scholarly journals The formation and function of extracellular enveloped vaccinia virus

2002 ◽  
Vol 83 (12) ◽  
pp. 2915-2931 ◽  
Author(s):  
Geoffrey L. Smith ◽  
Alain Vanderplasschen ◽  
Mansun Law
Keyword(s):  
Vaccinia Virus ◽  
Cell Biology ◽  
Neutralizing Antibody ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Virus Life Cycle ◽  
And Function ◽  
Mature Virus ◽  
Cell Spread

Vaccinia virus produces four different types of virion from each infected cell called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). These virions have different abundance, structure, location and roles in the virus life-cycle. Here, the formation and function of these virions are considered with emphasis on the EEV form and its precursors, IEV and CEV. IMV is the most abundant form of virus and is retained in cells until lysis; it is a robust, stable virion and is well suited to transmit infection between hosts. IEV is formed by wrapping of IMV with intracellular membranes, and is an intermediate between IMV and CEV/EEV that enables efficient virus dissemination to the cell surface on microtubules. CEV induces the formation of actin tails that drive CEV particles away from the cell and is important for cell-to-cell spread. Lastly, EEV mediates the long-range dissemination of virus in cell culture and, probably, in vivo. Seven virus-encoded proteins have been identified that are components of IEV, and five of them are present in CEV or EEV. The roles of these proteins in virus morphogenesis and dissemination, and as targets for neutralizing antibody are reviewed. The production of several different virus particles in the VV replication cycle represents a coordinated strategy to exploit cell biology to promote virus spread and to aid virus evasion of antibody and complement.

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 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.

scholarly journals Identification of the Orthopoxvirus p4c Gene, Which Encodes a Structural Protein That Directs Intracellular Mature Virus Particles into A-Type Inclusions

2002 ◽  
Vol 76 (22) ◽  
pp. 11216-11225 ◽  
Author(s):  
Terry A. McKelvey ◽  
Stanley C. Andrews ◽  
Sara E. Miller ◽  
Caroline A. Ray ◽  
David J. Pickup
Keyword(s):  
Virus Replication ◽  
Cowpox Virus ◽  
Structural Protein ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Viral Morphogenesis ◽  
Vaccinia Virus Strain ◽  
Mature Virus

ABSTRACT The orthopoxvirus gene p4c has been identified in the genome of the vaccinia virus strain Western Reserve. This gene encodes the 58-kDa structural protein P4c present on the surfaces of the intracellular mature virus (IMV) particles. The gene is disrupted in the genome of cowpox virus Brighton Red (BR), demonstrating that although the P4c protein may be advantageous for virus replication in vivo, it is not essential for virus replication in vitro. Complementation and recombination analyses with the p4c gene have shown that the P4c protein is required to direct the IMV into the A-type inclusions (ATIs) produced by cowpox virus BR. The p4c gene is highly conserved among most members of the orthopoxvirus genus, including viruses that produce ATIs, such as cowpox, ectromelia, and raccoonpox viruses, as well as those such as variola, monkeypox, vaccinia, and camelpox viruses, which do not. The conservation of the p4c gene among the orthopoxviruses, irrespective of their capacities to produce ATIs, suggests that the P4c protein provides functions in addition to that of directing IMV into ATIs. These findings, and the presence of the P4c protein in IMV but not extracellular enveloped virus (D. Ulaeto, D. Grosenbach, and D. E. Hruby, J. Virol. 70:3372-3377, 1996), suggest a model in which the P4c protein may play a role in the retrograde movement of IMV particles, thereby contributing to the retention of IMV particles within the cytoplasm and within ATIs when they are present. In this way, the P4c protein may affect both viral morphogenesis and processes of virus dissemination.

The vaccinia virus A27L protein is needed for the microtubule-dependent transport of intracellular mature virus particles

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 47-58 ◽  
Author(s):  
Christopher M. Sanderson ◽  
Michael Hollinshead ◽  
Geoffrey L. Smith
Keyword(s):  
Vaccinia Virus ◽  
Post Infection ◽  
Intracellular Transport ◽  
Specific Role ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Dependent Transport ◽  
Additional Role ◽  
Mature Virus ◽  
Virus Factory

The vaccinia virus (VV) A27L gene encodes a 14 kDa protein that is required for the formation of intracellular enveloped virus (IEV) and, consequently, normal sized plaques. Data presented here show that A27L plays an additional role in VV assembly. When cells were infected with the VV WR32-7/Ind 14K, under conditions that repress A27L expression, transport of intracellular mature virus (IMV) from virus factories was inhibited and some IMV was found in aberrant association with virus crescents. In contrast, other VV mutants (vΔB5R and vΔF13L) that are defective in IEV formation produce IMV particles that are transported out of virus factories. This indicated a specific role for A27L in IMV transport. Induction of A27L expression at 10 h post-infection promoted the dispersal of clustered IMV particles, but only when microtubules were intact. Formation of IEV particles was also impaired when cells were infected with WR32-7/14K, a VV strain expressing a mutated form of the A27L protein; however, this mutation did not inhibit intracellular transport of IMV particles. Collectively, these data define two novel aspects of VV morphogenesis. Firstly, A27L is required for both IMV transport and the process of envelopment that leads to IEV formation. Secondly, movement of IMV particles between the virus factory and the site of IEV formation is microtubule-dependent.

