scholarly journals Endoplasmic Reticulum-Golgi Intermediate Compartment Membranes and Vimentin Filaments Participate in Vaccinia Virus Assembly

2002 ◽  
Vol 76 (4) ◽  
pp. 1839-1855 ◽  
Author(s):  
Cristina Risco ◽  
Juan R. Rodríguez ◽  
Carmen López-Iglesias ◽  
José L. Carrascosa ◽  
Mariano Esteban ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Vaccinia Virus ◽  
Intermediate Filaments ◽  
Core Protein ◽  
Virus Assembly ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Intermediate Compartment ◽  
First Time ◽  
Vimentin Filaments

ABSTRACT Vaccinia virus (VV) has a complex morphogenetic pathway whose first steps are poorly characterized. We have studied the early phase of VV assembly, when viral factories and spherical immature viruses (IVs) form in the cytoplasm of the infected cell. After freeze-substitution numerous cellular elements are detected around assembling viruses: membranes, ribosomes, microtubules, filaments, and unidentified structures. A double membrane is clearly resolved in the VV envelope for the first time, and freeze fracture reveals groups of tubules interacting laterally on the surface of the viroplasm foci. These data strongly support the hypothesis of a cellular tubulovesicular compartment, related to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), as the origin of the first VV envelope. Moreover, the cytoskeletal vimentin intermediate filaments are found around viral factories and inside the viroplasm foci, where vimentin and the VV core protein p39 colocalize in the areas where crescents protrude. Confocal microscopy showed that ERGIC elements and vimentin filaments concentrate in the viral factories. We propose that modified cellular ERGIC membranes and vimentin intermediate filaments act coordinately in the construction of viral factories and the first VV form through a unique mechanism of viral morphogenesis from cellular elements.

scholarly journals Enigmatic origin of the poxvirus membrane from the endoplasmic reticulum shown by 3D imaging of vaccinia virus assembly mutants

2017 ◽  
Vol 114 (51) ◽  
pp. E11001-E11009 ◽  
Author(s):  
Andrea S. Weisberg ◽  
Liliana Maruri-Avidal ◽  
Himani Bisht ◽  
Bryan T. Hansen ◽  
Cindi L. Schwartz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Vaccinia Virus ◽  
Virus Assembly ◽  
Transmembrane Protein ◽  
Membrane Dynamics ◽  
Deletion Mutants ◽  
Viral Membrane ◽  
Transmission Electron

The long-standing inability to visualize connections between poxvirus membranes and cellular organelles has led to uncertainty regarding the origin of the viral membrane. Indeed, there has been speculation that viral membranes form de novo in cytoplasmic factories. Another possibility, that the connections are too short-lived to be captured by microscopy during a normal infection, motivated us to identify and characterize virus mutants that are arrested in assembly. Five conserved vaccinia virus proteins, referred to as Viral Membrane Assembly Proteins (VMAPs), that are necessary for formation of immature virions were found. Transmission electron microscopy studies of two VMAP deletion mutants had suggested retention of connections between viral membranes and the endoplasmic reticulum (ER). We now analyzed cells infected with each of the five VMAP deletion mutants by electron tomography, which is necessary to validate membrane continuity, in addition to conventional transmission electron microscopy. In all cases, connections between the ER and viral membranes were demonstrated by 3D reconstructions, supporting a role for the VMAPs in creating and/or stabilizing membrane scissions. Furthermore, coexpression of the viral reticulon-like transmembrane protein A17 and the capsid-like scaffold protein D13 was sufficient to form similar ER-associated viral structures in the absence of other major virion proteins. Determination of the mechanism of ER disruption during a normal VACV infection and the likely participation of both viral and cell proteins in this process may provide important insights into membrane dynamics.

scholarly journals Evidence against an Essential Role of COPII-Mediated Cargo Transport to the Endoplasmic Reticulum-Golgi Intermediate Compartment in the Formation of the Primary Membrane of Vaccinia Virus

2003 ◽  
Vol 77 (21) ◽  
pp. 11754-11766 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Vaccinia Virus ◽  
Essential Role ◽  
Recombinant Vaccinia Virus ◽  
Dominant Negative ◽  
Cargo Transport ◽  
Dominant Negative Form ◽  
Intermediate Compartment

