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Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding
J Klumperman1, J K Locker, A Meijer
1Department of Cell Biology, Utrecht University, The Netherlands.
Abstract:
The prevailing hypothesis is that the intracellular site of budding of coronaviruses is determined by the localization of its membrane protein M (previously called E1). We tested this by analyzing the site of budding of four different coronaviruses in relation to the intracellular localization of their M proteins. Mouse hepatitis virus (MHV) and infectious bronchitis virus (IBV) grown in Sac(-) cells, and feline infectious peritonitis virus (FIPV) and transmissible gastroenteritis virus (TGEV) grown in CrFK cells, all budded exclusively into smooth-walled, tubulovesicular membranes located intermediately between the rough endoplasmic reticulum and Golgi complex, identical to the so-called budding compartment previously identified for MHV. Indirect immunofluorescence staining of the infected cells showed that all four M proteins accumulated in a perinuclear region. Immunogold microscopy localized MHV M and IBV M in the budding compartment; in addition, a dense labeling in the Golgi complex occurred, MHV M predominantly in trans-Golgi cisternae and trans-Golgi reticulum and IBV M mainly in the cis and medial Golgi cisternae. The corresponding M proteins of the four viruses, when independently expressed in a recombinant vaccinia virus system, also accumulated in the perinuclear area. Quantitative pulse-chase analysis of metabolically labeled cells showed that in each case the majority of the M glycoproteins carried oligosaccharide side chains with Golgi-specific modifications within 4 h after synthesis. Immunoelectron microscopy localized recombinant MHV M and IBV M to the same membranes as the respective proteins in coronavirus-infected cells, with the same cis-trans distribution over the Golgi complex. Our results demonstrate that some of the M proteins of the four viruses are transported beyond the budding compartment and are differentially retained by intrinsic retention signals; in addition to M, other viral and/or cellular factors are probably required to determine the site of budding.
Insights
Coronavirus M proteins accumulate in the Golgi apparatus, influencing viral budding sites. This study shows M proteins are retained in the Golgi, suggesting other factors also control where coronaviruses bud.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- The intracellular site of coronavirus budding is hypothesized to be dictated by the M protein's localization.
- Understanding this process is crucial for comprehending viral assembly and pathogenesis.
Purpose of the Study:
- To investigate the intracellular localization of M proteins from four different coronaviruses.
- To determine the relationship between M protein localization and the site of viral budding.
Main Methods:
- Infection of Sac(-) and CrFK cells with four distinct coronaviruses (MHV, IBV, FIPV, TGEV).
- Indirect immunofluorescence staining and immunogold electron microscopy to visualize M protein localization.
- Expression of M proteins using a recombinant vaccinia virus system.
- Quantitative pulse-chase analysis to track glycoprotein modification and transport.
Main Results:
- All four coronaviruses budded into tubulovesicular membranes between the ER and Golgi.
- M proteins from all four viruses localized to a perinuclear region, including the Golgi complex.
- M proteins were found in the budding compartment and showed differential distribution within Golgi cisternae.
- Recombinant M proteins localized to similar membranes and Golgi compartments as in infected cells.
- M glycoproteins acquired Golgi-specific modifications within 4 hours of synthesis.
Conclusions:
- Coronavirus M proteins are transported beyond the initial budding compartment.
- Intrinsic retention signals within M proteins contribute to their localization.
- Additional viral and/or cellular factors likely play a role in determining the precise site of coronavirus budding.