scholarly journals Nuclear Localization of Tegument-Delivered pp71 in Human Cytomegalovirus-Infected Cells Is Facilitated by One or More Factors Present in Terminally Differentiated Fibroblasts

2010 ◽  
Vol 84 (19) ◽  
pp. 9853-9863 ◽  
Author(s):  
Rhiannon R. Penkert ◽  
Robert F. Kalejta
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Viral Entry ◽  
Viral Gene Expression ◽  
Cell Types ◽  
Lytic Replication ◽  
Viral Gene ◽  
Differentiated Cells ◽  
Virion Envelope ◽  
Infected Cells

ABSTRACT Herpesviral virions contain a tegument layer that consists primarily of viral proteins. The delivery of fully functional proteins to infected cells upon virion envelope fusion to the plasma membrane allows herpesviruses to modulate cellular activities prior to viral gene expression. Certain tegument proteins can also regulate viral processes. For example, the pp71 tegument protein encoded by the UL82 gene of human cytomegalovirus (HCMV) stimulates viral immediate early (IE) gene expression and thus acts to initiate the productive lytic infectious cycle. In terminally differentiated fibroblasts infected with HCMV, tegument-delivered pp71 traffics to the nucleus and degrades the cellular transcriptional corepressor Daxx to initiate viral IE gene expression and lytic replication. However, when HCMV infects incompletely differentiated cells, tegument-delivered pp71 remains in the cytoplasm, allowing the nucleus-localized Daxx protein to silence viral IE gene expression and promote the establishment of a latent infection in certain cell types. We sought to determine whether undifferentiated cells block the trafficking of tegument-delivered pp71 to the nucleus or whether differentiated cells facilitate the nuclear transport of tegument-delivered pp71. Heterogenous cell fusion experiments demonstrated that tegument-delivered pp71 found in the cytoplasm of undifferentiated NT2 cells could be driven into the nucleus by one or more factors provided by fully differentiated fibroblasts. Our data raise the intriguing possibility that latency is the default program launched by HCMV upon viral entry into cells and that lytic infection is initiated only in certain (differentiated) cells that can facilitate the delivery of incoming pp71 to the nucleus.

scholarly journals Human cytomegalovirus transcriptome activity differs during replication in human fibroblast, epithelial and astrocyte cell lines

2012 ◽  
Vol 93 (5) ◽  
pp. 1046-1058 ◽  
Author(s):  
James C. Towler ◽  
Bahram Ebrahimi ◽  
Brian Lane ◽  
Andrew J. Davison ◽  
Derrick J. Dargan
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Growth Kinetics ◽  
Viral Gene Expression ◽  
Cell Types ◽  
Viral Gene ◽  
Genome Replication ◽  
Cell Type ◽  
Low Passage ◽  
Temporal Expression Pattern

Broad cell tropism contributes to the pathogenesis of human cytomegalovirus (HCMV), but the extent to which cell type influences HCMV gene expression is unclear. A bespoke HCMV DNA microarray was used to monitor the transcriptome activity of the low passage Merlin strain of HCMV at 12, 24, 48 and 72 h post-infection, during a single round of replication in human fetal foreskin fibroblast cells (HFFF-2s), human retinal pigmented epithelial cells (RPE-1s) and human astrocytoma cells (U373MGs). In order to correlate transcriptome activity with concurrent biological responses, viral cytopathic effect, growth kinetics and genomic loads were examined in the three cell types. The temporal expression pattern of viral genes was broadly similar in HFFF-2s and RPE-1s, but dramatically different in U373MGs. Of the 165 known HCMV protein-coding genes, 41 and 48 were differentially regulated in RPE-1s and U373MGs, respectively, compared with HFFF-2s, and 22 of these were differentially regulated in both RPE-1s and U373MGs. In RPE-1s, all differentially regulated genes were downregulated, but, in U373MGs, some were down- and others upregulated. Differentially regulated genes were identified among the immediate-early, early, early late and true-late viral gene classes. Grouping of downregulated genes according to function at landmark stages of the replication cycle led to the identification of potential bottleneck stages (genome replication, virion assembly, and virion maturation and release) that may account for cell type-dependent viral growth kinetics. The possibility that cell type-specific differences in expressed cellular factors are responsible for modulation of viral gene expression is discussed.

