scholarly journals Phosphosite Analysis of the Cytomegaloviral mRNA Export Factor pUL69 Reveals Serines with Critical Importance for Recruitment of Cellular Proteins Pin1 and UAP56/URH49

2020 ◽  
Vol 94 (8) ◽  
Author(s):  
Marco Thomas ◽  
Regina Müller ◽  
Georg Horn ◽  
Boris Bogdanow ◽  
Koshi Imami ◽  
...  
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Posttranslational Modifications ◽  
Mrna Export ◽  
Critical Role ◽  
Alpha Helix ◽  
Adaptor Proteins ◽  
Arginine Methylation ◽  
Binding Motif ◽  
Phosphorylation Sites

ABSTRACT Human cytomegalovirus (HCMV) encodes the viral mRNA export factor pUL69, which facilitates the cytoplasmic accumulation of mRNA via interaction with the cellular RNA helicase UAP56 or URH49. We reported previously that pUL69 is phosphorylated by cellular CDKs and the viral CDK-like kinase pUL97. Here, we set out to identify phosphorylation sites within pUL69 and to characterize their importance. Mass spectrometry-based phosphosite mapping of pUL69 identified 10 serine/threonine residues as phosphoacceptors. Surprisingly, only a few of these sites localized to the N terminus of pUL69, which could be due to the presence of additional posttranslational modifications, like arginine methylation. As an alternative approach, pUL69 mutants with substitutions of putative phosphosites were analyzed by Phos-tag SDS-PAGE. This demonstrated that serines S46 and S49 serve as targets for phosphorylation by pUL97. Furthermore, we provide evidence that phosphorylation of these serines mediates cis/trans isomerization by the prolyl isomerase Pin1, thus forming a functional Pin1 binding motif. Surprisingly, while abrogation of the Pin1 motif did not affect the replication of recombinant cytomegaloviruses, mutation of serines next to the interaction site for UAP56/URH49 strongly decreased viral replication. This was correlated with a loss of UAP56/URH49 recruitment. Intriguingly, the critical serines S13 and S15 were located within a sequence resembling the UAP56 binding motif (UBM) of cellular mRNA adaptor proteins like REF and UIF. We propose that betaherpesviral mRNA export factors have evolved an extended UAP56/URH49 recognition sequence harboring phosphorylation sites to increase their binding affinities. This may serve as a strategy to successfully compete with cellular mRNA adaptor proteins for binding to UAP56/URH49. IMPORTANCE The multifunctional regulatory protein pUL69 of human cytomegalovirus acts as a viral RNA export factor with a critical role in efficient replication. Here, we identify serine/threonine phosphorylation sites for cellular and viral kinases within pUL69. We demonstrate that the pUL97/CDK phosphosites within alpha-helix 2 of pUL69 are crucial for its cis/trans isomerization by the cellular protein Pin1. Thus, we identified pUL69 as the first HCMV-encoded protein that is phosphorylated by cellular and viral serine/threonine kinases in order to serve as a substrate for Pin1. Furthermore, our study revealed that betaherpesviral mRNA export proteins contain extended binding motifs for the cellular mRNA adaptor proteins UAP56/URH49 harboring phosphorylated serines that are critical for efficient viral replication. Knowledge of the phosphorylation sites of pUL69 and the processes regulated by these posttranslational modifications is important in order to develop antiviral strategies based on a specific interference with pUL69 phosphorylation.

scholarly journals Revisiting the role of PML protein targeting and disruption of PML bodies in human cytomegalovirus infection

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Christina Paulus ◽  
Thomas Harwardt ◽  
Bernadette Walter ◽  
Andrea Marxreiter ◽  
Michael Nevels
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Critical Role ◽  
Wild Type ◽  
Early Protein ◽  
Type Protein ◽  
Wild Type Protein ◽  
Pml Bodies

