Genome Wide Nucleosome Mapping for HSV-1 Shows Nucleosomes Are Deposited at Preferred Positions during Lytic Infection

ABSTRACT The genomes of herpes simplex virus type 1 (HSV-1) are regularly chromatinized during latency such that their digestion with micrococcal nuclease (MCN) releases nucleosome-sized DNA fragments. In lytically infected cells, in contrast, MCN releases HSV-1 DNA in primarily heterogeneously sized fragments. Consistently, only a small percentage of this HSV-1 DNA coimmunoprecipitates with histones. Most current models propose that histones associate with HSV-1 DNA during lytic infections at low occupancy. However, histone modification or occupation is also proposed to regulate HSV-1 transcription. It remains unclear how the histones associated with a small percentage of HSV-1 DNA may regulate transcription globally. Moreover, the physical properties of the complexes containing histones and HSV-1 DNA are unknown. We evaluated the HSV-1 DNA-containing complexes at 5 h after (lytic) infection by biochemical fractionations. Nuclear HSV-1 DNA did not fractionate as protein-free HSV-1 DNA but as DNA in cellular nucleosomes. Moreover, MCN released HSV-1 DNA in complexes that fractionate as cellular mono- and dinucleosomes by centrifugation followed by sucrose gradients and size-exclusion chromatography. The HSV-1 DNA in such complexes was protected to heterogeneous sizes and was more accessible to MCN than DNA in most cellular chromatin. Using a modified MCN digestion to trap unstable digestion intermediates, HSV-1 DNA was quantitatively recovered in discrete mono- to polynucleosome sizes in complexes fractionating as cellular mono- to polynucleosomes. The HSV-1 DNAs in complexes fractionating as mono- to dinucleosomes were stabilized by cross-linking. Therefore, most HSV-1 DNA forms particularly unstable nucleosome-like complexes at 5 h of lytic infection.

Download Full-text

Herpes Simplex Virus Type 1 Genomes Are Associated with ND10 Nuclear Substructures in Quiescently Infected Human Fibroblasts

Journal of Virology ◽

10.1128/jvi.00705-07 ◽

2007 ◽

Vol 81 (20) ◽

pp. 10991-11004 ◽

Cited By ~ 84

Author(s):

Roger D. Everett ◽

Jill Murray ◽

Anne Orr ◽

Chris M. Preston

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Human Fibroblasts ◽

Lytic Infection ◽

Viral Genomes ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Herpes simplex virus type 1 (HSV-1) genomes become associated with structures related to cellular nuclear substructures known as ND10 or promyelocytic leukemia nuclear bodies during the early stages of lytic infection. This paper describes the relationship between HSV-1 genomes and ND10 in human fibroblasts that maintain the viral genomes in a quiescent state. We report that quiescent HSV-1 genomes detected by fluorescence in situ hybridization (FISH) are associated with enlarged ND10-like structures, frequently such that the FISH-defined viral foci are apparently enveloped within a sphere of PML and other ND10 proteins. The number of FISH viral foci in each quiescently infected cell is concordant with the input multiplicity of infection, with each structure containing no more than a small number of viral genomes. A proportion of the enlarged ND10-like foci in quiescently infected cells contain accumulations of the heterochromatin protein HP1 but not other common markers of heterochromatin such as histone H3 di- or trimethylated on lysine residue 9. Many of the virally induced enlarged ND10-like structures also contain concentrations of conjugated ubiquitin. Quiescent infections can be established in cells that are highly depleted for PML. However, during the initial stages of establishment of a quiescent infection in such cells, other ND10 proteins (Sp100, hDaxx, and ATRX) are recruited into virally induced foci that are likely to be associated with HSV-1 genomes. These observations illustrate that the intimate connections between HSV-1 genomes and ND10 that occur during lytic infection also extend to quiescent infections.

Download Full-text

Herpes Simplex Virus 1 Lytic Infection Blocks MicroRNA (miRNA) Biogenesis at the Stage of Nuclear Export of Pre-miRNAs

mBio ◽

10.1128/mbio.02856-18 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 6

Author(s):