scholarly journals General blockade of human cytomegalovirus immediate-early mRNA expression in the S/G2 phase by a nuclear, Daxx- and PML-independent mechanism

2011 ◽  
Vol 92 (12) ◽  
pp. 2757-2769 ◽  
Author(s):  
Martin Zydek ◽  
Ralf Uecker ◽  
Nina Tavalai ◽  
Thomas Stamminger ◽  
Christian Hagemeier ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Human Cytomegalovirus ◽  
Viral Entry ◽  
Nuclear Genome ◽  
Viral Gene Expression ◽  
Lytic Replication ◽  
Immediate Early ◽  
Viral Gene ◽  
Level Control ◽  
G2 Phase

The onset of human cytomegalovirus (HCMV) lytic replication is strictly controlled by the host cell division cycle. Although viral entry of S/G2-phase cells is unperturbed expression of major immediate-early (MIE) genes IE1 and IE2 is tightly blocked in these cells. Besides the finding that cyclin-dependent kinase (CDK) activity is required for IE1/IE2 repression little is known about the nature of this cell cycle-dependent block. Here, we show that the block occurs after nuclear entry of viral DNA and prevents the accumulation of IE1/IE2 mRNAs, suggesting an inhibition of transcription. Remarkably, the presence of cis-regulatory regions of the MIE locus is neither sufficient nor necessary for IE1/IE2 repression in the S/G2 phase. Furthermore, the block of viral mRNA expression also affects other immediate-early transcribed regions, i.e. the US3 and UL36–38 gene loci. This suggests a mechanism of repression that acts in a general and not a gene-specific fashion. Such a nuclear, genome-wide repression of HCMV is typically mediated by the intrinsic immune defence at nuclear domain 10 (ND10) structures. However, we found that neither Daxx nor PML, the main players of ND10-based immunity, are required for the block to viral gene expression in the S/G2 phase. In addition, the viral tegument protein pp71 (pUL82), a major antagonist of the intrinsic immunity at pre-immediate-early times of infection, proved to be functional in S-phase cells. This suggests the existence of a yet undiscovered, CDK-dependent mechanism exerting higher-level control over immediate-early mRNA expression in HCMV-infected cells.

scholarly journals Activation of p90 Ribosomal S6 Kinases by ORF45 of Kaposi's Sarcoma-Associated Herpesvirus Is Critical for Optimal Production of Infectious Viruses

2014 ◽  
Vol 89 (1) ◽  
pp. 195-207 ◽  
Author(s):  
Bishi Fu ◽  
Ersheng Kuang ◽  
Wenwei Li ◽  
Denis Avey ◽  
Xiaojuan Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacterial Artificial Chromosome ◽  
Critical Role ◽  
Viral Gene Expression ◽  
Artificial Chromosome ◽  
Lytic Replication ◽  
Viral Gene ◽  
Wild Type ◽  
Lytic Reactivation ◽  
Infected Cells

ABSTRACTWe have previously shown that ORF45, an immediate-early and tegument protein of Kaposi's sarcoma-associated herpesvirus (KSHV), causes sustained activation of p90 ribosomal S6 kinases (RSKs) and extracellular regulated kinase (ERK) (E. Kuang, Q. Tang, G. G. Maul, and F. Zhu, J Virol 82:1838–1850, 2008,http://dx.doi.org/10.1128/JVI.02119-07). We now have identified the critical region of ORF45 that is involved in RSK interaction and activation. Alanine scanning mutagenesis of this region revealed that a single F66A point mutation abolished binding of ORF45 to RSK or ERK and, consequently, its ability to activate the kinases. We introduced the F66A mutation into BAC16 (a bacterial artificial chromosome clone containing the entire infectious KSHV genome), producing BAC16-45F66A. In parallel, we also repaired the mutation and obtained a revertant, BAC16-45A66F. The reconstitution of these mutants in iSLK cells demonstrated that the ORF45-F66A mutant failed to cause sustained ERK and RSK activation during lytic reactivation, resulting in dramatic differences in the phosphoproteomic profile between the wild-type virus-infected cells and the mutant virus-infected cells. ORF45 mutation or deletion also was accompanied by a noticeable decreased in viral gene expression during lytic reactivation. Consequently, the ORF45-F66A mutant produced significantly fewer infectious progeny virions than the wild type or the revertant. These results suggest a critical role for ORF45-mediated RSK activation in KSHV lytic replication.IMPORTANCEKSHV is the causative agent of three human malignancies. KSHV pathogenesis is intimately linked to its ability to modulate the host cell microenvironment and to facilitate efficient production of progeny viral particles. We previously described the mechanism by which the KSHV lytic protein ORF45 activates the cellular kinases ERK and RSK. We now have mapped the critical region of ORF45 responsible for binding and activation of ERK/RSK to a single residue, F66. We mutated this amino acid of ORF45 (F66A) and introduced the mutation into a newly developed bacterial artificial chromosome containing the KSHV genome (BAC16). This system has provided us with a useful tool to characterize the functions of ORF45-activated RSK upon KSHV lytic reactivation. We show that viral gene expression and virion production are significantly reduced by F66A mutation, indicating a critical role for ORF45-activated RSK during KSHV lytic replication.

