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1.
Goldmacher VS 《Biochimie》2002,84(2-3):177-185
Human cytomegalovirus encodes a powerful cell death suppressor vMIA (viral mitochondria-localized inhibitor of apoptosis), also known as pUL37x1. vMIA, a product of the immediate early gene UL37 exon 1, is predominantly localized in mitochondria, where it appears to form a complex with adenine nucleotide translocator, believed to be a component of the mitochondrial transition pore complex. vMIA suppresses apoptosis by blocking permeabilization of the mitochondrial outer membrane. Expression of vMIA protects cells against apoptosis triggered by diverse stimuli, including ligation of death receptors, exposure to certain cytotoxic drugs, and infection with an adenovirus mutant deficient in E1B19K. Deletion mutagenesis of vMIA revealed two domains that are necessary and, together, sufficient for its anti-apoptotic activity. The first domain contains a mitochondrial targeting signal. The function of the second domain is still unknown. vMIA does not share any significant amino acid sequence homology with Bcl-2, and, unlike Bcl-2 or Bcl-x(L), it does not bind BAX or VDAC. These structural and functional differences between vMIA and Bcl-2 suggest that vMIA represents a separate class of cell death suppressors. Experiments with vMIA-deficient CMV (human cytomegalovirus) mutants provide strong evidence that the anti-apoptotic function of vMIA is required to prevent CMV-induced apoptosis, and is necessary for viral replication. In addition to vMIA, UL37 encodes two longer splice-variant proteins, gpUL37 and GP37(M). Biological functions of these proteins have not yet been identified, and may be unrelated to their anti-apoptotic activity. The identification of vMIA and the finding that its anti-apoptotic function is required for CMV replication provides a rationale for the development of anti-CMV pharmaceuticals that would inactivate vMIA and thus restore apoptosis in cells infected with CMV. 相似文献
2.
Mitochondrial cell death suppressors carried by human and murine cytomegalovirus confer resistance to proteasome inhibitor-induced apoptosis
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Human cytomegalovirus carries a mitochondria-localized inhibitor of apoptosis (vMIA) that is conserved in primate cytomegaloviruses. We find that inactivating mutations within UL37x1, which encodes vMIA, do not substantially affect replication in TownevarATCC (Towne-BAC), a virus that carries a functional copy of the betaherpesvirus-conserved viral inhibitor of caspase 8 activation, the UL36 gene product. In Towne-BAC infection, vMIA reduces susceptibility of infected cells to intrinsic death induced by proteasome inhibition. vMIA is sufficient to confer resistance to proteasome inhibition when expressed independent of viral infection. Murine cytomegalovirus m38.5, whose position in the viral genome is analogous to UL37x1, exhibits mitochondrial association and functions in much the same manner as vMIA in inhibiting intrinsic cell death. This work suggests a common role for vMIA in rodent and primate cytomegaloviruses, modulating the threshold of virus-infected cells to intrinsic cell death. 相似文献
3.
Cell death suppression by cytomegaloviruses 总被引:5,自引:0,他引:5
Goldmacher VS 《Apoptosis : an international journal on programmed cell death》2005,10(2):251-265
Cytomegaloviruses (CMVs), a subset of betaherpesviruses, employ multiple strategies to suppress apoptosis in infected cells and thus to delay their death. Human cytomegalovirus (HCMV) encodes at least two proteins that directly interfere with the apoptotic signaling pathways, viral inhibitor of caspase-8-induced apoptosis vICA (pUL36), and mitochondria-localized inhibitor of apoptosis vMIA (pUL37 × 1). vICA associates with pro-caspase-8 and appears to block its recruitment to the death-inducing signaling complex (DISC), a step preceding caspase-8 activation. vMIA binds and sequesters Bax at mitochondria, and interferes with BH3-only-death-factor/Bax-complex-mediated permeabilization of mitochondria. vMIA does not seem to either interact with Bak, a close structural and functional homologue of Bax, or to suppress Bak-mediated permeabilization of mitochondria and Bak-mediated apoptosis. All sequenced betaherpesviruses, including CMVs, encode close homologues of vICA, and those vICA homologues that have been tested, were found to be functional cell death suppressors. Overt sequence homologues of vMIA were found only in the genomes of primate CMVs, but recent observations made with murine CMV (MCMV) indicate that non-primate CMVs may also encode a cell death suppressor functionally resembling vMIA. The exact physiological rolesand relative contributions of vMIA and vICA in suppressing death of CMV-infected cells in vivo have not been elucidated. There is strong evidence that the cell death suppressing function of vMIA is indispensable, and that vICA is dispensable for replication of HCMV. In addition to suppressed caspase-8 activation and sequestered Bax, CMV-infected cells display several other phenomena, less well characterized, that may diminish, directly or indirectly the extent of cell death. 相似文献
4.
