scholarly journals Mutant Human Cytomegalovirus Lacking the Immediate-Early TRS1 Coding Region Exhibits a Late Defect

2002 ◽  
Vol 76 (23) ◽  
pp. 12290-12299 ◽  
Author(s):  
Catherine A. Blankenship ◽  
Thomas Shenk
Keyword(s):  
Human Cytomegalovirus ◽  
Infectious Virus ◽  
Human Fibroblasts ◽  
Reporter Genes ◽  
Immediate Early ◽  
Viral Gene ◽  
Wild Type ◽  
Viral Mrnas ◽  
Virus Particles ◽  
Infected Cells

ABSTRACT The human cytomegalovirus IRS1 and TRS1 open reading frames encode immediate-early proteins with identical N-terminal domains and divergent C-terminal regions. Both proteins have been shown previously to activate reporter genes in transfection assays in cooperation with other viral gene products. We have constructed two viruses carrying substitution mutations within either the IRS1 or TRS1 open reading frame. ADsubIRS1 failed to produce the related IRS1 and IRS1263 proteins, but it replicated with normal kinetics to produce a wild-type yield in human fibroblasts. The addition in trans of the IRS1263 protein, which antagonizes the ability of IRS1 and TRS1 proteins to activate reporter genes, did not inhibit the growth of the mutant virus. ADsubTRS1 failed to produce the TRS1 protein, and it generated an ∼200-fold-reduced yield of infectious virus in comparison to its wild-type parent. Viral DNA accumulated normally, as did a set of viral mRNAs that were monitored in ADsubTRS1-infected cells. However, two tegument proteins were partially mislocalized and infectious virus particles did not accumulate to normal levels within ADsubTRS1-infected cells. Further, infectious ADsubTRS1 particles sedimented abnormally in a glycerol-tartrate gradient, indicating that the structure of the mutant particles is aberrant. Our analysis of the ADsubTRS1 phenotype indicates that the TRS1 protein is required, either directly or indirectly, for efficient assembly of virus particles.

scholarly journals Exon 3 of the Human Cytomegalovirus Major Immediate-Early Region Is Required for Efficient Viral Gene Expression and for Cellular Cyclin Modulation

2005 ◽  
Vol 79 (12) ◽  
pp. 7438-7452 ◽  
Author(s):  
Elizabeth A. White ◽  
Deborah H. Spector
Keyword(s):  
Human Cytomegalovirus ◽  
Cyclin B1 ◽  
Mutant Virus ◽  
Immediate Early ◽  
Wild Type Virus ◽  
Viral Gene ◽  
Protein Accumulation ◽  
Wild Type ◽  
Altered Expression ◽  
Infected Cells

ABSTRACT The human cytomegalovirus (HCMV) major immediate-early (IE) proteins share an 85-amino-acid N-terminal domain specified by exons 2 and 3 of the major IE region, UL122-123. We have constructed IE Δ30-77, a recombinant virus that lacks the majority of IE exon 3 and consequently expresses smaller forms of both IE1 72- and IE2 86-kDa proteins. The mutant virus is viable but growth impaired at both high and low multiplicities of infection and exhibits a kinetic defect that is not rescued by growth in fibroblasts expressing IE1 72-kDa protein. The kinetics of mutant IE2 protein accumulation in IE Δ30-77 virus-infected cells are approximately normal compared to wild-type virus-infected cells, but the IE Δ30-77 virus is delayed in expression of early viral genes, including UL112-113 and UL44, and does not sustain expression of mutant IE1 protein as the infection progresses. Additionally, cells infected with IE Δ30-77 exhibit altered expression of cellular proteins compared to wild-type HCMV-infected cells. PML is not dispersed but is retained at ND10 sites following infection with IE Δ30-77 mutant virus. While the deletion mutant retains the ability to mediate the stabilization of cyclin B1, cdc6, and geminin in infected cells, its capacity to upregulate the expression of cyclin E has been reduced. These data indicate that the activity of one or both of the HCMV major IE proteins is required in vivo for the modulation of cell cycle proteins observed in cells infected with wild-type HCMV.

