Assembly of vaccinia virus: incorporation of p14 and p32 into the membrane of the intracellular mature virus.

The simpler of the two infectious forms of vaccinia virus, the intracellular mature virus (IMV) is known to infect cells less efficiently than the extracellular enveloped virus (EEV), which is surrounded by an additional, TGN-derived membrane. We show here that when the IMV binds HeLa cells, it activates a signaling cascade that is regulated by the GTPase rac1 and rhoA, ezrin, and both tyrosine and protein kinase C phosphorylation. These cascades are linked to the formation of actin and ezrin containing protrusions at the plasma membrane that seem to be essential for the entry of IMV cores. The identical cores of the EEV also appear to enter at the cell surface, but surprisingly, without the need for signaling and actin/membrane rearrangements. Thus, in addition to its known role in wrapping the IMV and the formation of intracellular actin comets, the membrane of the EEV seems to have evolved the capacity to enter cells silently, without a need for signaling.

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The vaccinia virus F12L protein is associated with intracellular enveloped virus particles and is required for their egress to the cell surface

Journal of General Virology ◽

10.1099/0022-1317-83-1-195 ◽

2002 ◽

Vol 83 (1) ◽

pp. 195-207 ◽

Cited By ~ 73

Author(s):

Henriette van Eijl ◽

Michael Hollinshead ◽

Gaener Rodger ◽

Wei-Hong Zhang ◽

Geoffrey L. Smith

Keyword(s):

Cell Surface ◽

Vaccinia Virus ◽

Virus Particles ◽

Enveloped Virus ◽

Virus Morphogenesis ◽

C Terminus ◽

Specific Proteins ◽

Infected Cells ◽

Mature Virus ◽

Confocal And Electron Microscopy

The vaccinia virus (VV) F12L gene encodes a 65 kDa protein that is expressed late during infection and is important for plaque formation, EEV production and virulence. Here we have used a recombinant virus (vF12LHA) in which the F12L protein is tagged at the C terminus with an epitope recognized by a monoclonal antibody to determine the location of F12L in infected cells and whether it associates with virions. Using confocal and electron microscopy we show that the F12L protein is located on intracellular enveloped virus (IEV) particles, but is absent from immature virions (IV), intracellular mature virus (IMV) and cell-associated enveloped virus (CEV). In addition, F12L shows co-localization with endosomal compartments and microtubules. F12L did not co-localize with virions attached to actin tails, providing further evidence that actin tails are associated with CEV but not IEV particles. In vΔF12L-infected cells, virus morphogenesis was arrested after the formation of IEV particles, so that the movement of these virions to the cell surface was inhibited and CEV particles were not found. Previously, virus mutants lacking IEV- or EEV-specific proteins were either unable to make IEV particles (vΔF13L and vΔB5R), or were unable to form actin tails after formation of CEV particles (vΔA36R, vΔA33R, vΔA34R). The F12L deletion mutant therefore defines a new stage in the morphogenic pathway and the F12L protein is implicated as necessary for microtubule-mediated egress of IEV particles to the cell surface.

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Structure and Assembly of Intracellular Mature Vaccinia Virus: Isolated-Particle Analysis

Journal of Virology ◽

10.1128/jvi.75.22.11034-11055.2001 ◽

2001 ◽

Vol 75 (22) ◽

pp. 11034-11055 ◽

Cited By ~ 48

Author(s):

Gareth Griffiths ◽

Roger Wepf ◽

Thomas Wendt ◽

Jacomine Krijnse Locker ◽

Marek Cyrklaff ◽

...

Keyword(s):

