Catch me if you can – the crosstalk of ZIKV and the restriction factor Tetherin

Zika virus (ZIKV) is a flavivirus that is mainly transmitted by Aedes mosquitos and normally causes mild symptoms. During the outbreak in the Americas in 2015, it was associated with more severe implications, like microcephaly in new-borns and the Gullain-Barré syndrome. The lack of specific vaccines and cures strengthen the need for a deeper understanding of the virus life cycle and virus-host interactions. The restriction factor tetherin (THN) is an interferon-inducible cellular protein with broad antiviral properties. It is known to inhibit the release of various enveloped viruses by tethering them to each other and to the cell membrane, thereby preventing their further spread. On the other hand, different viruses have developed various escape strategies against THN. Analysis of the crosstalk between ZIKV and THN revealed that in spite of a strong induction of THN mRNA expression in ZIKV-infected cells, this is not reflected by an elevated protein level of THN. Contrariwise, the THN protein level is decreased due to a reduced half-life. The increased degradation of THN in ZIKV infected cells involves the endo-lysosomal system, but does not depend on the early steps of autophagy. Enrichment of THN by depletion of the ESCRT-0 protein HRS diminishes ZIKV release and spread, which points out the capacity of THN to restrict ZIKV and explains the enhanced THN degradation in infected cells as an effective viral escape strategy. Importance Although tetherin expression is strongly induced by ZIKV infection there is a reduction in the amount of tetherin protein. This is due to an enhanced lysosomal degradation. However, if tetherin level is rescued release of ZIKV is impaired. This shows that tetherin is a restriction factor for ZIKV and the induction of an efficient degradation represents a viral escape strategy. To our knowledge this is the first study that describes and characterizes tetherin as an restriction factor for ZIKV life cycle.

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Role of Virally-Encoded Deubiquitinating Enzymes in Regulation of the Virus Life Cycle

International Journal of Molecular Sciences ◽

10.3390/ijms22094438 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4438

Author(s):

Jessica Proulx ◽

Kathleen Borgmann ◽

In-Woo Park

Keyword(s):

Immune Response ◽

Life Cycle ◽

Deubiquitinating Enzyme ◽

Deubiquitinating Enzymes ◽

Antiviral Immune Response ◽

Cellular Processes ◽

Virus Life Cycle ◽

Infected Cells ◽

Dependent Processes

The ubiquitin (Ub) proteasome system (UPS) plays a pivotal role in regulation of numerous cellular processes, including innate and adaptive immune responses that are essential for restriction of the virus life cycle in the infected cells. Deubiquitination by the deubiquitinating enzyme, deubiquitinase (DUB), is a reversible molecular process to remove Ub or Ub chains from the target proteins. Deubiquitination is an integral strategy within the UPS in regulating survival and proliferation of the infecting virus and the virus-invaded cells. Many viruses in the infected cells are reported to encode viral DUB, and these vial DUBs actively disrupt cellular Ub-dependent processes to suppress host antiviral immune response, enhancing virus replication and thus proliferation. This review surveys the types of DUBs encoded by different viruses and their molecular processes for how the infecting viruses take advantage of the DUB system to evade the host immune response and expedite their replication.

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Genome-Wide Expression Patterns of Rhoptry Kinases during the Eimeria tenella Life-Cycle

Microorganisms ◽

10.3390/microorganisms9081621 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1621

Author(s):

Adeline Ribeiro E Silva ◽

Alix Sausset ◽

Françoise I. Bussière ◽

Fabrice Laurent ◽

Sonia Lacroix-Lamandé ◽

...

