scholarly journals Role of Rubella Virus Glycoprotein Domains in Assembly of Virus-Like Particles

1999 ◽  
Vol 73 (5) ◽  
pp. 3524-3533 ◽  
Author(s):  
Mike Garbutt ◽  
Lok Man J. Law ◽  
Honey Chan ◽  
Tom C. Hobman
Keyword(s):  
Golgi Complex ◽  
Rubella Virus ◽  
Transmembrane Domain ◽  
Viral Envelope ◽  
Structural Proteins ◽  
Type I ◽  
Cytoplasmic Domains ◽  
Virus Like Particles ◽  
Positive Strand Rna

ABSTRACT Rubella virus is a small enveloped positive-strand RNA virus that assembles on intracellular membranes in a variety of cell types. The virus structural proteins contain all of the information necessary to mediate the assembly of virus-like particles in the Golgi complex. We have recently identified intracellular retention signals within the two viral envelope glycoproteins. E2 contains a Golgi retention signal in its transmembrane domain, whereas a signal for retention in the endoplasmic reticulum has been localized to the transmembrane and cytoplasmic domains of E1 (T. C. Hobman, L. Woodward, and M. G. Farquhar, Mol. Biol. Cell 6:7–20, 1995; T. C. Hobman, H. F. Lemon, and K. Jewell, J. Virol. 71:7670–7680, 1997). In the present study, we have analyzed the role of these retention signals in the assembly of rubella virus-like particles. Deletion or replacement of these domains with analogous regions from other type I membrane glycoproteins resulted in failure of rubella virus-like particles to be secreted from transfected cells. The E1 transmembrane and cytoplasmic domains were not required for targeting of the structural proteins to the Golgi complex and, surprisingly, assembly and budding of virus particles into the lumen of this organelle; however, the resultant particles were not secreted. In contrast, replacement or alteration of the E2 transmembrane or cytoplasmic domain, respectively, abrogated the targeting of the structural proteins to the budding site, and consequently, no virion formation was observed. These results indicate that the transmembrane and cytoplasmic domains of E2 and E1 are required for early and late steps respectively in the viral assembly pathway and that rubella virus morphogenesis is very different from that of the structurally similar alphaviruses.

scholarly journals Glycosphingolipids are required for sorting melanosomal proteins in the Golgi complex

2001 ◽  
Vol 155 (3) ◽  
pp. 369-380 ◽  
Author(s):  
Hein Sprong ◽  
Sophie Degroote ◽  
Tijs Claessens ◽  
Judith van Drunen ◽  
Viola Oorschot ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Protein Transport ◽  
Transmembrane Domain ◽  
Direct Pathway ◽  
Mouse Melanoma Cell ◽  
Multicellular Organisms ◽  
Rate Limiting ◽  
Tyrosinase Related Protein ◽  
Mouse Melanoma Cell Line

A;lthough glycosphingolipids are ubiquitously expressed and essential for multicellular organisms, surprisingly little is known about their intracellular functions. To explore the role of glycosphingolipids in membrane transport, we used the glycosphingolipid-deficient GM95 mouse melanoma cell line. We found that GM95 cells do not make melanin pigment because tyrosinase, the first and rate-limiting enzyme in melanin synthesis, was not targeted to melanosomes but accumulated in the Golgi complex. However, tyrosinase-related protein 1 still reached melanosomal structures via the plasma membrane instead of the direct pathway from the Golgi. Delivery of lysosomal enzymes from the Golgi complex to endosomes was normal, suggesting that this pathway is not affected by the absence of glycosphingolipids. Loss of pigmentation was due to tyrosinase mislocalization, since transfection of tyrosinase with an extended transmembrane domain, which bypassed the transport block, restored pigmentation. Transfection of ceramide glucosyltransferase or addition of glucosylsphingosine restored tyrosinase transport and pigmentation. We conclude that protein transport from Golgi to melanosomes via the direct pathway requires glycosphingolipids.

