Requirement of the N-terminal residues of human cytomegalovirus UL112-113 proteins for viral growth and oriLyt-dependent DNA replication

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.

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Role of Envelope Glycoprotein Complexes in Cell-Associated Spread of Human Cytomegalovirus

Viruses ◽

10.3390/v13040614 ◽

2021 ◽

Vol 13 (4) ◽

pp. 614

Author(s):

Nina Weiler ◽

Caroline Paal ◽

Kerstin Adams ◽

Christopher Calcaterra ◽

Dina Fischer ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Envelope Glycoprotein ◽

Infectious Virus ◽

Viral Envelope ◽

Accessory Proteins ◽

Viral Growth ◽

Cell Association ◽

Quantitative Immunoblotting ◽

Model Virus

The role of viral envelope glycoproteins, particularly the accessory proteins of trimeric and pentameric gH/gL-complexes, in cell-associated spread of human cytomegalovirus (HCMV) is unclear. We aimed to investigate their contribution in the context of HCMV variants that grow in a strictly cell-associated manner. In the genome of Merlin pAL1502, the glycoproteins gB, gH, gL, gM, and gN were deleted by introducing stop codons, and the mutants were analyzed for viral growth. Merlin and recent HCMV isolates were compared by quantitative immunoblotting for expression of accessory proteins of the trimeric and pentameric gH/gL-complexes, gO and pUL128. Isolates were treated with siRNAs against gO and pUL128 and analyzed regarding focal growth and release of infectious virus. All five tested glycoproteins were essential for growth of Merlin pAL1502. Compared with this model virus, higher gO levels were measured in recent isolates of HCMV, and its knockdown decreased viral growth. Knockdown of pUL128 abrogated the strict cell-association and led to release of infectivity, which allowed cell-free transfer to epithelial cells where the virus grew again strictly cell-associated. We conclude that both trimer and pentamer contribute to cell-associated spread of recent clinical HCMV isolates and downregulation of pentamer can release infectious virus into the supernatant.

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Interaction of the Putative Human Cytomegalovirus Portal Protein pUL104 with the Large Terminase Subunit pUL56 and Its Inhibition by Benzimidazole-d-Ribonucleosides

Journal of Virology ◽

10.1128/jvi.79.23.14660-14667.2005 ◽

2005 ◽

Vol 79 (23) ◽

pp. 14660-14667 ◽

Cited By ~ 48

Author(s):

Alexandra Dittmer ◽

John C. Drach ◽

Leroy B. Townsend ◽

Anke Fischer ◽

Elke Bogner

Keyword(s):

Dna Replication ◽

Human Cytomegalovirus ◽

Unit Length ◽

Large Subunit ◽

Intracellular Localization ◽

Direct Interaction ◽

Portal Protein ◽

Specific Association ◽

Carboxy Terminal

ABSTRACT Herpesvirus DNA replication leads to unit length genomes that are translocated into preformed procapsids through a unique portal vertex. The translocation is performed by the terminase that cleaves the DNA and powers the insertion by its ATPase activity. Recently, we demonstrated that the putative human cytomegalovirus (HCMV) portal protein, pUL104, also forms high-molecular-weight complexes. Analyses now have been performed to determine the intracellular localization and identification of interaction partners of pUL104. In infected cells, HCMV pUL104 was found to be predominantly localized throughout the nucleus as well as in cytoplasmic clusters at late times of infection. The latter localization was abolished by phosphonoacetic acid, an inhibitor of viral DNA replication. Immunofluorescence revealed that pUL104 colocalized with pUL56, the large subunit of the HCMV terminase. Specific association of in vitro translated pUL104 with the carboxy-terminal half of GST-UL56C was detected. By using coimmunoprecipitations a direct interaction with pUL56 was confirmed. In addition, this interaction was no longer detected when the benzimidazole-d-nucleosides BDCRB or Cl4RB were added, thus indicating that these HCMV inhibitors block the insertion of the DNA into the capsid by preventing a necessary interaction of pUL56 with the portal. Electron microscopy revealed that in the presence of Cl4RB DNA is not packaged into capsids and these capsids failed to egress from the nucleus. Furthermore, pulsed-field gel electrophoresis showed that DNA concatemers synthesized in the presence of the compound failed to be processed.