Electron Microscopy ◽

Endoplasmic Reticulum ◽

Vaccinia Virus ◽

Viral Entry ◽

Particle Analysis ◽

Assembly Process ◽

Novel Approach ◽

Complex Topology ◽

Scanning Em ◽

Mature Virus

ABSTRACT In a series of papers, we have provided evidence that during its assembly vaccinia virus is enveloped by a membrane cisterna that originates from a specialized, virally modified, smooth-membraned domain of the endoplasmic reticulum (ER). Recently, however, Hollinshead et al. (M. Hollinshead, A. Vanderplasschen, G. I. Smith, and D. J. Vaux, J. Virol. 73:1503–1517, 1999) argued against this hypothesis, based on their interpretations of thin-sectioned material. The present article is the first in a series of papers that describe a comprehensive electron microscopy (EM) analysis of the vaccinia Intracellular Mature Virus (IMV) and the process of its assembly in HeLa cells. In this first study, we analyzed the IMV by on-grid staining, cryo-scanning EM (SEM), and cryo-transmission EM. We focused on the structure of the IMV particle, both after isolation and in the context of viral entry. For the latter, we used high-resolution cryo-SEM combined with cryofixation, as well as a novel approach we developed for investigating vaccinia IMV bound to plasma membrane fragments adsorbed onto EM grids. Our analysis revealed that the IMV is made up of interconnected cisternal and tubular domains that fold upon themselves via a complex topology that includes an S-shaped fold. The viral tubules appear to be eviscerated from the particle during viral infection. Since the structure of the IMV is the result of a complex assembly process, we also provide a working model to explain how a specialized smooth-ER domain can be modulated to form the IMV. We also present theoretical arguments for why it is highly unlikely that the IMV is surrounded by only a single membrane.

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Protein B5 is required on extracellular enveloped vaccinia virus for repulsion of superinfecting virions

Journal of General Virology ◽

10.1099/vir.0.043943-0 ◽

2012 ◽

Vol 93 (9) ◽

pp. 1876-1886 ◽

Cited By ~ 21

Author(s):

Virginie Doceul ◽

Michael Hollinshead ◽

Adrien Breiman ◽

Kathlyn Laval ◽

Geoffrey L. Smith

Keyword(s):

Vaccinia Virus ◽

Actin Polymerization ◽

Protein S ◽

Viral Proteins ◽

Plaque Size ◽

Cell Monolayers ◽

Enveloped Virus ◽

Short Consensus Repeat ◽

Infected Cells ◽

Mature Virus

Vaccinia virus (VACV) spreads across cell monolayers fourfold faster than predicted from its replication kinetics. Early after infection, infected cells repulse some superinfecting extracellular enveloped virus (EEV) particles by the formation of actin tails from the cell surface, thereby causing accelerated spread to uninfected cells. This strategy requires the expression of two viral proteins, A33 and A36, on the surface of infected cells and upon contact with EEV this complex induces actin polymerization. Here we have studied this phenomenon further and investigated whether A33 and A36 expression in cell lines causes an increase in VACV plaque size, whether these proteins are able to block superinfection by EEV, and which protein(s) on the EEV surface are required to initiate the formation of actin tails from infected cells. Data presented show that VACV plaque size was not increased by expression of A33 and A36, and these proteins did not block entry of the majority of EEV binding to these cells. In contrast, expression of proteins A56 and K2 inhibited entry of both EEV and intracellular mature virus. Lastly, VACV protein B5 was required on EEV to induce the formation of actin tails at the surface of cells expressing A33 and A36, and B5 short consensus repeat 4 is critical for this induction.

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Vaccinia Virus WR53.5/F14.5 Protein Is a New Component of Intracellular Mature Virus and Is Important for Calcium-Independent Cell Adhesion and Vaccinia Virus Virulence in Mice

Journal of Virology ◽

10.1128/jvi.00816-08 ◽

2008 ◽

Vol 82 (20) ◽

pp. 10079-10087 ◽

Cited By ~ 10

Author(s):

Roza Izmailyan ◽

Wen Chang

Keyword(s):

Cell Adhesion ◽

Vaccinia Virus ◽

Transmembrane Protein ◽

Recombinant Vaccinia Virus ◽

Terminal Region ◽

Virus Growth ◽

C Terminus ◽

Infected Cells ◽

Mature Virus

ABSTRACT The vaccinia virus WR53.5L/F14.5L gene encodes a small conserved protein that was not detected previously. However, additional proteomic analyses of different vaccinia virus isolates and strains revealed that the WR53.5 protein was incorporated into intracellular mature virus (IMV). The WR53.5 protein contains a putative N-terminal transmembrane region and a short C-terminal region. Protease digestion removed the C terminus of WR53.5 protein from IMV particles, suggesting a similar topology to that of the IMV type II transmembrane protein. We generated a recombinant vaccinia virus, vi53.5L, that expressed WR53.5 protein under isopropyl-β-d-thiogalactopyranoside (IPTG) regulation and found that the vaccinia virus life cycle proceeded normally with or without IPTG, suggesting that WR53.5 protein is not essential for vaccinia virus growth in cell cultures. Interestingly, the C-terminal region of WR53.5 protein was exposed on the cell surface of infected cells and mediated calcium-independent cell adhesion. Finally, viruses with inactivated WR53.5L gene expression exhibited reduced virulence in mice when animals were inoculated intranasally, demonstrating that WR53.5 protein was required for virus virulence in vivo. In summary, we identified a new vaccinia IMV envelope protein, WR53.5, that mediates cell adhesion and is important for virus virulence in vivo.

