scholarly journals Phosphorylation by CK2 increases the SUMO-dependent activity of Cytomegalovirus transactivator IE2

2019 ◽  
Author(s):  
Vasvi Tripathi ◽  
Kiran Sankar Chatterjee ◽  
Ranabir Das
Keyword(s):  
Human Cytomegalovirus ◽  
Cross Talk ◽  
Viral Genome ◽  
Essential Gene ◽  
E3 Ligase ◽  
Host Protein ◽  
Genome Replication ◽  
Salt Bridges ◽  
Post Translational Modification ◽  
Viral Genes

AbstractViral factors manipulate the host post-translational modification (PTM) machinery for replication. Distinctly, phosphorylation and SUMOylation can regulate the activity of human cytomegalovirus (HCMV) protein IE2. However, the molecular mechanism of this process is unknown. Taking a structural, biochemical and cellular approach, we uncover a cross-talk of phosphorylation and SUMOylation exploited by IE2. A scan for the SUMO Interacting Motifs (SIMs) revealed two SIMs in IE2. A real-time SUMOylation assay indicated that the N-terminal SIM (IE2-SIM1) enhanced IE2 SUMOylation up to 4-fold. Kinetic analysis and structural studies proved that IE2 is a SUMO cis-E3 ligase. Two putative CK2 sites adjacent to IE2-SIM1 are phosphorylated in-vitro and in cellular conditions. Phosphorylation drastically increased the IE2/SUMO affinity, IE2-SUMOylation and cis-E3 activity of IE2. Additional salt-bridges between the phosphoserines and SUMO account for the higher IE2/SUMO affinity. Phosphorylation also enhances the SUMO-dependent transactivation activity and auto-repression activity of IE2. Together, our findings highlight a novel mechanism where SUMOylation and phosphorylation of the viral cis-E3 ligase and transactivator protein IE2, works in tandem to enable transcriptional regulation of viral genes.Author summaryThe host protein SUMO is a crucial regulator of cellular processes. Conjugation of other proteins to SUMO by a process called SUMOylation, can change the protein’s function or localization and regulate downstream cellular events. The SUMO pathway is exploited by viruses to transcribe viral genes and replicate the viral genome. IE2 is an essential gene of human Cytomegalovirus (HCMV), which acts as a transactivator and helps to transcribe other viral proteins required for viral genome replication and viral assembly. SUMOylation of IE2 is necessary for its function. Here, we have uncovered that IE2 functions as a cis-SUMO-E3 ligase, where a SUMO-Interacting Motif (SIM) in IE2 enhances its SUMOylation. Interestingly, phosphorylation of the SIM in IE2 augments its cis-E3 activity to further increase SUMOylation. Moreover, SIM phosphorylation also enhances the interaction between IE2 and SUMOylated binding partners. Thus, we uncover an exciting process, where phosphorylation enhances both covalent and non-covalent interaction of a protein (IE2) and SUMO. We also observe that the cross-talk of phosphorylation and SUMOylation has significant effects on the transactivation function of IE2. Overall, we discover how a viral protein IE2 exploits crosstalk between SUMOylation and Phosphorylation to enhance its activity and in turn, ensure efficient viral replication.

scholarly journals Histone H3 Lysine 4 Methylation Marks Postreplicative Human Cytomegalovirus Chromatin

2012 ◽  
Vol 86 (18) ◽  
pp. 9817-9827 ◽  
Author(s):  
Alexandra Nitzsche ◽  
Charlotte Steinhäußer ◽  
Katrin Mücke ◽  
Christina Paulus ◽  
Michael Nevels
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Histone Modification ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Histone H3 ◽  
Viral Dna ◽  
The Impact ◽  
Preferential Incorporation

