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 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 Assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the Golgi stacks.

1993 ◽  
Vol 121 (3) ◽  
pp. 521-541 ◽  
Author(s):  
B Sodeik ◽  
R W Doms ◽  
M Ericsson ◽  
G Hiller ◽  
C E Machamer ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
De Novo ◽  
Outer Envelope ◽  
Dna Virus ◽  
Phospholipid Analysis ◽  
Enveloped Virus ◽  
Assembly Pathway ◽  
One Step ◽  
Intermediate Compartment

Vaccinia virus, the prototype of the Poxviridae, is a large DNA virus which replicates in the cytoplasm of the host cell. The assembly pathway of vaccinia virus displays several unique features, such as the production of two structurally distinct, infectious forms. One of these, termed intracellular naked virus (INV), remains cells associated while the other, termed extracellular enveloped virus (EEV), is released from the cell. In addition, it has long been believed that INVs acquire their lipid envelopes by a unique example of de novo membrane biogenesis. To examine the structure and assembly of vaccinia virus we have used immunoelectron microscopy using antibodies to proteins of different subcellular compartments as well as a phospholipid analysis of purified INV and EEV. Our data are not consistent with the de novo model of viral membrane synthesis but rather argue that the vaccinia virus DNA becomes enwrapped by a membrane cisterna derived from the intermediate compartment between the ER and the Golgi stacks, thus acquiring two membranes in one step. Phospholipid analysis of purified INV supports its derivation from an early biosynthetic compartment. This unique assembly process is repeated once more when the INV becomes enwrapped by an additional membrane cisterna, in agreement with earlier reports. The available data suggest that after fusion between the outer envelope and the plasma membrane, mature EEV is released from the cell.

scholarly journals Phototracking Vaccinia Virus Transport Reveals Dynamics of Cytoplasmic Dispersal and a Requirement for A36R and F12L for Exit from the Site of Wrapping

Viruses ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 390
Author(s):  
Helena Lynn ◽  
Liam Howell ◽  
Russell Diefenbach ◽  
Timothy Newsome
Keyword(s):  
Vaccinia Virus ◽  
Viral Infections ◽  
Cell Periphery ◽  
Virus Transport ◽  
Anterograde Transport ◽  
Recombinant Viruses ◽  
Virus Particles ◽  
Viral Particles ◽  
Viral Morphogenesis ◽  
Golgi Network

The microtubule cytoskeleton is a primary organizer of viral infections for delivering virus particles to their sites of replication, establishing and maintaining subcellular compartments where distinct steps of viral morphogenesis take place, and ultimately dispersing viral progeny. One of the best characterized examples of virus motility is the anterograde transport of the wrapped virus form of vaccinia virus (VACV) from the trans-Golgi network (TGN) to the cell periphery by kinesin-1. Yet many aspects of this transport event are elusive due to the speed of motility and the challenges of imaging this stage at high resolution over extended time periods. We have established a novel imaging technology to track virus transport that uses photoconvertible fluorescent recombinant viruses to track subsets of virus particles from their site of origin and determine their destination. Here we image virus exit from the TGN and their rate of egress to the cell periphery. We demonstrate a role for kinesin-1 engagement in regulating virus exit from the TGN by removing A36 and F12 function, critical viral mediators of kinesin-1 recruitment to virus particles. Phototracking viral particles and components during infection is a powerful new imaging approach to elucidate mechanisms of virus replication.

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 Movements of vaccinia virus intracellular enveloped virions with GFP tagged to the F13L envelope protein

2001 ◽  
Vol 82 (11) ◽  
pp. 2747-2760 ◽  
Author(s):  
María M. Geada ◽  
Inmaculada Galindo ◽  
María M. Lorenzo ◽  
Beatriz Perdiguero ◽  
Rafael Blasco
Keyword(s):  
Cell Surface ◽  
Vaccinia Virus ◽  
Intracellular Transport ◽  
Virus Transport ◽  
Enveloped Virus ◽  
Vaccinia Viruses ◽  
Butanedione Monoxime ◽  
Golgi Network ◽  
Dependent Mechanism