Promyelocytic leukaemia (PML) bodies are nuclear organelles implicated in post-translational modification by small ubiquitin-like modifier (SUMO) proteins and in the antiviral host cell response to infection. The 72-kDa immediate-early protein 1 (IE1) is considered the principal antagonist of PML bodies encoded by the human cytomegalovirus, one of eight human herpesviruses. Previous work has suggested that the interaction between IE1 and PML proteins, the central organisers of PML bodies, and the subsequent disruption of these organelles serve a critical role in viral replication by counteracting intrinsic antiviral immunity and the induction of interferon (IFN)-stimulated genes. However, this picture has emerged largely from studying mutant IE1 proteins known or predicted to be globally misfolded und metabolically unstable. We systematically screened for stable IE1 mutants by clustered charge-to-alanine scanning. We identified a mutant protein (IE1cc172-176) selectively defective for PML interaction. Functional comparisons between the mutant and wild-type protein revealed that IE1 can undergo modification by mixed polymeric SUMO chains and that it targets PML and Sp100, the two main constituents of PML bodies, via distinct mechanisms. Unexpectedly, IE1cc172-176 supported viral replication almost as efficiently as wild-type IE1. Moreover, lower instead of higher (as expected) levels of tumor necrosis factor alpha, IFN-beta, IFN-lambda and IFN-stimulated gene expression were observed with the mutant compared to the wild-type protein and virus. These results suggest that the disruption of PML bodies is linked to induction rather than inhibition of antiviral gene expression. Our findings challenge current views regarding the role of PML bodies in viral infection.

scholarly journals pUL69 of Human Cytomegalovirus Recruits the Cellular Protein Arginine Methyltransferase 6 via a Domain That Is Crucial for mRNA Export and Efficient Viral Replication

2015 ◽  
Vol 89 (18) ◽  
pp. 9601-9615 ◽  
Author(s):  
Marco Thomas ◽  
Eric Sonntag ◽  
Regina Müller ◽  
Stefanie Schmidt ◽  
Barbara Zielke ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Human Cytomegalovirus ◽  
Posttranslational Modification ◽  
Mrna Export ◽  
Regulatory Protein ◽  
Arginine Methylation ◽  
Arginine Methyltransferase ◽  
Export Activity ◽  
N Terminus ◽  
Rna Export

ABSTRACTThe regulatory protein pUL69 of human cytomegalovirus acts as a viral mRNA export factor, facilitating the cytoplasmic accumulation of unspliced RNA via interaction with the cellular mRNA export factor UAP56. Here we provide evidence for a posttranslational modification of pUL69 via arginine methylation within the functionally important N terminus. First, we demonstrated a specific immunoprecipitation of full-length pUL69 as well as pUL69aa1-146 by a mono/dimethylarginine-specific antibody. Second, we observed a specific electrophoretic mobility shift upon overexpression of the catalytically active protein arginine methyltransferase 6 (PRMT6). Third, a direct interaction of pUL69 and PRMT6 was confirmed by yeast two-hybrid and coimmunoprecipitation analyses. We mapped the PRMT6 interaction motif to the pUL69 N terminus and identified critical amino acids within the arginine-rich R1 box of pUL69 that were crucial for PRMT6 and/or UAP56 recruitment. In order to test the impact of putative methylation substrates on the functions of pUL69, we constructed various pUL69 derivatives harboring arginine-to-alanine substitutions and tested them for RNA export activity. Thus, we were able to discriminate between arginines within the R1 box of pUL69 that were crucial for UAP56/PRMT6-interaction and/or mRNA export activity. Remarkably, nuclear magnetic resonance (NMR) analyses revealed the same α-helical structures for pUL69 sequences encoding either the wild type R1/R2 boxes or a UAP56/PRMT6 binding-deficient derivative, thereby excluding the possibility that R/A amino acid substitutions within R1 affected the secondary structure of pUL69. We therefore conclude that the pUL69 N terminus is methylated by PRMT6 and that this critically affects the functions of pUL69 for efficient mRNA export and replication of human cytomegalovirus.IMPORTANCEThe UL69 protein of human cytomegalovirus is a multifunctional regulatory protein that acts as a viral RNA export factor with a critical role for efficient replication. Here, we demonstrate that pUL69 is posttranslationally modified via arginine methylation and that the protein methyltransferase PRMT6 mediates this modification. Furthermore, arginine residues with a crucial function for RNA export and for binding of the cellular RNA export factor UAP56 as well as PRMT6 were mapped within the arginine-rich R1 motif of pUL69. Importantly, we demonstrated that mutation of those arginines did not alter the secondary structure of R1, suggesting that they may serve as critical methylation substrates. In summary, our study reveals a novel posttranslational modification of pUL69 which has a significant impact on the function of this important viral regulatory protein. Since PRMTs appear to be amenable to selective inhibition by small molecules, this may constitute a novel target for antiviral therapy.

