Role of the Transmembrane Domain of Marburg Virus Surface Protein GP in Assembly of the Viral Envelope

ABSTRACT The major protein constituents of the filoviral envelope are the matrix protein VP40 and the surface transmembrane protein GP. While VP40 is recruited to the sites of budding via the late retrograde endosomal transport route, GP is suggested to be transported via the classical secretory pathway involving the endoplasmic reticulum, Golgi apparatus, and trans-Golgi network until it reaches the plasma membrane where most filoviral budding takes place. Since both transport routes target the plasma membrane, it was thought that GP and VP40 join there to form the viral envelope. However, it was recently shown that, upon coexpression of both proteins, GP is partially recruited into peripheral VP40-enriched multivesicular bodies, which contained markers of the late endosome. Accumulation of GP and VP40 in this compartment was presumed to play an important role in the formation of the filoviral envelope. Using a domain-swapping approach, we were able to show that the transmembrane domain of GP was essential and sufficient for (i) partial recruitment of chimeric glycoproteins into VP40-enriched multivesicular bodies and (ii) incorporation into virus-like particles (VLPs) that were released upon expression of VP40. Only those chimeric glycoproteins which were targeted to VP40-enriched endosomal multivesicular bodies were subsequently recruited into VLPs. These data show that the transmembrane domain of GP is critical for the mixing of VP40 and GP in multivesicular bodies and incorporation of GP into the viral envelope. Results further suggest that trapping of GP in the VP40-enriched late endosomal compartment is important for the formation of the viral envelope.

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Multivesicular Bodies as a Platform for Formation of the Marburg Virus Envelope

Journal of Virology ◽

10.1128/jvi.78.22.12277-12287.2004 ◽

2004 ◽

Vol 78 (22) ◽

pp. 12277-12287 ◽

Cited By ~ 79

Author(s):

Larissa Kolesnikova ◽

Beate Berghöfer ◽

Sandra Bamberg ◽

Stephan Becker

Keyword(s):

Intracellular Transport ◽

Matrix Protein ◽

Lipid Membrane ◽

Marburg Virus ◽

Viral Envelope ◽

Multivesicular Bodies ◽

Virus Envelope ◽

Transport Pathways ◽

Virus Like Particles

ABSTRACT The Marburg virus (MARV) envelope consists of a lipid membrane and two major proteins, the matrix protein VP40 and the glycoprotein GP. Both proteins use different intracellular transport pathways: GP utilizes the exocytotic pathway, while VP40 is transported through the retrograde late endosomal pathway. It is currently unknown where the proteins combine to form the viral envelope. In the present study, we identified the intracellular site where the two major envelope proteins of MARV come together as peripheral multivesicular bodies (MVBs). Upon coexpression with VP40, GP is redistributed from the trans-Golgi network into the VP40-containing MVBs. Ultrastructural analysis of MVBs suggested that they provide the platform for the formation of membrane structures that bud as virus-like particles from the cell surface. The virus-like particles contain both VP40 and GP. Single expression of GP also resulted in the release of particles, which are round or pleomorphic. Single expression of VP40 led to the release of filamentous structures that closely resemble viral particles and contain traces of endosomal marker proteins. This finding indicated a central role of VP40 in the formation of the filamentous structure of MARV particles, which is similar to the role of the related Ebola virusVP40. In MARV-infected cells, VP40 and GP are colocalized in peripheral MVBs as well. Moreover, intracellular budding of progeny virions into MVBs was frequently detected. Taken together, these results demonstrate an intracellular intersection between GP and VP40 pathways and suggest a crucial role of the late endosomal compartment for the formation of the viral envelope.

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Phosphorylation of Marburg virus matrix protein VP40 triggers assembly of nucleocapsids with the viral envelope at the plasma membrane

Cellular Microbiology ◽

10.1111/j.1462-5822.2011.01709.x ◽

2011 ◽

Vol 14 (2) ◽

pp. 182-197 ◽

Cited By ~ 28

Author(s):

Larissa Kolesnikova ◽

Eva Mittler ◽

Gordian Schudt ◽

Hosam Shams-Eldin ◽

Stephan Becker

Keyword(s):

Plasma Membrane ◽

Matrix Protein ◽

Marburg Virus ◽

Viral Envelope

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Vaccinia Virus A26 and A27 Proteins Form a Stable Complex Tethered to Mature Virions by Association with the A17 Transmembrane Protein

Journal of Virology ◽

10.1128/jvi.01524-08 ◽

2008 ◽

Vol 82 (24) ◽

pp. 12384-12391 ◽

Cited By ~ 30

Author(s):

