scholarly journals Redistribution of Cyclophilin A to Viral Factories during Vaccinia Virus Infection and Its Incorporation into Mature Particles

2003 ◽  
Vol 77 (16) ◽  
pp. 9052-9068 ◽  
Author(s):  
Ana Paula V. Castro ◽  
Técia M. U. Carvalho ◽  
Nissin Moussatché ◽  
Clarissa R. A. Damaso
Keyword(s):  
Confocal Microscopy ◽  
Virus Infection ◽  
Vaccinia Virus ◽  
Conformational Changes ◽  
Cyclophilin A ◽  
Cellular Systems ◽  
Cell Cytoplasm ◽  
Electron Microscopic ◽  
The Core ◽  
Infected Cells

ABSTRACT Cyclophilins are peptidyl-prolyl cis-trans isomerases involved in catalyzing conformational changes and accelerating the rate of protein folding and refolding in several cellular systems. In the present study, we analyzed the expression pattern and intracellular distribution of the cellular isomerase cyclophilin A (CypA) during vaccinia virus (VV) infection. An impressive increase in CypA stability was observed, leading to a practically unchanged accumulation of CypA during infection, although its synthesis was completely inhibited at late times. By confocal microscopy, we observed that CypA went through an intense reorganization in the cell cytoplasm and colocalized with the virosomes late in infection. CypA relocation to viral factories required the synthesis of viral postreplicative proteins, and treatment of infected cells with cyclosporine (CsA) prevented CypA relocation, clearly excluding the virosomes from CypA staining. Immunoelectron microscopy of VV-infected cells showed that CypA was incorporated into VV particles during morphogenesis. Biochemical and electron microscopic assays with purified virions confirmed that CypA was encapsidated within the virus particle and localized specifically in the core. This work suggests that CypA may develop an important role in VV replication.

scholarly journals Vaccinia Virus Infection Disarms the Mitochondrion-Mediated Pathway of the Apoptotic Cascade by Modulating the Permeability Transition Pore

2001 ◽  
Vol 75 (23) ◽  
pp. 11437-11448 ◽  
Author(s):  
Shawn T. Wasilenko ◽  
Adrienne F. A. Meyers ◽  
Kathleen Vander Helm ◽  
Michele Barry
Keyword(s):  
Virus Infection ◽  
Vaccinia Virus ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Jurkat Cells ◽  
Caspase 8 ◽  
Apoptotic Pathway ◽  
Vaccinia Virus Infection ◽  
Infected Cells ◽  
Apoptotic Cascade

ABSTRACT Many viruses have evolved strategies that target crucial components within the apoptotic cascade. One of the best studied is the caspase 8 inhibitor, crmA/Spi-2, encoded by members of the poxvirus family. Since many proapoptotic stimuli induce apoptosis through a mitochondrion-dependent, caspase 8-independent pathway, we hypothesized that vaccinia virus would encode a mechanism to directly modulate the mitochondrial apoptotic pathway. In support of this, we observed that Jurkat cells, which undergo Fas-mediated apoptosis exclusively through the mitochondrial route, were resistant to Fas-induced death following infection with a crmA/Spi-2-deficient strain of vaccinia virus. In addition, vaccinia virus-infected cells subjected to the proapoptotic stimulus staurosporine exhibited decreased levels of both cytochromec released from the mitochondria and caspase 3 activation. In all cases we found that the loss of the mitochondrial membrane potential, which occurs as a result of opening the multimeric permeability transition pore complex, was prevented in vaccinia virus-infected cells. Moreover, vaccinia virus infection specifically inhibited opening of the permeability transition pore following treatment with the permeability transition pore ligand atractyloside and t-butylhydroperoxide. These studies indicate that vaccinia virus infection directly impacts the mitochondrial apoptotic cascade by influencing the permeability transition pore.

scholarly journals Genetic Analysis of the Vaccinia Virus I6 Telomere-Binding Protein Uncovers a Key Role in Genome Encapsidation