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Involvement of the N-Terminal Deubiquitinating Protease Domain of Human Cytomegalovirus UL48 Tegument Protein in Autoubiquitination, Virion Stability, and Virus Entry

Journal of Virology ◽

10.1128/jvi.02766-15 ◽

2016 ◽

Vol 90 (6) ◽

pp. 3229-3242 ◽

Cited By ~ 11

Author(s):

Young-Eui Kim ◽

Se Eun Oh ◽

Ki Mun Kwon ◽

Chan Hee Lee ◽

Jin-Hyun Ahn

Keyword(s):

Human Cytomegalovirus ◽

Virus Entry ◽

Terminal Region ◽

Mutant Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Viral Growth ◽

Internal Region ◽

Deubiquitinating Protease

ABSTRACTHuman cytomegalovirus (HCMV) protein pUL48 is closely associated with the capsid and has a deubiquitinating protease (DUB) activity in its N-terminal region. Although this DUB activity moderately increases virus replication in cultured fibroblast cells, the requirements of the N-terminal region of pUL48 in the viral replication cycle are not fully understood. In this study, we characterized the recombinant viruses encoding UL48(ΔDUB/NLS), which lacks the DUB domain and the adjacent nuclear localization signal (NLS), UL48(ΔDUB), which lacks only the DUB, and UL48(Δ360–1200), which lacks the internal region (amino acids 360 to 1200) downstream of the DUB/NLS. While ΔDUB/NLS and Δ360–1200 mutant viruses did not grow in fibroblasts, the ΔDUB virus replicated to titers 100-fold lower than those for wild-type virus and showed substantially reduced viral gene expression at low multiplicities of infection. The DUB domain contained ubiquitination sites, and DUB activity reduced its own proteasomal degradation intrans. Deletion of the DUB domain did not affect the nuclear and cytoplasmic localization of pUL48, whereas the internal region (360–1200) was necessary for cytoplasmic distribution. In coimmunoprecipitation assays, pUL48 interacted with three tegument proteins (pUL47, pUL45, and pUL88) and two capsid proteins (pUL77 and pUL85) but the DUB domain contributed to only pUL85 binding. Furthermore, we found that the ΔDUB virus showed reduced virion stability and less efficiently delivered its genome into the cell than the wild-type virus. Collectively, our results demonstrate that the N-terminal DUB domain of pUL48 contributes to efficient viral growth by regulating its own stability and promoting virion stabilization and virus entry.IMPORTANCEHCMV pUL48 and its herpesvirus homologs play key roles in virus entry, regulation of immune signaling pathways, and virion assembly. The N terminus of pUL48 contains the DUB domain, which is well conserved among all herpesviruses. Although studies using the active-site mutant viruses revealed that the DUB activity promotes viral growth, the exact role of this region in the viral life cycle is not fully understood. In this study, using the mutant virus lacking the entire DUB domain, we demonstrate that the DUB domain of pUL48 contributes to viral growth by regulating its own stability via autodeubiquitination and promoting virion stability and virus entry. This report is the first to demonstrate the characteristics of the mutant virus with the entire DUB domain deleted, which, along with information on the functions of this region, is useful in dissecting the functions associated with pUL48.

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Infection of Cells with Human Cytomegalovirus during S Phase Results in a Blockade to Immediate-Early Gene Expression That Can Be Overcome by Inhibition of the Proteasome

Journal of Virology ◽

10.1128/jvi.76.11.5369-5379.2002 ◽

2002 ◽

Vol 76 (11) ◽

pp. 5369-5379 ◽

Cited By ~ 54

Author(s):

Elizabeth A. Fortunato ◽

Veronica Sanchez ◽

Judy Y. Yen ◽

Deborah H. Spector

Keyword(s):

Gene Expression ◽

Dna Replication ◽

Human Cytomegalovirus ◽

Dna Content ◽

Laser Scanning ◽

Small Population ◽

S Phase ◽

Early Gene ◽

Immediate Early ◽

Laser Scanning Cytometry

ABSTRACT Cells infected with human cytomegalovirus (HCMV) after commencing DNA replication do not initiate viral immediate-early (IE) gene expression and divide before arresting. To determine the nature of this blockade, we examined cells that were infected 24 h after release from G0 using immunofluorescence, laser scanning cytometry, and fluorescence-activated cell sorting (FACS) analysis. Approximately 40 to 50% of the cells had 2N DNA content, became IE+ in the first 12 h, and arrested. Most but not all of the cells with >2N DNA content did not express IE antigens until after mitosis. To define the small population of IE+ cells that gradually accumulated within the S and G2/M compartments, cells were pulsed with bromodeoxyuridine (BrdU) just prior to S-phase infection and analyzed at 12 h postinfection for IE gene expression, BrdU positivity, and cell cycle position. Most of the BrdU+ cells were IE− and had progressed into G2/M or back to G1. The majority of the IE+ cells in S and G2/M were BrdU−. Only a few cells were IE+ BrdU+, and they resided in G2/M. Multipoint BrdU pulse-labeling revealed that, compared to cells actively synthesizing DNA at the beginning of the infection, a greater percentage of the cells that initiated DNA replication 4 h later could express IE antigens and proceed into S. Synchronization of the cells with aphidicolin also indicated that the blockade to the activation of IE gene expression was established in cells soon after initiation of DNA replication. It appears that a short-lived protein in S-phase cells may be required for IE gene expression, as it is partially restored by treatment with the proteasome inhibitor MG132.