scholarly journals HCMV miRNA Targets Reveal Important Cellular Pathways for Viral Replication, Latency, and Reactivation

2018 ◽  
Vol 4 (4) ◽  
pp. 29 ◽  
Author(s):  
Nicole Diggins ◽  
Meaghan Hancock
Keyword(s):  
Gene Expression ◽  
Latent Infection ◽  
Current Knowledge ◽  
Critical Role ◽  
Viral Gene Expression ◽  
Lytic Replication ◽  
Progeny Virus ◽  
Viral Gene ◽  
Cellular Targets ◽  
Differentiated Cells

It is now well appreciated that microRNAs (miRNAs) play a critical role in the lifecycles of many herpes viruses. The human cytomegalovirus (HCMV) replication cycle varies significantly depending on the cell type infected, with lytic replication occurring in fully-differentiated cells such as fibroblasts, endothelial cells, or macrophages, and latent infection occurring in less-differentiated CD14+ monocytes and CD34+ hematopoietic progenitor cells where viral gene expression is severely diminished and progeny virus is not produced. Given their non-immunogenic nature and their capacity to target numerous cellular and viral transcripts, miRNAs represent a particularly advantageous means for HCMV to manipulate viral gene expression and cellular signaling pathways during lytic and latent infection. This review will focus on our current knowledge of HCMV miRNA viral and cellular targets, and discuss their importance in lytic and latent infection, highlight the challenges of studying HCMV miRNAs, and describe how viral miRNAs can help us to better understand the cellular processes involved in HCMV latency.

scholarly journals Human Cytomegalovirus Subverts the Functions of Monocytes, Impairing Chemokine-Mediated Migration and Leukocyte Recruitment

2006 ◽  
Vol 80 (15) ◽  
pp. 7578-7589 ◽  
Author(s):  
Giada Frascaroli ◽  
Stefania Varani ◽  
Barbara Moepps ◽  
Christian Sinzger ◽  
Maria Paola Landini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Chemokine Receptors ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Host Immune System ◽  
Infected Cells ◽  
And Function ◽  
A Site

ABSTRACT Despite their role in innate and adaptive immunity, during human cytomegalovirus (HCMV) infection, monocytes are considered to be an important target of infection, a site of latency, and vehicles for virus dissemination. Since chemokine receptors play crucial roles in monocyte activation and trafficking, we investigated the effects of HCMV on their expression and function. By using endotheliotropic strains of HCMV, we obtained high rates (roughly 50%) of in vitro-infected monocytes but only restricted viral gene expression. At 24 h after infection, while the chemokine receptors CX3CR and CCR7 were unaffected, CCR1, CCR2, CCR5, and CXCR4 were downmodulated on the cell surface and retained intracellularly. Structural components of the viral particles, but not viral gene expression or soluble factors released from infected cells, accounted for the changed localization of the receptor molecules and for the block of chemokine-driven migration. HCMV-infected monocytes indeed became unresponsive to inflammatory and homeostatic chemokines, although the basal cell motility and responsiveness to N-formyl-Met-Leu-Phe were unaffected or slightly increased. The production of inflammatory mediators responsible for the recruitment of other immune cells was also hampered by HCMV. Whereas endothelial and fibroblast cells infected by HCMV efficiently recruited leukocytes, infected monocytes were unable to recruit lymphocytes, monocytes, and neutrophils. Our data further highlight the complex level of interference exerted by HCMV on the host immune system.

