scholarly journals Preparation of the Human Cytomegalovirus Nuclear Egress Complex and Associated Proteins

2016 ◽  
pp. 517-526 ◽  
Author(s):  
Mayuri Sharma ◽  
Jeremy P. Kamil ◽  
Donald M. Coen
Keyword(s):  
Human Cytomegalovirus ◽  
Nuclear Egress ◽  
Associated Proteins

scholarly journals RASCAL Is a New Human Cytomegalovirus-Encoded Protein That Localizes to the Nuclear Lamina and in Cytoplasmic Vesicles at Late Times Postinfection

2010 ◽  
Vol 84 (13) ◽  
pp. 6483-6496 ◽  
Author(s):  
Matthew S. Miller ◽  
Wendy E. Furlong ◽  
Leesa Pennell ◽  
Marc Geadah ◽  
Laura Hertel
Keyword(s):  
Amino Acids ◽  
Nuclear Envelope ◽  
Human Cytomegalovirus ◽  
Nuclear Membrane ◽  
Nuclear Lamina ◽  
Open Reading Frames ◽  
Short Version ◽  
Nuclear Egress ◽  
Cytoplasmic Vesicles ◽  
Associated Proteins

ABSTRACT The products of numerous open reading frames (ORFs) present in the genome of human cytomegalovirus (CMV) have not been characterized. Here, we describe the identification of a new CMV protein localizing to the nuclear envelope and in cytoplasmic vesicles at late times postinfection. Based on this distinctive localization pattern, we called this new protein nuclear r im- as sociated c ytomegalovir al protein, or RASCAL. Two RASCAL isoforms exist, a short version of 97 amino acids encoded by the majority of CMV strains and a longer version of 176 amino acids encoded by the Towne, Toledo, HAN20, and HAN38 strains. Both isoforms colocalize with lamin B in deep intranuclear invaginations of the inner nuclear membrane (INM) and in novel cytoplasmic vesicular structures possibly derived from the nuclear envelope. INM infoldings have been previously described as sites of nucleocapsid egress, which is mediated by the localized disruption of the nuclear lamina, promoted by the activities of viral and cellular kinases recruited by the lamina-associated proteins UL50 and UL53. RASCAL accumulation at the nuclear membrane required the presence of UL50 but not of UL53. RASCAL and UL50 also appeared to specifically interact, suggesting that RASCAL is a new component of the nuclear egress complex (NEC) and possibly involved in mediating nucleocapsid egress from the nucleus. Finally, the presence of RASCAL within cytoplasmic vesicles raises the intriguing possibility that this protein might participate in additional steps of virion maturation occurring after capsid release from the nucleus.

scholarly journals Human Cytomegalovirus Drives WDR5 to the Virion Assembly Compartment to Facilitate Virion Assembly

2020 ◽  
Author(s):  
Bo Yang ◽  
YongXuan Yao ◽  
Hui Wu ◽  
Hong Yang ◽  
Xue-Hui Ma ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Antiviral Treatment ◽  
Critical Role ◽  
Virion Assembly ◽  
Potential Target ◽  
Nuclear Egress ◽  
Early Endosomes ◽  
Hcmv Replication ◽  
Assembly Compartment

AbstractWe previously reported that human cytomegalovirus (HCMV) utilizes the cellular protein WDR5 to facilitate capsid nuclear egress. Here, we further show that HCMV infection drives WDR5 to the perinuclear region by a mechanism that requires viral replication and intact microtubules. WDR5 accumulated in the virion assembly compartment (vAC) and co-localized with vAC markers of gamma-tubulin (γ-tubulin), early endosomes, and viral vAC marker proteins pp65, pp28, and glycoprotein B (gB). WDR5 interacted with multiple virion proteins, including MCP, pp150, pp65, pIRS1, and pTRS1, which may explain the increasing WDR5 accumulation in the vAC during infection. WDR5 was then incorporated into HCMV virions and localized to the tegument layer, as demonstrated by fractionation and immune-gold electron microscopy. Thus, WDR5 is driven to the vAC and incorporated into virions, suggesting that WDR5 facilitates HCMV replication at later stage of virion assembly besides the capsid nuclear egress stage. These data highlight that WDR5 is a potential target for antiviral therapy.ImportanceHuman cytomegalovirus (HCMV) has a large (~235-kb) genome that contains over 170 ORFs and exploits numerous cellular factors to facilitate its replication. In the late phase of HCMV infection cytoplasmic membranes are profoundly reconfigured to establish the virion assembly compartment (vAC), which is important for efficient assembly of progeny virions. We previously reported that WDR5 promotes HCMV nuclear egress. Here, we show that WDR5 is further driven to the vAC and incorporated into virions, perhaps to facilitate efficient virion maturation. This work identified potential roles for WDR5 in HCMV replication in the cytoplasmic stages of virion assembly. Taken together, WDR5 plays a critical role in HCMV capsid nuclear egress and is important for virion assembly, and thus is a potential target for antiviral treatment of HCMV-associated diseases.