Viruses encode suppressors of cell death to block intrinsic and extrinsic host-initiated death pathways that reduce viral yield as well as control the termination of infection. Cytomegalovirus (CMV) infection terminates by a caspase-independent cell fragmentation process after an extended period of continuous virus production. The viral mitochondria-localized inhibitor of apoptosis (vMIA; a product of the UL37x1 gene) controls this fragmentation process. UL37x1 mutant virus-infected cells fragment three to four days earlier than cells infected with wt virus. Here, we demonstrate that infected cell death is dependent on serine proteases. We identify mitochondrial serine protease HtrA2/Omi as the initiator of this caspase-independent death pathway. Infected fibroblasts develop susceptibility to death as levels of mitochondria-resident HtrA2/Omi protease increase. Cell death is suppressed by the serine protease inhibitor TLCK as well as by the HtrA2-specific inhibitor UCF-101. Experimental overexpression of HtrA2/Omi, but not a catalytic site mutant of the enzyme, sensitizes infected cells to death that can be blocked by vMIA or protease inhibitors. Uninfected cells are completely resistant to HtrA2/Omi induced death. Thus, in addition to suppression of apoptosis and autophagy, vMIA naturally controls a novel serine protease-dependent CMV-infected cell-specific programmed cell death (cmvPCD) pathway that terminates the CMV replication cycle. 相似文献
5.
Many viruses have evolved strategies to either evade or hijack host cell immune programs, as a means of promoting their own reproduction. For example, the human cytomegalovirus (HCMV) immediate-early protein vMIA/UL37ex1 inhibits host cell apoptosis, and its expression during infection aids virus replication. Here it is shown that stable expression of vMIA/UL37ex1 reduces cleavage of the innate immune response-proteins MAVS and RIG-I by caspases during apoptosis. Unexpectedly, it is demonstrated that RIG-I, but not MAVS, is degraded during HCMV infection. This process occurs in a non-apoptotic manner, and provides new evidence that HCMV may have evolved a unique strategy to evade RIG-I-mediated immune responses. 相似文献
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7.
Disruption of mitochondrial networks by the human cytomegalovirus UL37 gene product viral mitochondrion-localized inhibitor of apoptosis
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By 24 h after infection with human cytomegalovirus, the reticular mitochondrial network characteristic of uninfected fibroblasts was disrupted as mitochondria became punctate and dispersed. These alterations were associated with expression of the immediate-early (alpha) antiapoptotic UL37x1 gene product viral mitochondrion-localized inhibitor of apoptosis (vMIA). Similar alterations in mitochondrial morphology were induced directly by vMIA in transfected cells. A 68-amino-acid antiapoptotic derivative of vMIA containing the mitochondrial localization and antiapoptotic domains also induced disruption, whereas a mutant lacking the antiapoptotic domain failed to cause disruption. These data suggest that the fission and/or fusion process that normally controls mitochondrial networks is altered by vMIA. Mitochondrial fission has been implicated in the induction of apoptosis and vMIA-mediated inhibition of apoptosis may occur subsequent to this event. 相似文献
8.