scholarly journals Multiple roles of PP2A binding motif in hepatitis B virus core linker and PP2A in regulating core phosphorylation state and viral replication

2021 ◽  
Vol 17 (1) ◽  
pp. e1009230
Author(s):  
Ji Xi ◽  
Laura Luckenbaugh ◽  
Jianming Hu
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Replication ◽  
Critical Role ◽  
Binding Motif ◽  
Phosphorylation State ◽  
Terminal Domain ◽  
Linker Peptide ◽  
B Virus ◽  
Ctd Phosphorylation

Hepatitis B virus (HBV) capsid or core protein (HBc) contains an N-terminal domain (NTD) and a C-terminal domain (CTD) connected by a short linker peptide. HBc plays a critical role in virtually every step of viral replication, which is further modulated by dynamic phosphorylation and dephosphorylation of its CTD. While several cellular kinases have been identified that mediate HBc CTD phosphorylation, there is little information on the cellular phosphatases that mediate CTD dephosphorylation. Herein, a consensus binding motif for the protein phosphatase 2A (PP2A) regulatory subunit B56 was recognized within the HBc linker peptide. Mutations within this motif designed to block or enhance B56 binding showed pleiotropic effects on CTD phosphorylation state as well as on viral RNA packaging, reverse transcription, and virion secretion. Furthermore, linker mutations affected the HBV nuclear episome (the covalently closed circular or CCC DNA) differentially during intracellular amplification vs. infection. The effects of linker mutations on CTD phosphorylation state varied with different phosphorylation sites and were only partially consistent with the linker motif serving to recruit PP2A-B56, specifically, to dephosphorylate CTD, suggesting that multiple phosphatases and/or kinases may be recruited to modulate CTD (de)phosphorylation. Furthermore, pharmacological inhibition of PP2A could decrease HBc CTD dephosphorylation and increase the nuclear HBV episome. These results thus strongly implicate the HBc linker in recruiting PP2A and other host factors to regulate multiple stages of HBV replication.

Exploring Proteomic Drug Targets, Therapeutic Strategies and Protein - Protein Interactions in Cancer: Mechanistic View

2019 ◽  
Vol 19 (6) ◽  
pp. 430-448 ◽  
Author(s):  
Khalid Bashir Dar ◽  
Aashiq Hussain Bhat ◽  
Shajrul Amin ◽  
Syed Anjum ◽  
Bilal Ahmad Reshi ◽  
...  
Keyword(s):  
Protein Interactions ◽  
High Throughput Screening ◽  
Critical Role ◽  
Cancer Therapeutics ◽  
Adaptor Proteins ◽  
Therapeutic Strategies ◽  
Small Gtpases ◽  
Beta Catenin ◽  
Protein Protein Interactions ◽  
Apoptosis Protein

Protein-Protein Interactions (PPIs) drive major signalling cascades and play critical role in cell proliferation, apoptosis, angiogenesis and trafficking. Deregulated PPIs are implicated in multiple malignancies and represent the critical targets for treating cancer. Herein, we discuss the key protein-protein interacting domains implicated in cancer notably PDZ, SH2, SH3, LIM, PTB, SAM and PH. These domains are present in numerous enzymes/kinases, growth factors, transcription factors, adaptor proteins, receptors and scaffolding proteins and thus represent essential sites for targeting cancer. This review explores the candidature of various proteins involved in cellular trafficking (small GTPases, molecular motors, matrix-degrading enzymes, integrin), transcription (p53, cMyc), signalling (membrane receptor proteins), angiogenesis (VEGFs) and apoptosis (BCL-2family), which could possibly serve as targets for developing effective anti-cancer regimen. Interactions between Ras/Raf; X-linked inhibitor of apoptosis protein (XIAP)/second mitochondria-derived activator of caspases (Smac/DIABLO); Frizzled (FRZ)/Dishevelled (DVL) protein; beta-catenin/T Cell Factor (TCF) have also been studied as prospective anticancer targets. Efficacy of diverse molecules/ drugs targeting such PPIs although evaluated in various animal models/cell lines, there is an essential need for human-based clinical trials. Therapeutic strategies like the use of biologicals, high throughput screening (HTS) and fragment-based technology could play an imperative role in designing cancer therapeutics. Moreover, bioinformatic/computational strategies based on genome sequence, protein sequence/structure and domain data could serve as competent tools for predicting PPIs. Exploring hot spots in proteomic networks represents another approach for developing targetspecific therapeutics. Overall, this review lays emphasis on a productive amalgamation of proteomics, genomics, biochemistry, and molecular dynamics for successful treatment of cancer.

