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. 2001 Jan;75(2):1061-4.
doi: 10.1128/JVI.75.2.1061-1064.2001.

Recombinant expression of lymphocytic choriomeningitis virus strain WE glycoproteins: a single amino acid makes the difference

W R Beyer  1 , H Miletic, W Ostertag, D von Laer
Affiliations

Recombinant expression of lymphocytic choriomeningitis virus strain WE glycoproteins: a single amino acid makes the difference

W R Beyer et al. J Virol. 2001 Jan.

Abstract

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.

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Figures

FIG. 1
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.
FIG. 2
FIG. 2
Twelve different amino acids are encoded by the recloned LCMV GP(WE-HPI) cDNA and the originally published LCMV GP(WE) sequence (15).
See this image and copyright information in PMC

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