2003 ◽  
Vol 77 (20) ◽  
pp. 10929-10942 ◽  
Author(s):  
Olivera Grubisha ◽  
Paula Traktman
Keyword(s):  
Vaccinia Virus ◽  
Proteolytic Processing ◽  
Temperature Sensitive ◽  
Electron Microscopic ◽  
Recombinant Viruses ◽  
Telomere Binding Protein ◽  
Microscopic Studies ◽  
Infected Cells ◽  
Genome Encapsidation

ABSTRACT The linear, double-stranded DNA genome of vaccinia virus contains covalently closed hairpin termini. These hairpin termini comprise a terminal loop and an A+T-rich duplex stem that has 12 extrahelical bases. DeMasi et al. have shown previously that proteins present in infected cells and in virions form distinct complexes with the telomeric hairpins and that these interactions require the extrahelical bases. The vaccinia virus I6 protein was identified as the protein showing the greatest specificity and affinity for interaction with the viral hairpins (J. DeMasi, S. Du, D. Lennon, and P. Traktman, J. Virol. 75:10090-10105, 2001). To gain insight into the role of I6 in vivo, we generated eight recombinant viruses bearing altered alleles of I6 in which clusters of charged amino acids were changed to alanine residues. One allele (temperature-sensitive I6-12 [tsI6-12]) conferred a tight ts phenotype and was used to examine the stage(s) of the viral life cycle that was affected at the nonpermissive temperature. Gene expression, DNA replication, and genome resolution proceeded normally in this mutant. However, proteolytic processing of structural proteins, which accompanies virus maturation, was incomplete. Electron microscopic studies confirmed a severe block in morphogenesis in which immature, but no mature, virions were observed. Instead, aberrant spherical virions and large crystalloids were seen. When purified, these aberrant virions were found to have normal protein content but to be devoid of viral DNA. We propose that the binding of I6 to viral telomeres directs genome encapsidation into the virus particle.

scholarly journals Characterization of Vaccinia Virus Intracellular Cores: Implications for Viral Uncoating and Core Structure

2000 ◽  
Vol 74 (8) ◽  
pp. 3525-3536 ◽  
Author(s):  
Ketil Pedersen ◽  
Eric J. Snijder ◽  
Sibylle Schleich ◽  
Norbert Roos ◽  
Gareth Griffiths ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Vaccinia Virus ◽  
Dna Labeling ◽  
The Core ◽  
Double Stranded Dna ◽  
Core Proteins ◽  
Transcription Inhibitor ◽  
Infected Cells ◽  
Combined Data

ABSTRACT The entry of vaccinia virus (VV) into the host cell results in the delivery of the double-stranded DNA genome-containing core into the cytoplasm. The core is disassembled, releasing the viral DNA in order to initiate VV cytoplasmic transcription and DNA replication. Core disassembly can be prevented using the VV early transcription inhibitor actinomycin D (actD), since early VV protein synthesis is required for core uncoating. In this study, VV intracellular cores were accumulated in the presence of actD and isolated from infected cells. The content of these cores was analyzed by negative staining EM and by Western blotting using a collection of antibodies to VV core and membrane proteins. By Western blot analyses, intracellular actD cores, as well as cores prepared by NP-40–dithiothreitol treatment of purified virions (NP-40/DTT cores), contained the core proteins p25 (encoded by L4R), 4a (A10L), 4b (A3L), and p39 (A4L) as well as small amounts of the VV membrane proteins p32 (D8L) and p35 (H3L). While NP-40/DTT cores contained the major putative DNA-binding protein p11 (F17R), actD cores entirely lacked this protein. Labeled cryosections of cells infected for different periods of time in the presence or absence of actD were subsequently used to follow the fate of VV core proteins by EM. These EM images confirmed that p11 was lost at the plasma membrane upon core penetration. The cores that accumulated in the presence of actD were labeled with antibodies to 4a, p39, p25, and DNA at all times examined. In the absence of the drug the cores gradually lost their electron-dense inner part, concomitant with the loss of p25 and DNA labeling. The remaining core shell still labeled with antibodies to p39 and 4a/4b, implying that these proteins are part of this structure. These combined data are discussed with respect to the structure of VV as well as core disassembly.