The human cytomegalovirus (HCMV) protein UL37 exon 1 (pUL37x1), also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), sequentially traffics from the endoplasmic reticulum (ER) through mitochondrion-associated membranes (MAMs) to the outer mitochondrial membrane (OMM), where it robustly inhibits apoptosis. Here, we report the association of pUL37x1/vMIA with internal lipid rafts (LRs) in the ER/MAM. The MAM, which serves as a site for lipid transfer and calcium signaling to mitochondria, is enriched in detergent-resistant membrane (DRM)-forming lipids, including cholesterol and ceramide, which are found in lower concentrations in the bulk ER. Sigma 1 receptor (Sig-1R), a MAM chaperone affecting calcium signaling to mitochondria, is anchored in the MAM by its LR association. Because of its trafficking through the MAM and partial colocalization with Sig-1R, we tested whether pUL37x1/vMIA associates with MAM LRs. Extraction with methyl-β-cyclodextrin (MβCD) removed pUL37x1/vMIA from lysed but not intact cells, indicating its association with internal LRs. Furthermore, the isolation of DRMs from purified intracellular organelles independently verified the localization of pUL37x1/vMIA within ER/MAM LRs. However, pUL37x1/vMIA was not detected in DRMs from mitochondria. pUL37x1/vMIA associated with LRs during all temporal phases of HCMV infection, indicating the likely importance of this location for HCMV growth. Although detected during its sequential trafficking to the OMM, the pUL37x1/vMIA LR association was independent of its mitochondrial targeting signals. Rather, it was dependent upon cholesterol binding. These studies suggest a conserved ability of UL37 proteins to interact with cholesterol and LRs, which is functionally distinguishable from their sequential trafficking to mitochondria. 相似文献
9.
Cyclin-dependent kinase activity is required at early times for accurate processing and accumulation of the human cytomegalovirus UL122-123 and UL37 immediate-early transcripts and at later times for virus production 总被引:3,自引:0,他引:3
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Sanchez V McElroy AK Yen J Tamrakar S Clark CL Schwartz RA Spector DH 《Journal of virology》2004,78(20):11219-11232
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Cytomegalovirus-mediated induction of antisense mRNA expression to UL44 inhibits virus replication in an astrocytoma cell line: identification of an essential gene. 总被引:6,自引:5,他引:1
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We have used an antisense RNA approach in the analysis of gene function in human cytomegalovirus (HCMV). An astrocytoma cell line (U373-MG) that is permissive for virus replication was permanently transfected with a construct bearing sequence from HCMV UL44 (coding for the major late DNA-binding protein, ppUL44, also known as pp52 or ICP36) in an antisense orientation and under the control of the immediate-early enhancer-promoter element. Upon HCMV infection at a high multiplicity, we found a marked reduction in UL44 protein products (the ICP36 family of proteins) in established cell transfectants and a strong inhibition of virus yield in infected-cell supernatants at two weeks postinfection, while herpes simplex virus replication was not affected. In infected cells, viral DNA replication was strongly inhibited. While gene products such as pUS22 and pUL32 were also inhibited, pUL123 and pUL82 accumulated in the infected cells over time. Our data suggest an essential role for the UL44 family of proteins in HCMV replication and represent a model of virus inhibition by virus-induced antisense RNA synthesis in genetically modified cells. 相似文献
12.