scholarly journals Human Cytomegalovirus pUS24 Is a Virion Protein That Functions Very Early in the Replication Cycle

2006 ◽  
Vol 80 (17) ◽  
pp. 8371-8378 ◽  
Author(s):  
Xuyan Feng ◽  
Jörg Schröer ◽  
Dong Yu ◽  
Thomas Shenk
Keyword(s):  
Human Cytomegalovirus ◽  
Mutant Virus ◽  
Replication Cycle ◽  
Immediate Early ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virion Protein ◽  
Viral Dna ◽  
Infected Cells

ABSTRACT We have characterized the function of the human cytomegalovirus US24 gene, a US22 gene family member. Two US24-deficient mutants (BADinUS24 and BADsubUS24) exhibited a 20- to 30-fold growth defect, compared to their wild-type parent (BADwt), after infection at a relatively low (0.01 PFU/cell) or high (1 PFU/cell) input multiplicity. Representative virus-encoded proteins and viral DNA accumulated with normal kinetics to wild-type levels after infection with mutant virus when cells received equal numbers of mutant and wild-type infectious units. Further, the proteins were properly localized and no ultrastructural differences were found by electron microscopy in mutant-virus-infected cells compared to wild-type-virus-infected cells. However, virions produced by US24-deficient mutants had a 10-fold-higher genome-to-PFU ratio than wild-type virus. When infections were performed using equal numbers of input virus particles, the expression of immediate-early, early, and late viral proteins was substantially delayed and decreased in the absence of US24 protein. This delay is not due to inefficient virus entry, since two tegument proteins and viral DNA moved to the nucleus equally well in mutant- and wild-type-virus-infected cells. In summary, US24 is a virion protein and virions produced by US24-deficient viruses exhibit a block to the human cytomegalovirus replication cycle after viral DNA reaches the nucleus and before immediate-early mRNAs are transcribed.

scholarly journals A Strong Negative Transcriptional Regulatory Region between the Human Cytomegalovirus UL127 Gene and the Major Immediate-Early Enhancer

1999 ◽  
Vol 73 (11) ◽  
pp. 9039-9052 ◽  
Author(s):  
Christopher A. Lundquist ◽  
Jeffrey L. Meier ◽  
Mark F. Stinski
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Genome ◽  
De Novo ◽  
Human Fibroblasts ◽  
Regulatory Region ◽  
Fold Increase ◽  
Immediate Early ◽  
Wild Type ◽  
Viral Protein Synthesis ◽  
Unique Region

ABSTRACT The region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer is referred to as the unique region. DNase I protection analysis with human cell nuclear extracts demonstrated multiple protein binding sites in this region of the viral genome (P. Ghazal, H. Lubon, C. Reynolds-Kohler, L. Hennighausen, and J. A. Nelson, Virology 174:18–25, 1990). However, the function of this region in the context of the viral genome is not known. In wild-type human CMV-infected human fibroblasts, cells permissive for viral replication, there is little to no transcription from UL127. We determined that the unique region prevented transcription from the UL127 promoter but had no effect on the divergent MIE promoter. In transient-transfection assays, the basal level of expression from the UL127 promoter increased significantly when the wild-type unique sequences were mutated. In recombinant viruses with similar mutations in the unique region, expression from the UL127 promoter occurred only after de novo viral protein synthesis, typical of an early viral promoter. A 111-bp deletion-substitution of the unique sequence caused approximately a 20-fold increase in the steady-state level of RNA from the UL127 promoter and a 245-fold increase in the expression of a downstream indicator gene. This viral negative regulatory region was also mutated at approximately 50-bp regions proximal and distal to the UL127 promoter. Although some repressive effects were detected in the distal region, mutations of the region proximal to the UL127 promoter had the most significant effects on transcription. Within the proximal and distal regions, there are potential cis sites for known eucaryotic transcriptional repressor proteins. This region may also bind unknown viral proteins. We propose that the unique region upstream of the UL127 promoter and the MIE enhancer negatively regulates the expression from the UL127 promoter in permissive human fibroblast cells. This region may be a regulatory boundary preventing the effects of the very strong MIE enhancer on this promoter.

