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Open accessJournal ArticleDOI: 10.1080/15548627.2020.1732686

Autophagy interferes with human cytomegalovirus genome replication, morphogenesis, and progeny release.

04 Mar 2021-Autophagy (Taylor & Francis)-Vol. 17, Iss: 3, pp 779-795
Abstract: Viral infections are often accompanied by the induction of autophagy as an intrinsic cellular defense mechanism. Herpesviruses have developed strategies to evade autophagic degradation and to manipulate autophagy of the host cells to their benefit. Here we addressed the role of macroautophagy/autophagy in human cytomegalovirus replication and for particle morphogenesis. We found that proteins of the autophagy machinery localize to cytoplasmic viral assembly compartments and enveloped virions in the cytoplasm. Surprisingly, the autophagy receptor SQSTM1/p62 was also found to colocalize with HCMV capsids in the nucleus of infected cells. This finding indicates that the autophagy machinery interacts with HCMV already at the early nuclear stages of particle morphogenesis. The membrane-bound form of LC3 and several autophagy receptors were packaged into extracellular HCMV virions. This suggested that autophagic membranes were included during secondary envelopment of HCMV virions. To further address the importance of autophagy in HCMV infection, we generated an HCMV mutant that expressed a dominant-negative version of the protease ATG4B (BAD-ATG4BC74A). The proteolytic activity of ATG4B is required for LC3 cleavage, priming it for membrane conjugation. Surprisingly, both genome replication and virus release were enhanced in cells infected with BAD-ATG4BC74A, compared to control strains. These results show that autophagy operates as an antiviral process during HCMV infection but is dispensable for secondary HCMV particle envelopment.Abbreviations: ATG: autophagy-related; BAC: bacterial artificial chromosome; BECN1: beclin 1; CPE: cytopathic effect; cVACs: cytoplasmic viral assembly compartments; d.p.i.: days post-infection; DB: dense body; EBV: Epstein-Barr virus; galK: galactokinase; HCMV: human cytomegalovirus; HFF: human foreskin fibroblasts; IE: immediate-early; IRS: internal repeat short; LC3: MAP1LC3A/B; m.o.i.; multiplicity of infection; MCP: major capsid protein; Pp: phosphoprotein; sCP/UL48a: smallest capsid protein; TRS: terminal repeat short; UL: unique long; US: unique short.

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Topics: BECN1 (62%), Autophagy (55%), Human cytomegalovirus (55%) ... read more
Citations
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7 results found


Open accessJournal ArticleDOI: 10.1128/JVI.02346-20
Abstract: Autophagy is a catabolic process contributing to intrinsic cellular defense by degrading viral particles or proteins; however, several viruses hijack this pathway for their own benefit. The role of autophagy during human cytomegalovirus (HCMV) replication has not been definitely clarified yet. Utilizing small interfering RNA (siRNA)-based screening, we observed that depletion of many autophagy-related proteins resulted in reduced virus release, suggesting a requirement of autophagy-related factors for efficient HCMV replication. Additionally, we could show that the autophagy-initiating serine/threonine protein kinase ULK1 as well as other constituents of the ULK1 complex were upregulated at early times of infection and stayed upregulated throughout the replication cycle. We demonstrate that indirect interference with ULK1 through inhibition of the upstream regulator AMP-activated protein kinase (AMPK) impaired virus release. Furthermore, this result was verified by direct abrogation of ULK1 kinase activity utilizing the ULK1-specific kinase inhibitors SBI-0206965 and ULK-101. Analysis of viral protein expression in the presence of ULK-101 revealed a connection between the cellular kinase ULK1 and the viral tegument protein pp28 (pUL99), and we identified pp28 as a novel viral substrate of ULK1 by in vitro kinase assays. In the absence of ULK1 kinase activity, large pp28- and pp65-positive structures could be detected in the cytoplasm at late time points of infection. Transmission electron microscopy demonstrated that these structures represent large perinuclear protein accumulations presumably representing aggresomes. Our results indicate that HCMV manipulates ULK1 and further components of the autophagic machinery to ensure the efficient release of viral particles.IMPORTANCE The catabolic program of autophagy represents a powerful immune defense against viruses that is, however, counteracted by antagonizing viral factors. Understanding the exact interplay between autophagy and HCMV infection is of major importance since autophagy-related proteins emerged as promising targets for pharmacologic intervention. Our study provides evidence for a proviral role of several autophagy-related proteins suggesting that HCMV has developed strategies to usurp components of the autophagic machinery for its own benefit. In particular, we observed strong upregulation of the autophagy-initiating protein kinase ULK1 and further components of the ULK1 complex during HCMV replication. In addition, both siRNA-mediated depletion of ULK1 and interference with ULK1 protein kinase activity by two chemically different inhibitors resulted in impaired viral particle release. Thus, we propose that ULK1 kinase activity is required for efficient HCMV replication and thus represents a promising novel target for future antiviral drug development.