Keyword(s):

Life Cycle ◽

Expression Patterns ◽

Housekeeping Genes ◽

Catalytic Triad ◽

Eimeria Tenella ◽

Apicomplexan Parasites ◽

Genome Wide ◽

Infected Cells ◽

First And Second Generation

Kinome from apicomplexan parasites is composed of eukaryotic protein kinases and Apicomplexa specific kinases, such as rhoptry kinases (ROPK). Ropk is a gene family that is known to play important roles in host–pathogen interaction in Toxoplasma gondii but is still poorly described in Eimeria tenella, the parasite responsible for avian coccidiosis worldwide. In the E. tenella genome, 28 ropk genes are predicted and could be classified as active (n = 7), inactive (incomplete catalytic triad, n = 12), and non-canonical kinases (active kinase with a modified catalytic triad, n = 9). We characterized the ropk gene expression patterns by real-time quantitative RT-PCR, normalized by parasite housekeeping genes, during the E. tenella life-cycle. Analyzed stages were: non-sporulated oocysts, sporulated oocysts, extracellular and intracellular sporozoites, immature and mature schizonts I, first- and second-generation merozoites, and gametes. Transcription of all those predicted ropk was confirmed. The mean intensity of transcription was higher in extracellular stages and 7–9 ropk were specifically transcribed in merozoites in comparison with sporozoites. Transcriptional profiles of intracellular stages were closely related to each other, suggesting a probable common role of ROPKs in hijacking signaling pathways and immune responses in infected cells. These results provide a solid basis for future functional analysis of ROPK from E. tenella.

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Mutation of Putative N-Glycosylation Sites on Dengue Virus NS4B Decreases RNA Replication

Journal of Virology ◽

10.1128/jvi.00423-15 ◽

2015 ◽

Vol 89 (13) ◽

pp. 6746-6760 ◽

Cited By ~ 26

Author(s):

Nenavath Gopal Naik ◽

Huey-Nan Wu

Keyword(s):

Life Cycle ◽

Recombinant Protein ◽

Dengue Virus ◽

Protein Expression ◽

Nonstructural Protein ◽

Recombinant Protein Expression ◽

Rna Replication ◽

Viral Production ◽

Glycosylation Sites ◽

Infected Cells

ABSTRACTDengue virus (DENV) nonstructural protein 4B (NS4B) is an endoplasmic reticulum (ER) membrane-associated protein, and mutagenesis studies have revealed its significance in viral genome replication. In this work, we demonstrated that NS4B is an N-glycosylated protein in virus-infected cells as well as in recombinant protein expression. NS4B is N glycosylated at residues 58 and 62 and exists in two forms, glycosylated and unglycosylated. We manipulated full-length infectious RNA clones and subgenomic replicons to generate N58Q, N62Q, and N58QN62Q mutants. Each of the single mutants had distinct effects, but the N58QN62Q mutation resulted in dramatic reduction of viral production efficiency without affecting secretion or infectivity of the virion in mammalian and mosquito C6/36 hosts. Real-time quantitative PCR (qPCR), subgenomic replicon, andtrans-complementation assays indicated that the N58QN62Q mutation affected RNA replication possibly by the loss of glycans. In addition, four intragenic mutations (S59Y, S59F, T66A, and A137T) were obtained from mammalian and/or mosquito C6/36 cell culture systems. All of these second-site mutations compensated for the replication defect of the N58QN62Q mutant without creating novel glycosylation sites.In vivoprotein stability analyses revealed that the N58QN62Q mutation alone or plus a compensatory mutation did not affect the stability of NS4B. Overall, our findings indicated that mutation of putative N-glycosylation sites affected the biological function of NS4B in the viral replication complex.IMPORTANCEThis is the first report to identify and reveal the biological significance of dengue virus (DENV) nonstructural protein 4B (NS4B) posttranslation N-glycosylation to the virus life cycle. The study demonstrated that NS4B is N glycosylated in virus-infected cells and in recombinant protein expression. NS4B is modified by glycans at Asn-58 and Asn-62. Functional characterization implied that DENV NS4B utilizes the glycosylation machinery in both mammalian and mosquito hosts. Four intragenic mutations were found to compensate for replication and subsequent viral production deficiencies without creating novel N-glycosylation sites or modulating the stabilities of the protein, suggesting that glycans may be involved in maintaining the NS4B protein conformation. NS4B glycans may be necessary elements of the viral life cycle, but compensatory mutations can circumvent their requirement. This novel finding may have broader implications in flaviviral biology as the most likely glycan at Asn-62 of NS4B is conserved in DENV serotypes and in some related flaviviruses.