scholarly journals Recognition of Vaccinia Virus-Infected Cells by Human Natural Killer Cells Depends on Natural Cytotoxicity Receptors

2006 ◽  
Vol 80 (5) ◽  
pp. 2225-2233 ◽  
Author(s):  
Susan E. Chisholm ◽  
Hugh T. Reyburn
Keyword(s):  
Virus Infection ◽  
Vaccinia Virus ◽  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Target Cells ◽  
Natural Cytotoxicity ◽  
Vaccinia Virus Infection ◽  
Infected Cells ◽  
Natural Cytotoxicity Receptors

ABSTRACT Natural Killer (NK) cells are important in the immune response to a number of viruses; however, the mechanisms used by NK cells to discriminate between healthy and virus-infected cells are only beginning to be understood. Infection with vaccinia virus provokes a marked increase in the susceptibility of target cells to lysis by NK cells, and we show that recognition of the changes in the target cell induced by vaccinia virus infection depends on the natural cytotoxicity receptors NKp30, NKp44, and NKp46. Vaccinia virus infection does not induce expression of ligands for the activating NKG2D receptor, nor does downregulation of major histocompatibility complex class I molecules appear to be of critical importance for altered target cell susceptibility to NK cell lysis. The increased susceptibility to lysis by NK cells triggered upon poxvirus infection depends on a viral gene, or genes, transcribed early in the viral life cycle and present in multiple distinct orthopoxviruses. The more general implications of these data for the processes of innate immune recognition are discussed.

scholarly journals Vaccinia Virus Infection Attenuates Innate Immune Responses and Antigen Presentation by Epidermal Dendritic Cells

2006 ◽  
Vol 80 (20) ◽  
pp. 9977-9987 ◽  
Author(s):  
Liang Deng ◽  
Peihong Dai ◽  
Wanhong Ding ◽  
Richard D. Granstein ◽  
Stewart Shuman
Keyword(s):  
Virus Infection ◽  
Vaccinia Virus ◽  
Poly I:C ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Vaccinia Virus Infection ◽  
Tnf Α ◽  
Infected Cells ◽  
Virus Mutant

ABSTRACT Langerhans cells (LCs) are antigen-presenting cells in the skin that play sentinel roles in host immune defense by secreting proinflammatory molecules and activating T cells. Here we studied the interaction of vaccinia virus with XS52 cells, a murine epidermis-derived dendritic cell line that serves as a surrogate model for LCs. We found that vaccinia virus productively infects XS52 cells, yet this infection displays an atypical response to anti-poxvirus agents. Whereas adenosine N1-oxide blocked virus production and viral protein synthesis during a synchronous infection, cytosine arabinoside had no effect at concentrations sufficient to prevent virus replication in BSC40 monkey kidney cells. Vaccinia virus infection of XS52 cells not only failed to elicit the production of various cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, IL-12 p40, alpha interferon (IFN-α), and IFN-γ, it actively inhibited the production of proinflammatory cytokines TNF-α and IL-6 by XS52 cells in response to exogenous lipopolysaccharide (LPS) or poly(I:C). Infection with a vaccinia virus mutant lacking the E3L gene resulted in TNF-α secretion in the absence of applied stimuli. Infection of XS52 cells or BSC40 cells with the ΔE3L virus, but not wild-type vaccinia virus, triggered proteolytic decay of IκBα. These results suggest a novel role for the E3L protein as an antagonist of the NF-κB signaling pathway. ΔE3L-infected XS52 cells secreted higher levels of TNF-α and IL-6 in response to LPS and poly(I:C) than did cells infected with the wild-type virus. XS52 cells were productively infected by a vaccinia virus mutant lacking the K1L gene. ΔK1L-infected cells secreted higher levels of TNF-α and IL-6 in response to LPS than wild-type virus-infected cells. Vaccinia virus infection of primary LCs harvested from mouse epidermis was nonpermissive, although a viral reporter protein was expressed in the infected LCs. Vaccinia virus infection of primary LCs strongly inhibited their capacity for antigen-specific activation of T cells. Our results highlight suppression of the skin immune response as a feature of orthopoxvirus infection.

