The human cytomegalovirus regulatory protein UL69 and its effect on mRNA export

10.2741/2899 ◽  
2008 ◽  
Vol 13 (13) ◽  
pp. 2939 ◽  
Author(s):  
Zsolt Toth
Keyword(s):  
Human Cytomegalovirus ◽  
Mrna Export ◽  
Regulatory Protein

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 The UL69 Transactivator Protein of Human Cytomegalovirus Interacts with DEXD/H-Box RNA Helicase UAP56 To Promote Cytoplasmic Accumulation of Unspliced RNA

2006 ◽  
Vol 26 (5) ◽  
pp. 1631-1643 ◽  
Author(s):  
Peter Lischka ◽  
Zsolt Toth ◽  
Marco Thomas ◽  
Regina Mueller ◽  
Thomas Stamminger
Keyword(s):  
Human Cytomegalovirus ◽  
Nuclear Export ◽  
Mrna Export ◽  
Regulatory Protein ◽  
Nuclear Export Signal ◽  
Cellular Transport ◽  
Nucleocytoplasmic Shuttling ◽  
Rna Helicases ◽  
Cytoplasmic Accumulation ◽  
Acid Domain

ABSTRACT The UL69 gene product of human cytomegalovirus belongs to a family of regulatory proteins conserved among all herpesviruses that have in part been characterized as posttranscriptional transactivators participating in the nuclear export of RNA. Recent experiments suggested that pUL69 also acts as a posttranscriptional activator since it was demonstrated that nucleocytoplasmic shuttling via a CRM1-independent nuclear export signal is a prerequisite for its stimulatory effect on gene expression. Based on these findings we initiated studies to investigate the role of pUL69 in mRNA export and demonstrate that pUL69 efficiently promotes the cytoplasmic accumulation of unspliced RNA. Furthermore, we show that this pUL69 activity is linked to the cellular mRNA export machinery by direct protein interaction with the highly related DEXD/H-box RNA helicases UAP56 and URH49. Particularly, we identified a 12-amino-acid domain within the N terminus of pUL69 which is required for binding to UAP56 and URH49, and we could demonstrate that UAP56 interaction and nucleocytoplasmic shuttling are both prerequisites for pUL69-mediated mRNA export. Thus, we identified a novel cellular target which provides a herpesviral regulatory protein with access to a conserved cellular transport system in order to promote nuclear export of unspliced RNA.

scholarly journals Human Cytomegalovirus UL84 Protein Contains Two Nuclear Export Signals and Shuttles between the Nucleus and the Cytoplasm

2006 ◽  
Vol 80 (20) ◽  
pp. 10274-10280 ◽  
Author(s):  
Peter Lischka ◽  
Claudia Rauh ◽  
Regina Mueller ◽  
Thomas Stamminger
Keyword(s):  
Human Cytomegalovirus ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Signal Sequence ◽  
Regulatory Protein ◽  
Nucleocytoplasmic Shuttling ◽  
Viral Dna ◽  
Nuclear Localization Signal Sequence ◽  
Localization Signal

ABSTRACT Previous studies defined pUL84 of human cytomegalovirus as an essential regulatory protein with nuclear localization that was proposed to act during initiation of viral-DNA synthesis. Recently, we demonstrated that a complex domain of 282 amino acids within pUL84 functions as a nonconventional nuclear localization signal. Sequence inspection of this domain revealed the presence of motifs with homology to leucine-rich nuclear export signals. Here, we report the identification of two functional, autonomous nuclear export signals and show that pUL84 acts as a CRM-1-dependent nucleocytoplasmic shuttling protein. This suggests an unexpected cytoplasmic role for this essential viral regulatory protein.

scholarly journals Loss of the Human Cytomegalovirus US16 Protein Abrogates Virus Entry into Endothelial and Epithelial Cells by Reducing the Virion Content of the Pentamer

2017 ◽  
Vol 91 (11) ◽  
Author(s):  
Anna Luganini ◽  
Noemi Cavaletto ◽  
Stefania Raimondo ◽  
Stefano Geuna ◽  
Giorgio Gribaudo
Keyword(s):  
Epithelial Cells ◽  
Human Cytomegalovirus ◽  
Virus Entry ◽  
Regulatory Protein ◽  
Direct Consequence ◽  
Cell Types ◽  
Target Cells ◽  
C Terminus ◽  
Viral Particles ◽  
Low Passage