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Bub1 in Complex with LANA Recruits PCNA To Regulate Kaposi's Sarcoma-Associated Herpesvirus Latent Replication and DNA Translesion Synthesis

Journal of Virology ◽

10.1128/jvi.01524-15 ◽

2015 ◽

Vol 89 (20) ◽

pp. 10206-10218 ◽

Cited By ~ 13

Author(s):

Zhiguo Sun ◽

Hem Chandra Jha ◽

Erle S. Robertson

Keyword(s):

Dna Replication ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Kinase Domain ◽

Cellular Protein ◽

Nuclear Antigen ◽

Infected Cells ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Molecular Bridge

ABSTRACTLatent DNA replication of Kaposi's sarcoma-associated herpesvirus (KSHV) initiates at the terminal repeat (TR) element and requirestrans-acting elements, both viral and cellular, such as ORCs, MCMs, and latency-associated nuclear antigen (LANA). However, how cellular proteins are recruited to the viral genome is not very clear. Here, we demonstrated that the host cellular protein, Bub1, is involved in KSHV latent DNA replication. We show that Bub1 constitutively interacts with proliferating cell nuclear antigen (PCNA) via a highly conserved PIP box motif within the kinase domain. Furthermore, we demonstrated that Bub1 can form a complex with LANA and PCNA in KSHV-positive cells. This strongly indicated that Bub1 serves as a scaffold or molecular bridge between LANA and PCNA. LANA recruited PCNA to the KSHV genome via Bub1 to initiate viral replication in S phase and interacted with PCNA to promote its monoubiquitination in response to UV-induced damage for translesion DNA synthesis. This resulted in increased survival of KSHV-infected cells.IMPORTANCEDuring latency in KSHV-infected cells, the viral episomal DNA replicates once each cell cycle. KSHV does not express DNA replication proteins during latency. Instead, KSHV LANA recruits the host cell DNA replication machinery to the replication origin. However, the mechanism by which LANA mediates replication is uncertain. Here, we show that LANA is able to form a complex with PCNA, a critical protein for viral DNA replication. Furthermore, our findings suggest that Bub1, a spindle checkpoint protein, serves as a scaffold or molecular bridge between LANA and PCNA. Our data further support a role for Bub1 and LANA in PCNA-mediated cellular DNA replication processes as well as monoubiquitination of PCNA in response to UV damage. These data reveal a therapeutic target for inhibition of KSHV persistence in malignant cells.

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Cellular Zinc Finger Protein 622 Hinders Human Adenovirus Lytic Growth and Limits Binding of the Viral pVII Protein to Virus DNA

Journal of Virology ◽

10.1128/jvi.01628-18 ◽

2018 ◽

Vol 93 (3) ◽

Cited By ~ 2

Author(s):

Kwangchol Mun ◽

Tanel Punga

Keyword(s):

