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Assays to Assess Arenaviral Glycoprotein Function
Junjie Shao1, Xiaoying Liu1, Yuying Liang1
1Department of Veterinary and Biomedical Sciences, University of Minnesota, Twin Cities, 1988 Fitch Ave., 295 AS/VM Bldg., Saint Paul, MN, 55108, USA.
Abstract:
Arenaviruses, such as Lassa virus (LASV) and Pichindé virus (PICV), are enveloped viruses with a bi-segmented ambisense RNA genome. The large (L) genomic segment encodes the Z matrix protein and the L RNA-dependent RNA polymerase, whereas the small (S) genomic segment encodes the nucleoprotein (NP) and the glycoprotein precursor complex (GPC). GPC is processed by signal peptidase in the endoplasmic reticulum into the stable signal peptide (SSP) and GP1/GP2, which is further cleaved by the Golgi-resident subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) into the cellular receptor-recognition subunit GP1 and the transmembrane subunit GP2, which helps promote the membrane fusion reaction to allow virus entry into the cell. This article describes assays to assess PICV GPC expression, proteolytic processing, fusion function, and GPC-mediated virus-like particle (VLP) entry into cells under tissue-culture conditions.
Insights
This study details assays for evaluating Pichindé virus glycoprotein complex (GPC) expression, processing, and function. These methods assess GPC
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Arenaviruses possess a bi-segmented ambisense RNA genome encoding key viral proteins.
- The glycoprotein precursor complex (GPC) is crucial for viral entry, undergoing complex processing.
- Understanding GPC function is vital for studying arenavirus pathogenesis, including Lassa virus (LASV).
Purpose of the Study:
- To establish and describe assays for assessing Pichindé virus (PICV) GPC expression.
- To evaluate the proteolytic processing of PICV GPC.
- To determine the fusion function and cell entry mediated by PICV GPC using virus-like particles (VLPs).
Main Methods:
- Development of assays to measure PICV GPC expression levels.
- Analysis of GPC proteolytic cleavage by endoplasmic reticulum and Golgi-resident proteases.
- Assessment of GPC-mediated membrane fusion and VLP entry into host cells in vitro.
Main Results:
- Established functional assays for PICV GPC.
- Characterized the proteolytic processing pathway of PICV GPC.
- Demonstrated GPC's role in mediating VLP entry into cells.
Conclusions:
- The described assays provide a framework for studying PICV GPC.
- These methods facilitate further research into arenavirus entry mechanisms.
- This work aids in understanding the molecular basis of arenavirus infections.
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