Uncoupling GP1 and GP2 Expression in the Lassa Virus Glycoprotein Complex: Implications for GP1 Ectodomain Shedding
BIOFACTURA INC ROCKVILLE MD
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Background Sera from convalescent Lassa fever patients often contains antibodies to Lassa virus LASV glycoprotein 1 GP1, and glycoprotein 2 GP2 Immunization of non-human primates with viral vectors expressing the arenaviral glycoprotein complex GPC confers full protective immunity against a lethal challenge with LASV. Thus, the development of native or quasi native recombinant LASV GP1 and GP2 as soluble, uncoupled proteins will improve current diagnostics, treatment, and prevention of Lassa fever. To this end, mammalian expression systems were engineered for production and purification of secreted forms of soluble LASV GP1 and GP2 proteins. Results Determinants for mammalian cell expression of secreted uncoupled Lassa virus LASV glycoprotein 1 GP1 and glycoprotein 2 GP2 were established. Soluble GP1 was generated using either the native glycoprotein precursor GPC signal peptide SP or human IgG signal sequences s.s.. GP2 was secreted from cells only when 1 the transmembrane TM domain was deleted, the intracellular domain IC was fused to the ectodomain, and the gene was co-expressed with a complete GP1 gene in cis 2 the TM and IC domains were deleted and GP1 was co-expressed in cis 3 expression of GP1 was driven by the native GPC SP. These data implicate GP1 as a chaperone for processing and shuttling GP2 to the cell surface. The soluble forms of GP1 and GP2 generated through these studies were secreted as homogeneously glycosylated proteins that contained high mannose glycans. Furthermore, observation of GP1 ectodomain shedding from cells expressing wild type LASV GPC represents a novel aspect of arenaviral glycoprotein expression. Conclusion These results implicate GP1 as a chaperone for the correct processing and shuttling of GP2 to the cell surface, and suggest that native GPC SP plays a role in this process.
- Genetic Engineering and Molecular Biology
- Medicine and Medical Research