Life Cycle ◽

Zinc Finger ◽

Zinc Finger Protein ◽

Human Adenovirus ◽

Antiviral Protein ◽

Viral Dna ◽

Finger Protein ◽

Wide Range ◽

Infected Cells ◽

Cellular Zinc

ABSTRACTHuman adenovirus (HAdV) encodes a multifunctional DNA-binding protein pVII, which is involved in virus DNA packaging and extracellular immune signaling regulation. Although the pVII is an essential viral protein, its exact role in the virus life cycle and interplay with cellular proteins have remained to a large extent unclear. We have recently identified the cellular zinc finger protein 622 (ZNF622) as a potential pVII-interacting protein. In this study, we describe the functional consequences of the ZNF622-pVII interplay and the role of ZNF622 in the HAdV life cycle. ZNF622 protein expression increased, and it accumulated similarly to the pVII protein in the nuclei of virus-infected cells. The lack of the ZNF622 protein specifically increased pVII binding to viral DNA in the infected cells and elevated the pVII protein levels in the purified virions. In addition, ZNF622 knockout cells showed an increased cell lysis and enhanced accumulation of the infectious virus particles. Protein interaction studies revealed that ZNF622 forms a trimeric complex with the pVII protein and the cellular histone chaperon protein nucleophosmin 1 (NPM1). The integrity of this complex is important since ZNF622 mutations and NPM1 deficiency changed pVII ability to bind viral DNA. Collectively, our results implicate that ZNF622 may act as a cellular antiviral protein hindering lytic HAdV growth and limiting pVII protein binding to viral DNA.IMPORTANCEHuman adenoviruses (HAdVs) are common human pathogens causing a wide range of acute infections. To counteract viral pathogenicity, cells encode a variety of antiviral proteins and noncoding RNAs to block virus growth. In this study, we show that the cellular zinc finger protein 622 (ZNF622) interacts with an essential HAdV protein known as pVII. This mutual interaction limits pVII binding to viral DNA. Further, ZNF622 has a role in HAdV life cycle since the lack of ZNF622 correlates with increased lysis of the infected cells and accumulation of the infectious virions. Together, our study reveals a novel cellular antiviral protein ZNF622, which may impede lytic HAdV growth.

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Dynamic proteomics of HSV1 infection reveals molecular events that govern non-stochastic infection outcomes

10.1101/081653 ◽

2016 ◽

Cited By ~ 2

Author(s):

Nir Drayman ◽

Omer Karin ◽

Avi Mayo ◽

Tamar Danon ◽

Lev Shapira ◽

...

Keyword(s):

Single Cells ◽

Infection Process ◽

Human Pathogen ◽

Herpes Simplex Virus 1 ◽

Host Interactions ◽

Protein Levels ◽

Molecular Events ◽

Proteomics Approach ◽

Infected Cells ◽

Simplex Virus

AbstractViral infection is usually studied at the level of cell populations, averaging over hundreds of thousands of individual cells. Moreover, measurements are typically done by analyzing a few time points along the infection process. While informative, such measurements are limited in addressing how cell variability affects infection outcome. Here we employ dynamic proteomics to study virus-host interactions, using the human pathogen Herpes Simplex virus 1 as a model. We tracked >50,000 individual cells as they respond to HSV1 infection, allowing us to model infection kinetics and link infection outcome (productive or not) with the cell state at the time of initial infection. We find that single cells differ in their preexisting susceptibility to HSV1, and that this is partially mediated by their cell-cycle position. We also identify specific changes in protein levels and localization in infected cells, attesting to the power of the dynamic proteomics approach for studying virus-host interactions.

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Virus–host interactions in persistently FMDV-infected cells derived from bovine pharynx

Virology ◽

10.1016/j.virol.2014.08.004 ◽

2014 ◽

Vol 468-470 ◽

pp. 185-196 ◽

Cited By ~ 11

Author(s):

V. O’Donnell ◽

J.M. Pacheco ◽

Michael Larocco ◽

D.P. Gladue ◽

S.J. Pauszek ◽

...