scholarly journals Renal Manifestations of Covid-19: Physiology and Pathophysiology

2021 ◽  
Vol 10 (6) ◽  
pp. 1216
Author(s):  
Zaher Armaly ◽  
Safa Kinaneh ◽  
Karl Skorecki
Keyword(s):  
Viral Replication ◽  
Virus Disease ◽  
Multiorgan Failure ◽  
Critical Role ◽  
Kidney Injury ◽  
Small Sample ◽  
Renal Physiology ◽  
High Morbidity ◽  
Enzymatic Production ◽  
High Incidence

Corona virus disease 2019 (COVID-19) imposes a serious public health pandemic affecting the whole world, as it is spreading exponentially. Besides its high infectivity, SARS-CoV-2 causes multiple serious derangements, where the most prominent is severe acute respiratory syndrome as well as multiple organ dysfunction including heart and kidney injury. While the deleterious impact of SARS-CoV-2 on pulmonary and cardiac systems have attracted remarkable attention, the adverse effects of this virus on the renal system is still underestimated. Kidney susceptibility to SARS-CoV-2 infection is determined by the presence of angiotensin-converting enzyme 2 (ACE2) receptor which is used as port of the viral entry into targeted cells, tissue tropism, pathogenicity and subsequent viral replication. The SARS-CoV-2 cellular entry receptor, ACE2, is widely expressed in proximal epithelial cells, vascular endothelial and smooth muscle cells and podocytes, where it supports kidney integrity and function via the enzymatic production of Angiotensin 1-7 (Ang 1-7), which exerts vasodilatory, anti-inflammatory, antifibrotic and diuretic/natriuretic actions via activation of the Mas receptor axis. Loss of this activity constitutes the potential basis for the renal damage that occurs in COVID-19 patients. Indeed, several studies in a small sample of COVID-19 patients revealed relatively high incidence of acute kidney injury (AKI) among them. Although SARS-CoV-1 -induced AKI was attributed to multiorgan failure and cytokine release syndrome, as the virus was not detectable in the renal tissue of infected patients, SARS-CoV-2 antigens were detected in kidney tubules, suggesting that SARS-CoV-2 infects the human kidney directly, and eventually induces AKI characterized with high morbidity and mortality. The mechanisms underlying this phenomenon are largely unknown. However, the fact that ACE2 plays a crucial role against renal injury, the deprivation of the kidney of this advantageous enzyme, along with local viral replication, probably plays a central role. The current review focuses on the critical role of ACE2 in renal physiology, its involvement in the development of kidney injury during SARS-CoV-2 infection, renal manifestations and therapeutic options. The latter includes exogenous administration of Ang (1-7) as an appealing option, given the high incidence of AKI in this ACE2-depleted disorder, and the benefits of ACE2/Ang1-7 including vasodilation, diuresis, natriuresis, attenuation of inflammation, oxidative stress, cell proliferation, apoptosis and coagulation.

scholarly journals Targeting novel LSD1-dependent ACE2 demethylation domains inhibits SARS-CoV-2 replication

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Wen Juan Tu ◽  
Robert D. McCuaig ◽  
Michelle Melino ◽  
Daniel J. Rawle ◽  
Thuy T. Le ◽  
...  
Keyword(s):  
Viral Replication ◽  
Treatment Options ◽  
Critical Role ◽  
Post Exposure Prophylaxis ◽  
Nuclear Entry ◽  
Importin Α ◽  
Active Transcription ◽  
Nuclear Shuttling ◽  
Asymptomatic Phase ◽  
Exposure Prophylaxis