In the nuclei of permissive cells, human cytomegalovirus genomes form nucleosomal structures initially resembling heterochromatin but gradually switching to a euchromatin-like state. This switch is characterized by a decrease in histone H3 K9 methylation and a marked increase in H3 tail acetylation and H3 K4 methylation across the viral genome. We used ganciclovir and a mutant virus encoding a reversibly destabilized DNA polymerase to examine the impact of DNA replication on histone modification dynamics at the viral chromatin. The changes in H3 tail acetylation and H3 K9 methylation proceeded in a DNA replication-independent fashion. In contrast, the increase in H3 K4 methylation proved to depend widely on viral DNA synthesis. Consistently, labeling of nascent DNA using “click chemistry” revealed preferential incorporation of methylated H3 K4 into viral (but not cellular) chromatin during or following DNA replication. This study demonstrates largely selective epigenetic tagging of postreplicative human cytomegalovirus chromatin.

scholarly journals Targeting Hedgehog Signalling through the Ubiquitylation Process: The Multiple Roles of the HECT-E3 Ligase Itch

Cells ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 98 ◽  
Author(s):  
Paola Infante ◽  
Ludovica Lospinoso Severini ◽  
Flavia Bernardi ◽  
Francesca Bufalieri ◽  
Lucia Di Marcotullio
Keyword(s):  
E3 Ligase ◽  
Multiple Roles ◽  
Post Translational Modification ◽  
Therapeutic Approaches ◽  
Cellular Functions ◽  
Hedgehog Signalling ◽  
Promising Target ◽  
Important Pathway ◽  
Multiple Levels

Hedgehog signalling (Hh) is a developmental conserved pathway strongly involved in cancers when deregulated. This important pathway is orchestrated by numerous regulators, transduces through distinct routes and is finely tuned at multiple levels. In this regard, ubiquitylation processes stand as essential for controlling Hh pathway output. Although this post-translational modification governs proteins turnover, it is also implicated in non-proteolytic events, thereby regulating the most important cellular functions. The HECT E3 ligase Itch, well known to control immune response, is emerging to have a pivotal role in tumorigenesis. By illustrating Itch specificities on Hh signalling key components, here we review the role of this HECT E3 ubiquitin ligase in suppressing Hh-dependent tumours and explore its potential as promising target for innovative therapeutic approaches.

scholarly journals The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation

2016 ◽  
Vol 90 (13) ◽  
pp. 5860-5875 ◽  
Author(s):  
Eva Maria Borst ◽  
Rudolf Bauerfeind ◽  
Anne Binz ◽  
Thomas Min Stephan ◽  
Sebastian Neuber ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Fluorescent Protein ◽  
Unit Length ◽  
Start Codon ◽  
Nuclear Targeting ◽  
Reading Frame ◽  
A Genome ◽  
Genome Encapsidation ◽  
Insight Into

ABSTRACTSeveral essential viral proteins are proposed to participate in genome encapsidation of human cytomegalovirus (HCMV), among them pUL77 and pUL93, which remain largely uncharacterized. To gain insight into their properties, we generated an HCMV mutant expressing a pUL77-monomeric enhanced green fluorescent protein (mGFP) fusion protein and a pUL93-specific antibody. Immunoblotting demonstrated that both proteins are incorporated into capsids and virions. Conversely to data suggesting internal translation initiation sites within the UL93 open reading frame (ORF), we provide evidence that pUL93 synthesis commences at the first start codon. In infected cells, pUL77-mGFP was found in nuclear replication compartments and dot-like structures, colocalizing with capsid proteins. Immunogold labeling of nuclear capsids revealed that pUL77 is present on A, B, and C capsids. Pulldown of pUL77-mGFP revealed copurification of pUL93, indicating interaction between these proteins, which still occurred when capsid formation was prevented. Correct subnuclear distribution of pUL77-mGFP required pUL93 as well as the major capsid protein (and thus probably the presence of capsids), but not the tegument protein pp150 or the encapsidation protein pUL52, demonstrating that pUL77 nuclear targeting occurs independently of the formation of DNA-filled capsids. When pUL77 or pUL93 was missing, generation of unit-length genomes was not observed, and only empty B capsids were produced. Taken together, these results show that pUL77 and pUL93 are capsid constituents needed for HCMV genome encapsidation. Therefore, the task of pUL77 seems to differ from that of its alphaherpesvirus orthologue pUL25, which exerts its function subsequent to genome cleavage-packaging.IMPORTANCEThe essential HCMV proteins pUL77 and pUL93 were suggested to be involved in viral genome cleavage-packaging but are poorly characterized both biochemically and functionally. By producing a monoclonal antibody against pUL93 and generating an HCMV mutant in which pUL77 is fused to a fluorescent protein, we show that pUL77 and pUL93 are capsid constituents, with pUL77 being similarly abundant on all capsid types. Each protein is required for genome encapsidation, as the absence of either pUL77 or pUL93 results in a genome packaging defect with the formation of empty capsids only. This distinguishes pUL77 from its alphaherpesvirus orthologue pUL25, which is enriched on DNA-filled capsids and exerts its function after the viral DNA is packaged. Our data for the first time describe an HCMV mutant with a fluorescent capsid and provide insight into the roles of pUL77 and pUL93, thus contributing to a better understanding of the HCMV encapsidation network.