scholarly journals Reversal of Human Cytomegalovirus Major Immediate-Early Enhancer/Promoter Silencing in Quiescently Infected Cells via the Cyclic AMP Signaling Pathway

2007 ◽  
Vol 81 (12) ◽  
pp. 6669-6681 ◽  
Author(s):  
Michael J. Keller ◽  
Allen W. Wu ◽  
Janet I. Andrews ◽  
Patrick W. McGonagill ◽  
Eric E. Tibesar ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Cyclic Amp ◽  
Signaling Pathway ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Lytic Cycle ◽  
Immediate Early ◽  
Infected Cells ◽  
Ifn Γ ◽  
Stimulation Of

ABSTRACT The human cytomegalovirus (HCMV) major immediate-early (MIE) enhancer contains five functional cyclic AMP (cAMP) response elements (CRE). Because the CRE in their native context do not contribute appreciably to MIE enhancer/promoter activity in lytically infected human fibroblasts and NTera2 (NT2)-derived neurons, we postulated that they might have a role in MIE enhancer/promoter reactivation in quiescently infected cells. Here, we show that stimulation of the cAMP signaling pathway by treatment with forskolin (FSK), an adenylyl cyclase activator, greatly alleviates MIE enhancer/promoter silencing in quiescently infected NT2 neuronal precursors. The effect is immediate, independent of de novo protein synthesis, associated with the phosphorylation of ATF-1 serine 63 and CREB serine 133, dependent on protein kinase A (PKA) and the enhancer's CRE, and linked to viral-lytic-cycle advancement. Coupling of FSK treatment with the inhibition of either histone deacetylases or protein synthesis synergistically activates MIE gene expression in a manner suggesting that MIE enhancer/promoter silencing is optimally relieved by an interplay of multiple regulatory mechanisms. In contrast, MIE enhancer/promoter silence is not overcome by stimulation of the gamma interferon (IFN-γ) signaling pathway, despite the enhancer having two IFN-γ-activated-site-like elements. We conclude that stimulation of the cAMP/PKA signaling pathway drives CRE-dependent MIE enhancer/promoter activation in quiescently infected cells, thus exposing a potential mode of regulation in HCMV reactivation.

scholarly journals Human Cytomegalovirus Infection Leads to Accumulation of Geminin and Inhibition of the Licensing of Cellular DNA Replication

2003 ◽  
Vol 77 (4) ◽  
pp. 2369-2376 ◽  
Author(s):  
Nilima Biswas ◽  
Veronica Sanchez ◽  
Deborah H. Spector
Keyword(s):  
Dna Replication ◽  
Host Cell ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Early Protein ◽  
Infected Cells ◽  
Mcm Proteins ◽  
Cellular Dna

ABSTRACT Previous studies have shown that infection of G0-synchronized human fibroblasts by human cytomegalovirus (HCMV) results in a block to cellular DNA synthesis. In this study, we have examined the effect of viral infection on the formation of the host cell DNA prereplication complex (pre-RC). We found that the Cdc6 protein level was significantly upregulated in the virus-infected cells and that there was a delay in the expression of the Mcm family of proteins. The loading of the Mcm proteins onto the DNA pre-RC complex also appeared to be defective in the virus-infected cells. This inhibition of DNA replication licensing was associated with the accumulation of geminin, a replication inhibitor. Cdt1, which participates in the loading of the Mcm proteins, was also downregulated and modified differentially in the infected cells. Early viral gene expression was sufficient for the virus-induced alteration of the pre-RC, and the immediate-early protein IE1 was not required. These studies show that the inhibition of replication licensing in HCMV-infected cells is one of the multiple pathways by which the virus dysregulates the host cell cycle.

scholarly journals Characterization of phenyl pyrimidine derivatives that inhibit cytomegalovirus immediate-early gene expression

2018 ◽  
Vol 26 ◽  
pp. 204020661876319 ◽  
Author(s):  
Koh-Hei Yamada ◽  
Ryuichi Majima ◽  
Toyofumi Yamaguchi ◽  
Naoki Inoue
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Early Gene ◽  
Immediate Early ◽  
Murine Cytomegalovirus ◽  
Viral Attachment ◽  
Varicella Zoster ◽  
Infected Cells