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The vaccinia virus 4c and A-type inclusion proteins are specific markers for the intracellular mature virus particle.

Journal of Virology ◽

10.1128/jvi.70.6.3372-3377.1996 ◽

1996 ◽

Vol 70 (6) ◽

pp. 3372-3377 ◽

Cited By ~ 37

Author(s):

D Ulaeto ◽

D Grosenbach ◽

D E Hruby

Keyword(s):

Vaccinia Virus ◽

Virus Particle ◽

Mature Virus Particle ◽

Type Inclusion ◽

Mature Virus

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Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

Journal of Virology ◽

10.1128/jvi.00209-15 ◽

2015 ◽

Vol 89 (16) ◽

pp. 8365-8382 ◽

Cited By ~ 15

Author(s):

Jye-Chian Hsiao ◽

Li-Wei Chu ◽

Yung-Tsun Lo ◽

Sue-Ping Lee ◽

Tzu-Jung Chen ◽

...

Keyword(s):

Vaccinia Virus ◽

Membrane Fusion ◽

Hela Cells ◽

Fluid Phase ◽

Content Type ◽

Virus Core ◽

Early Endosomes ◽

Fluid Phase Endocytosis ◽

Mature Virus ◽

In Cells

ABSTRACTVaccinia virus, the prototype of theOrthopoxvirusgenus in the familyPoxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm.IMPORTANCEVaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex executes endosomal membrane fission and cargo sorting to the Rab11-positive and Rab22-positive recycling pathway, resulting in membrane fusion and virus core uncoating in the cytoplasm.

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The Block in Assembly of Modified Vaccinia Virus Ankara in HeLa Cells Reveals New Insights into Vaccinia Virus Morphogenesis

Journal of Virology ◽

10.1128/jvi.76.16.8318-8334.2002 ◽

2002 ◽

Vol 76 (16) ◽

pp. 8318-8334 ◽

Cited By ~ 40

Author(s):

M. Carmen Sancho ◽

Sibylle Schleich ◽

Gareth Griffiths ◽

Jacomine Krijnse-Locker

Keyword(s):

Vaccinia Virus ◽

Hela Cells ◽

Immunofluorescence Microscopy ◽

Close Association ◽

Viral Dna ◽

Virus Morphogenesis ◽

Modified Vaccinia Virus Ankara ◽

Replication Sites ◽

Mature Virus ◽

H 30

ABSTRACT It has previously been shown that upon infection of HeLa cells with modified vaccinia virus Ankara (MVA), assembly is blocked at a late stage of infection and immature virions (IVs) accumulate (G. Sutter and B. Moss, Proc. Natl. Acad. Sci. USA 89:10847-10851, 1992). In the present study the morphogenesis of MVA in HeLa cells was studied in more detail and compared to that under two conditions that permit the production of infectious particles: infection of HeLa cells with the WR strain of vaccinia virus (VV) and infection of BHK cells with MVA. Using several quantitative and qualitative assays, we show that early in infection, MVA in HeLa cells behaves in a manner identical to that under the permissive conditions. By immunofluorescence microscopy (IF) at late times of infection, the labelings for an abundant membrane protein of the intracellular mature virus, p16/A14L, and the viral DNA colocalize under permissive conditions, whereas in HeLa cells infected with MVA these two structures do not colocalize to the same extent. In both permissive and nonpermissive infection, p16-labeled IVs first appear at 5 h postinfection. In HeLa cells infected with MVA, IVs accumulated predominantly outside the DNA regions, whereas under permissive conditions they were associated with the viral DNA. At 4 h 30 min, the earliest time at which p16 is detected, the p16 labeling was found predominantly in a small number of distinct puncta by IF, which were distinct from the sites of DNA in both permissive and nonpermissive infection. By electron microscopy, no crescents or IVs were found at this time, and the p16-labeled structures were found to consist of membrane-rich vesicles that were in continuity with the cellular endoplasmic reticulum. Over the next 30 min of infection, a large number of p16-labeled crescents and IVs appeared abruptly under both permissive and nonpermissive conditions. Under permissive conditions, these IVs were in close association with the sites of DNA, and a significant amount of these IVs engulfed the viral DNA. In contrast, under nonpermissive conditions, the IVs and DNA were mostly in separate locations and relatively few IVs acquired DNA. Our data show that in HeLa cells MVA forms normal DNA replication sites and normal viral precursor membranes but the transport between these two structures is inhibited.