Amanda R. Howard ◽

Tatiana G. Senkevich ◽

Bernard Moss

Keyword(s):

Vaccinia Virus ◽

Matrix Protein ◽

Transmembrane Domain ◽

Noncovalent Complex ◽

Transmembrane Protein ◽

Amino Acid Identity ◽

Noncovalent Interaction ◽

Terminal Segment ◽

Stable Complex ◽

Cowpox Virus

ABSTRACT During vaccinia virus replication, mature virions (MVs) are wrapped with cellular membranes, transported to the periphery, and exported as extracellular virions (EVs) that mediate spread. The A26 protein is unusual in that it is present in MVs but not EVs. This distribution led to a proposal that A26 negatively regulates wrapping. A26 also has roles in the attachment of MVs to the cell surface and incorporation of MVs into proteinaceous A-type inclusions in some orthopoxvirus species. However, A26 lacks a transmembrane domain, and nothing is known regarding how it associates with the MV, regulates incorporation of the MV into inclusions, and possibly prevents EV formation. Here, we provide evidence that A26 forms a disulfide-bonded complex with A27 that is anchored to the MV through a noncovalent interaction with the A17 transmembrane protein. In the absence of A27, A26 was unstable, and only small amounts were detected. The interaction of A26 with A27 depended on a C-terminal segment of A26 with 45% amino acid identity to A27. Deletion of A26 failed to enhance EV formation by vaccinia virus, as had been predicted. Nevertheless, the interaction of A26 and A27 may have functional significance, since each is thought to mediate binding to cells through interaction with laminin and heparan sulfate, respectively. We also found that A26 formed a noncovalent complex with A25, a truncated form of the cowpox virus A-type inclusion matrix protein. The latter association suggests a mechanism for incorporation of virions into A-type inclusions in other orthopoxvirus strains.

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Ykt6 membrane-to-cytosol cycling regulates exosomal Wnt secretion

10.1101/485565 ◽

2018 ◽

Cited By ~ 2

Author(s):

Karen Linnemannstöns ◽

Pradhipa Karuna M ◽

Leonie Witte ◽

Jeanette Clarissa Kittel ◽

Adi Danieli ◽

...

Keyword(s):

Plasma Membrane ◽

Wnt Signaling ◽

Signaling Pathways ◽

Long Range ◽

Protein Trafficking ◽

Secretory Pathway ◽

Multivesicular Bodies ◽

Phosphorylation Sites ◽

Wnt Proteins ◽

C Terminus

Protein trafficking in the secretory pathway, for example the secretion of Wnt proteins, requires tight regulation. These ligands activate Wnt signaling pathways and are crucially involved in development and disease. Wnt is transported to the plasma membrane by its cargo receptor Evi, where Wnt/Evi complexes are endocytosed and sorted onto exosomes for long-range secretion. However, the trafficking steps within the endosomal compartment are not fully understood. The promiscuous SNARE Ykt6 folds into an auto-inhibiting conformation in the cytosol, but a portion associates with membranes by its farnesylated and palmitoylated C-terminus. Here, we demonstrate that membrane detachment of Ykt6 is essential for exosomal Wnt secretion. We identified conserved phosphorylation sites within the SNARE domain of Ykt6, which block Ykt6 cycling from the membrane to the cytosol. In Drosophila, Ykt6-RNAi mediated block of Wg secretion is rescued by wildtype but not phosphomimicking Ykt6. The latter accumulates at membranes, while wildtype Ykt6 regulates Wnt trafficking between the plasma membrane and multivesicular bodies. Taken together, we show that a regulatory switch in Ykt6 fine-tunes sorting of Wnts in endosomes.

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Structural basis for cholesterol transport-like activity of the Hedgehog receptor Patched

10.1101/352443 ◽

2018 ◽

Cited By ~ 3

Author(s):

Yunxiao Zhang ◽

David P. Bulkley ◽

Kelsey J. Roberts ◽

Yao Xin ◽

Daniel E. Asarnow ◽

...

Keyword(s):

Plasma Membrane ◽

Basal Cell Carcinoma ◽

Cell Carcinoma ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Extracellular Domain ◽

Structural Basis ◽

Cholesterol Transport ◽

Common Cause ◽

Common Receptor

AbstractHedgehog protein signals mediate tissue patterning and maintenance via binding to and inactivation of their common receptor Patched, a twelve-transmembrane protein that otherwise would suppress activity of the seven-transmembrane protein, Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. The extracellular domain mediates association of Patched monomers in an unusual dimeric architecture that implies curvature in the associated membrane. A central conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Patched expression indeed reduces cholesterol activity in the inner leaflet of the plasma membrane, in a manner antagonized by Hedgehog stimulation and with implications for regulation of Smoothened.

