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

2002 ◽  
Vol 83 (1) ◽  
pp. 195-207 ◽  
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.

scholarly journals Entry of the Two Infectious Forms of Vaccinia Virus at the Plasma Membane Is Signaling-Dependent for the IMV but Not the EEV

2000 ◽  
Vol 11 (7) ◽  
pp. 2497-2511 ◽  
Author(s):  
Jacomine Krijnse Locker ◽  
Annett Kuehn ◽  
Sibylle Schleich ◽  
Gaby Rutter ◽  
Heinrich Hohenberg ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Vaccinia Virus ◽  
Hela Cells ◽  
Signaling Cascade ◽  
Enveloped Virus ◽  
Kinase C ◽  
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.

scholarly journals Primary Envelopment of Pseudorabies Virus at the Nuclear Membrane Requires the UL34 Gene Product

2000 ◽  
Vol 74 (21) ◽  
pp. 10063-10073 ◽  
Author(s):  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Thomas C. Mettenleiter
Keyword(s):  
Gene Product ◽  
Nuclear Membrane ◽  
Pseudorabies Virus ◽  
Tegument Protein ◽  
Outer Nuclear Membrane ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Perinuclear Space ◽  
Infected Cells ◽  
Mature Virus

ABSTRACT Primary envelopment of several herpesviruses has been shown to occur by budding of intranuclear capsids through the inner nuclear membrane. By subsequent fusion of the primary envelope with the outer nuclear membrane, capsids are released into the cytoplasm and gain their final envelope by budding into vesicles in thetrans-Golgi area. We show here that the product of the UL34 gene of pseudorabies virus, an alphaherpesvirus of swine, is localized in transfected and infected cells in the nuclear membrane. It is also detected in the envelope of virions in the perinuclear space but is undetectable in intracytoplasmic and extracellular enveloped virus particles. Conversely, the tegument protein UL49 is present in mature virus particles and absent from perinuclear virions. In the absence of the UL34 protein, acquisition of the primary envelope is blocked and neither virus particles in the perinuclear space nor intracytoplasmic capsids or virions are observed. However, light particles which label with the anti-UL49 serum are formed in the cytoplasm. We conclude that the UL34 protein is required for primary envelopment, that the primary envelope is biochemically different from the final envelope in that it contains the UL34 protein, and that perinuclear virions lack the tegument protein UL49, which is present in mature virions. Thus, we provide additional evidence for a two-step envelopment process in herpesviruses.

scholarly journals An investigation of incorporation of cellular antigens into vaccinia virus particles

2002 ◽  
Vol 83 (10) ◽  
pp. 2347-2359 ◽  
Author(s):  
Oliver Krauss ◽  
Ruth Hollinshead ◽  
Michael Hollinshead ◽  
Geoffrey L. Smith
Keyword(s):  
Vaccinia Virus ◽  
Specific Cell ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Membrane Compartments ◽  
Specific Proteins ◽  
Intermediate Compartment ◽  
Low Levels ◽  
Golgi Network ◽  
Exocytic Pathway

Vaccinia virus (VV) infection produces several types of virus particle called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). Some cellular antigens are associated with EEV and these vary with the cell type used to grow the virus. To investigate if specific cell antigens are associated with VV particles, and to address the origin of membranes used to envelope IMV and IEV/CEV/EEV, we have studied whether cell antigens and foreign antigens expressed by recombinant VVs are incorporated into VV particles. Membrane proteins that are incorporated into the endoplasmic reticulum (ER), intermediate compartment (IC), cis/medial-Golgi, trans-Golgi network (TGN) or plasma membrane were not detected in purified IMV particles. In contrast, proteins present in the TGN or membrane compartments further downstream in the exocytic pathway co-purify with EEV particles when analysed by immunoblotting. Immunoelectron microscopy found only low levels of these proteins in IEV, CEV/EEV. The incorporation of foreign antigens into VV particles was not affected by loss of individual IEV or EEV-specific proteins or by redirection of B5R to the ER. These data suggest that (i) host cell antigens are excluded from the lipid envelope surrounding the IMV particle and (ii) membranes of the ER, IC and cis/medial-Golgi are not used to wrap IMV particles to form IEV. Lastly, the VV haemagglutinin was absent from one-third of IEV and CEV/EEV particles, whereas other EEV antigens were present in all these virions.

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

2012 ◽  
Vol 93 (9) ◽  
pp. 1876-1886 ◽  
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.

scholarly journals 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

2008 ◽  
Vol 82 (20) ◽  
pp. 10079-10087 ◽  
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.

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

2005 ◽  
Vol 86 (5) ◽  
pp. 1279-1290 ◽  
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.

scholarly journals Vaccinia Virus A34 Glycoprotein Determines the Protein Composition of the Extracellular Virus Envelope

2007 ◽  
Vol 82 (5) ◽  
pp. 2150-2160 ◽  
Author(s):  
Beatriz Perdiguero ◽  
María M. Lorenzo ◽  
Rafael Blasco
Keyword(s):  
Vaccinia Virus ◽  
Protein Composition ◽  
Recombinant Vaccinia Virus ◽  
Virus Envelope ◽  
Enveloped Virus ◽  
Extracellular Virus ◽  
Transmembrane Glycoprotein ◽  
C Terminus ◽  
Vaccinia Viruses ◽  
Infected Cells