ABSTRACT The human cytomegalovirus (HCMV) US12 gene family encodes a group of predicted seven-transmembrane proteins whose functions have yet to be established. While inactivation of individual US12 members in laboratory strains of HCMV does not affect viral replication in fibroblasts, disruption of the US16 gene in the low-passage-number TR strain prevents viral growth in endothelial and epithelial cells. In these cells, the US16-null viruses fail to express immediate early (IE), early (E), and late (L) viral proteins due to a defect which occurs prior to IE gene expression. Here, we show that this defective phenotype is a direct consequence of deficiencies in the entry of US16-null viruses in these cell types due to an impact on the gH/gL/UL128/UL130/UL131A (pentamer) complex. Indeed, viral particles released from fibroblasts infected with US16-null viruses were defective for the pentamer, thus preventing entry during infections of endothelial and epithelial cells. A link between pUS16 and the pentamer was further supported by the colocalization of pUS16 and pentamer proteins within the cytoplasmic viral assembly compartment (cVAC) of infected fibroblasts. Deletion of the C-terminal tail of pUS16 reproduced the defective growth phenotype and alteration of virion composition as US16-null viruses. However, the pentamer assembly and trafficking to the cVAC were not affected by the lack of the C terminus of pUS16. Coimmunoprecipitation results then indicated that US16 interacts with pUL130 but not with the mature pentamer or gH/gL/gO. Together, these results suggest that pUS16 contributes to the tropism of HCMV by influencing the content of the pentamer into virions. IMPORTANCE Human cytomegalovirus (HCMV) is major pathogen in newborns and immunocompromised individuals. A hallmark of HCMV pathogenesis is its ability to productively replicate in an exceptionally broad range of target cells. The virus infects a variety of cell types by exploiting different forms of the envelope glycoprotein gH/gL hetero-oligomers, which allow entry into many cell types through different pathways. For example, incorporation of the pentameric gH/gL/UL128/UL130/UL131A complex into virions is a prerequisite for infection of endothelial and epithelial cells. Here, we show that the absence of US16, a thus far uncharacterized HCMV multitransmembrane protein, abrogates virus entry into endothelial and epithelial cells and that this defect is due to the lack of adequate amounts of the pentameric complex in extracellular viral particles. Our study suggests pUS16 as a novel viral regulatory protein important for shaping virion composition in a manner that influences HCMV cell tropism.

scholarly journals Intrinsic Immune Mechanisms Restricting Human Cytomegalovirus Replication

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 179
Author(s):  
Eva-Maria Schilling ◽  
Myriam Scherer ◽  
Thomas Stamminger
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Entry ◽  
Hcmv Infection ◽  
Current Knowledge ◽  
Cytidine Deaminase ◽  
Regulatory Protein ◽  
Detailed Knowledge ◽  
Restriction Factors ◽  
Immune Mechanisms ◽  
Early Protein

Cellular restriction factors (RFs) act as important constitutive innate immune barriers against viruses. In 2006, the promyelocytic leukemia protein was described as the first RF against human cytomegalovirus (HCMV) infection which is antagonized by the viral immediate early protein IE1. Since then, at least 15 additional RFs against HCMV have been identified, including the chromatin regulatory protein SPOC1, the cytidine deaminase APOBEC3A and the dNTP triphosphohydrolase SAMHD1. These RFs affect distinct steps of the viral replication cycle such as viral entry, gene expression, the synthesis of progeny DNA or egress. This review summarizes our current knowledge on intrinsic immune mechanisms restricting HCMV replication as well as on the viral strategies to counteract the inhibitory effects of RFs. Detailed knowledge on the interplay between host RFs and antagonizing viral factors will be fundamental to develop new approaches to combat HCMV infection.

scholarly journals Human Cytomegalovirus UL84 Insertion Mutant Defective for Viral DNA Synthesis and Growth

2004 ◽  
Vol 78 (19) ◽  
pp. 10360-10369 ◽  
Author(s):  
Yiyang Xu ◽  
Sylvia A. Cei ◽  
Alicia Rodriguez Huete ◽  
Gregory S. Pari
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Real Time ◽  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Infectious Virus ◽  
Regulatory Protein ◽  
Insertion Mutant ◽  
Viral Dna ◽  
Transfected Cells