Dongli Pan ◽

Gang Li ◽

Jenna Morris-Love ◽

Shuyuan Qi ◽

Lei Feng ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Nuclear Export ◽

Latent Infection ◽

Mature Mirnas ◽

Lytic Infection ◽

Mirna Biogenesis ◽

Herpes Simplex Virus 1 ◽

Simplex Virus ◽

Hsv 1

ABSTRACTHerpes simplex virus 1 (HSV-1) switches between two infection programs, productive (“lytic”) and latent infection. Some HSV-1 microRNAs (miRNAs) have been hypothesized to help control this switch, and yet little is known about regulation of their expression. Using Northern blot analyses, we found that, despite inherent differences in biogenesis efficiency among six HSV-1 miRNAs, all six exhibited high pre-miRNA/miRNA ratios during lytic infection of different cell lines and, when detectable, in acutely infected mouse trigeminal ganglia. In contrast, considerably lower ratios were observed in latently infected ganglia and in cells transduced with lentiviral vectors expressing the miRNAs, suggesting that HSV-1 lytic infection blocks miRNA biogenesis. This phenomenon is not specific to viral miRNAs, as a host miRNA expressed from recombinant HSV-1 also exhibited high pre-miRNA/miRNA ratios late during lytic infection. The levels of most of the mature miRNAs remained stable during infection in the presence of actinomycin D, indicating that the high ratios are due to inefficient pre-miRNA conversion to miRNA. Cellular fractionation experiments showed that late (but not early) during infection, pre-miRNAs were enriched in the nucleus and depleted in the cytoplasm, indicating that nuclear export was blocked. A mutation eliminating ICP27 expression or addition of acyclovir reduced pre-miRNA/miRNA ratios, but mutations drastically reducing Us11 expression did not. Thus, HSV-1 lytic infection inhibits miRNA biogenesis at the step of nuclear export and does so in an ICP27- and viral DNA synthesis-dependent manner. This mechanism may benefit the virus by reducing expression of repressive miRNAs during lytic infection while permitting elevated expression during latency.IMPORTANCEVarious mechanisms have been identified by which viruses target host small RNA biogenesis pathways to achieve optimal infection outcomes. Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen whose successful persistence in the host entails both productive (“lytic”) and latent infection. Although many HSV-1 miRNAs have been discovered and some are thought to help control the lytic/latent switch, little is known about regulation of their biogenesis. By characterizing expression of both pre-miRNAs and mature miRNAs under various conditions, this study revealed striking differences in miRNA biogenesis between lytic and latent infection and uncovered a regulatory mechanism that blocks pre-miRNA nuclear export and is dependent on viral protein ICP27 and viral DNA synthesis. This mechanism represents a new virus-host interaction that could limit the repressive effects of HSV-1 miRNAs hypothesized to promote latency and may shed light on the regulation of miRNA nuclear export, which has been relatively unexplored.

Download Full-text

Analysis of the Functions of Herpes Simplex Virus Type 1 Regulatory Protein ICP0 That Are Critical for Lytic Infection and Derepression of Quiescent Viral Genomes

Journal of Virology ◽

10.1128/jvi.02593-08 ◽

2009 ◽

Vol 83 (10) ◽

pp. 4963-4977 ◽

Cited By ~ 66

Author(s):

Roger D. Everett ◽

Marie-Laure Parsy ◽

Anne Orr

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Regulatory Protein ◽

Lytic Infection ◽

Viral Genomes ◽

Protein Icp0 ◽

Simplex Virus ◽

Mutant Forms ◽

Hsv 1

ABSTRACT Herpes simplex virus type 1 (HSV-1) immediate-early regulatory protein ICP0 is important for stimulating the initiation of the lytic cycle and efficient reactivation of latent or quiescent infection. Extensive investigation has suggested several potential functions for ICP0, including interference in the interferon response, disruption of functions connected with PML nuclear bodies (ND10), and inhibition of cellular histone deacetylase (HDAC) activity through an interaction with the HDAC-1 binding partner CoREST. Analysis of the significance of these potential functions and whether they are direct or indirect effects of ICP0 is complicated because HSV-1 mutants expressing mutant forms of ICP0 infect cells with widely differing efficiencies. On the other hand, transfection approaches for ICP0 expression do not allow studies of whole cell populations because of their limited efficiency. To overcome these problems, we have established a cell line in which ICP0 expression can be induced at levels pertaining during the early stages of HSV-1 infection in virtually all cells in the culture. Such cells enable 100% complementation of ICP0-null mutant HSV-1. Using cells expressing the wild type and a variety of mutant forms of ICP0, we have used this system to analyze the role of defined domains of the protein in stimulating lytic infection and derepression from quiescence. Activity in these core functions correlated well the ability of ICP0 to disrupt ND10 and inhibit the recruitment of ND10 proteins to sites closely associated with viral genomes at the onset of infection, whereas the CoREST binding region was neither sufficient nor necessary for ICP0 function in lytic and reactivating infections.