ABSTRACT The outer envelope of the extracellular form of vaccinia virus contains five virus-encoded proteins, F13, A33, A34, A56, and B5, that, with the exception of A56, are implicated in virus egress or infectivity. A34, a type II transmembrane glycoprotein, is involved in the induction of actin tails, the release of enveloped virus from the surfaces of infected cells, and the disruption of the virus envelope after ligand binding prior to virus entry. To investigate interactions between A34 and other envelope proteins, a recombinant vaccinia virus (vA34RHA) expressing an epitope-tagged version of A34 (A34HA) was constructed by appending an epitope from influenza virus hemagglutinin to the C terminus of A34. Complexes of A34HA with B5 and A36, but not with A33 or F13, were detected in vA34RHA-infected cells. A series of vaccinia viruses expressing mutated versions of the B5 protein was used to investigate the domain(s) of B5 required for interaction with A34. Both the cytoplasmic and the transmembrane domains of B5 were dispensable for binding to A34. Most of the extracellular domain of B5, which contains four short consensus repeats homologous to complement control proteins, was sufficient for A34 interaction, indicating that both proteins interact through their ectodomains. Immunofluorescence experiments on cells infected with A34-deficient virus indicated that A34 is required for efficient targeting of B5, A36, and A33 into wrapped virions. Consistent with this observation, the envelope of A34-deficient virus contained normal amounts of F13 but decreased amounts of A33 and B5 with respect to the parental WR virus. These results point to A34 as a major determinant in the protein composition of the vaccinia virus envelope.

scholarly journals The Vaccinia Virus A14.5L Gene Encodes a Hydrophobic 53-Amino-Acid Virion Membrane Protein That Enhances Virulence in Mice and Is Conserved among Vertebrate Poxviruses

2000 ◽  
Vol 74 (9) ◽  
pp. 4085-4092 ◽  
Author(s):  
Tatiana Betakova ◽  
Elizabeth J. Wolffe ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Recombinant Vaccinia Virus ◽  
Mutant Virus ◽  
Open Reading Frames ◽  
Epitope Tag ◽  
Virus Particles ◽  
C Terminus ◽  
Nonionic Detergent ◽  
Infected Cells ◽  
Triton X

ABSTRACT A short sequence, located between the A14L and A15L open reading frames (ORFs) of vaccinia virus, was predicted to encode a hydrophobic protein of 53 amino acids that is conserved in orthopoxviruses, leporipoxviruses, yatapoxiruses, and molluscipoxviruses. We constructed a recombinant vaccinia virus with a 10-codon epitope tag appended to the C terminus of the A14.5L ORF. Synthesis of the tagged protein occurred at late times and was blocked by an inhibitor of DNA replication, consistent with regulation by a predicted late promoter just upstream of the A14.5L ORF. Hydrophobicity of the protein was demonstrated by extraction into the detergent phase of Triton X-114. The protein was associated with purified vaccinia virus particles and with membranes of immature and mature virions that were visualized by electron microscopy of infected cells. Efficient release of the protein from purified virions occurred after treatment with a nonionic detergent and reducing agent. A mutant virus, in which the A14.5L ORF was largely deleted, produced normal-size plaques in several cell lines, and the yields of infectious intra- and extracellular viruses were similar to those of the parent. In contrast, with a mouse model, mutant viruses with the A14.5L ORF largely deleted were attenuated relative to that of the parental virus or a mutant virus with a restored A14.5L gene.

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

2002 ◽  
Vol 83 (1) ◽  
pp. 209-222 ◽  
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.

scholarly journals The Envelope G3L Protein Is Essential for Entry of Vaccinia Virus into Host Cells

2006 ◽  
Vol 80 (17) ◽  
pp. 8402-8410 ◽  
Author(s):  
Ruzan A. Izmailyan ◽  
Cheng-Yen Huang ◽  
Shamim Mohammad ◽  
Stuart N. Isaacs ◽  
Wen Chang
Keyword(s):  
Life Cycle ◽  
Vaccinia Virus ◽  
Transmembrane Domain ◽  
Recombinant Vaccinia Virus ◽  
Host Cells ◽  
Virus Growth ◽  
Enveloped Virus ◽  
Virus Life Cycle ◽  
Infected Cells ◽  
Mature Virus

ABSTRACT The vaccinia virus G3L/WR079 gene encodes a conserved protein with a predicted transmembrane domain. Our proteomic analyses of vaccinia virus revealed that G3L protein is incorporated into intracellular mature virus; however, the function of G3L protein in the vaccinia virus life cycle has not been investigated. In this study, a recombinant vaccinia virus, viG3L, expressing G3L protein under IPTG (isopropyl-β-d-thiogalactopyranoside) regulation was constructed. Under permissive conditions when G3L protein was expressed, the vaccinia virus life cycle proceeded normally, resulting in plaque formation in BSC40 cells. In contrast, under nonpermissive conditions when G3L protein expression was repressed, no plaques were formed, showing that G3L protein is essential for vaccinia virus growth in cell cultures. In infected cells when G3L protein was not expressed, the formation of intracellular mature virus (IMV) and cell-associated enveloped virus occurred normally, showing that G3L protein is not required for virion morphogenesis. IMV particles containing (G3L+) or lacking (G3L−) G3L protein were purified and were found to be indistinguishable on microscopic examination. Both G3L+ and G3L− IMV bound to HeLa cells; however, G3L− IMV failed to enter the cells, showing that G3L protein is required for IMV penetration into cells. Finally, G3L protein was required for fusion of the infected cells under low-pH treatment. Thus, our results provide direct evidence that G3L is an essential component of the vaccinia virus fusion complex, in addition to the previously reported A28, H2, L5, A21, and A16 proteins.

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