The murine cytomegalovirus cell death suppressor m38.5 binds Bax and blocks Bax-mediated mitochondrial outer membrane permeabilization 总被引:1,自引:0,他引:1
Arnoult D Skaletskaya A Estaquier J Dufour C Goldmacher VS 《Apoptosis : an international journal on programmed cell death》2008,13(9):1100-1110
Apoptosis is increasingly implicated as an early line of defense against viral infections. Viruses have devised numerous strategies
to delay apoptosis of infected cells. Many viruses encode cell death suppressors that target mitochondrial apoptotic signaling
pathway, indicating the importance of this pathway in the anti-viral response. Human and primate cytomegaloviruses encode
the viral mitochondria-localized inhibitor of apoptosis vMIA, but no overt homologue of vMIA was identified in any non-primate
cytomegalovirus. Here we report that m38.5 protein encoded by murine cytomegalovirus, which is unrelated to vMIA in its amino
acid sequence, delays death receptor ligation-induced cell death, and that m38.5 associates with Bax, recruits it to mitochondria,
and blocks Bax-mediated but not Bak-mediated mitochondrial outer membrane permeabilization. Thus, primate and murine cytomegaloviruses
have evolved non-homologous but functionally similar cell death suppressors selectively targeting the Bax-mediated branch
of the mitochondrial apoptotic signaling pathway, indicating the importance of this branch in the response of diverse host
organisms against cytomegalovirus infections. 相似文献
13.
Rhesus cytomegalovirus (RhCMV) is an emerging model for human cytomegalovirus (HCMV) pathogenesis that facilitates experimental CMV infection of a natural primate host closely related to humans. We have generated a library of RhCMV mutants with lesions in genes whose HCMV orthologues have been characterized as nonessential for replication in human fibroblasts, and we characterized their replication in rhesus fibroblasts and epithelial cells. The RhCMV mutants grew well in fibroblasts, as predicted by earlier studies with HCMV. However, mutations in four genes caused replication defects in rhesus retinal pigment epithelial cells: Rh01 (an HCMV TRL1 orthologue), Rh159 (HCMV UL148), Rh160 (HCMV UL132), and Rh203 (HCMV US22). Growth of the Rh01-deficient mutant was examined in detail. After entry into epithelial cells, the mutant expressed representative viral proteins, accumulated viral DNA, and generated infectious virus, but it failed to spread efficiently. We conclude that Rh01 is a cell tropism determinant that has the potential to dramatically affect virus spread and pathogenesis. 相似文献
14.
Poncet D Larochette N Pauleau AL Boya P Jalil AA Cartron PF Vallette F Schnebelen C Bartle LM Skaletskaya A Boutolleau D Martinou JC Goldmacher VS Kroemer G Zamzami N 《The Journal of biological chemistry》2004,279(21):22605-22614
The viral mitochondria-localized inhibitor of apoptosis (vMIA), encoded by the UL37 gene of human cytomegalovirus, inhibits apoptosis-associated mitochondrial membrane permeabilization by a mechanism different from that of Bcl-2. Here we show that vMIA induces several changes in Bax that resemble those found in apoptotic cells yet take place in unstimulated, non-apoptotic vMIA-expressing cells. These changes include the constitutive localization of Bax at mitochondria, where it associates tightly with the mitochondrial membrane, forming high molecular weight aggregates that contain vMIA. vMIA recruits Bax to mitochondria but delays relocation of caspase-8-activated truncated Bid-green fluorescent protein (GFP) (t-Bid-GFP) to mitochondria. The ability of vMIA and its deletion mutants to associate with Bax and to induce relocation of Bax to mitochondria correlates with their anti-apoptotic activity and with their ability to suppress mitochondrial membrane permeabilization. Taken together, our data indicate that vMIA blocks apoptosis via its interaction with Bax. vMIA neutralizes Bax by recruiting it to mitochondria and "freezing" its pro-apoptotic activity. These data unravel a novel strategy of subverting an intrinsic pathway of apoptotic signaling. 相似文献
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Goldner T Hewlett G Ettischer N Ruebsamen-Schaeff H Zimmermann H Lischka P 《Journal of virology》2011,85(20):10884-10893
Human cytomegalovirus (HCMV) remains the leading viral cause of birth defects and life-threatening disease in transplant recipients. All approved antiviral drugs target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance. Attempts to discover improved anti-HCMV drugs led to the identification of the small-molecular-weight compound AIC246 (Letermovir). AIC246 exhibits outstanding anti-HCMV activity in vitro and in vivo and currently is undergoing a clinical phase IIb trial. The initial mode-of-action studies suggested that the drug acts late in the HCMV replication cycle via a mechanism distinct from that of polymerase inhibitors. Here, we extend our mode-of-action analyses and report that AIC246 blocks viral replication without inhibiting the synthesis of progeny HCMV DNA or viral proteins. The genotyping of mutant viruses that escaped AIC246 inhibition uncovered distinct point mutations in the UL56 subunit of the viral terminase complex. Marker transfer analyses confirmed that these mutations were sufficient to mediate AIC246 resistance. The mapping of drug resistance to open reading frame UL56 suggests that viral DNA processing and/or packaging is targeted by AIC246. In line with this, we demonstrate that AIC246 affects the formation of proper unit-length genomes from viral DNA concatemers and interferes with virion maturation. However, since AIC246-resistant viruses do not exhibit cross-resistance to previously published terminase inhibitors, our data suggest that AIC246 interferes with HCMV DNA cleavage/packaging via a molecular mechanism that is distinct from that of other compound classes known to target the viral terminase. 相似文献
18.
