scholarly journals Human Cytomegalovirus Tegument Proteins ppUL82 (pp71) and ppUL35 Interact and Cooperatively Activate the Major Immediate-Early Enhancer

2004 ◽  
Vol 78 (17) ◽  
pp. 9512-9523 ◽  
Author(s):  
Karina Schierling ◽  
Thomas Stamminger ◽  
Thomas Mertens ◽  
Michael Winkler
Keyword(s):  
Human Cytomegalovirus ◽  
Early Gene ◽  
Immediate Early ◽  
Tegument Protein ◽  
Tegument Proteins ◽  
Infected Cells ◽  
Early Transcription ◽  
Cooperative Activation ◽  
Two Hybrid

ABSTRACT The tegument protein ppUL82 (pp71) of human cytomegalovirus (HCMV) has previously been shown to activate the immediate-early transcription of HCMV and to enhance the infectivity of viral DNA. This is concordant with its localization adjacent to promyelocytic leukemia oncogenic domains (PODs) immediately after infection. In a yeast two-hybrid screen, we identified the tegument protein ppUL35 as an interacting partner of ppUL82. The interaction could be confirmed in transfected and infected cells. The domain responsible for interaction was narrowed down to amino acids 447 to 516 within ppUL35, thus allowing both forms of ppUL35 to interact with ppUL82. Immunofluorescence experiments showed a relocalization of ppUL35 from a diffuse nuclear pattern when expressed alone to PODs when expressed together with ppUL82. In accordance with this observation and the role of ppUL82 as a transactivator, we observed a cooperative activation of the HCMV major immediate-early enhancer but not of heterologous viral enhancer elements. These results suggest an important role for this interaction in the stimulation of viral immediate-early gene expression and viral infection.

scholarly journals UL26-Deficient Human Cytomegalovirus Produces Virions with Hypophosphorylated pp28 Tegument Protein That Is Unstable within Newly Infected Cells

2006 ◽  
Vol 80 (7) ◽  
pp. 3541-3548 ◽  
Author(s):  
Joshua Munger ◽  
Dong Yu ◽  
Thomas Shenk
Keyword(s):  
Human Cytomegalovirus ◽  
Deletion Mutant ◽  
Open Reading Frame ◽  
Early Gene ◽  
Immediate Early ◽  
Tegument Protein ◽  
Protein Accumulation ◽  
Reading Frame ◽  
Tegument Proteins ◽  
Infected Cells

ABSTRACT The human cytomegalovirus UL26 open reading frame encodes proteins of 21 and 27 kDa that result from the use of two different in-frame initiation codons. The UL26 protein is a constituent of the virion and thus is delivered to cells upon viral entry. We have characterized a mutant of human cytomegalovirus in which the UL26 open reading frame has been deleted. The UL26 deletion mutant has a profound growth defect, the magnitude of which is dependent on the multiplicity of infection. Two very early defects were discovered. First, even though they were present in normal amounts within mutant virions, the UL99-coded pp28 and UL83-coded pp65 tegument proteins were present in reduced amounts at the earliest times assayed within newly infected cells; second, there was a delay in immediate-early mRNA and protein accumulation. Further analysis revealed that although wild-type levels of the pp28 tegument protein were present in UL26 deletion mutant virions, the protein was hypophosphorylated. We conclude that the UL26 protein influences the normal phosphorylation of at least pp28 in virions and possibly additional tegument proteins. We propose that the hypophosphorylation of tegument proteins causes their destabilization within newly infected cells, perhaps disrupting the normal detegumentation process and leading to a delay in the onset of immediate-early gene expression.

scholarly journals Cyclin-Dependent Kinase Activity Is Required for Efficient Expression and Posttranslational Modification of Human Cytomegalovirus Proteins and for Production of Extracellular Particles

2006 ◽  
Vol 80 (12) ◽  
pp. 5886-5896 ◽  
Author(s):  
Veronica Sanchez ◽  
Deborah H. Spector
Keyword(s):  
Steady State ◽  
Human Cytomegalovirus ◽  
Virus Titer ◽  
State Level ◽  
Early Gene ◽  
Tegument Protein ◽  
Cyclin Dependent Kinase ◽  
Viral Dna ◽  
Infected Cells ◽  
Steady State Levels

