Uncoupling GP1 and GP2 expression in the Lassa virus glycoprotein complex: implications for GP1 ectodomain shedding

Area of Science:

• Virology
• Protein biochemistry
• Mammalian cell expression systems

Background:

• Lassa fever patient sera contain antibodies to Lassa virus (LASV) glycoproteins GP1 and GP2.
• Immunization with viral vectors expressing the glycoprotein complex (GPC) provides protection against LASV.
• Development of soluble, uncoupled LASV GP1 and GP2 is crucial for improved diagnostics, treatment, and prevention of Lassa fever.

Purpose of the Study:

• To engineer mammalian expression systems for the production and purification of secreted, soluble Lassa virus (LASV) glycoprotein 1 (GP1) and glycoprotein 2 (GP2).
• To establish determinants for the mammalian cell expression of secreted, uncoupled LASV GP1 and GP2.
• To generate quasi-native recombinant LASV GP1 and GP2 proteins for potential therapeutic and diagnostic applications.

Main Methods:

• Engineered mammalian expression systems to produce secreted forms of soluble LASV GP1 and GP2.
• Utilized native GPC signal peptide or human IgG signal sequences for GP1 secretion.
• Modified GP2 constructs by deleting transmembrane and intracellular domains, and co-expressing with GP1.

Main Results:

• Established conditions for mammalian cell expression of secreted, uncoupled LASV GP1 and GP2.
• Demonstrated that GP1 acts as a chaperone for GP2 processing and cell surface transport.
• Generated homogeneously glycosylated, soluble GP1 and GP2 proteins with high mannose glycans.
• Observed GP1 ectodomain shedding from cells expressing wild-type LASV GPC, a novel finding.

Conclusions:

• GP1 is essential for the proper processing and cell surface shuttling of GP2, with the native GPC signal peptide playing a role.
• Alternative pathways may lead to heterogeneous glycosylation or incomplete maturation of GP2 in the absence of GP1 or GPC SP.
• Successfully generated and purified soluble, uncoupled GP1 and GP2 proteins with quasi-native properties from mammalian cells.
• GP1 ectodomain shedding provides new insights into arenaviral glycoprotein expression and potential early diagnostic markers for Lassa fever.