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 A non-enveloped arbovirus released in lysosome-derived extracellular vesicles induces super-infection exclusion

2020 ◽  
Author(s):  
Thomas Labadie ◽  
Polly Roy
Keyword(s):  
Extracellular Vesicles ◽  
Defense Mechanism ◽  
Degradation Process ◽  
Free Virus ◽  
Enveloped Viruses ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Super Infection

AbstractRecent developments on extracellular vesicles (EVs) containing multiple virus particles challenge the rigid definition of non-enveloped viruses. However, how non-enveloped viruses hijack cell machinery to promote non-lytic release in EVs, and their functional roles, remain to be clarified. Here we used Bluetongue virus (BTV) as a model of a non-enveloped arthropod-borne virus and observed that the majority of viruses are released in EVs, both in vitro and in the blood of infected animals. Based on the cellular proteins detected in these EVs, and use of inhibitors targeting the cellular degradation process, we demonstrated that these extracellular vesicles are derived from secretory lysosomes, in which the acidic pH is neutralized upon the infection. Moreover, we report that secreted EVs are more efficient than free-viruses for initiating infections, but that they trigger super-infection exclusion that only free-viruses can overcome.Author summaryRecent discoveries of non-enveloped virus secreted in EVs opened the door to new developments in our understanding of the transmission and pathogenicity of these viruses. In particular, how these viruses hijack the host cellular secretion machinery, and the role of these EVs compared with free-virus particles remained to be explored. Here, we tackled these two aspects, by studying BTV, an emerging arthropod-borne virus causing epidemics worldwide. We showed that this virus is mainly released in EVs, in vivo and in the blood of infected animals, and that inhibition of the cell degradation machinery decreases the release of infectious EVs, but not free-virus particles. We found that BTV must neutralize the pH of lysosomes, which are important organelles of the cell degradation machinery, for efficient virus release in EVs. Our results highlight unique features for a virus released in EVs, explaining how BTV transits in lysosomes without being degraded. Interestingly, we observed that EVs are more infectious than free-virus particles, but only free-viruses are able to overcome the super-infection exclusion, which is a common cellular defense mechanism. In conclusion, our study stresses the dual role played by both forms, free and vesicular, in the virus life cycle.

scholarly journals Functional Role of N-Linked Glycosylation in Pseudorabies Virus Glycoprotein gH

2018 ◽  
Vol 92 (9) ◽  
pp. e00084-18 ◽  
Author(s):  
Melina Vallbracht ◽  
Sascha Rehwaldt ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter ◽  
Walter Fuchs
Keyword(s):  
Pseudorabies Virus ◽  
Viral Envelope ◽  
Fusion Activity ◽  
Virus Particles ◽  
Glycosylation Sites ◽  
Functional Relevance ◽  
And Function ◽  
Cell Spread

ABSTRACTMany viral envelope proteins are modified by asparagine (N)-linked glycosylation, which can influence their structure, physicochemical properties, intracellular transport, and function. Here, we systematically analyzed the functional relevance of N-linked glycans in the alphaherpesvirus pseudorabies virus (PrV) glycoprotein H (gH), which is an essential component of the conserved core herpesvirus fusion machinery. Upon gD-mediated receptor binding, the heterodimeric complex of gH and gL activates gB to mediate fusion of the viral envelope with the host cell membrane for viral entry. gH contains five potential N-linked glycosylation sites at positions 77, 162, 542, 604, and 627, which were inactivated by conservative mutations (asparagine to glutamine) singly or in combination. The mutated proteins were tested for correct expression and fusion activity. Additionally, the mutated gH genes were inserted into the PrV genome for analysis of function during virus infection. Our results demonstrate that all five sites are glycosylated. Inactivation of the PrV-specific N77 or the conserved N627 resulted in significantly reducedin vitrofusion activity, delayed penetration kinetics, and smaller virus plaques. Moreover, substitution of N627 greatly affected transport of gH in transfected cells, resulting in endoplasmic reticulum (ER) retention and reduced surface expression. In contrast, mutation of N604, which is conserved in theVaricellovirusgenus, resulted in enhancedin vitrofusion activity and viral cell-to-cell spread. These results demonstrate a role of the N-glycans in proper localization and function of PrV gH. However, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles.IMPORTANCEHerpesvirus infection requires fusion of the viral envelope with cellular membranes, which involves the conserved fusion machinery consisting of gB and the heterodimeric gH/gL complex. The bona fide fusion protein gB depends on the presence of the gH/gL complex for activation. Viral envelope glycoproteins, such as gH, usually contain N-glycans, which can have a strong impact on their folding, transport, and functions. Here, we systematically analyzed the functional relevance of all five predicted N-linked glycosylation sites in the alphaherpesvirus pseudorabies virus (PrV) gH. Despite the fact that mutation of specific sites affected gH transport,in vitrofusion activity, and cell-to-cell spread and resulted in delayed penetration kinetics, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles. Thus, our results demonstrate a modulatory but nonessential role of N-glycans for gH function.

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.

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