ABSTRACT We have previously shown that the addition of the cyclin-dependent kinase (cdk) inhibitor Roscovitine at the beginning of infection of cells with human cytomegalovirus (HCMV) significantly disrupts immediate-early gene expression and the progression of the infection. In the present study, we have examined the effects of cdk inhibition on late viral events by delaying addition of Roscovitine until 24 h postinfection. Although viral DNA replication was inhibited two- to threefold by treatment of infected cells with Roscovitine, the drop did not correspond to the 1- to 2-log-unit decrease in virus titer. Quantification of viral DNA in the supernatant from cells revealed that there was a significant reduction in the production or release of extracellular particles. We observed a lag in the expression of several viral proteins but there was a significant decrease in the steady-state levels of IE2-86. Likewise, the steady-state level of the essential tegument protein UL32 (pp150) was reduced. The levels of pp150 and IE2-86 mRNA were not greatly affected by treatment with Roscovitine and thus did not correlate with the reduced levels of protein. In contrast, the expression of the tegument protein ppUL69 was higher in drug-treated samples, and the protein accumulated in a hyperphosphorylated form. ppUL69 localized to intranuclear aggregates that did not overlap with viral replication centers in cells treated with Roscovitine. Taken together, these data indicate that cdk activity is required at multiple steps during HCMV infection, including the expression, modification, and localization of virus-encoded proteins.

scholarly journals Dysregulation of Cyclin E Gene Expression in Human Cytomegalovirus-Infected Cells Requires Viral Early Gene Expression and Is Associated with Changes in the Rb-Related Protein p130

2000 ◽  
Vol 74 (9) ◽  
pp. 4192-4206 ◽  
Author(s):  
Anita K. McElroy ◽  
Roopashree S. Dwarakanath ◽  
Deborah H. Spector
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Human Cytomegalovirus ◽  
Cyclin E ◽  
Early Gene ◽  
Immediate Early ◽  
Mrna Levels ◽  
Early Gene Expression ◽  
Cell Extracts ◽  
Infected Cells

ABSTRACT We have previously shown that many cell cycle regulatory gene products are markedly affected by infection of primary fibroblasts with human cytomegalovirus (HCMV) (F. M. Jault, J. M. Jault, F. Ruchti, E. A. Fortunato, C. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697–6704, 1995). One of these proteins, cyclin E, is a key determinant of cell cycle progression during G1, and its mRNA levels are significantly increased in HCMV-infected fibroblasts (B. S. Salvant, E. A. Fortunato, and D. H. Spector, J. Virol. 72:3729–3741, 1998). To determine the molecular basis of this effect, we have examined the events that occur at the endogenous cyclin E promoter during the course of infection. In vivo dimethyl sulfate footprinting of the cyclin E promoter revealed several regions of protection and hypersensitivity that were unique to infected cells. In accord with this observation, we find that the virus-induced cyclin E transcripts initiate downstream of the start site identified in mock-infected cells, in regions where these newly appearing protected and hypersensitive sites occur. Viral gene expression is required for this induction. However, the viral immediate-early proteins IE1-72 and IE2-86, either alone or in combination, cannot induce expression of the endogenous cyclin E. The virus must progress past the immediate-early phase and express an early gene product(s) for activation of cyclin E expression. Moreover, IE1-72 does not appear to be required, as infection of cells with an HCMV mutant containing a deletion in the IE1-72 gene leads to full upregulation of cyclin E expression. Using electrophoretic mobility shift assays with infected cell extracts and a region of the cyclin E promoter that includes two previously defined E2F sites as the probe, we detected the appearance of an infection-specific banding pattern. One of the infection-specific bands contained the proteins E2F-4, DP-1, and p130, which were maintained in the infected cells as uniquely phosphorylated species. These results suggest that an altered E2F-4–DP-1–p130 complex along with viral early gene expression may play a role in the transcriptional regulation of cyclin E mRNA during HCMV infection.

scholarly journals Interaction between the Human Cytomegalovirus Tegument Proteins UL94 and UL99 Is Essential for Virus Replication

2012 ◽  
Vol 86 (18) ◽  
pp. 9995-10005 ◽  
Author(s):  
Stacia L. Phillips ◽  
Daniel Cygnar ◽  
Alexandra Thomas ◽  
Wade A. Bresnahan
Keyword(s):  
Amino Acids ◽  
Human Cytomegalovirus ◽  
Infectious Virus ◽  
Viral Proteins ◽  
Tegument Protein ◽  
Dependent Manner ◽  
Tegument Proteins ◽  
Cytoplasmic Structure ◽  
Infected Cells ◽  
Secretory Apparatus