Alternative translation and alternative RNA processing mechanism in parvovirus RNA processing

2014 ◽  
Author(s):  
◽  
Olufemi Fasina
Keyword(s):  
Gene Expression ◽  
Rna Processing ◽  
Viral Genome ◽  
Transcription Initiation ◽  
Alternative Polyadenylation ◽  
Open Reading Frames ◽  
Genome Replication ◽  
University Of Missouri ◽  
Diverse Range ◽  
Viral Genome Replication

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Viruses as obligate intracellular metabolic parasite require the capacity to orchestrate and modulate the host environment either in the nucleus or cytoplasm for their efficient reproductive life cycle. This warrants the use of diverse range of proteins expressed from the viral genome with the ability of regulating viral genome replication, transcription and translation, in addition antagonizing host factors inhibitory to the virus. Therefore, in order to achieve these goals, viruses utilizes gene expression strategies to expand their coding capacity. Gene expression mechanism such as transcription initiation, capping, splicing and 3�-end processing afford viruses the opportunities to utilize the eukaryotic metabolic machineries for generating proteome diversity. Parvoviruses and other DNA viruses effectively capitalize on their use of nuclear eukaryotic metabolic machineries to co-opt host cell factors for optimal replication and gene expression. Parvoviruses with small genome size and overlapping open reading frames utilize alternative transcription initiation, alternative splicing and alternative polyadenylation to co-ordinate the expression of its non-structural and structural proteins. In this work, we have characterized how two parvoviruses; Dependovirus AAV5 and Bocavirus Minute virus of canine (MVC) utilize alternative gene expression mechanisms and strategies to optimize expression of viral proteins from their genome.

scholarly journals BothcisandtransActivities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication

2016 ◽  
Vol 90 (15) ◽  
pp. 6864-6883 ◽  
Author(s):  
Morgan R. Herod ◽  
Cristina Ferrer-Orta ◽  
Eleni-Anna Loundras ◽  
Joseph C. Ward ◽  
Nuria Verdaguer ◽  
...  
Keyword(s):  
Disease Virus ◽  
Viral Genome ◽  
Foot And Mouth Disease ◽  
Rna Replication ◽  
Mouth Disease ◽  
Viral Rna ◽  
Genome Replication ◽  
Replication Complex ◽  
Mouth Disease Virus ◽  
Foot And Mouth