Keyword(s):

Host Interactions ◽

Infected Cells

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System-Based Approaches to Delineate the Antiviral Innate Immune Landscape

Viruses ◽

10.3390/v12101196 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1196

Author(s):

Karsten Krey ◽

Aleksandra W. Babnis ◽

Andreas Pichlmair

Keyword(s):

Large Scale ◽

Innate Immune ◽

Host Interactions ◽

Interferon Stimulated Genes ◽

Cellular Processes ◽

Virus Inhibition ◽

Cellular Factors ◽

Functional Factors ◽

Infected Cells ◽

Screening Approaches

Viruses pose substantial challenges for society, economy, healthcare systems, and research. Their distinctive pathologies are based on specific interactions with cellular factors. In order to develop new antiviral treatments, it is of central importance to understand how viruses interact with their host and how infected cells react to the virus on a molecular level. Invading viruses are commonly sensed by components of the innate immune system, which is composed of a highly effective yet complex network of proteins that, in most cases, mediate efficient virus inhibition. Central to this process is the activity of interferons and other cytokines that coordinate the antiviral response. So far, numerous methods have been used to identify how viruses interact with cellular processes and revealed that the innate immune response is highly complex and involves interferon-stimulated genes and their binding partners as functional factors. Novel approaches and careful experimental design, combined with large-scale, high-throughput methods and cutting-edge analysis pipelines, have to be utilized to delineate the antiviral innate immune landscape at a global level. In this review, we describe different currently used screening approaches, how they contributed to our knowledge on virus–host interactions, and essential considerations that have to be taken into account when planning such experiments.

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Abundant constitutive expression of the immediate-early 94K protein from cytomegalovirus (Colburn) in a DNA-transfected mouse cell line.

Molecular and Cellular Biology ◽

10.1128/mcb.4.10.2214 ◽

1984 ◽

Vol 4 (10) ◽

pp. 2214-2223 ◽

Cited By ~ 8

Author(s):

K T Jeang ◽

M S Cho ◽

G S Hayward

Keyword(s):

Cell Line ◽

Mammalian Cells ◽

Regulatory Region ◽

Constitutive Expression ◽

Regulatory Elements ◽

Mouse Cell ◽

Cellular Protein ◽

Immediate Early ◽

Early Protein ◽

Infected Cells

A 94-kilodalton phosphoprotein known as IE94 is the only viral polypeptide synthesized in abundance under immediate-early conditions after infection by cytomegalovirus (CMV) strain Colburn in either permissive primate or nonpermissive rodent cells. The IE94 gene, which maps at coordinates 0.71 to 0.73 in the viral genome, contains a large intron in the 5' leader sequence, and its promoter regulatory region contains novel, multiple-palindromic, repeated elements. Two recombinant plasmids (pTJ148 and pTJ198) containing the 10.5-kilobase-pair HindIII-H DNA fragment from CMV (Colburn) were transfected into mouse Ltk- cells, by either linked or unlinked coselection in hypoxanthine-aminopterin-thymidine medium, together with herpes simplex virus thymidine kinase genes. With both procedures, constitutive synthesis of the IE94 immediate-early protein was detected in pools of Ltk+ cells by immunoprecipitation. Subsequently, we isolated a clonal Ltk+ cell line which expressed the [35S]methionine-labeled IE94 polypeptide in sufficient abundance to be visualized directly in autoradiographs after gel electrophoresis of total-cell-culture protein extracts. The IE94 polypeptide synthesized in the transfected cells was indistinguishable in size and overall net charge from that produced in virus-infected cells. In addition, the IE94 protein expressed in LH2p198-3 cells was phosphorylated (presumably by a cellular protein kinase) and generated similar phosphopeptide patterns after partial tryptic digestion to those obtained with the CMV IE94 protein from infected cells. The cell line contained two to four stably integrated copies of the IE94 gene and synthesized a single virus-specific mRNA of 2.5 kilobases detectable on Northern blots. A new antigen, detectable by indirect anticomplement immunofluorescence with monoclonal antibody against the human CMV IE68 protein, was present in the nuclei of more than 95% of the LH2p198-3 cells. This evidence suggests that (unlike most herpesvirus genes) the CMV IE94 gene, together with its complex promoter and spliced mRNA structure, may contain all of the regulatory elements necessary for strong constitutive expression in mammalian cells in the absence of other viral factors.

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