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The Equine Herpesvirus 1 US2 Homolog Encodes a Nonessential Membrane-Associated Virion Component

Journal of Virology ◽

10.1128/jvi.73.4.3430-3437.1999 ◽

1999 ◽

Vol 73 (4) ◽

pp. 3430-3437 ◽

Cited By ~ 27

Author(s):

Alexandra Meindl ◽

Nikolaus Osterrieder

Keyword(s):

Amino Acids ◽

Plasma Membrane ◽

Secretory Pathway ◽

Fluorescent Protein ◽

Viral Envelope ◽

Equine Herpesvirus ◽

Equine Herpesvirus 1 ◽

Amino Terminal ◽

Infected Cells ◽

Herpesvirus 1

ABSTRACT Experiments were conducted to analyze the equine herpesvirus 1 (EHV-1) gene 68 product which is encoded by the EHV-1 US2 homolog. An antiserum directed against the amino-terminal 206 amino acids of the EHV-1 US2 protein specifically detected a protein with an M r of 34,000 in cells infected with EHV-1 strain RacL11. EHV-1 strain Ab4 encodes a 44,000-M r Us2 protein, whereas vaccine strain RacH, a high-passage derivative of RacL11, encodes a 31,000-M r Us2 polypeptide. Irrespective of its size, the US2 protein was incorporated into virions. The EHV-1 US2 protein localized to membrane and nuclear fractions of RacL11-infected cells and to the envelope fraction of purified virions. To monitor intracellular trafficking of the protein, the green fluorescent protein (GFP) was fused to the carboxy terminus of the EHV-1 US2 protein or to a truncated US2 protein lacking a stretch of 16 hydrophobic amino acids at the extreme amino terminus. Both fusion proteins were detected at the plasma membrane and accumulated in the vicinity of nuclei of transfected cells. However, trafficking of either GFP fusion protein through the secretory pathway could not be demonstrated, and the EHV-1 US2 protein lacked detectable N- and O-linked carbohydrates. Consistent with the presence of the US2 protein in the viral envelope and plasma membrane of infected cells, a US2-negative RacL11 mutant (L11ΔUS2) exhibited delayed penetration kinetics and produced smaller plaques compared with either wild-type RacL11 or a US2-repaired virus. After infection of BALB/c mice with L11ΔUS2, reduced pathogenicity compared with the parental RacL11 virus and the repaired virus was observed. It is concluded that the EHV-1 US2 protein modulates virus entry and cell-to-cell spread and appears to support sustained EHV-1 replication in vivo.

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Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells

Journal of Virology ◽

10.1128/jvi.01668-08 ◽

2009 ◽

Vol 83 (6) ◽

pp. 2611-2622 ◽

Cited By ~ 42

Author(s):

Subash C. Das ◽

Debasis Panda ◽

Debasis Nayak ◽

Asit K. Pattnaik

Keyword(s):

Plasma Membrane ◽

Vesicular Stomatitis Virus ◽

Matrix Protein ◽

Fluorescent Protein ◽

Dynamic Imaging ◽

Viral Envelope ◽

M Protein ◽

Recombinant Viruses ◽

Infected Cells ◽

Vesicular Stomatitis

ABSTRACT A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-ΔM-Mtc and VSV-ΔM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-ΔM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-ΔM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min.

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A Preliminary Bioinformatics Analysis of Cation Diffusion Facilitator (CDF) Proteins in Different Plants

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.282-283.453 ◽

2011 ◽

Vol 282-283 ◽

pp. 453-456

Author(s):

Hua Zhong ◽

Bao Ping Sun ◽

Fang Ying Zhao

Keyword(s):

Secretory Pathway ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Populus Trichocarpa ◽

Alpha Helix ◽

Random Coil ◽

Post Translational Modification ◽

Cation Diffusion ◽

Cation Diffusion Facilitator ◽

Arabidopsis Lyrata

The amino acid sequence of Cation Diffusion Facilitator from Populus trichocarpa, Thlaspi goesingense, Arabidopsis lyrata subsp. Lyrata, Brassica juncea and Medicago sativa, registered in GenBank, were analyzed and researched by the bioinformatic tools in the several aspects, including hydrophobicity / hydrophilicity properties, post-translational modification, secondary structures prediction and transmembrane domain. The results showed that Cation Diffusion Facilitator is a hydrophobic and transmembrane protein, which exists in endoplasmic reticulum and other secretory pathway. The main motifs of predicted secondary structure of Cation Diffusion Facilitator are alpha helix and random coil.