ABSTRACTThePicornaviridaeis a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided intrans(i.e., via expression from a separate RNA molecule), while others are required incis(i.e., expressed from the template RNA molecule).In vitrostudies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymaticcis-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that thiscis-acting role of 3D is distinct from the catalytic activity, which is predominantlytransacting. Immunofluorescence studies suggest that bothcis- andtrans-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts inciswith RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further.IMPORTANCEFoot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within replication complexes, and understanding this process can facilitate the development of novel therapeutic strategies. Many of the nonstructural proteins involved in replication possess multiple functions in the viral life cycle, some of which can be supplied to the replication complex from a separate genome (i.e., intrans) while others must originate from the template (i.e., incis). Here, we present an analysis ofcisandtransactivities of the RNA-dependent RNA polymerase 3D. We demonstrate a novelcis-acting role of 3D in replication. Our data suggest that this role is distinct from its enzymatic functions and requires interaction with the viral genome. Our data further the understanding of genome replication of this important pathogen.

scholarly journals Human cytomegalovirus transcriptome activity differs during replication in human fibroblast, epithelial and astrocyte cell lines

2012 ◽  
Vol 93 (5) ◽  
pp. 1046-1058 ◽  
Author(s):  
James C. Towler ◽  
Bahram Ebrahimi ◽  
Brian Lane ◽  
Andrew J. Davison ◽  
Derrick J. Dargan
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Growth Kinetics ◽  
Viral Gene Expression ◽  
Cell Types ◽  
Viral Gene ◽  
Genome Replication ◽  
Cell Type ◽  
Low Passage ◽  
Temporal Expression Pattern

Broad cell tropism contributes to the pathogenesis of human cytomegalovirus (HCMV), but the extent to which cell type influences HCMV gene expression is unclear. A bespoke HCMV DNA microarray was used to monitor the transcriptome activity of the low passage Merlin strain of HCMV at 12, 24, 48 and 72 h post-infection, during a single round of replication in human fetal foreskin fibroblast cells (HFFF-2s), human retinal pigmented epithelial cells (RPE-1s) and human astrocytoma cells (U373MGs). In order to correlate transcriptome activity with concurrent biological responses, viral cytopathic effect, growth kinetics and genomic loads were examined in the three cell types. The temporal expression pattern of viral genes was broadly similar in HFFF-2s and RPE-1s, but dramatically different in U373MGs. Of the 165 known HCMV protein-coding genes, 41 and 48 were differentially regulated in RPE-1s and U373MGs, respectively, compared with HFFF-2s, and 22 of these were differentially regulated in both RPE-1s and U373MGs. In RPE-1s, all differentially regulated genes were downregulated, but, in U373MGs, some were down- and others upregulated. Differentially regulated genes were identified among the immediate-early, early, early late and true-late viral gene classes. Grouping of downregulated genes according to function at landmark stages of the replication cycle led to the identification of potential bottleneck stages (genome replication, virion assembly, and virion maturation and release) that may account for cell type-dependent viral growth kinetics. The possibility that cell type-specific differences in expressed cellular factors are responsible for modulation of viral gene expression is discussed.

scholarly journals Structural and Functional Consequences of Age-Related Isomerization in α-Crystallins

2018 ◽  
Author(s):  
Yana A. Lyon ◽  
Dylan L. Riggs ◽  
Miranda P. Collier ◽  
Matteo T. Degiacomi ◽  
Justin L.P. Benesch ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Dynamics ◽  
Amino Acid ◽  
Native Mass Spectrometry ◽  
Salt Bridges ◽  
Loss Of Function ◽  
Post Translational Modification ◽  
Age Related ◽  
Αb Crystallin ◽  
Functional Consequences