Human cytomegalovirus (HCMV) virions are structurally complex, and the mechanisms by which they are assembled are poorly understood, especially with respect to the cytoplasmic phase of assembly, during which the majority of the tegument is acquired and final envelopment occurs. These processes occur at a unique cytoplasmic structure called the assembly complex, which is formed through a reorganization of the cellular secretory apparatus. The HCMV tegument protein UL99 (pp28) is essential for viral replication at the stage of secondary envelopment. We previously demonstrated that UL99 interacts with the essential tegument protein UL94 in infected cells as well as in the absence of other viral proteins. Here we show that UL94 and UL99 alter each other's localization and that UL99 stabilizes UL94 in a binding-dependent manner. We have mapped the interaction between UL94 and UL99 to identify the amino acids of each protein that are required for their interaction. Mutation of these amino acids in the context of the viral genome demonstrates that HCMV is completely defective for replication in the absence of the interaction between UL94 and UL99. Further, we demonstrate that in the absence of their interaction, both UL94 and UL99 exhibit aberrant localization and do not accumulate at the assembly complex during infection. Taken together, our data suggest that the interaction between UL94 and UL99 is essential for the proper localization of each protein to the assembly complex and thus for the production of infectious virus.

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 The UL48 Tegument Protein of Pseudorabies Virus Is Critical for Intracytoplasmic Assembly of Infectious Virions

2002 ◽  
Vol 76 (13) ◽  
pp. 6729-6742 ◽  
Author(s):  
Walter Fuchs ◽  
Harald Granzow ◽  
Barbara G. Klupp ◽  
Martina Kopp ◽  
Thomas C. Mettenleiter
Keyword(s):  
Pseudorabies Virus ◽  
Early Gene ◽  
Tegument Protein ◽  
Western Blots ◽  
Tegument Proteins ◽  
Extracellular Virus ◽  
Virion Morphogenesis ◽  
Infected Cells ◽  
Virion Formation ◽  
Hsv 1

ABSTRACT The pseudorabies virus (PrV) homolog of the tegument protein encoded by the UL48 gene of herpes simplex virus type 1 (HSV-1) was identified by using a monospecific rabbit antiserum against a bacterial fusion protein. UL48-related polypeptides of 53, 55, and 57 kDa were detected in Western blots of infected cells and purified virions. Immunofluorescence studies demonstrated that the PrV UL48 protein is predominantly localized in the cytoplasm but is also found in the nuclei of infected cells. Moreover, it is a constituent of extracellular virus particles but is absent from primary enveloped perinuclear virions. In noncomplementing cells, a UL48-negative PrV mutant (PrV-ΔUL48) exhibited delayed growth and significantly reduced plaque sizes and virus titers, deficiencies which were corrected in UL48-expressing cells. RNA analyses indicated that, like its HSV-1 homolog, the PrV UL48 protein is involved in regulation of immediate-early gene expression. However, the most salient effect of the UL48 gene deletion was a severe defect in virion morphogenesis. Late after infection, electron microscopy of cells infected with PrV-ΔUL48 revealed retention of newly formed nucleocapsids in the cytoplasm, whereas enveloped intracytoplasmic or extracellular complete virions were only rarely observed. In contrast, capsidless particles were produced and released in great amounts. Remarkably, the intracytoplasmic capsids were labeled with antibodies against the UL36 and UL37 tegument proteins, whereas the capsidless particles were labeled with antisera directed against the UL46, UL47, and UL49 tegument proteins. These findings suggested that the UL48 protein is involved in linking capsid and future envelope-associated tegument proteins during virion formation. Thus, like its HSV-1 homolog, the UL48 protein of PrV functions in at least two different steps of the viral life cycle. The drastic inhibition of virion formation in the absence of the PrV UL48 protein indicates that it plays an important role in virion morphogenesis prior to secondary envelopment of intracytoplasmic nucleocapsids. However, the UL48 gene of PrV is not absolutely essential, and concomitant deletion of the adjacent tegument protein gene UL49 also did not abolish virus replication in cell culture.

scholarly journals Human Cytomegalovirus pUL47 Modulates Tegumentation and Capsid Accumulation at the Viral Assembly Complex