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Topics: Kinase activity (64%), Protein kinase A (61%), Viral protein (59%) ... read more

6 Citations


Journal ArticleDOI: 10.1002/MED.21772
Fan Xia1, Peiqing Liu1, Min Li1Institutions (1)
Abstract: Macroautophagy (autophagy) is an evolutionarily conserved and dynamic degradation/recycling pathway in which portions of the cytoplasm, such as dysfunctional proteins and surplus organelles, are engulfed by double-membrane bound vesicles through a lysosome-dependent process. As the only proteolytic enzyme of the core mammalian autophagy proteins, autophagy-related protein 4 (ATG4) primes newly synthesized pro-light chain 3 (LC3) to form LC3-I that attaches to phosphatidylethanolamine and delipidates LC3-PE to LC3-I for recycling. Besides autophagy, ATG4 has been shown to be involved in regulating various biological and pathological processes. The roles of ATG4 in cancer therapy, a methodology for ATG4 activity detection, and the discovery of chemical modulators have been well-reviewed. However, a comprehensive summary on how ATG4 is regulated by multiple factors and, thereby, how ATG4 influences autophagy or other pathways remains lacking. In this paper, we summarize multiple processes and molecules that regulate the activity of ATG4, such as micro-RNAs, posttranslational modifications, and small molecules. Additionally, we focus on the relationship between ATG4 and diverse diseases, including cancer, neurodegeneration, microbial infection, and other diseases. It provides insight regarding potential ATG4-targeted therapeutic opportunities, which could be beneficial for future studies and human health.

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Topics: Autophagy (56%), Proteolytic enzymes (54%)

4 Citations


Journal ArticleDOI: 10.1002/JMV.26357
Xinyan Zhang1, Ting Xi1, Lin-Lin Zhang1, Yidan Bi1  +4 moreInstitutions (1)
Abstract: The purpose of this study was to determine whether autophagy regulates the expression of human cytomegalovirus (HCMV) immediately early two viral protein (IE2). Rapamycin and 3-methyladenine (3-MA) were used to stimulate or suppress autophagy during HCMV infection. UL122 recombinant plasmid was transfected to overexpress IE2 and small interference RNA against autophagy-related protein 3 (ATG3) was used to knockdown ATG3. Western blot was performed to measure the expression of viral proteins and autophagy levels. Immunofluorescence was used to detect the immediately early 1 viral protein (IE1) expression. In human embryonic lung fibroblasts, infection of HCMV promotes the lipidation of light chain 3 (LC3) at 6 and 24 hours post infection (hpi), which was accompanied by the increased expression of viral protein IE2. When only IE2 was overexpressed via UL122 recombinant plasmid transfection without HCMV infection, the autophagy hallmarks LC3II and ATG3 were upregulated. Furthermore, viral protein IE2 expression was reduced at 24 and 48 hpi either by the treatment of autophagy inducer rapamycin or by the inhibitor 3-MA before HCMV infection. At the same time, small interference ATG3 transient transfection, used to suppress autophagy, significantly inhibited IE2 expression. However, when 3-MA was used to regulate autophagy levels after HCMV infection, expression of IE2 and IE1 were both decreased, while autophagy inducer rapamycin treatment after HCMV infection increased IE2 expression slightly. IE2 was involved in autophagy induced by HCMV infection and blocking autophagy could inhibit the expression of HCMV viral protein IE2, which might be one way for autophagy to restrict HCMV replication.

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Topics: Autophagy (57%), Human cytomegalovirus (56%), Gene knockdown (51%) ... read more

1 Citations


Book ChapterDOI: 10.1007/978-3-030-67989-7_3
Saqib Mahmood1, Tariq Mahmood, Naeem Iqbal1, Samina Sabir1  +2 moreInstitutions (3)
01 Jan 2021-
Abstract: Keeping in view the pandemic situation and whose recommendations to search antiviral medicines, biologists, pathologists, and microbiologists started working on that Chinese government has recommended therapeutic application of traditional Chinese medicine (TCM) against COVID-19 in the third edition of COVID-19 Treatment Guidelines (2020) published in January From the day onward, hundreds of herbs have been practiced all over China This chapter has been planned to compile existing information for future utilization of TCM all over the world with better understanding and minimal chances of side effects For well understanding of TCM as reliable treatment (for COVID-19), it is aimed to pool data of TCM from its history of usage against other viruses and against related viruses of SARS-CoV, collection of the data regarding case studies with positive outcomes, detailed pool of knowledge about most practiced (approved/proposed) decoctions, frequently used components of these decoctions, their active ingredients, possible mechanism to inhibit viruses, similarity of SARS-CoV with SARS-CoV-2, and possibility to treat COVID-19 with antiviral drugs previously used against SARS-CoV Additional relevant meta-analyses have been reviewed that may guide about practical implementation of these TCM in the future round the globe

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1 Citations



References
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81 results found


Journal ArticleDOI: 10.1038/NPROT.2006.468
Andrej Shevchenko1, Henrik Tomas1, Jan Havliš1, Jesper V. Olsen1  +1 moreInstitutions (1)
01 Dec 2006-Nature Protocols
Abstract: In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.