AbstractLong-lived proteins are subject to spontaneous degradation and may accumulate a range of modifications over time, including subtle alterations such as isomerization. Recently, tandem-mass spectrometry approaches have enabled the identification and detailed characterization of such peptide isomers, including those differing only in chirality. However, the structural and functional consequences of these perturbations remain largely unexplored. Here we examine the site-specific impact of isomerization of aspartic acid and epimerization of serine in human αA- and αB-crystallin. From a total of 81 sites of modification identified in aged eye lenses, four (αBSer59, αASer162, αBAsp62, αBAsp109) map to crucial oligomeric interfaces. To characterize the effect of isomerization on quaternary assembly, molecular dynamics calculations and native mass spectrometry experiments were performed on recombinant forms of αA- and αB-crystallin that incorporate, or mimic, isomerized residues. In all cases, oligomerization is significantly affected, with epimerization of a single serine residue (αASer162) sufficing to weaken inter-subunit binding dramatically. Furthermore, phosphorylation of αBSer59, known to play an important regulatory role in oligomerization, is severely inhibited by serine epimerization and altered by isomerization of nearby αBAsp62. Similarly, isomerization of αBAsp109 disrupts a vital salt-bridge with αBArg120, a loss previously shown to yield aberrant oligomerization and aggregation in several disease variants. Our results illustrate how isomerization of amino-acid residues, which may seem like a minor structural perturbation, can have profound consequences on protein assembly and activity by disrupting specific hydrogen bonds and salt bridges.Significance StatementProteins play numerous critical roles in our bodies but suffer damage with increasing age. For example, isomerization is a spontaneous post-translational modification that alters the three-dimensional connectivity of an amino acid, yet remains invisible to traditional proteomic experiments. Herein, radical-based fragmentation was used for isomer identification while molecular dynamics and native mass spectrometry were utilized to assess structural consequences. The results demonstrate that isomerization disrupts both oligomeric assembly and phosphorylation in the α-crystallins, which are long-lived proteins in the lens of the eye. The loss of function associated with these modifications is likely connected to age-related diseases such as cataract and neurodegenerative disorders, while the methodologies we present represent a framework for structure-function studies on other isomerized proteins.

scholarly journals ALotus japonicusE3 ligase interacts with the Nod factor receptor 5 and positively regulates nodulation

2018 ◽  
Author(s):  
Daniela Tsikou ◽  
Estrella E. Ramirez ◽  
Ioanna S. Psarrakou ◽  
Jaslyn E. Wong ◽  
Dorthe B. Jensen ◽  
...  
Keyword(s):  
Lotus Japonicus ◽  
E3 Ligase ◽  
Kinase Domain ◽  
Plant Interactions ◽  
Post Translational Modification ◽  
Direct Role ◽  
Nod Factor ◽  
Mesorhizobium Loti ◽  
Signaling Systems ◽  
Nodulation Capacity

SUMMARYPost-translational modification of receptor proteins is involved in activation and de-activation of signaling systems in plants. Both ubiquitination and deubiquitination have been implicated in plant interactions with pathogens and symbionts. Here we presentLjPUB13, a PUB-ARMADILLO repeat E3 ligase that specifically ubiquitinates the kinase domain of the Nod Factor receptor NFR5 and has a direct role in nodule organogenesis events inLotus japonicus. Phenotypic analyses of three LORE1 retroelement insertion plant lines revealed thatpub13plants display delayed and reduced nodulation capacity and retarded growth.LjPUB13expression is spatially regulated during symbiosis withMesorhizobium loti, with increased levels in young developing nodules. Thus,LjPUB13 is an E3 ligase with a positive regulatory role during the initial stages of nodulation inL. japonicus.

scholarly journals Dimerisation of the influenza virus RNA polymerase during viral genome replication

2019 ◽  
Vol 1 (1A) ◽  
Author(s):  
Alexander Walker ◽  
Haitian Fan ◽  
Loic Carrique ◽  
Jeremy Keown ◽  
David Bauer ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Viral Genome ◽  
Genome Replication ◽  
Virus Rna ◽  
Viral Genome Replication

The formation and modification of chromatin-like structure of human parvovirus B19 regulate viral genome replication and RNA processing

2017 ◽  
Vol 232 ◽  
pp. 134-138 ◽  
Author(s):  
Huanzhou Xu ◽  
Sujuan Hao ◽  
Junmei Zhang ◽  
Zhen Chen ◽  
Hanzhong Wang ◽  
...  
Keyword(s):  
Rna Processing ◽  
Viral Genome ◽  
Parvovirus B19 ◽  
Genome Replication ◽  
Human Parvovirus B19 ◽  
Viral Genome Replication
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