Download Full-text

The Histone Variant H3.3 Regulates Gene Expression during Lytic Infection with Herpes Simplex Virus Type 1

Journal of Virology ◽

10.1128/jvi.01276-08 ◽

2008 ◽

Vol 83 (3) ◽

pp. 1416-1421 ◽

Cited By ~ 69

Author(s):

Brandon J. Placek ◽

Jing Huang ◽

Jennifer R. Kent ◽

Jean Dorsey ◽

Lyndi Rice ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Histone Variant ◽

Lytic Infection ◽

A Genome ◽

Simplex Virus ◽

Hsv 1

ABSTRACTIt has been proposed that incorporation of the histone variant H3.3 within actively transcribed regions of a genome helps to facilitate transcription. In this report we use lytic infection by herpes simplex virus type 1 (HSV-1) as a model to examine the temporal profile of histone H3 incorporation and to determine whether the variant histone H3.3 has a direct effect on transcription. We find that canonical H3.1 and variant H3.3 exhibit distinct temporal associations with the genome in cell lines expressing equal amounts of epitope-tagged H3 variants. At the earliest times examined after infection, the HSV-1 genome is incorporated into chromatin that predominantly contains the variant H3.3, whereas incorporation of canonical H3.1 occurs later in infection and is dependent on replication of the HSV-1 genome. Further, inhibition of H3.3 association, via reduced expression of the H3.3 chaperone HIRA, significantly reduces the levels of HSV-1 mRNA. These findings show that incorporation of H3.3 facilitates transcription, and they provide new evidence for a regulatory role of chromatin composition during HSV-1 acute infection.

Download Full-text

Circularization of the Herpes Simplex Virus Type 1 Genome upon Lytic Infection

Journal of Virology ◽

10.1128/jvi.79.19.12487-12494.2005 ◽

2005 ◽

Vol 79 (19) ◽

pp. 12487-12494 ◽

Cited By ~ 63

Author(s):

Blair L. Strang ◽

Nigel D. Stow

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Blot Hybridization ◽

Lytic Infection ◽

Kinase Gene ◽

Simplex Virus ◽

Hsv 1

ABSTRACT For many years, the generally accepted model for the replication of the double-stranded DNA genome of herpes simplex virus type 1 (HSV-1) incorporated initial circularization of linear molecules in the cell nucleus. Ensuing DNA synthesis resulted in the generation of head-to-tail concatemers which were subsequently cleaved into monomeric units and packaged into the nascent viral capsid. Recently, however, it has been proposed that circularization of HSV-1 genomes does not occur at the onset of lytic infection and moreover that this event is specifically inhibited by the HSV-1 transcriptional transactivator, ICP0 (S.A. Jackson and N.A. DeLuca, Proc. Natl. Acad. Sci. USA 100:7871-7876, 2003). To further investigate genome circularization, we have generated HSV-1 derivatives in which the viral a sequences, which contain the cleavage-packaging signals, have been replaced by a minimal packaging element located in the thymidine kinase gene. In contrast to wild-type HSV-1, fusion of the genomic termini of these viruses produces a novel fragment in circular or concatemeric DNA which can be detected by Southern blot hybridization. Utilizing these viruses, we demonstrate that fusion of the genomic termini occurred rapidly upon infection and in the presence of inhibitors of viral DNA or protein synthesis. We provide evidence indicating that the end joining represented circularization rather than concatemerization of input molecules and that circularized molecules functioned as templates for replication. Since the termini of these viruses lack direct repeats, our findings indicate that circularization can be mediated by direct end-to-end ligation of linear input genomes.

Download Full-text

The latency-associated transcript locus of herpes simplex virus 1 is a virulence determinant in human skin

PLoS Pathogens ◽

10.1371/journal.ppat.1009166 ◽

2020 ◽

Vol 16 (12) ◽

pp. e1009166

Author(s):

Emilia A. H. Vanni ◽

Joseph W. Foley ◽

Andrew J. Davison ◽

Marvin Sommer ◽

Dongmei Liu ◽

...