Protein-RNA interactome analysis reveals wide association of KSHV ORF57 with host non-coding RNAs and polysomes

2021 ◽  
Author(s):  
Beatriz Alvarado-Hernandez ◽  
Yanping Ma ◽  
Nishi R. Sharma ◽  
Vladimir Majerciak ◽  
Alexei Lobanov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Splicing ◽  
Rna Binding ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
28S Rrna ◽  
Protein Coding ◽  
Infected Cells ◽  
Non Coding Rnas ◽  
Rna Interaction

Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF57 is an RNA-binding post-transcriptional regulator. We recently applied an affinity-purified anti-ORF57 antibody to conduct ORF57-CLIP (Cross-linking Immunoprecipitation) in combination with RNA-sequencing (CLIP-seq) and analyzed the genome-wide host RNA transcripts in association with ORF57 in BCBL-1 cells with lytic KSHV infection. Mapping of the CLIPed RNA reads to the human genome (GRCh37) revealed that most of the ORF57-associated RNA reads were from rRNAs. The remaining RNA reads mapped to several classes of host non-coding and protein-coding mRNAs. We found ORF57 binds and regulates expression of a subset of host lncRNAs, including LINC00324, LINC00355, and LINC00839 which are involved in cell growth. ORF57 binds snoRNAs responsible for 18S and 28S rRNA modifications, but does not interact with fibrillarin and NOP58. We validated ORF57 interactions with 67 snoRNAs by ORF57-RNA immunoprecipitation (RIP)-snoRNA-array assays. Most of the identified ORF57 rRNA binding sites (BS) overlap with the sites binding snoRNAs. We confirmed ORF57-snoRA71B RNA interaction in BCBL-1 cells by ORF57-RIP and Northern blot analyses using a 32 P-labeled oligo probe from the 18S rRNA region complementary to snoRA71B. Using RNA oligos from the rRNA regions that ORF57 binds for oligo pulldown-Western blot assays, we selectively verified ORF57 interactions with 5.8S and 18S rRNAs. Polysome profiling revealed that ORF57 associates with both monosomes and polysomes and its association with polysomes increases PABPC1 binding to, but prevent Ago2 from polysomes. Our data indicate a functional correlation with ORF57 binding and suppression of Ago2 activities for ORF57 promotion of gene expression. Significance As an RNA-binding protein, KSHV ORF57 regulates RNA splicing, stability, and translation and inhibits host innate immunity by blocking the formation of RNA granules in virus infected cells. In this report, ORF57 was found to interact many host non-coding RNAs, including lncRNAs, snoRNAs and ribosomal RNAs to carry out additional unknown functions. ORF57 binds a group of lncRNAs via the identified RNA motifs by ORF57 CLIP-seq to regulate their expression. ORF57 associates with snoRNAs independently of fibrillarin and NOP58 proteins, and with ribosomal RNA in the regions that commonly bind snoRNAs. Knockdown of fibrillarin expression decreases the expression of snoRNAs and CDK4, but not affect viral gene expression. More importantly, we found that ORF57 binds translationally active polysomes and enhances PABPC-1 but prevents Ago2 association with polysomes. Data provide a compelling evidence on how ORF57 in KSHV infected cells might regulate protein synthesis by blocking Ago2’s hostile activities on translation.

scholarly journals Adenovirus Virus-Associated RNA Is Processed to Functional Interfering RNAs Involved in Virus Production

2006 ◽  
Vol 80 (3) ◽  
pp. 1376-1384 ◽  
Author(s):  
Oscar Aparicio ◽  
Nerea Razquin ◽  
Mikel Zaratiegui ◽  
Iñigo Narvaiza ◽  
Puri Fortes
Keyword(s):  
Gene Expression ◽  
Virus Production ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Small Interfering Rnas ◽  
Control Of Gene Expression ◽  
Posttranscriptional Gene Silencing ◽  
Argonaute 2 ◽  
Complementary Sequences ◽  
Infected Cells