AbstractTreatment options for COVID-19 remain limited, especially during the early or asymptomatic phase. Here, we report a novel SARS-CoV-2 viral replication mechanism mediated by interactions between ACE2 and the epigenetic eraser enzyme LSD1, and its interplay with the nuclear shuttling importin pathway. Recent studies have shown a critical role for the importin pathway in SARS-CoV-2 infection, and many RNA viruses hijack this axis to re-direct host cell transcription. LSD1 colocalized with ACE2 at the cell surface to maintain demethylated SARS-CoV-2 spike receptor-binding domain lysine 31 to promote virus–ACE2 interactions. Two newly developed peptide inhibitors competitively inhibited virus–ACE2 interactions, and demethylase access to significantly inhibit viral replication. Similar to some other predominantly plasma membrane proteins, ACE2 had a novel nuclear function: its cytoplasmic domain harbors a nuclear shuttling domain, which when demethylated by LSD1 promoted importin-α-dependent nuclear ACE2 entry following infection to regulate active transcription. A novel, cell permeable ACE2 peptide inhibitor prevented ACE2 nuclear entry, significantly inhibiting viral replication in SARS-CoV-2-infected cell lines, outperforming other LSD1 inhibitors. These data raise the prospect of post-exposure prophylaxis for SARS-CoV-2, either through repurposed LSD1 inhibitors or new, nuclear-specific ACE2 inhibitors.

scholarly journals Prolonged activation of NF-κB by human cytomegalovirus promotes efficient viral replication and late gene expression

Virology ◽  
2006 ◽  
Vol 346 (1) ◽  
pp. 15-31 ◽  
Author(s):  
Ian B. DeMeritt ◽  
Jagat P. Podduturi ◽  
A. Michael Tilley ◽  
Maciej T. Nogalski ◽  
Andrew D. Yurochko
Keyword(s):  
Gene Expression ◽  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Late Gene ◽  
Late Gene Expression ◽  
Efficient Viral Replication

Viral replication in HeLa/fibroblast hybrid cells infected with human cytomegalovirus

1987 ◽  
Vol 95 (1-2) ◽  
pp. 29-40 ◽  
Author(s):  
Y. Tsutsui ◽  
S. Sonta ◽  
A. Kashiwai ◽  
T. Nogami ◽  
T. Furukawa
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Hybrid Cells

scholarly journals The Carboxy-Terminal Domain of Glycoprotein N of Human Cytomegalovirus Is Required for Virion Morphogenesis

2007 ◽  
Vol 81 (10) ◽  
pp. 5212-5224 ◽  
Author(s):  
Michael Mach ◽  
Karolina Osinski ◽  
Barbara Kropff ◽  
Ursula Schloetzer-Schrehardt ◽  
Magdalena Krzyzaniak ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Critical Role ◽  
Point Mutations ◽  
Cysteine Residue ◽  
Cysteine Residues ◽  
Type I ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Assembly Compartment

ABSTRACT Glycoproteins M and N (gM and gN, respectively) are among the few proteins that are conserved across the herpesvirus family. The function of the complex is largely unknown. Whereas deletion from most alphaherpesviruses has marginal effects on the replication of the respective viruses, both proteins are essential for replication of human cytomegalovirus (HCMV). We have constructed a series of mutants in gN to study the function of this protein. gN of HCMV is a type I glycoprotein containing a short carboxy-terminal domain of 14 amino acids, including two cysteine residues directly adjacent to the predicted transmembrane anchor at positions 125 and 126. Deletion of the entire carboxy-terminal domain as well as substitution with the corresponding region from alpha herpesviruses or mutations of both cysteine residues resulted in a replication-incompetent virus. Recombinant viruses containing point mutations of either cysteine residue could be generated. These viruses were profoundly defective for replication. Complex formation of the mutant gNs with gM and transport of the complex to the viral assembly compartment appeared unaltered compared to the wild type. However, in infected cells, large numbers of capsids accumulated in the cytoplasm that failed to acquire an envelope. Transiently expressed gN was shown to be modified by palmitic acid at both cysteine residues. In summary, our data suggest that the carboxy-terminal domain of gN plays a critical role in secondary envelopment of HCMV and that palmitoylation of gN appears to be essential for function in secondary envelopment of HCMV and virus replication.

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