Keyword(s):

Gene Expression ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Human Skin ◽

Lytic Infection ◽

Growth Defect ◽

Viral Gene ◽

Herpes Simplex Virus 1 ◽

Simplex Virus ◽

Hsv 1

Herpes simplex virus 1 (HSV-1) infects skin and mucosal epithelial cells and then travels along axons to establish latency in the neurones of sensory ganglia. Although viral gene expression is restricted during latency, the latency-associated transcript (LAT) locus encodes many RNAs, including a 2 kb intron known as the hallmark of HSV-1 latency. Here, we studied HSV-1 infection and the role of the LAT locus in human skin xenografts in vivo and in cultured explants. We sequenced the genomes of our stock of HSV-1 strain 17syn+ and seven derived viruses and found nonsynonymous mutations in many viral proteins that had no impact on skin infection. In contrast, deletions in the LAT locus severely impaired HSV-1 replication and lesion formation in skin. However, skin replication was not affected by impaired intron splicing. Moreover, although the LAT locus has been implicated in regulating gene expression in neurones, we observed only small changes in transcript levels that were unrelated to the growth defect in skin, suggesting that its functions in skin may be different from those in neurones. Thus, although the LAT locus was previously thought to be dispensable for lytic infection, we show that it is a determinant of HSV-1 virulence during lytic infection of human skin.

Download Full-text

Rapid and Inexpensive Preparation of Genome-Wide Nucleosome Footprints from Model and Non-Model Organisms

10.1101/870659 ◽

2019 ◽

Author(s):

Laura E McKnight ◽

Johnathan G Crandall ◽

Thomas B Bailey ◽

Orion GB Banks ◽

Kona N Orlandi ◽

...

Keyword(s):

Leukemia Cell ◽

Leukemia Cell Line ◽

Model Organisms ◽

Dna Fragments ◽

Chemical Waste ◽

Genome Wide ◽

Solid Agar ◽

Eukaryotic Dna ◽

Nucleosome Mapping ◽

The Relationship

AbstractEukaryotic DNA is packaged into nucleosomes, the smallest repeating unit of chromatin. The positions of nucleosomes determine the relative accessibility of genomic DNA. Several protocols exist for mapping nucleosome positions in eukaryotic genomes in order to study the relationship between chromatin structure and DNA-dependent processes. These nucleosome mapping protocols can be laborious and, at minimum, require two to three days to isolate nucleosome-protected DNA fragments. We have developed a streamlined protocol for mapping nucleosomes from S. cerevisiae liquid culture or from patches on solid agar. This method isolates nucleosome-sized footprints in three hours using 1.5 ml tubes with minimal chemical waste. We validate that these footprints match those produced by previously published methods and we demonstrate that our protocol works for N. crassa and S. pombe. A slightly modified protocol can be used for isolation of nucleosome-protected DNA fragments from a variety of wild fungal specimens thereby providing a simple, easily multiplexed and unified strategy to map nucleosome positions in model and non-model fungi. Finally, we demonstrate recovery of nucleosome footprints from the diploid myeloid leukemia cell line PLB-985 in less than three hours using an abbreviated version of the same protocol. With reduced volume and incubation times and a streamlined workflow, the described method should be compatible with high-throughput, automated creation of MNase-seq libraries. We believe this simple validated method for rapidly producing sequencing-ready nucleosome footprints from a variety of organisms will make nucleosome mapping studies widely accessible to researchers globally.

Download Full-text

Comprehensive nucleosome mapping of the human genome in cancer progression

10.1101/021618 ◽

2015 ◽

Author(s):

Brooke Druliner ◽

Daniel Vera ◽

Ruth Johnson ◽

Xiaoyang Ruan ◽

Lynne Apone ◽

...

Keyword(s):

Cancer Progression ◽

Chromatin Structure ◽

Colon Adenocarcinoma ◽

Transcription Start ◽

Transcription Start Sites ◽

Genome Wide ◽

Early Adenocarcinoma ◽

Capture Method ◽

Nucleosome Mapping

Altered chromatin structure is a hallmark of cancer, and inappropriate regulation of chromatin structure may represent the origin of transformation. Important studies have mapped human nucleosome distributions genome wide, but the role of chromatin structure in cancer progression has not been addressed. We developed a MNase-Transcription Start Site Sequence Capture method (mTSS-seq) to map the nucleosome distribution at human transcription start sites genome-wide in primary human lung and colon adenocarcinoma tissue. Here, we confirm that nucleosome redistribution is an early, widespread event in lung (LAC) and colon (CRC) adenocarcinoma. These altered nucleosome architectures are consistent between LAC and CRC patient samples indicating that they may serve as important early adenocarcinoma markers. We demonstrate that the nucleosome alterations are driven by the underlying DNA sequence and potentiate transcription factor binding. We conclude that DNA-directed nucleosome redistributions are widespread early in cancer progression. We have proposed an entirely new hierarchical model for chromatin-mediated genome regulation.

Download Full-text