ABSTRACT Human cytomegalovirus (HCMV) UL84 is required for oriLyt-dependent DNA replication, and evidence from transient transfection assays suggests that UL84 directly participates in DNA synthesis. In addition, because of its apparent interaction with IE2, UL84 is implicated as a possible regulatory protein. To address the role of UL84 in the context of the viral genome, we generated a recombinant HCMV bacterial artificial chromosome (BAC) construct that did not express the UL84 gene product. This construct, BAC-IN84/Ep, displayed a null phenotype in that it failed to produce infectious virus after transfection into human fibroblast cells, whereas a revertant virus readily produced viral plaques and, subsequently, infectious virus. Real-time quantitative PCR showed that BAC-IN84/Ep was defective for DNA synthesis in that no increase in the accumulation of viral DNA was observed in transfected cells. We were unable to complement BAC-IN84/Ep in trans; however, oriLyt-dependent DNA replication was observed by the cotransfection of UL84 and BAC-IN84/Ep. An analysis of viral mRNA by real-time PCR indicated that, even in the absence of DNA synthesis, all representative kinetic classes of genes were expressed in cells transfected with BAC-IN84/Ep. The detection of UL44 and IE2 by immunofluorescence in BAC-IN84/Ep-transfected cells showed that these proteins failed to partition into replication compartments, indicating that UL84 expression is essential for the formation of these proteins into replication centers within the context of the viral genome. These results show that UL84 provides an essential DNA replication function and influences the subcellular localization of other viral proteins.

scholarly journals Functional Interaction between Pleiotropic Transactivator pUL69 of Human Cytomegalovirus and the Human Homolog of Yeast Chromatin Regulatory Protein SPT6

2000 ◽  
Vol 74 (17) ◽  
pp. 8053-8064 ◽  
Author(s):  
Michael Winkler ◽  
Thomas aus dem Siepen ◽  
Thomas Stamminger
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Protein ◽  
Regulatory Protein ◽  
Single Amino Acid ◽  
Human Homolog ◽  
Carboxy Terminus ◽  
Homologous Proteins ◽  
Interaction Domain ◽  
Central Domain

ABSTRACT The phosphoprotein pUL69 of human cytomegalovirus (HCMV), which is a herpesvirus of considerable medical importance in immunosuppressed patients and newborns, has previously been identified as an early-late viral protein that can stimulate several viral and cellular promoters and thus exerts a rather broad activation pattern. To gain insight into the mechanism of this transactivation process, we looked for cellular factors interacting with pUL69 in a yeast two-hybrid screen. Using a B-lymphocyte cDNA library fused to the GAL4 activation domain, we identified 34 clones, 11 of which comprised one distinct gene. Interaction with this gene turned out to be very strong, producing β-galactosidase levels 100-fold greater than the background as measured in an ONPG (o-nitrophenyl-β-d-galactopyranoside) assay. Sequencing identified this gene as the human homolog of the yeast factor SPT6, which is thought to be involved in the regulation of chromatin structure. A direct interaction of pUL69 and the carboxy terminus of hSPT6 could be demonstrated using in vitro pull-down experiments. After having generated a specific antiserum that is able to detect the endogenous hSPT6 protein, we were able to observe an in vivo interaction of both proteins by coimmunoprecipitation analysis. The interaction domain within pUL69 was mapped to a central domain of this viral protein that is conserved within the homologous proteins of other herpesviruses such as the ICP27 protein of herpes simplex virus. Internal deletions within this central domain, as well as a single amino acid exchange at position C495, resulted in a loss of interaction. This correlated with a loss of the transactivation potential of the respective mutants, suggesting that the hSPT6 interaction of pUL69 is essential for stimulating gene expression. Furthermore, we demonstrate that the carboxy terminus of hSPT6 also binds to histon H3 and that this interaction can be antagonized by pUL69. This allows the deduction of a model by which pUL69 acts as an antirepressor by competing for binding of histones to hSPT6, thereby antagonizing the chromatin remodeling function of this cellular protein.

Sign in / Sign up

Export Citation Format

Share Document