Vaccinia virus produces several forms of infectious virions. Intracellular mature virions (IMV) assemble in areas close to the cell nucleus. Some IMV acquire an envelope from intracellular membranes derived from the trans-Golgi network, producing enveloped forms found in the cytosol (intracellular enveloped virus; IEV), on the cell surface (cell-associated enveloped virus) or free in the medium (extracellular enveloped virus; EEV). Blockage of IMV envelopment inhibits transport of virions to the cell surface, indicating that enveloped virus forms are required for virion movement from the Golgi area. To date, the induction of actin tails that propel IEV is the only well-characterized mechanism for enveloped virus transport. However, enveloped virus transport and release occur under conditions where actin tails are not formed. In order to study these events, recombinant vaccinia viruses were constructed with GFP fused to the most abundant protein in the EEV envelope, P37 (F13L). The P37–GFP fusion, like normal P37, accumulated in the Golgi area and was incorporated efficiently into enveloped virions. These recombinants allowed the monitoring of enveloped virus movements in vivo. In addition to a variety of relatively slow movements (<0·4 μm/s), faster, saltatory movements both towards and away from the Golgi area were observed. These movements were different from those dependent on actin tails and were inhibited by the microtubule-disrupting drug nocodazole, but not by the myosin inhibitor 2,3-butanedione monoxime. Video microscopy (5 frames per s) revealed that saltatory movements had speeds of up to, and occasionally more than, 3 μm/s. These results suggest that a second, microtubule-dependent mechanism exists for intracellular transport of enveloped vaccinia virions.

scholarly journals Vaccinia virus intracellular enveloped virions move to the cell periphery on microtubules in the absence of the A36R protein

2005 ◽  
Vol 86 (11) ◽  
pp. 2961-2968 ◽  
Author(s):  
Esteban Herrero-Martínez ◽  
Kim L. Roberts ◽  
Michael Hollinshead ◽  
Geoffrey L. Smith
Keyword(s):  
Vaccinia Virus ◽  
Fluorescent Protein ◽  
Core Protein ◽  
Time Lapse ◽  
Cell Periphery ◽  
Wild Type ◽  
Enveloped Virus ◽  
Microtubule Motors ◽  
Specific Proteins ◽  
Green Fluorescent

Vaccinia virus (VACV) intracellular enveloped virus (IEV) particles are transported to the cell periphery on microtubules where they fuse with the plasma membrane to form cell-associated enveloped virus (CEV). Two IEV-specific proteins, F12L and A36R, are implicated in mediating transport of IEV. Without F12L, virus morphogenesis halts after formation of IEV, and CEV is not formed, whereas without A36R, IEV was reported not to be transported, yet CEV was formed, To address the roles of A36R and F12L in IEV transport, viruses with deletions of either F12L (vΔF12L) or A36R (vΔA36R) were labelled with enhanced green fluorescent protein (EGFP) fused to the core protein A5L, and used to follow CEV production with time. Without F12L, CEV production was inhibited by >99 %, whereas without A36R, CEV were produced at ∼60 % of wild-type levels at 24 h post-infection. Depolymerization of microtubules, but not actin, inhibited CEV formation in vΔA36R-infected cells. Moreover, vΔA36R IEV labelled with EGFP fused to the B5R protein co-localized with microtubules, showing that the A36R protein is not required for the interaction of IEV with microtubules. Time-lapse confocal microscopy confirmed that both wild-type and vΔA36R IEV moved in a stop–start manner at speeds consistent with microtubular movement, although the mean length of vΔA36R IEV movement was shorter. These data demonstrate that VACV IEV is transported to the cell surface using microtubules in the absence of A36R, and therefore IEV must attach to microtubule motors using at least one protein other than A36R.

scholarly journals The Molluscum Contagiosum Gene MC021L Partially Compensates for the Loss of Its Vaccinia Virus Homolog, F13L

2020 ◽  
Vol 94 (20) ◽  
Author(s):  
Stephanie R. Monticelli ◽  
Peter Bryk ◽  
Brian M. Ward
Keyword(s):  
Vaccinia Virus ◽  
Molluscum Contagiosum ◽  
Molluscum Contagiosum Virus ◽  
Virion Protein ◽  
Small Plaque ◽  
Reading Frame ◽  
Extracellular Virus ◽  
Specific Proteins ◽  
Plaque Phenotype ◽  
Golgi Network