ABSTRACT Vaccinia virus assembles two distinct lipoprotein membranes. The primary membrane contains nonglycosylated proteins, appears as crescents in the cytoplasm, and delimits immature and mature intracellular virions. The secondary or wrapping membrane contains glycoproteins, is derived from virus-modified trans-Golgi or endosomal cisternae, forms a loose coat around some intracellular mature virions, and becomes the envelope of extracellular virions. Although the mode of formation of the wrapping membrane is partially understood, we know less about the primary membrane. Recent reports posit that the primary membrane originates from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). According to this model, viral primary membrane proteins are cotranslationally inserted into the ER and accumulate in the ERGIC. To test the ERGIC model, we employed Sar1H79G, a dominant negative form of the Sar1 protein, which is an essential component of coatomer protein II (COPII)-mediated cargo transport from the ER to the ERGIC and other post-ER compartments. Overexpression of Sar1H79G by transfection or by a novel recombinant vaccinia virus with an inducible Sar1H79G gene resulted in retention of ERGIC 53 in the ER but did not interfere with localization of viral primary membrane proteins in factory regions or with formation of viral crescent membranes and infectious intracellular mature virions. Wrapping of intracellular mature virions and formation of extracellular virions did not occur, however, because some proteins that are essential for the secondary membrane were retained in the ER as a consequence of Sar1H79G overexpression. Our data argue against an essential role of COPII-mediated cargo transport and the ERGIC in the formation of the viral primary membrane. Instead, viral membranes may be derived directly from the ER or by a novel mechanism.

scholarly journals Avipoxvirus phylogenetics: identification of a PCR length polymorphism that discriminates between the two major clades

2006 ◽  
Vol 87 (8) ◽  
pp. 2191-2201 ◽  
Author(s):  
Susan Jarmin ◽  
Ruth Manvell ◽  
Richard E. Gough ◽  
Stephen M. Laidlaw ◽  
Michael A. Skinner
Keyword(s):  
Vaccinia Virus ◽  
Core Protein ◽  
Sequence Data ◽  
Fragment Size ◽  
Taxonomic Structure ◽  
Avian Host ◽  
Virus Core ◽  
Phylogenetic Studies ◽  
Tight Correlation ◽  
First Time

Avipoxvirus infections have been observed in an extensive range of wild, captive and domesticated avian hosts, yet little is known about the genome diversity and host-range specificity of the causative agent(s). Genome-sequence data are largely restricted to Fowlpox virus (FWPV) and Canarypox virus (CNPV), which have been sequenced completely, showing considerable divergence between them. It is therefore proving difficult, by empirical approaches, to identify pan-genus, avipoxvirus-specific oligonucleotide probes for PCR and sequencing to support phylogenetic studies. A previous preliminary study used the fpv167 locus, which encodes orthologues of vaccinia virus core protein P4b (A3). PCR per se did not discriminate between viruses, but restriction-enzyme or sequence analysis indicated that the avipoxviruses clustered either with FWPV or with CNPV. Here, further study of the P4b locus demonstrated a third cluster, from psittacine birds. A newly identified locus, flanking fpv140 (orthologue of vaccinia virus H3L), confirms the taxonomic structure. This locus is particularly useful in that viruses from the fowlpox-like and canarypox-like clusters can be discriminated by PCR on the basis of fragment size, whilst sequence comparison allows discrimination for the first time between Pigeonpox virus and Turkeypox virus. Except within the psittacines, virus and avian host taxonomies do not show tight correlation, with viruses from the same species located in very different clades. Nor are all the existing recognized avipoxvirus species, defined primarily by avian host species (such as CNPV and Sparrowpox virus), resolved within the present structure.

scholarly journals Differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins S, M and E

2005 ◽  
Vol 86 (5) ◽  
pp. 1423-1434 ◽  
Author(s):  
Béatrice Nal ◽  
Cheman Chan ◽  
Francois Kien ◽  
Lewis Siu ◽  
Jane Tse ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Severe Acute Respiratory Syndrome ◽  
Subcellular Localization ◽  
Secretory Pathway ◽  
Virus Assembly ◽  
Protein Translation ◽  
Surface Proteins ◽  
E Protein ◽  
Intermediate Compartment ◽  
Post Entry