Marsh AK Willer DO Ambagala AP Dzamba M Chan JK Pilon R Fournier J Sandstrom P Brudno M MacDonald KS 《Journal of virology》2011,85(24):12995-13009
Cytomegalovirus (CMV) infection is the most common opportunistic infection in immunosuppressed individuals, such as transplant recipients or people living with HIV/AIDS, and congenital CMV is the leading viral cause of developmental disabilities in infants. Due to the highly species-specific nature of CMV, animal models that closely recapitulate human CMV (HCMV) are of growing importance for vaccine development. Here we present the genomic sequence of a novel nonhuman primate CMV from cynomolgus macaques (Macaca fascicularis; CyCMV). CyCMV (Ottawa strain) was isolated from the urine of a healthy, captive-bred, 4-year-old cynomolgus macaque of Philippine origin, and the viral genome was sequenced using next-generation Illumina sequencing to an average of 516-fold coverage. The CyCMV genome is 218,041 bp in length, with 49.5% G+C content and 84% protein-coding density. We have identified 262 putative open reading frames (ORFs) with an average coding length of 789 bp. The genomic organization of CyCMV is largely colinear with that of rhesus macaque CMV (RhCMV). Of the 262 CyCMV ORFs, 137 are homologous to HCMV genes, 243 are homologous to RhCMV 68.1, and 200 are homologous to RhCMV 180.92. CyCMV encodes four ORFs that are not present in RhCMV strain 68.1 or 180.92 but have homologies with HCMV (UL30, UL74A, UL126, and UL146). Similar to HCMV, CyCMV does not produce the RhCMV-specific viral homologue of cyclooxygenase-2. This newly characterized CMV may provide a novel model in which to study CMV biology and HCMV vaccine development. 相似文献
19.
人巨细胞病毒(HCMV) UL23基因编码病毒皮层蛋白,该基因缺失时,病毒在人包皮成纤维细胞(HFF)中的繁殖速度加快.为进一步阐述HCMV UL23基因编码产物 pUL23的功能及调控机制,采用鸟枪法构建了融合于GAL4活性区域的HCMV Towne株 基因组随机表达文库.利用酵母双杂交技术,以pGBKT7 -UL23为诱饵质粒,从构建 的HCMV基因组表达文库中筛选到与pUL23相互作用的病毒编码蛋白pUL24. GST-pull down实验和免疫共沉淀实验进一步确认两种病毒蛋白之间的相互作用.结果 表明,构建的HCMV基因组表达文库能够用于GAL4酵母双杂交系统筛选与诱饵蛋白相互作用的病毒自身编码蛋白.病毒蛋白pUL23和pUL24之间具有相互作用,这为进一 步阐述pUL23在HCMV感染过程中的功能提供依据.该研究为揭示HCMV病毒感染机制奠定了基础. 相似文献