ABSTRACT Orthopoxviruses produce two antigenically distinct infectious enveloped virions termed intracellular mature virions and extracellular virions (EV). EV have an additional membrane compared to intracellular mature virions due to a wrapping process at the trans-Golgi network and are required for cell-to-cell spread and pathogenesis. Specific to the EV membrane are a number of proteins highly conserved among orthopoxviruses, including F13, which is required for the efficient wrapping of intracellular mature virions to produce EV and which plays a role in EV entry. The distantly related molluscipoxvirus, molluscum contagiosum virus, is predicted to encode several vaccinia virus homologs of EV-specific proteins, including the homolog of F13L, MC021L. To study the function of MC021, we replaced the F13L open reading frame in vaccinia virus with an epitope-tagged version of MC021L. The resulting virus (vMC021L-HA) had a small-plaque phenotype compared to vF13L-HA but larger than vΔF13L. The localization of MC021-HA was markedly different from that of F13-HA in infected cells, but MC021-HA was still incorporated in the EV membrane. Similar to F13-HA, MC021-HA was capable of interacting with both A33 and B5. Although MC021-HA expression did not fully restore plaque size, vMC021L-HA produced amounts of EV similar to those produced by vF13L-HA, suggesting that MC021 retained some of the functionality of F13. Further analysis revealed that EV produced from vMC021L-HA exhibit a marked reduction in target cell binding and an increase in dissolution, both of which correlated with a small-plaque phenotype. IMPORTANCE The vaccinia virus extracellular virion protein F13 is required for the production and release of infectious extracellular virus, which in turn is essential for the subsequent spread and pathogenesis of orthopoxviruses. Molluscum contagiosum virus infects millions of people worldwide each year, but it is unknown whether EV are produced during infection for spread. Molluscum contagiosum virus contains a homolog of F13L termed MC021L. To study the potential function of this homolog during infection, we utilized vaccinia virus as a surrogate and showed that a vaccinia virus expressing MC021L-HA in place of F13L-HA exhibits a small-plaque phenotype but produces similar levels of EV. These results suggest that MC021-HA can compensate for the loss of F13-HA by facilitating wrapping to produce EV and further delineates the dual role of F13 during infection.

The envelope of vaccinia virus reveals an unusual phospholipid in Golgi complex membranes

1996 ◽  
Vol 109 (8) ◽  
pp. 2121-2131 ◽  
Author(s):  
E.B. Cluett ◽  
C.E. Machamer
Keyword(s):  
Vaccinia Virus ◽  
Hela Cells ◽  
High Performance ◽  
Normal Component ◽  
Phosphatidyl Inositol ◽  
Subcellular Fractionation ◽  
Trans Golgi Network ◽  
Enveloped Virus ◽  
Lipid Species ◽  
Golgi Network

We isolated forms of enveloped vaccinia virus from infected HeLa cells to obtain membranes for the analysis of lipids of the cis-Golgi network and trans-Golgi network. The intracellular mature virus obtains its envelope by wrapping itself in the membranes of the cis-Golgi network. A fraction of these virions then acquires a second envelope by enwrapping trans-Golgi network membranes to form the intracellular enveloped virus. Lipids were analyzed by high performance thin layer chromatography and digital densitometry to establish a steady-state lipid profile of viral membranes, which should reflect the compositions of the cis-Golgi network and trans-Golgi network. Phosphatidyl-inositol was slightly enriched in the cis-Golgi network of HeLa cells, whereas the trans-Golgi network showed a minor increase in phosphatidylserine and sphingomyelin. Similarly, cholesterol was only slightly more abundant in the trans-Golgi compared to the cis-Golgi. An unusual lipid, semilysobisphosphatidic acid, was present in significant amounts in vaccinia envelopes. Semilysobisphosphatidic acid was present in similar levels in infected and uninfected cells, and was therefore not induced by vaccinia infection. Subcellular fractionation of HeLa cells indicated that the recovery of semilysobisphosphatidic acid paralleled the recovery of a Golgi marker. Furthermore, a lipid species that comigrated with semilysobisphosphatidic acid was also present in lipids extracted from highly purified, intact Golgi complexes from rat liver. Together, these results suggest that semilysobisphosphatidic acid is a normal component of Golgi membranes.

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