Post-translational modifications and correct subcellular localization of viral structural proteins are prerequisites for assembly and budding of enveloped viruses. Coronaviruses, like the severe acute respiratory syndrome-associated virus (SARS-CoV), bud from the endoplasmic reticulum-Golgi intermediate compartment. In this study, the subcellular distribution and maturation of SARS-CoV surface proteins S, M and E were analysed by using C-terminally tagged proteins. As early as 30 min post-entry into the endoplasmic reticulum, high-mannosylated S assembles into trimers prior to acquisition of complex N-glycans in the Golgi. Like S, M acquires high-mannose N-glycans that are subsequently modified into complex N-glycans in the Golgi. The N-glycosylation profile and the absence of O-glycosylation on M protein relate SARS-CoV to the previously described group 1 and 3 coronaviruses. Immunofluorescence analysis shows that S is detected in several compartments along the secretory pathway from the endoplasmic reticulum to the plasma membrane while M predominantly localizes in the Golgi, where it accumulates, and in trafficking vesicles. The E protein is not glycosylated. Pulse-chase labelling and confocal microscopy in the presence of protein translation inhibitor cycloheximide revealed that the E protein has a short half-life of 30 min. E protein is found in bright perinuclear patches colocalizing with endoplasmic reticulum markers. In conclusion, SARS-CoV surface proteins S, M and E show differential subcellular localizations when expressed alone suggesting that additional cellular or viral factors might be required for coordinated trafficking to the virus assembly site in the endoplasmic reticulum-Golgi intermediate compartment.

scholarly journals Adaptive mutations promote hepatitis C virus assembly by accelerating Core translocation to the endoplasmic reticulum

2020 ◽  
pp. jbc.RA120.016010
Author(s):  
Fuxiang Zheng ◽  
Ni Li ◽  
Yi Xu ◽  
Yuanping Zhou ◽  
Yi-Ping Li
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Virus Assembly ◽  
Buoyant Density ◽  
Virus Production ◽  
Adaptive Mutations ◽  
Huh7 Cells ◽  
Density Analysis

The envelopment of hepatitis C virus (HCV) is believed to occur primarily in the endoplasmic reticulum (ER)-associated membrane, and the translocation of viral Core protein from lipid droplets (LDs) to the ER is essential for the envelopment of viral particles. However, the factors involved in are not completely understood. Herein, we identified eight adaptive mutations that enhanced virus spread and infectivity of genotype 1a clone TNcc in hepatoma Huh7 cells through long-term culture adaptation and reverse genetic study. Of eight mutations, I853V in NS2 and C2865F in NS5B were found to be minimal mutation sets that enabled an increase in virus production without apparently affecting RNA replication, thus suggesting its roles in the post-replication stage of the HCV life cycle. Using a protease K protection and confocal microscopy analysis, we demonstrated that C2865F and the combination of I853V/C2865F enhanced virus envelopment by facilitating Core translocation from LDs to the ER. Buoyant density analysis revealed that I853V/C2865F contributed to the release of virion with a density of ~1.10 g/ml. Moreover, we demonstrated that NS5B directly interacted with NS2 at the protease domain, and that mutations I853V, C2865F, I853V/C2865F enhanced the interaction. In addition, C2865F also enhanced the interaction between NS5B and Core. In conclusion, this study demonstrated that adaptive mutations in NS2 and NS5B promoted HCV envelopment by accelerating Core translocation from LDs to the ER and reinforced the interaction between NS2 and NS5B. The findings facilitate our understanding of the assembly of HCV morphogenesis.

Scanning electron microscopy of the septal pore cap of the basidiomycete Schizophyllum commune

1994 ◽  
Vol 40 (10) ◽  
pp. 879-883 ◽  
Author(s):  
Wally H. Müller ◽  
Adriaan C. van Aelst ◽  
Theo P. van der Krift ◽  
Teun Boekhout
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Schizophyllum Commune ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Transmission Electron ◽  
And Function ◽  
Tubular Endoplasmic Reticulum ◽  
Scanning Electron