Background Previously, we established a reporter cell line for human cytomegalovirus and screened anti-human cytomegalovirus compounds using the cell line. In this study, we characterized one of the identified compounds, 2,4-diamino-6–(4-methoxyphenyl)pyrimidine (coded as 35C10). Methods 50% Effective concentrations (EC50s) and 50% cytotoxic concentrations (CC50s) of 35C10 and its derivatives in human fibroblasts were determined by X-gal staining of the cells infected with human cytomegalovirus Towne strain expressing β-galactosidase. Results EC50 and CC50 of 35C10 were 4.3 µM and >200 µM, respectively. Among several 35C10 derivatives, only one lacking 4-amino group of pyrimidine showed a similar EC50. 35C10 weakly inhibited murine cytomegalovirus, herpes simplex virus type 1, and varicella-zoster virus. A “time of addition” experiment suggested that 35C10 inhibited an early phase of the infection. Although 35C10 did not inhibit viral attachment to the cells nor the delivery of viral DNA to the nuclei, it decreased the number of infected cells expressing immediate-early 1 and 2 (IE1/IE2) proteins. 35C10 also inhibited the activation of a promoter for TRL4 in the reporter cells upon human cytomegalovirus infection, but not in the same reporter cells transfected with a plasmid expressing IE2. Conclusion Our findings suggest that 35C10 is a novel compound that inhibits IE gene expression in human cytomegalovirus-infected cells.

scholarly journals Recruitment of Human Cytomegalovirus Immediate-Early 2 Protein onto Parental Viral Genomes in Association with ND10 in Live-Infected Cells

2007 ◽  
Vol 81 (18) ◽  
pp. 10123-10136 ◽  
Author(s):  
George Sourvinos ◽  
Nina Tavalai ◽  
Anja Berndt ◽  
Demetrios A. Spandidos ◽  
Thomas Stamminger
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Spatial Organization ◽  
Human Fibroblasts ◽  
Nuclear Bodies ◽  
Immediate Early ◽  
Viral Dna ◽  
Subnuclear Localization ◽  
Pml Nuclear Bodies ◽  
Infected Cells

ABSTRACT The human cytomegalovirus (HCMV) immediate-early 2 (IE2) transactivator has previously been shown to form intranuclear, dot-like accumulations in association with subnuclear structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10. We recently observed that IE2 can form dot-like structures even after infection of PML knockdown cells, which lack genuine ND10. To further analyze the determinants of IE2 subnuclear localization, a recombinant HCMV expressing IE2 fused to the enhanced green fluorescent protein was constructed. We infected primary human fibroblasts expressing Sp100 fused to the autofluorescent protein mCherry while performing live-cell imaging experiments. These experiments revealed a very dynamic association of IE2 dots with ND10 structures during the first hours postinfection: juxtaposed structures rapidly fused to precise colocalizations, followed by segregation, and finally, the dispersal of ND10 accumulations. Furthermore, by infecting PML knockdown cells we determined that the number of IE2 accumulations was dependent on the multiplicity of infection. Since time-lapse microscopy in live-infected cells revealed that IE2 foci developed into viral replication compartments, we hypothesized that viral DNA could act as a determinant of IE2 accumulations. Direct evidence that IE2 molecules are associated with viral DNA early after HCMV infection was obtained using fluorescence in situ hybridization. Finally, a DNA-binding-deficient IE2 mutant could no longer be recruited into viral replication centers, suggesting that the association of IE2 with viral DNA is mediated by a direct DNA contact. Thus, we identified viral DNA as an important determinant of IE2 subnuclear localization, which suggests that the formation of a virus-induced nucleoprotein complex and its spatial organization is likely to be critical at the early stages of a lytic infection.

scholarly journals Human Cytomegalovirus 5-Kilobase Immediate-Early RNA Is a Stable Intron