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

2004 ◽  
Vol 78 (22) ◽  
pp. 12277-12287 ◽  
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.

scholarly journals Role of Capsid Anchor in the Morphogenesis of Zika Virus

2018 ◽  
Vol 92 (22) ◽  
Author(s):  
Jyoti Rana ◽  
José Luis Slon Campos ◽  
Gabriella Leccese ◽  
Maura Francolini ◽  
Marco Bestagno ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Zika Virus ◽  
Protein C ◽  
Transmembrane Domain ◽  
Viral Envelope ◽  
Signal Peptidase ◽  
C Terminus ◽  
Cytosolic Side ◽  
The One

ABSTRACTThe flavivirus capsid protein (C) is separated from the downstream premembrane (PrM) protein by a hydrophobic sequence named capsid anchor (Ca). During polyprotein processing, Ca is sequentially cleaved by the viral NS2B/NS3 protease on the cytosolic side and by signal peptidase on the luminal side of the endoplasmic reticulum (ER). To date, Ca is considered important mostly for directing translocation of PrM into the ER lumen. In this study, the role of Ca in the assembly and secretion of Zika virus was investigated using a pseudovirus-based approach. Our results show that, while Ca-mediated anchoring of C to the ER membrane is not needed for the production of infective particles, Ca expression inciswith respect to PrM is strictly required to allow proper assembly of infectious particles. Finally, we show that the presence of heterologous, but not homologous, Ca induces degradation of E through the autophagy/lysosomal pathway.IMPORTANCEThe capsid anchor (Ca) is a single-pass transmembrane domain at the C terminus of the capsid protein (C) known to function as a signal for the translocation of PrM into the ER lumen. The objective of this study was to further examine the role of Ca in Zika virus life cycle, whether involved in the formation of nucleocapsid through association with C or in the formation of viral envelope. In this study, we show that Ca has a function beyond the one of translocation signal, controlling protein E stability and therefore its availability for assembly of infectious particles.

Cellular and molecular biology of HCV infection and hepatitis

2009 ◽  
Vol 117 (2) ◽  
pp. 49-65 ◽  
Author(s):  
Hengli Tang ◽  
Henry Grisé
Keyword(s):  
Hepatitis C ◽  
Rna Replication ◽  
Viral Proteins ◽  
The United States ◽  
Hcv Infection ◽  
Host Cells ◽  
Structural Proteins ◽  
Particle Assembly ◽  
Positive Strand Rna

HCV (hepatitis C virus) infects nearly 3% of the population worldwide and has emerged as a major causative agent of liver disease, resulting in acute and chronic infections that can lead to fibrosis, cirrhosis and hepatocellular carcinoma. Hepatitis C represents the leading cause of liver transplantation in the United States and Europe. A positive-strand RNA virus of the Flaviviridae family, HCV contains a single-stranded RNA genome of approx. 9600 nucleotides. The genome RNA serves as both mRNA for translation of viral proteins and the template for RNA replication. Cis-acting RNA elements within the genome regulate RNA replication by forming secondary structures that interact with each other and trans-acting factors. Although structural proteins are clearly dispensable for RNA replication, recent evidence points to an important role of several non-structural proteins in particle assembly and release, turning their designation on its head. HCV enters host cells through receptor-mediated endocytosis, and the process requires the co-ordination of multiple cellular receptors and co-receptors. RNA replication takes place at specialized intracellular membrane structures called ‘membranous webs’ or ‘membrane-associated foci’, whereas viral assembly probably occurs on lipid droplets and endoplasmic reticulum. Liver inflammation plays a central role in the liver damage seen in hepatitis C, but many HCV proteins also directly contribute to HCV pathogenesis. In the present review, the molecular and cellular aspects of the HCV life cycle and the role of viral proteins in pathological liver conditions caused by HCV infection are described.

scholarly journals Mutational Analysis, Using a Full-Length Rubella Virus cDNA Clone, of Rubella Virus E1 Transmembrane and Cytoplasmic Domains Required for Virus Release

1999 ◽  
Vol 73 (6) ◽  
pp. 4622-4630 ◽  
Author(s):  
Jiansheng Yao ◽  
Shirley Gillam
Keyword(s):  
Cdna Clone ◽  
Rubella Virus ◽  
Transmembrane Domain ◽  
Virus Assembly ◽  
Cytoplasmic Domain ◽  
Full Length ◽  
Virus Yield ◽  
Virus Release ◽  
Cytoplasmic Domains ◽  
Virus Budding