2015 ◽  
Vol 89 (14) ◽  
pp. 7314-7328 ◽  
Author(s):  
Ilaria Cappadona ◽  
Clarissa Villinger ◽  
Gabi Schutzius ◽  
Thomas Mertens ◽  
Jens von Einem
Keyword(s):  
Human Cytomegalovirus ◽  
Nuclear Localization ◽  
Protein Interactions ◽  
Viral Assembly ◽  
Tegument Protein ◽  
Cell Periphery ◽  
Morphological Alterations ◽  
Tegument Proteins ◽  
Enveloped Virus ◽  
Infected Cells

ABSTRACTHuman cytomegalovirus (HCMV) tegument protein pUL47 is an interaction partner of pUL48 and highly conserved among herpesviruses. It is closely associated with the capsid and has an important function early in infection. Here, we report a specific role of pUL47 in the tegumentation of capsids in the cytoplasm. A newly generated mutant virus (TB-47stop), in which expression of pUL47 is blocked, exhibited a severe impairment in cell-to-cell spread and release of infectivity from infected cells. Ultrastructural analysis of TB-47stop-infected cells clearly showed cytoplasmic accumulations of nonenveloped capsids that were only partially tegumented, indicating that these capsids failed to complete tegumentation. Nevertheless, these accumulations were positive for HCMV inner tegument proteins pp150 and pUL48, suggesting that their attachment to capsids occurs independently of pUL47. Despite these morphological alterations, fully enveloped virus particles were found in the extracellular space and at the viral assembly complex (vAC) of TB-47stop-infected cells, indicating that pUL47 is not essential for the generation of virions. We confirmed findings that incorporation of pUL48 into virions is impaired in the absence of pUL47. Interestingly, pUL47 exhibited a strong nuclear localization in transfected cells, whereas it was found exclusively at the vAC in the context of virus infection. Colocalization of pUL47 and pUL48 at the vAC is consistent with their interaction. We also found a shift to a more nuclear localization of pUL47 when the expression of pUL48 was reduced. Summarizing our results, we hypothesize that pUL48 directs pUL47 to the vAC to promote tegumentation and secondary envelopment of capsids.IMPORTANCEGeneration of infectious HCMV particles requires an organized and multistep process involving the action of several viral and cellular proteins as well as protein-protein interactions. A better understanding of these processes is important for understanding the biology of HCMV and may help to identify targets for antiviral intervention. Here, we identified tegument protein pUL47 to function in tegumentation and proper trafficking of capsids during late phases of infection. Although pUL47 is not essential for the generation and release of infectious virions, its absence led to massive accumulations of partially tegumented capsids at the cell periphery. Detection of pUL48 at these accumulations indicated a pUL47-independent attachment of pUL48 to the capsid. On the other hand, localization of pUL47 to the vAC during infection appeared to be dependent on tegument protein pUL48, which suggests an intricate interplay of these proteins for normal generation of infectious virus progeny.

scholarly journals Inactivating a Cellular Intrinsic Immune Defense Mediated by Daxx Is the Mechanism through Which the Human Cytomegalovirus pp71 Protein Stimulates Viral Immediate-Early Gene Expression

2006 ◽  
Vol 80 (8) ◽  
pp. 3863-3871 ◽  
Author(s):  
Ryan T. Saffert ◽  
Robert F. Kalejta
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Immediate Early Gene ◽  
Nuclear Body ◽  
Immune Defense ◽  
Early Gene ◽  
Immediate Early ◽  
Innate Immune Responses ◽  
Early Gene Expression ◽  
Infected Cells

ABSTRACT Human cytomegalovirus (HCMV) masterfully evades adaptive and innate immune responses, allowing infection to be maintained and periodically reactivated for the life of the host. Here we show that cells also possess an intrinsic immune defense against HCMV that is disarmed by the virus. In HCMV-infected cells, the promyelocytic leukemia nuclear body (PML-NB) protein Daxx silences viral immediate-early gene expression through the action of a histone deacetylase. However, this antiviral tactic is efficiently neutralized by the viral pp71 protein, which is incorporated into virions, delivered to cells upon infection, and mediates the proteasomal degradation of Daxx. This work demonstrates the mechanism through which pp71 activates viral immediate-early gene expression in HCMV-infected cells. Furthermore, it provides insight into how a PML-NB protein institutes an intrinsic immune defense against a DNA virus and how HCMV pp71 inactivates this defense.

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