ABSTRACT Posttranscriptional gene silencing allows sequence-specific control of gene expression. Specificity is guaranteed by small antisense RNAs such as microRNAs (miRNAs) or small interfering RNAs (siRNAs). Functional miRNAs derive from longer double-stranded RNA (dsRNA) molecules that are cleaved to pre-miRNAs in the nucleus and are transported by exportin 5 (Exp 5) to the cytoplasm. Adenovirus-infected cells express virus-associated (VA) RNAs, which are dsRNA molecules similar in structure to pre-miRNAs. VA RNAs are also transported by Exp 5 to the cytoplasm, where they accumulate. Here we show that small RNAs derived from VA RNAs (svaRNAs), similar to miRNAs, can be found in adenovirus-infected cells. VA RNA processing to svaRNAs requires neither viral replication nor viral protein expression, as evidenced by the fact that svaRNA accumulation can be detected in cells transfected with VA sequences. svaRNAs are efficiently bound by Argonaute 2, the endonuclease of the RNA-induced silencing complex, and behave as functional siRNAs, in that they inhibit the expression of reporter genes with complementary sequences. Blocking svaRNA-mediated inhibition affects efficient adenovirus production, indicating that svaRNAs are required for virus viability. Thus, svaRNA-mediated silencing could represent a novel mechanism used by adenoviruses to control cellular or viral gene expression.

scholarly journals Principal Role of TRAP/Mediator and SWI/SNF Complexes in Kaposi's Sarcoma-Associated Herpesvirus RTA-Mediated Lytic Reactivation

2003 ◽  
Vol 23 (6) ◽  
pp. 2055-2067 ◽  
Author(s):  
Yousang Gwack ◽  
Hwa Jin Baek ◽  
Hiroyuki Nakamura ◽  
Sun Hwa Lee ◽  
Michael Meisterernst ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kaposi's Sarcoma ◽  
Viral Gene Expression ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Viral Gene ◽  
Transcription Activator ◽  
Lytic Reactivation ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT An important step in the herpesvirus life cycle is the switch from latency to lytic reactivation. The RTA transcription activator of Kaposi's sarcoma-associated herpesvirus (KSHV) acts as a molecular switch for lytic reactivation. Here we demonstrate that KSHV RTA recruits CBP, the SWI/SNF chromatin remodeling complex, and the TRAP/Mediator coactivator into viral promoters through interactions with a short acidic sequence in the carboxyl region and that this recruitment is essential for RTA-dependent viral gene expression. The Brg1 subunit of SWI/SNF and the TRAP230 subunit of TRAP/Mediator were shown to interact directly with RTA. Consequently, genetic ablation of these interactions abolished KSHV lytic replication. These results demonstrate that the recruitment of CBP, SWI/SNF, and TRAP/Mediator complexes by RTA is the principal mechanism to direct well-controlled viral gene expression and thereby viral lytic reactivation.

scholarly journals Lytic infection of permissive cells with human cytomegalovirus is regulated by an intrinsic ‘pre-immediate-early’ repression of viral gene expression mediated by histone post-translational modification

2009 ◽  
Vol 90 (10) ◽  
pp. 2364-2374 ◽  
Author(s):  
Ian J. Groves ◽  
Matthew B. Reeves ◽  
John H. Sinclair
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Chromatin Structure ◽  
Viral Gene Expression ◽  
Lytic Infection ◽  
Immediate Early ◽  
Late Gene ◽  
Viral Gene ◽  
Gene Promoters ◽  
Late Gene Expression

Human cytomegalovirus (HCMV) lytic gene expression occurs in a regulated cascade, initiated by expression of the viral major immediate-early (IE) proteins. Transcribed from the major IE promoter (MIEP), the major IE genes regulate viral early and late gene expression. This study found that a substantial proportion of infecting viral genomes became associated with histones immediately upon infection of permissive fibroblasts at low m.o.i. and these histones bore markers of repressed chromatin. As infection progressed, however, the viral MIEP became associated with histone marks, which correlate with the known transcriptional activity of the MIEP at IE time points. Interestingly, this chromatin-mediated repression of the MIEP at ‘pre-IE’ times of infection could be overcome by inhibition of histone deacetylases, as well as by infection at high m.o.i., and resulted in a temporal advance of the infection cycle by inducing premature viral early and late gene expression and DNA replication. As well as the MIEP, and consistent with previous observations, the viral early and late promoters were also initially associated with repressive chromatin. However, changes in histone modifications around these promoters also occurred as infection progressed, and this correlated with the known temporal regulation of the viral early and late gene expression cascade. These data argue that the chromatin structure of all classes of viral genes are initially repressed on infection of permissive cells and that the chromatin structure of HCMV gene promoters plays an important role in regulating the time course of viral gene expression during lytic infection.

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