein expression constructs were generated
using the XbaI and EcoRI sites in GP (Fig.
2). For both restriction sites, the expression plasmids encoding LCMV GP with the N-terminal portion of the
GP(WE-HPI) sequence and the C-terminal part of the original GP(WE)
sequence led to cell surface expression, whereas the inverse constructs
did not. Therefore, the essential difference is upstream of the
EcoRI site, where four missense mutations are located (Fig. 2).
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Nucleotide sequence accession number. The cDNA sequence of the recloned LCMV GP(WE-HPI) has been submitted to the EMBL database under the accession no. AJ297484.
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ACKNOWLEDGMENTS |
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We thank B. Abel for excellent technical assistance and M. Bruns for providing monoclonal antibodies, virus, and plasmids.
The work was supported by the Deutsche Forschungsgemeinschaft (grant LA 1135/3-1) and the Bundesministerium für Bildung und Forschung and CellTec Biotechnologie GmbH (grant 00312173).
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FOOTNOTES |
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* Corresponding author. Present address: Georg-Speyer-Haus, Paul-Ehrlich-Strasse 42-44, D-60596 Frankfurt, Germany. Phone: 49-69-63395232. Fax: 49-69-63395297. E-mail: laer{at}em.uni-frankfurt.de.
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