scholarly journals An Unconventional Role for Cytoplasmic Disulfide Bonds in Vaccinia Virus Proteins

1999 ◽  
Vol 144 (2) ◽  
pp. 267-279 ◽  
Author(s):  
Jacomine Krijnse Locker ◽  
Gareth Griffiths
Keyword(s):  
Membrane Proteins ◽  
Vaccinia Virus ◽  
Disulfide Bonds ◽  
Virus Assembly ◽  
Infection Process ◽  
Disulfide Bonding ◽  
The Core ◽  
Core Proteins ◽  
Unique System ◽  
Infected Cells

Previous data have shown that reducing agents disrupt the structure of vaccinia virus (vv). Here, we have analyzed the disulfide bonding of vv proteins in detail. In vv-infected cells cytoplasmically synthesized vv core proteins became disulfide bonded in the newly assembled intracellular mature viruses (IMVs). vv membrane proteins also assembled disulfide bonds, but independent of IMV formation and to a large extent on their cytoplasmic domains. If disulfide bonding was prevented, virus assembly was only partially impaired as shown by electron microscopy as well as a biochemical assay of IMV formation. Under these conditions, however, the membranes around the isolated particles appeared less stable and detached from the underlying core. During the viral infection process the membrane proteins remained disulfide bonded, whereas the core proteins were reduced, concomitant with delivery of the cores into the cytoplasm. Our data show that vv has evolved an unique system for the assembly of cytoplasmic disulfide bonds that are localized both on the exterior and interior parts of the IMV.

scholarly journals Envelope Formation Is Blocked by Mutation of a Sequence Related to the HKD Phospholipid Metabolism Motif in the Vaccinia Virus F13L Protein

1999 ◽  
Vol 73 (2) ◽  
pp. 1108-1117 ◽  
Author(s):  
Rachel L. Roper ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Phospholipid Metabolism ◽  
Electron Microscopic ◽  
Golgi Membranes ◽  
Golgi Compartment ◽  
Extracellular Form ◽  
Infected Cells ◽  
And Function ◽  
Triton X ◽  
Intracellular Virus

ABSTRACT The outer envelope of the extracellular form of vaccinia virus is derived from Golgi membranes that have been modified by the insertion of specific viral proteins, of which the major component is the 37-kDa, palmitylated, nonglycosylated product of the F13L gene. The F13L protein contains a variant of the HKD (His-Lys-Asp) motif, which is conserved in numerous enzymes of phospholipid metabolism. Vaccinia virus mutants with a conservative substitution of either the K (K314R) or the D (D319E) residue of the F13L protein formed only tiny plaques similar to those produced by an F13L deletion mutant, were unable to produce extracellular enveloped virions, and failed to mediate low-pH-induced fusion of infected cells. Membrane-wrapped forms of intracellular virus were rarely detected in electron microscopic images of cells infected with either of the mutants. Western blotting and pulse-chase experiments demonstrated that the D319E protein was less stable than either the K314R or wild-type F13L protein. Most striking, however, was the failure of either of the two mutated proteins to concentrate in the Golgi compartment. Palmitylation, oleation, and partitioning of the F13L protein in Triton X-114 detergent were unaffected by the K314R substitution. These results indicated that the F13L protein must retain the K314 and D319 for it to localize in the Golgi compartment and function in membrane envelopment of vaccinia virus.

scholarly journals Vaccinia Virus 15-Kilodalton (A14L) Protein Is Essential for Assembly and Attachment of Viral Crescents to Virosomes

1998 ◽  
Vol 72 (2) ◽  
pp. 1287-1296 ◽  
Author(s):  
Juan Ramón Rodríguez ◽  
Cristina Risco ◽  
José L. Carrascosa ◽  
Mariano Esteban ◽  
Dolores Rodríguez
Keyword(s):  
Vaccinia Virus ◽  
Electron Microscopic Examination ◽  
Viral Proteins ◽  
Wild Type ◽  
Electron Microscopic ◽  
Small Plaque ◽  
Virion Assembly ◽  
Plaque Phenotype ◽  
Infected Cells ◽  
Intermediate Compartment