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Mutation of Hydrophobic Residues in the C-Terminal Domain of the Marburg Virus Matrix Protein VP40 Disrupts Trafficking to the Plasma Membrane

Viruses ◽

10.3390/v12040482 ◽

2020 ◽

Vol 12 (4) ◽

pp. 482 ◽

Cited By ~ 2

Author(s):

Kaveesha J. Wijesinghe ◽

Luke McVeigh ◽

Monica L. Husby ◽

Nisha Bhattarai ◽

Jia Ma ◽

...

Keyword(s):

Amino Acids ◽

Plasma Membrane ◽

Matrix Protein ◽

Marburg Virus ◽

Membrane Localization ◽

Hydrophobic Residues ◽

Plasma Membrane Localization ◽

Terminal Domain ◽

Loop Region

Marburg virus (MARV) is a lipid-enveloped negative sense single stranded RNA virus, which can cause a deadly hemorrhagic fever. MARV encodes seven proteins, including VP40 (mVP40), a matrix protein that interacts with the cytoplasmic leaflet of the host cell plasma membrane. VP40 traffics to the plasma membrane inner leaflet, where it assembles to facilitate the budding of viral particles. VP40 is a multifunctional protein that interacts with several host proteins and lipids to complete the viral replication cycle, but many of these host interactions remain unknown or are poorly characterized. In this study, we investigated the role of a hydrophobic loop region in the carboxy-terminal domain (CTD) of mVP40 that shares sequence similarity with the CTD of Ebola virus VP40 (eVP40). These conserved hydrophobic residues in eVP40 have been previously shown to be critical to plasma membrane localization and membrane insertion. An array of cellular experiments and confirmatory in vitro work strongly suggests proper orientation and hydrophobic residues (Phe281, Leu283, and Phe286) in the mVP40 CTD are critical to plasma membrane localization. In line with the different functions proposed for eVP40 and mVP40 CTD hydrophobic residues, molecular dynamics simulations demonstrate large flexibility of residues in the EBOV CTD whereas conserved mVP40 hydrophobic residues are more restricted in their flexibility. This study sheds further light on important amino acids and structural features in mVP40 required for its plasma membrane localization as well as differences in the functional role of CTD amino acids in eVP40 and mVP40.

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Apical Budding of a Recombinant Influenza A Virus Expressing a Hemagglutinin Protein with a Basolateral Localization Signal

Journal of Virology ◽

10.1128/jvi.76.7.3544-3553.2002 ◽

2002 ◽

Vol 76 (7) ◽

pp. 3544-3553 ◽

Cited By ~ 40

Author(s):

Rosalia Mora ◽

Enrique Rodriguez-Boulan ◽

Peter Palese ◽

Adolfo García-Sastre

Keyword(s):

Plasma Membrane ◽

Influenza A ◽

Mdck Cells ◽

Viral Envelope ◽

Apical Plasma Membrane ◽

Major Protein ◽

Mutant Form ◽

Apical Surface ◽

Cytoplasmic Domains ◽

Viral Budding

ABSTRACT Influenza virions bud preferentially from the apical plasma membrane of infected epithelial cells, by enveloping viral nucleocapsids located in the cytosol with its viral integral membrane proteins, i.e., hemagglutinin (HA), neuraminidase (NA), and M2 proteins, located at the plasma membrane. Because individually expressed HA, NA, and M2 proteins are targeted to the apical surface of the cell, guided by apical sorting signals in their transmembrane or cytoplasmic domains, it has been proposed that the polarized budding of influenza virions depends on the interaction of nucleocapsids and matrix proteins with the cytoplasmic domains of HA, NA, and/or M2 proteins. Since HA is the major protein component of the viral envelope, its polarized surface delivery may be a major force that drives polarized viral budding. We investigated this hypothesis by infecting MDCK cells with a transfectant influenza virus carrying a mutant form of HA (C560Y) with a basolateral sorting signal in its cytoplasmic domain. C560Y HA was expressed nonpolarly on the surface of infected MDCK cells. Interestingly, viral budding remained apical in C560Y virus-infected cells, and so did the location of NP and M1 proteins at late times of infection. These results are consistent with a model in which apical viral budding is a shared function of various viral components rather than a role of the major viral envelope glycoprotein HA.

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