scholarly journals The Human Cytomegalovirus Transmembrane Protein pUL50 Induces Loss of VCP/p97 and Is Regulated by a Small Isoform of pUL50

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Myoung Kyu Lee ◽  
Seokhwan Hyeon ◽  
Jin-Hyun Ahn
Keyword(s):  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Amino Acid Position ◽  
Mutant Virus ◽  
Early Gene ◽  
Immediate Early ◽  
Nuclear Egress ◽  
The Unfolded Protein Response

ABSTRACT The human cytomegalovirus (HCMV) UL50 gene encodes a transmembrane protein, pUL50, which acts as a core component of the nuclear egress complex (NEC) for nucleocapsids. Recently, pUL50 has been shown to have NEC-independent activities: downregulation of IRE1 to repress the unfolded protein response and degradation of UBE1L to inhibit the protein ISG15 modification pathway. Here, we demonstrate that a 26-kDa N-terminal truncated isoform of pUL50 (UL50-p26) is expressed from an internal methionine at amino acid position 199 and regulates the activity of pUL50 to induce the loss of valosin-containing protein (VCP/p97). A UL50(M199V) mutant virus expressing pUL50(M199V) but not UL50-p26 showed delayed growth at a low multiplicity of infection. There was also delayed accumulation of the viral immediate early 2 (IE2) protein in the mutant virus, and this correlated with the reduced expression of VCP/p97, which promotes IE2 expression. Infection with mutant virus did not significantly alter ISGylation levels. In transient expression assays, pUL50 induced VCP/p97 loss posttranscriptionally, and this was dependent on the presence of its transmembrane domain. In contrast, UL50-p26 did not destabilize VCP/p97 but, rather, inhibited pUL50-mediated VCP/p97 loss and the associated major IE gene suppression. Both pUL50 and UL50-p26 interacted with VCP/p97, although UL50-p26 did so more weakly than pUL50. UL50-p26 interacted with pUL50, and this interaction was much stronger than the pUL50 self-interaction. Furthermore, UL50-p26 was able to interfere with the pUL50-VCP/p97 interaction. Our study newly identifies UL50-p26 expression during HCMV infection and suggests a regulatory role for UL50-p26 in blocking pUL50-mediated VCP/p97 loss by associating with pUL50. IMPORTANCE Targeting the endoplasmic reticulum (ER) by viral proteins may affect ER-associated protein homeostasis. During human cytomegalovirus (HCMV) infection, pUL50 targets the ER through its transmembrane domain and moves to the inner nuclear membrane (INM) to form the nuclear egress complex (NEC), which facilitates capsid transport from the nucleus to the cytoplasm. Here, we demonstrate that pUL50 induces the loss of valosin-containing protein (VCP/p97), which promotes the expression of viral major immediate early gene products, in a manner dependent on its membrane targeting but that a small isoform of pUL50 is expressed to negatively regulate this pUL50 activity. This study reports a new NEC-independent function of pUL50 and highlights the fine regulation of pUL50 activity by a smaller isoform for efficient viral growth.

scholarly journals Transmembrane Protein pUL50 of Human Cytomegalovirus Inhibits ISGylation by Downregulating UBE1L

2018 ◽  
Vol 92 (15) ◽  
Author(s):  
Myoung Kyu Lee ◽  
Ye Ji Kim ◽  
Young-Eui Kim ◽  
Tae-Hee Han ◽  
Jens Milbradt ◽  
...  
Keyword(s):  
Viral Infection ◽  
Human Cytomegalovirus ◽  
Transmembrane Protein ◽  
Protein A ◽  
E3 Ligase ◽  
Proteasomal Degradation ◽  
Ubiquitin E3 Ligase ◽  
Nuclear Egress ◽  
Isg15 Expression ◽  
Tm Domain