ABSTRACT Early stages in vaccinia virus (VV) assembly involve the recruitment of cellular membranes from the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) to virus factories (or virosomes). The key viral factors involved in this process are not yet known. We have previously identified and characterized two viral proteins, of 21 kDa (A17L gene) and 15 kDa (A14L gene), that associate with tubulovesicular elements related to the ERGIC and are localized in viral membranes at all stages of virion assembly. We showed that the 21-kDa protein is not responsible for the recruitment of membranes from the ERGIC to viral factories. However, it appears to be essential for the organization of viral membranes. In this investigation we have generated a VV recombinant, VVindA14L, in which the expression of the A14L gene is inducibly regulated by the Escherichia coli lacIoperator-repressor system. Repression of 15-kDa protein synthesis has a dramatic effect on virus yields and severely impairs plaque formation. Compared to wild-type VV, reduced amounts of 15-kDa protein are produced in VVindA14L-infected cells in the presence of IPTG (isopropyl-β-d-thiogalactoside), and this correlates with a small-plaque phenotype and reduced VVindA14L yields under these conditions. In the absence of the 15-kDa protein, early and late viral protein syntheses proceed normally; however, proteolytic cleavage of the major core precursors is inhibited. Electron microscopic examination of cells infected with VVindA14L under nonpermissive conditions reveals the presence of numerous membranous elements that look like unfinished or disassembled crescents interespersed between electron-dense masses. These abnormal membrane elements are usually well separated from the surfaces of the dense structures. These findings show that the 15-kDa protein is essential for VV morphogenesis and indicate that this polypeptide is necessary both for the correct assembly of viral crescents and for their stable attachment to the surfaces of viral factories.

scholarly journals Fowlpox Virus Encodes a Bcl-2 Homologue That Protects Cells from Apoptotic Death through Interaction with the Proapoptotic Protein Bak

2007 ◽  
Vol 81 (20) ◽  
pp. 11032-11045 ◽  
Author(s):  
Logan Banadyga ◽  
Jenna Gerig ◽  
Tara Stewart ◽  
Michele Barry
Keyword(s):  
Virus Infection ◽  
Vaccinia Virus ◽  
Recombinant Vaccinia Virus ◽  
Virus Protein ◽  
Necrosis Factor Alpha ◽  
Fowlpox Virus ◽  
Proapoptotic Protein ◽  
Infected Cells ◽  
Factor Alpha ◽  
Transient Overexpression

ABSTRACT Poxviruses are renowned for encoding numerous immunomodulatory proteins capable of undermining potent immune defenses. One effective barrier against infection is apoptosis, a process controlled at the mitochondria by pro- and antiapoptotic members of the highly conserved Bcl-2 family of proteins. Although poxviruses are known to encode an array of effective inhibitors of apoptosis, members of the Avipoxvirus genus, which includes fowlpox virus, encode proteins with Bcl-2 homology. Here, we show that FPV039, a fowlpox virus protein with limited Bcl-2 homology, inhibited apoptosis in response to a variety of cytotoxic stimuli, including virus infection itself. Similar to other antiapoptotic Bcl-2 proteins, FPV039 localized predominantly to the mitochondria in both human and chicken cells and protected human cells from tumor necrosis factor alpha-induced loss of the mitochondrial membrane potential. In addition, coimmunoprecipitation revealed that FPV039 interacted constitutively with the proapoptotic Bcl-2 protein, Bak, in both human and chicken cells. Concordantly, FPV039 also inhibited apoptosis induced by the transient overexpression of Bak. To confirm these results in the context of virus infection, we generated a recombinant vaccinia virus lacking F1L, the endogenous apoptotic inhibitor in vaccinia virus, and expressing FPV039. In the context of vaccinia virus infection, FPV039 retained the ability to localize to the mitochondria and interacted with Bak. Moreover, FPV039 prevented the activation of Bak and protected infected cells from apoptosis induced by staurosporine and virus infection. Together, our data indicate that FPV039 is a functional Bcl-2 homologue that inhibits apoptosis by neutralizing the proapoptotic Bcl-2 family member Bak.

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