As part of a comparative study of the structure and function of pore structures in heterobasidiomycetous yeasts, dikaryotic hyphae of Schizophyllum commune were subjected to chemical fixation, freeze fracturing, maceration, and freeze substitution, and were subsequently prepared for scanning electron microscopy. The interior of the hyphal cell was visualized and revealed the perforated septal pore cap or parenthesome, mitochondria, vacuoles, and tubular endoplasmic reticulum. The septal pore cap showed connections with tubular endoplasmic reticulum. This tubular endoplasmic reticulum covered the dolipore septal surface. The results presented here complement and extend the ultrastructural image of the septal pore cap obtained from transmission electron micrographs.Key words: septal pore cap, Schizophyllum commune, freeze fracture, maceration, scanning electron microscopy.

scholarly journals The Cytoplasmic Tail of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein Contains a Novel Endoplasmic Reticulum Retrieval Signal That Binds COPI and Promotes Interaction with Membrane Protein

2006 ◽  
Vol 81 (5) ◽  
pp. 2418-2428 ◽  
Author(s):  
Corrin E. McBride ◽  
Jie Li ◽  
Carolyn E. Machamer
Keyword(s):  
Endoplasmic Reticulum ◽  
Severe Acute Respiratory Syndrome ◽  
Virus Assembly ◽  
Cytoplasmic Tail ◽  
Viral Envelope ◽  
S Protein ◽  
Golgi Region ◽  
Vitro Binding ◽  
Intermediate Compartment

ABSTRACT Like other coronaviruses, severe acute respiratory syndrome coronavirus (SARS CoV) assembles at and buds into the lumen of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC). Accumulation of the viral envelope proteins at this compartment is a prerequisite for virus assembly. Previously, we reported the identification of a dibasic motif (KxHxx) in the cytoplasmic tail of the SARS CoV spike (S) protein that was similar to a canonical dilysine ER retrieval signal. Here we demonstrate that this motif is a novel and functional ER retrieval signal which reduced the rate of traffic of the full-length S protein through the Golgi complex. The KxHxx motif also partially retained two different reporter proteins in the ERGIC region and reduced their rates of trafficking, although the motif was less potent than the canonical dilysine signal. The dibasic motif bound the coatomer complex I (COPI) in an in vitro binding assay, suggesting that ER retrieval may contribute to the accumulation of SARS CoV S protein near the virus assembly site for interaction with other viral structural proteins. In support of this, we found that the dibasic motif on the SARS S protein was required for its localization to the ERGIC/Golgi region when coexpressed with SARS membrane (M) protein. Thus, the cycling of SARS S through the ER-Golgi system may be required for its incorporation into assembling virions in the ERGIC.

scholarly journals Role of the I7 Protein in Proteolytic Processing of Vaccinia Virus Membrane and Core Components

2004 ◽  
Vol 78 (12) ◽  
pp. 6335-6343 ◽  
Author(s):  
Camilo Ansarah-Sobrinho ◽  
Bernard Moss
Keyword(s):  
Membrane Protein ◽  
Vaccinia Virus ◽  
Core Protein ◽  
Virus Assembly ◽  
Proteolytic Processing ◽  
Temperature Sensitive ◽  
Lethal Mutant ◽  
Core Proteins ◽  
Core Components

ABSTRACT Certain core and membrane proteins of vaccinia virus undergo proteolytic cleavage at consensus AG/X sites. The processing of core proteins is coupled to morphogenesis and is inhibited by the drug rifampin, whereas processing of the A17 membrane protein occurs at an earlier stage of assembly and is unaffected by the drug. A temperature-sensitive mutant with a lesion in the I7L gene exhibits blocks in morphogenesis and in cleavage of core proteins. We found that the mutant also failed to cleave the A17 membrane protein. To further investigate the role of the putative I7 protease, we constructed a conditional lethal mutant in which the I7L gene was regulated by the Escherichia coli lac repressor. In the absence of an inducer, the synthesis of I7 was repressed, proteolytic processing of the A17 membrane protein and the L4 core protein was inhibited, and virus morphogenesis was blocked. Under these conditions, expression of the wild-type I7 protein in trans restored protein processing. In contrast, rescue did not occur when the putative protease active site residue histidine 241 or cysteine 328 of I7 was converted to alanine. The mutation of an authentic AG/A and an alternative AG/S motif of L4 prevented substrate cleavage. Similarly, when AG/X sites of A17 were mutated, I7-induced cleavages at the N and C termini failed to occur. In conclusion, we provide evidence that I7 is a viral protease that is required for AG/X-specific cleavages of viral membrane and core proteins, which occur at early and late stages of virus assembly, respectively.

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