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Entry of the vaccinia virus intracellular mature virion and its interactions with glycosaminoglycans

Journal of General Virology ◽

10.1099/vir.0.80831-0 ◽

2005 ◽

Vol 86 (5) ◽

pp. 1279-1290 ◽

Cited By ~ 104

Author(s):

Gemma C. Carter ◽

Mansun Law ◽

Michael Hollinshead ◽

Geoffrey L. Smith

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Vaccinia Virus ◽

Lipid Membrane ◽

Surface Proteins ◽

Enveloped Virus ◽

Virus Core ◽

Cell Type Specific ◽

Mature Virus ◽

Entry Mechanism

Vaccinia virus (VACV) produces two distinct enveloped virions, the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV), but the entry mechanism of neither virion is understood. Here, the binding and entry of IMV particles have been investigated. The cell receptors for IMV are unknown, but it was proposed that IMV can bind to glycosaminoglycans (GAGs) on the cell surface and three IMV surface proteins have been implicated in this. In this study, the effect of soluble GAGs on IMV infectivity was reinvestigated and it was demonstrated that GAGs affected IMV infectivity partially in some cells, but not at all in others. Therefore, binding of IMV to GAGs is cell type-specific and not essential for IMV entry. By using electron microscopy, it is demonstrated that IMV from strains Western Reserve and modified virus Ankara enter cells by fusion with the plasma membrane. After an IMV particle bound to the cell, the IMV membrane fused with the plasma membrane and released the virus core into the cytoplasm. IMV surface antigen became incorporated into the plasma membrane and was not left outside the cell, as claimed in previous studies. Continuity between the IMV membrane and the plasma membrane was confirmed by tilt-series analysis to orientate membranes perpendicularly to the beam of the electron microscope. This analysis shows unequivocally that IMV is surrounded by a single lipid membrane and enters by fusion at the cell surface.

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Antibody-sensitive and antibody-resistant cell-to-cell spread by vaccinia virus: role of the A33R protein in antibody-resistant spread

Journal of General Virology ◽

10.1099/0022-1317-83-1-209 ◽

2002 ◽

Vol 83 (1) ◽

pp. 209-222 ◽

Cited By ~ 58

Author(s):

Mansun Law ◽

Ruth Hollinshead ◽

Geoffrey L. Smith

Keyword(s):

Vaccinia Virus ◽

Neutralizing Antibodies ◽

Resistant Cell ◽

Enveloped Virus ◽

Associated Proteins ◽

Specific Proteins ◽

Range Spread ◽

Mature Virus ◽

Cell Spread

The roles of vaccinia virus (VV) intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV) and their associated proteins in virus spread were investigated. The plaques made by VV mutants lacking individual IEV- or EEV-specific proteins (vΔA33R, vΔA34R, vΔA36R, vΔA56R, vΔB5R, vΔF12L and vΔF13L) were compared in the presence of IMV- or EEV-neutralizing antibodies (Ab). Data presented show that for long-range spread, the comet-shaped plaques of VV were caused by the unidirectional spread of EEV probably by convection currents, and for cell-to-cell spread, VV uses a combination of Ab-resistant and Ab-sensitive pathways. Actin tails play a major role in the Ab-resistant pathway, but mutants such as vΔA34R and vΔA36R that do not make actin tails still spread from cell to cell in the presence of Ab. Most strikingly, the Ab-resistant pathway was abolished when the A33R gene was deleted. This effect was not due to alterations in the efficiency of neutralization of EEV made by this mutant, nor due to a deficiency in IMV wrapping to form IEV, which was indispensable for EEV formation by vΔA33R and vΔA34R. We suggest a role for A33R in promoting Ab-resistant cell-to-cell spread of virus. The roles of the different virus forms in the VV life-cycle are discussed.

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