SARS-CoV-2 envelope protein topology in eukaryotic membranes

Coronavirus E protein is a small membrane protein found in the virus envelope. Different coronavirus E proteins share striking biochemical and functional similarities, but sequence conservation is limited. In this report, we studied the E protein topology from the new SARS-CoV-2 virus both in microsomal membranes and in mammalian cells. Experimental data reveal that E protein is a single-spanning membrane protein with the N-terminus being translocated across the membrane, while the C-terminus is exposed to the cytoplasmic side (Nt lum /Ct cyt ). The defined membrane protein topology of SARS-CoV-2 E protein may provide a useful framework to understand its interaction with other viral and host components and contribute to establish the basis to tackle the pathogenesis of SARS-CoV-2.

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SARS-CoV-2 envelope protein topology in eukaryotic membranes

10.1101/2020.05.27.118752 ◽

2020 ◽

Author(s):

Gerard Duart ◽

Ma Jesús García-Murria ◽

Brayan Grau ◽

José M. Acosta-Cáceres ◽

Luis Martínez-Gil ◽

...

Keyword(s):

Membrane Protein ◽

Mammalian Cells ◽

E Proteins ◽

Virus Envelope ◽

Protein Topology ◽

Membrane Protein Topology ◽

E Protein ◽

C Terminus ◽

Microsomal Membranes ◽

Cytoplasmic Side

ABSTRACTCoronavirus E protein is a small membrane protein found in the virus envelope. Different coronavirus E proteins share striking biochemical and functional similarities, but sequence conservation is limited. In this report, we studied the E protein topology from the new SARS-CoV-2 virus both in microsomal membranes and in mammalian cells. Experimental data reveal that E protein is a single-spanning membrane protein with the N-terminus being translocated across the membrane, while the C-terminus is exposed to the cytoplasmic side (Ntlum/Ctcyt). The defined membrane protein topology of SARS-CoV-2 E protein may provide a useful framework to understand its interaction with other viral and host components and establish the basis to tackle the pathogenesis of SARS-CoV-2.

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Regulation of multispanning membrane protein topology via post-translational annealing

eLife ◽

10.7554/elife.08697 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 28

Author(s):

Reid C Van Lehn ◽

Bin Zhang ◽

Thomas F Miller

Keyword(s):

Membrane Protein ◽

Annealing Process ◽

Coarse Grained ◽

Fully Integrated ◽

Protein Topology ◽

Membrane Protein Topology ◽

C Terminus ◽

Coarse Grained Simulations ◽

Positively Charged

The canonical mechanism for multispanning membrane protein topogenesis suggests that protein topology is established during cotranslational membrane integration. However, this mechanism is inconsistent with the behavior of EmrE, a dual-topology protein for which the mutation of positively charged loop residues, even close to the C-terminus, leads to dramatic shifts in its topology. We use coarse-grained simulations to investigate the Sec-facilitated membrane integration of EmrE and its mutants on realistic biological timescales. This work reveals a mechanism for regulating membrane-protein topogenesis, in which initially misintegrated configurations of the proteins undergo post-translational annealing to reach fully integrated multispanning topologies. The energetic barriers associated with this post-translational annealing process enforce kinetic pathways that dictate the topology of the fully integrated proteins. The proposed mechanism agrees well with the experimentally observed features of EmrE topogenesis and provides a range of experimentally testable predictions regarding the effect of translocon mutations on membrane protein topogenesis.

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The Length of and Nonhydrophobic Residues in the Transmembrane Domain of Dengue Virus Envelope Protein Are Critical for Its Retention and Assembly in the Endoplasmic Reticulum

Journal of Virology ◽

10.1128/jvi.01963-09 ◽

2010 ◽

Vol 84 (9) ◽

pp. 4782-4797 ◽

Cited By ~ 25

Author(s):

Szu-Chia Hsieh ◽

Wen-Yang Tsai ◽

Wei-Kung Wang

Keyword(s):

Endoplasmic Reticulum ◽

Dengue Virus ◽

Secretory Pathway ◽

Transmembrane Domain ◽

E Proteins ◽

Virus Envelope ◽

Enveloped Viruses ◽

E Protein ◽

C Terminus ◽

Er Retention

ABSTRACT The morphogenesis of many enveloped viruses, in which viral nucleocapsid complex interacts with envelope (E) protein, is known to take place at various sites along the secretory pathway. How viral E protein retains in a particular intracellular organelle for assembly remains incompletely understood. In this study, we investigated determinants in the E protein of dengue virus (DENV) for its retention and assembly in the endoplasmic reticulum (ER). A chimeric experiment between CD4 and DENV precursor membrane/E constructs suggested that the transmembrane domain (TMD) of E protein contains an ER retention signal. Substitutions of three nonhydrophobic residues at the N terminus of the first helix (T1) and at either the N or C terminus of the second helix of the TMD with three hydrophobic residues, as well as an increase in the length of T1, led to the release of chimeric CD4 and E protein from the ER, suggesting that short length and certain nonhydrophobic residues of the TMD are critical for ER retention. The analysis of enveloped viruses assembled at the plasma membrane and of those assembled in the Golgi complex and ER revealed a trend of decreasing length and increasing nonhydrophobic residues of the TMD of E proteins. Taken together, these findings support a TMD-dependent sorting for viral E proteins along the secretory pathway. Moreover, similar mutations introduced into the TMD of DENV E protein resulted in the increased production of virus-like particles (VLPs), suggesting that modifications of TMD facilitate VLP production and have implications for utilizing flaviviral VLPs as serodiagnostic antigens and vaccine candidates.

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