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Human cytomegalovirus UL141 protein interacts with CELF5 and affects viral DNA replication

Molecular Medicine Reports ◽

10.3892/mmr.2018.8419 ◽

2018 ◽

Cited By ~ 2

Author(s):

Fei Zou ◽

Zhi‑Tao Lu ◽

Shuang Wang ◽

Si Wu ◽

Ying‑Ying Wu ◽

...

Keyword(s):

Dna Replication ◽

Human Cytomegalovirus ◽

Viral Dna ◽

Viral Dna Replication

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Human Cytomegalovirus UL84 Interacts with an RNA Stem-Loop Sequence Found within the RNA/DNA Hybrid Region of oriLyt

Journal of Virology ◽

10.1128/jvi.00058-07 ◽

2007 ◽

Vol 81 (13) ◽

pp. 7077-7085 ◽

Cited By ~ 30

Author(s):

Kelly S. Colletti ◽

Kate E. Smallenburg ◽

Yiyang Xu ◽

Gregory S. Pari

Keyword(s):

Dna Replication ◽

Human Cytomegalovirus ◽

Specific Interaction ◽

Simian Virus 40 ◽

Loop Structure ◽

Stem Loop ◽

Stem Loop Structure ◽

Core Proteins ◽

Essential Region

ABSTRACT Human cytomegalovirus (HCMV) lytic DNA replication is initiated at the complex cis-acting oriLyt region, which spans nearly 3 kb. DNA synthesis requires six core proteins together with UL84 and IE2. Previously, two essential regions were identified within oriLyt. Essential region I (nucleotides [nt] 92209 to 92573) can be replaced with the constitutively active simian virus 40 promoter, which in turn eliminates the requirement for IE2 in the origin-dependent transient-replication assay. Essential region II (nt 92979 to 93513) contains two elements of interest: an RNA/DNA hybrid domain and an inverted repeat sequence capable of forming a stem-loop structure. Our studies now reveal for the first time that UL84 interacts with a stem-loop RNA oligonucleotide in vitro, and although UL84 interacted with other nucleic acid substrates, a specific interaction occurred only with the RNA stem-loop. Increasing concentrations of purified UL84 produced a remarkable downward-staircase pattern, which is not due to a nuclease activity but is dependent upon the presence of secondary structures, suggesting that UL84 modifies the conformation of the RNA substrate. Cross-linking experiments show that UL84 possibly changes the conformation of the RNA substrate. The addition of purified IE2 to the in vitro binding reaction did not affect binding to the stem-loop structure. Chromatin immunoprecipitation assays performed using infected cells and purified virus show that UL84 is bound to oriLyt in a region adjacent to the RNA/DNA hybrid and the stem-loop structure. These results solidify UL84 as the potential initiator of HCMV DNA replication through a unique interaction with a conserved RNA stem-loop structure within oriLyt.

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Inhibition of human cytomegalovirus DNA replication by small interfering RNAs targeted to UL49

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmt025 ◽

2013 ◽

Vol 45 (5) ◽

pp. 401-407 ◽

Cited By ~ 4

Author(s):

K. Wang ◽

Y. Li ◽

G. Zhao ◽

Y. Wu ◽

X. Zhang ◽

...

Keyword(s):

Dna Replication ◽

Human Cytomegalovirus ◽

Small Interfering Rnas

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Role of Homodimerization of Human Cytomegalovirus DNA Polymerase Accessory Protein UL44 in Origin-Dependent DNA Replication in Cells

Journal of Virology ◽

10.1128/jvi.01193-08 ◽

2008 ◽

Vol 82 (24) ◽

pp. 12574-12579 ◽

Cited By ~ 17

Author(s):

Elisa Sinigalia ◽

Gualtiero Alvisi ◽

Beatrice Mercorelli ◽

Donald M. Coen ◽

Gregory S. Pari ◽

...

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Human Cytomegalovirus ◽

Nuclear Localization ◽

Accessory Protein ◽

C Terminus ◽

Cellular Context ◽

In Cells

ABSTRACT The presumed processivity subunit of human cytomegalovirus (HCMV) DNA polymerase, UL44, forms homodimers. The dimerization of UL44 is important for binding to DNA in vitro; however, whether it is also important for DNA replication in a cellular context is unknown. Here we show that UL44 point mutants that are impaired for dimerization, but not for nuclear localization or interaction with the C terminus of the polymerase catalytic subunit, are not capable of supporting HCMV oriLyt-dependent DNA replication in cells. These data suggest that the disruption of UL44 homodimers could represent a novel anti-HCMV strategy.

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