ABSTRACT We report on the construction of a full-length cDNA clone, pBRM33, derived from wild-type rubella virus M33 strain. The RNA transcripts synthesized in vitro from pBRM33 are highly infectious, and the viruses produced retain the phenotypic characteristics of the parental M33 virus in growth rate and plaque size. This cDNA clone was used to study the role of E1 transmembrane and cytoplasmic domains in virus assembly by site-directed mutagenesis. Three different alanine substitutions were introduced in the transmembrane domain of E1. These included substitution of leucine 464, cysteine 466, cysteine 467, and both cysteines 466 and 467 to alanine. In the E1 cytoplasmic domain, cysteine 470 and leucine 471 were altered to alanine. We found that these mutations did not significantly affect viral RNA replication, viral structural protein synthesis and transport, or E2/E1 heterodimer formation. Except for the substitution of cysteine 470, these mutations did, however, lead to a reduction in virus release. Substitution of cysteine 467 in the transmembrane region and of leucine 471 in the cytoplasmic domain dramatically reduced virus yield, resulting in the production of only 1 and 10% of the parental virus yield, respectively, in a parallel infection. These data show that E1 transmembrane and cytoplasmic domains play an important role in late stages of virus assembly, possibly during virus budding, consistent with earlier studies indicating that the E1 cytoplasmic domain may interact with nucleocapsids and that this interaction drives virus budding.

Assembly of Rubella Virus Structural Proteins into Virus-like Particles in Transfected Cells

Virology ◽  
1994 ◽  
Vol 202 (2) ◽  
pp. 574-585 ◽  
Author(s):  
Tom C. Hobman ◽  
Marita L. Lundstrom ◽  
Chris A. Mauracher ◽  
Luann Woodward ◽  
Shirley Gillam ◽  
...  
Keyword(s):  
Rubella Virus ◽  
Structural Proteins ◽  
Transfected Cells ◽  
Virus Like Particles

scholarly journals Gain, Preservation, and Loss of a Group 1a Coronavirus Accessory Glycoprotein

2008 ◽  
Vol 82 (20) ◽  
pp. 10312-10317 ◽  
Author(s):  
Alessio Lorusso ◽  
Nicola Decaro ◽  
Pepijn Schellen ◽  
Peter J. M. Rottier ◽  
Canio Buonavoglia ◽  
...  
Keyword(s):  
Evolutionary History ◽  
Rna Viruses ◽  
Structural Proteins ◽  
Type I ◽  
Accessory Gene ◽  
Multiple Group ◽  
Genetic Complexity ◽  
History Of ◽  
Positive Strand Rna ◽  
Insight Into

ABSTRACT Coronaviruses are positive-strand RNA viruses of extraordinary genetic complexity and diversity. In addition to a common set of genes for replicase and structural proteins, each coronavirus may carry multiple group-specific genes apparently acquired through relatively recent heterologous recombination events. Here we describe an accessory gene, ORF3, unique to canine coronavirus type I (CCoV-I) and characterize its product, glycoprotein gp3. Whereas ORF3 is conserved in CCoV-I, only remnants remain in CCoV-II and CCoV-II-derived porcine and feline coronaviruses. Our findings provide insight into the evolutionary history of coronavirus group 1a and into the dynamics of gain and loss of accessory genes.

scholarly journals The rubella virus E2 and E1 spike glycoproteins are targeted to the Golgi complex.

1993 ◽  
Vol 121 (2) ◽  
pp. 269-281 ◽  
Author(s):  
T C Hobman ◽  
L Woodward ◽  
M G Farquhar
Keyword(s):  
Membrane Proteins ◽  
Golgi Complex ◽  
Rubella Virus ◽  
Cell Types ◽  
Vero Cells ◽  
Type I ◽  
Surface Transport ◽  
Golgi Stack ◽  
Golgi Region ◽  
Different Cell Types

Rubella virus (RV) has been reported to bud from intracellular membranes in certain cell types. In this study the intracellular site of targeting of RV envelope E2 and E1 glycoproteins has been investigated in three different cell types (CHO, BHK-21 and Vero cells) transfected with a cDNA encoding the two glycoproteins. By indirect immunofluorescence, E2 and E1 were localized to the Golgi region of all three cell types, and their distribution was disrupted by treatment with BFA or nocodazole. Immunogold labeling demonstrated that E2 and E1 were localized to Golgi cisternae and indicated that the glycoproteins were distributed across the Golgi stack. Analysis of immunoprecipitates obtained from stably transfected CHO cells revealed that E2 and E1 become endo H resistant and undergo sialylation without being transported to the cell surface. Transport of RV glycoproteins to the Golgi complex was relatively slow (t1/2 = 60-90 min). Coprecipitation experiments indicated that E2 and E1 form a heterodimer in the RER. E1 was found to fold much more slowly than E2, suggesting that the delay in transport of the heterodimer to the Golgi may be due to the slow maturation of E1 in the ER. These results indicate that RV glycoproteins behave as integral membrane proteins of the Golgi complex and thus provide a useful model to study targeting and turnover of type I membrane proteins in this organelle.

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