ABSTRACT Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that can be conjugated to proteins via an enzymatic cascade involving the E1, E2, and E3 enzymes. ISG15 expression and protein ISGylation modulate viral infection; however, the viral mechanisms regulating the function of ISG15 and ISGylation are not well understood. We recently showed that ISGylation suppresses the growth of human cytomegalovirus (HCMV) at multiple steps of the virus life cycle and that the virus-encoded pUL26 protein inhibits protein ISGylation. In this study, we demonstrate that the HCMV UL50-encoded transmembrane protein, a component of the nuclear egress complex, also inhibits ISGylation. pUL50 interacted with UBE1L, an E1-activating enzyme for ISGylation, and (to a lesser extent) with ISG15, as did pUL26. However, unlike pUL26, pUL50 caused proteasomal degradation of UBE1L. The UBE1L level induced in human fibroblast cells by interferon beta treatment or virus infection was reduced by pUL50 expression. This activity of pUL50 involved the transmembrane (TM) domain within its C-terminal region, although pUL50 could interact with UBE1L in a manner independent of the TM domain. Consistently, colocalization of pUL50 with UBE1L was observed in cells treated with a proteasome inhibitor. Furthermore, we found that RNF170, an endoplasmic reticulum (ER)-associated ubiquitin E3 ligase, interacted with pUL50 and promoted pUL50-mediated UBE1L degradation via ubiquitination. Our results demonstrate a novel role for the pUL50 transmembrane protein of HCMV in the regulation of protein ISGylation. IMPORTANCE Proteins can be conjugated covalently by ubiquitin or ubiquitin-like proteins, such as SUMO and ISG15. ISG15 is highly induced in viral infection, and ISG15 conjugation, termed ISGylation, plays important regulatory roles in viral growth. Although ISGylation has been shown to negatively affect many viruses, including human cytomegalovirus (HCMV), viral countermeasures that might modulate ISGylation are not well understood. In the present study, we show that the transmembrane protein encoded by HCMV UL50 inhibits ISGylation by causing proteasomal degradation of UBE1L, an E1-activating enzyme for ISGylation. This pUL50 activity requires membrane targeting. In support of this finding, RNF170, an ER-associated ubiquitin E3 ligase, interacts with pUL50 and promotes UL50-mediated UBE1L ubiquitination and degradation. Our results provide the first evidence, to our knowledge, that viruses can regulate ISGylation by directly targeting the ISGylation E1 enzyme.

scholarly journals Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97

Viruses ◽  
2018 ◽  
Vol 10 (1) ◽  
pp. 35 ◽  
Author(s):  
Jens Milbradt ◽  
Eric Sonntag ◽  
Sabrina Wagner ◽  
Hanife Strojan ◽  
Christina Wangen ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Human Cytomegalovirus ◽  
Viral Protein ◽  
The Core ◽  
Nuclear Egress ◽  
Viral Protein Kinase

scholarly journals The Essential Human Cytomegalovirus Gene UL52 Is Required for Cleavage-Packaging of the Viral Genome

2007 ◽  
Vol 82 (5) ◽  
pp. 2065-2078 ◽  
Author(s):  
Eva Maria Borst ◽  
Karen Wagner ◽  
Anne Binz ◽  
Beate Sodeik ◽  
Martin Messerle
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Essential Role ◽  
Unit Length ◽  
Viral Dna ◽  
Subnuclear Localization ◽  
Viral Genomes ◽  
Nuclear Egress ◽  
C Terminus ◽  
Infected Cells

ABSTRACT Replication of human cytomegalovirus (HCMV) produces large DNA concatemers of head-to-tail-linked viral genomes that upon packaging into capsids are cut into unit-length genomes. The mechanisms underlying cleavage-packaging and the subsequent steps prior to nuclear egress of DNA-filled capsids are incompletely understood. The hitherto uncharacterized product of the essential HCMV UL52 gene was proposed to participate in these processes. To investigate the function of pUL52, we constructed a ΔUL52 mutant as well as a complementing cell line. We found that replication of viral DNA was not impaired in noncomplementing cells infected with the ΔUL52 virus, but viral concatemers remained uncleaved. Since the subnuclear localization of the known cleavage-packaging proteins pUL56, pUL89, and pUL104 was unchanged in ΔUL52-infected fibroblasts, pUL52 does not seem to act via these proteins. Electron microscopy studies revealed only B capsids in the nuclei of ΔUL52-infected cells, indicating that the mutant virus has a defect in encapsidation of viral DNA. Generation of recombinant HCMV genomes encoding epitope-tagged pUL52 versions showed that only the N-terminally tagged pUL52 supported viral growth, suggesting that the C terminus is crucial for its function. pUL52 was expressed as a 75-kDa protein with true late kinetics. It localized preferentially to the nuclei of infected cells and was found to enclose the replication compartments. Taken together, our results demonstrate an essential role for pUL52 in cleavage-packaging of HCMV DNA. Given its unique subnuclear localization, the function of pUL52 might be distinct from that of other cleavage-packaging proteins.

scholarly journals Novel Mode of Phosphorylation-triggered Reorganization of the Nuclear Lamina during Nuclear Egress of Human Cytomegalovirus

2010 ◽  
Vol 285 (18) ◽  
pp. 13979-13989 ◽  
Author(s):  
Jens Milbradt ◽  
Rike Webel ◽  
Sabrina Auerochs ◽  
Heinrich Sticht ◽  
Manfred Marschall
Keyword(s):  
Human Cytomegalovirus ◽  
Nuclear Lamina ◽  
Nuclear Egress

scholarly journals Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus

2017 ◽  
Vol 98 (10) ◽  
pp. 2569-2581 ◽  
Author(s):  
Eric Sonntag ◽  
Jens Milbradt ◽  
Adriana Svrlanska ◽  
Hanife Strojan ◽  
Sigrun Häge ◽  
...  
Keyword(s):  
Protein Kinases ◽  
Human Cytomegalovirus ◽  
Core Complex ◽  
Nuclear Egress
Sign in / Sign up

Export Citation Format

Share Document