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Topics: Bottom-up proteomics (65%), In-gel digestion (59%), Gel electrophoresis (55%) ... read more

4,008 Citations


Open accessJournal ArticleDOI: 10.1016/J.CELL.2005.07.002
Sophie Pattingre1, Sophie Pattingre2, Amina T. Tassa2, Xueping Qu1  +8 moreInstitutions (4)
23 Sep 2005-Cell
Abstract: Apoptosis and autophagy are both tightly regulated biological processes that play a central role in tissue homeostasis, development, and disease. The anti-apoptotic protein, Bcl-2, interacts with the evolutionarily conserved autophagy protein, Beclin 1. However, little is known about the functional significance of this interaction. Here, we show that wild-type Bcl-2 antiapoptotic proteins, but not Beclin 1 binding defective mutants of Bcl-2, inhibit Beclin 1-dependent autophagy in yeast and mammalian cells and that cardiac Bcl-2 transgenic expression inhibits autophagy in mouse heart muscle. Furthermore, Beclin 1 mutants that cannot bind to Bcl-2 induce more autophagy than wild-type Beclin 1 and, unlike wild-type Beclin 1, promote cell death. Thus, Bcl-2 not only functions as an antiapoptotic protein, but also as an antiautophagy protein via its inhibitory interaction with Beclin 1. This antiautophagy function of Bcl-2 may help maintain autophagy at levels that are compatible with cell survival, rather than cell death.

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Topics: BECN1 (66%), Autophagy (62%), BAG3 (62%) ... read more

3,139 Citations


Journal ArticleDOI: 10.1038/NPROT.2007.261
01 Jan 2007-Nature Protocols
Abstract: Mass spectrometry (MS)-based proteomics measures peptides derived from proteins by proteolytic cleavage. Before performing the analysis by matrix-assisted laser desorption/ionization-tandem mass spectrometry (MALDI-MS/MS), nanoelectrospray-MS/MS (NanoES-MS/MS) or liquid chromatography-MS/MS (LC-MS/MS), the peptide mixtures need to be cleaned, concentrated and often selectively enriched or pre-fractionated, for which we employ simple, self-made and extremely economical stop-and-go-extraction tips (StageTips). StageTips are ordinary pipette tips containing very small disks made of beads with reversed phase, cation-exchange or anion-exchange surfaces embedded in a Teflon mesh. The fixed nature of the beads allows flexible combination of disks with different surfaces to obtain multi-functional tips. Disks containing different surface functionalities and loose beads such as titania and zirconia for phosphopeptide enrichment can be combined. Incorporation into an automated workflow has also been demonstrated. Desalting and concentration takes approximately 5 min while fractionation or enrichment takes approximately 30 min.

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Topics: Mass spectrometry (51%)

2,803 Citations


Journal ArticleDOI: 10.1038/NCB1007-1102
Zhiping Xie1, Daniel J. Klionsky1Institutions (1)
Abstract: Eukaryotic cells employ autophagy to degrade damaged or obsolete organelles and proteins. Central to this process is the formation of autophagosomes, double-membrane vesicles responsible for delivering cytoplasmic material to lysosomes. In the past decade many autophagy-related genes, ATG, have been identified that are required for selective and/or nonselective autophagic functions. In all types of autophagy, a core molecular machinery has a critical role in forming sequestering vesicles, the autophagosome, which is the hallmark morphological feature of this dynamic process. Additional components allow autophagy to adapt to the changing needs of the cell.

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Topics: Autophagy database (62%), Autophagosome (62%), Atg1 (60%) ... read more

1,903 Citations


Open accessJournal ArticleDOI: 10.1093/NAR/GNI035
Abstract: Recombineering allows DNA cloned in Escherichia coli to be modified via lambda (l) Red-mediated homologous recombination, obviating the need for restriction enzymes and DNA ligases to modify DNA Here, we describe the construction of three new recombineering strains (SW102, SW105 and SW106) that allow bacterial artificial chromosomes (BACs) to be modified using galK positive/negative selection This two-step selection procedure allows DNA to be modified without introducing an unwanted selectable marker at the modification site All three strains contain an otherwise complete galactose operon, except for a precise deletion of the galK gene, and a defective temperature-sensitive l prophage that makes recombineering possible SW105 and SW106 cells in addition carry L-arabinose-inducible Cre or Flp genes, respectively The galK function can be selected both for and against This feature greatly reduces the background seen in other negative-selection schemes, and galK selection is considerably more efficient than other related selection methods published We also show how galK selection can be used to rapidly introduce point mutations, deletions and loxP sites into BAC DNA and thus facilitate functional studies of SNP and/or disease-causing point mutations, the identification of long-range regulatory elements and the construction of conditional targeting vectors

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Topics: Recombineering (66%), Selectable marker (52%), Negative selection (52%)

1,216 Citations