Deletion of AcMNPV AC16 and AC17 results in delayed viral gene expression in budded virus infected cells but not transfected cells

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.

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

Journal of Virology ◽

10.1128/jvi.80.3.1376-1384.2006 ◽

2006 ◽

Vol 80 (3) ◽

pp. 1376-1384 ◽

Cited By ~ 108

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.

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The Immune Evasion Paradox: Immunoevasins of Murine Cytomegalovirus Enhance Priming of CD8 T Cells by Preventing Negative Feedback Regulation

Journal of Virology ◽

10.1128/jvi.01510-08 ◽

2008 ◽

Vol 82 (23) ◽

pp. 11637-11650 ◽

Cited By ~ 50

Author(s):

Verena Böhm ◽

Christian O. Simon ◽

Jürgen Podlech ◽

Christof K. Seckert ◽

Dorothea Gendig ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

Negative Feedback ◽

Cd8 T Cells ◽

Viral Gene Expression ◽

Cd8 T Cell ◽

Murine Cytomegalovirus ◽

Viral Gene ◽

Infected Cells

ABSTRACT Cytomegaloviruses express glycoproteins that interfere with antigen presentation to CD8 T cells. Although the molecular modes of action of these “immunoevasins” differ between cytomegalovirus species, the convergent biological outcome is an inhibition of the recognition of infected cells. In murine cytomegalovirus, m152/gp40 retains peptide-loaded major histocompatibility complex class I molecules in a cis-Golgi compartment, m06/gp48 mediates their vesicular sorting for lysosomal degradation, and m04/gp34, although not an immunoevasin in its own right, appears to assist in the concerted action of all three molecules. Using the Ld-restricted IE1 epitope YPHFMPTNL in the BALB/c mouse model as a paradigm, we provide here an explanation for the paradox that immunoevasins enhance CD8 T-cell priming although they inhibit peptide presentation in infected cells. Adaptive immune responses are initiated in the regional lymph node (RLN) draining the site of pathogen exposure. In particular for antigens that are not virion components, the magnitude of viral gene expression providing the antigens is likely a critical parameter in priming efficacy. We have therefore focused on the events in the RLN and have related priming to intranodal viral gene expression. We show that immunoevasins enhance priming by downmodulating an early CD8 T-cell-mediated “negative feedback” control of the infection in the cortical region of the RLN, thus supporting the model that immunoevasins improve antigen supply for indirect priming by uninfected antigen-presenting cells. As an important consequence, these findings predict that deletion of immunoevasin genes in a replicative vaccine virus is not a favorable option but may, rather, be counterproductive.

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Cell Cycle-Independent Expression of Immediate-Early Gene 3 Results in G1 and G2 Arrest in Murine Cytomegalovirus-Infected Cells

Journal of Virology ◽

10.1128/jvi.01212-08 ◽

2008 ◽

Vol 82 (20) ◽

pp. 10188-10198 ◽

Cited By ~ 27

Author(s):

Lüder Wiebusch ◽

Anke Neuwirth ◽

Linus Grabenhenrich ◽

Sebastian Voigt ◽

Christian Hagemeier

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Cell Cycle Arrest ◽

Viral Gene Expression ◽

Immediate Early Gene ◽

Early Gene ◽

Immediate Early ◽

Viral Gene ◽

Cycle Arrest ◽

Infected Cells

ABSTRACT The infectious cycle of human cytomegalovirus (HCMV) is intricately linked to the host's cell cycle. Viral gene expression can be initiated only in G0/G1 phase. Once expressed, the immediate-early gene product IE2 prevents cellular DNA synthesis, arresting infected cells with a G1 DNA content. This function is required for efficient viral replication in vitro. A prerequisite for addressing its in vivo relevance is the characterization of cell cycle-regulatory activities of CMV species for which animal models have been established. Here, we show that murine CMV (MCMV), like HCMV, has a strong antiproliferative capacity and arrests cells in G1. Unexpectedly, and in contrast to HCMV, MCMV can also block cells that have passed through S phase by arresting them in G2. Moreover, MCMV can also replicate in G2 cells. This is made possible by the cell cycle-independent expression of MCMV immediate-early genes. Transfection experiments show that of several MCMV candidate genes, only immediate-early gene 3 (ie3), the homologue of HCMV IE2, exhibits cell cycle arrest activity. Accordingly, an MCMV ie3 deletion mutant has lost the ability to arrest cells in either G1 or G2. Thus, despite interspecies variations in the cell cycle dependence of viral gene expression, the central theme of HCMV IE2-induced cell cycle arrest is conserved in the murine counterpart, raising the possibility of studying its physiological relevance at the level of the whole organism.