2004 ◽  
Vol 78 (23) ◽  
pp. 13182-13189 ◽  
Author(s):  
Caroline A. Kulesza ◽  
Thomas Shenk
Keyword(s):  
Viral Infection ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Donor Site ◽  
Immediate Early ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Viral Gene ◽  
Acceptor Site ◽  
Splice Donor

ABSTRACT Immediate-early viral gene products of human cytomegalovirus (HCMV) are derived from several genomic loci and largely serve to establish a cellular environment conducive to viral replication. We have further examined an unusual immediate-early transcript known as the 5-kb RNA, concluding that it is a stable intron encoded by HCMV. The 5-kb RNA is highly AT rich in sequence and lacks open reading frames likely to be translated into protein. We confirmed the absence of polyadenylation of the transcript and showed that it is primarily nuclear localized during viral infection. We mapped the 5′ end of the 5-kb RNA to a consensus splice donor site and localized the 3′ end in the vicinity of a splice acceptor site. In transfection studies, we showed that the 5-kb RNA can be spliced from a heterologous primary transcript. Using bacterial artificial chromosome technology, we constructed a viral recombinant containing a mutation in the 5′ splice donor site that defines the 5′ end of the RNA and found that this mutation eliminates expression of the 5-kb RNA during viral infection. This mutant grows in human fibroblasts without complementation. Taken together, these data support the conclusion that the 5-kb RNA is a stable intron expressed by HCMV.

scholarly journals Human Cytomegalovirus UL47 Tegument Protein Functions after Entry and before Immediate-Early Gene Expression

2002 ◽  
Vol 76 (3) ◽  
pp. 1043-1050 ◽  
Author(s):  
Jill T. Bechtel ◽  
Thomas Shenk
Keyword(s):  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Open Reading Frame ◽  
Early Gene ◽  
Immediate Early ◽  
Wild Type Virus ◽  
Marker Genes ◽  
Type Virus ◽  
Wild Type ◽  
Reading Frame

ABSTRACT The human cytomegalovirus UL47 open reading frame encodes a 110-kDa protein that is a component of the virion tegument. We have constructed a cytomegalovirus mutant, ADsubUL47, in which the central portion of the UL47 open reading frame has been replaced by two marker genes. The mutant replicated to titers 100-fold lower than those for wild-type virus after infection at either a high or a low input multiplicity in primary human fibroblasts but was substantially complemented on cells expressing UL47 protein. A revertant virus in which the mutation was repaired, ADrevUL47, replicated with wild-type kinetics. Mutant virions lacked UL47 protein and contained reduced amounts of UL48 protein. The mutant was found to be less infectious than wild-type virus, and a defect very early in the replication cycle was observed. Transcription of the viral immediate-early 1 gene was delayed by 8 to 10 h. However, this delay was not the result of a defect in virus entry or of the inability of virion proteins to transactivate the major immediate-early promoter. We also show that the UL47 protein coprecipitated with the UL48 and UL69 tegument proteins and the UL86-encoded major capsid protein. We propose that a UL47-containing complex is involved in the release of viral DNA from the disassembling virus particle and that the loss of UL47 protein causes this process to be delayed.

scholarly journals Characterization of a Human Cytomegalovirus with Phosphorylation Site Mutations in the Immediate-Early 2 Protein

2002 ◽  
Vol 76 (2) ◽  
pp. 928-932 ◽  
Author(s):  
Julie A. Heider ◽  
Yongjun Yu ◽  
Thomas Shenk ◽  
James C. Alwine
Keyword(s):  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Phosphorylation Site ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Immediate Early ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type

ABSTRACT A human cytomegalovirus mutant (TNsubIE2P) was constructed with alanine substitutions of four residues (T27, S144, T233, and S234) previously shown to be phosphorylated in the immediate-early 2 (IE2) protein. This mutant grew as well as the wild type at both low and high multiplicities of infection. The mutant activated the major immediate-early, UL4, and UL44 promoters to similar levels, and with similar kinetics, as wild-type virus. However, the TNsubIE2P mutant virus transactivated an endogenous simian virus 40 early promoter 4 h earlier and to higher levels than the wild-type virus in infected human fibroblasts. The modification of the IE2 protein by SUMO-1 (i.e., its sumoylated state) was also examined.

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