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Cellular immune response to adenoviral vector infected cells does not require de novo viral gene expression: Implications for gene therapy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.95.19.11377 ◽

1998 ◽

Vol 95 (19) ◽

pp. 11377-11382 ◽

Cited By ~ 188

Author(s):

T. Kafri ◽

D. Morgan ◽

T. Krahl ◽

N. Sarvetnick ◽

L. Sherman ◽

...

Keyword(s):

Gene Expression ◽

Gene Therapy ◽

Immune Response ◽

Cellular Immune Response ◽

Adenoviral Vector ◽

De Novo ◽

Viral Gene Expression ◽

Viral Gene ◽

Infected Cells ◽

Cellular Immune

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

Journal of Virology ◽

10.1128/jvi.00500-10 ◽

2010 ◽

Vol 84 (19) ◽

pp. 9853-9863 ◽

Cited By ~ 21

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.

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Epigenomic characterization of latent HIV infection identifies latency regulating transcription factors

PLoS Pathogens ◽

10.1371/journal.ppat.1009346 ◽

2021 ◽

Vol 17 (2) ◽

pp. e1009346

Author(s):

Stuart R. Jefferys ◽

Samuel D. Burgos ◽

Jackson J. Peterson ◽

Sara R. Selitsky ◽

Anne-Marie W. Turner ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

Transcription Factors ◽

Hiv Infection ◽

Cd4 T Cells ◽

Viral Gene Expression ◽

Viral Gene ◽

Hiv Latency ◽

Latently Infected ◽

Infected Cells

Transcriptional silencing of HIV in CD4 T cells generates a reservoir of latently infected cells that can reseed infection after interruption of therapy. As such, these cells represent the principal barrier to curing HIV infection, but little is known about their characteristics. To further our understanding of the molecular mechanisms of latency, we characterized a primary cell model of HIV latency in which infected cells adopt heterogeneous transcriptional fates. In this model, we observed that latency is a stable, heritable state that is transmitted through cell division. Using Assay of Transposon-Accessible Chromatin sequencing (ATACseq) we found that latently infected cells exhibit greatly reduced proviral accessibility, indicating the presence of chromatin-based structural barriers to viral gene expression. By quantifying the activity of host cell transcription factors, we observe elevated activity of Forkhead and Kruppel-like factor transcription factors (TFs), and reduced activity of AP-1, RUNX and GATA TFs in latently infected cells. Interestingly, latency reversing agents with different mechanisms of action caused distinct patterns of chromatin reopening across the provirus. We observe that binding sites for the chromatin insulator CTCF are highly enriched in the differentially open chromatin of infected CD4 T cells. Furthermore, depletion of CTCF inhibited HIV latency, identifying this factor as playing a key role in the initiation or enforcement of latency. These data indicate that HIV latency develops preferentially in cells with a distinct pattern of TF activity that promotes a closed proviral structure and inhibits viral gene expression. Furthermore, these findings identify CTCF as a novel regulator of HIV latency.

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HSV-1 single cell analysis reveals anti-viral and developmental programs activation in distinct sub-populations

10.1101/566489 ◽

2019 ◽

Author(s):

Nir Drayman ◽

Parthiv Patel ◽

Luke Vistain ◽

Savaş Tay

Keyword(s):

Gene Expression ◽

Single Cell ◽

Single Cell Analysis ◽

Viral Gene Expression ◽

Population Level ◽

Cellular Reprogramming ◽

Viral Gene ◽

Beta Catenin ◽

Infected Cells ◽

Hsv 1

ABSTRACTViral infection is usually studied at the population level by averaging over millions of cells. However, infection at the single-cell level is highly heterogeneous. Here, we combine live-cell imaging and single-cell RNA sequencing to characterize viral and host transcriptional heterogeneity during HSV-1 infection of primary human cells. We find extreme variability in the level of viral gene expression among individually infected cells and show that they cluster into transcriptionally distinct sub-populations. We find that anti-viral signaling is initiated in a rare group of abortively infected cells, while highly infected cells undergo cellular reprogramming to an embryonic-like transcriptional state. This reprogramming involves the recruitment of beta-catenin to the host nucleus and viral replication compartments and is required for late viral gene expression and progeny production. These findings uncover the transcriptional differences in cells with variable infection outcomes and shed new light on the manipulation of host pathways by HSV-1.

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

Journal of Virology ◽

10.1128/jvi.01937-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 195-207 ◽

Cited By ~ 18

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.

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