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Direct RNA sequencing on nanopore arrays redefines the transcriptional complexity of a viral pathogen
Depledge, Daniel P; Srinivas, Kalanghad Puthankalam; Sadaoka, Tomohiko; Bready, Devin; Mori, Yasuko; Placantonakis, Dimitris G; Mohr, Ian; Wilson, Angus C
Characterizing complex viral transcriptomes by conventional RNA sequencing approaches is complicated by high gene density, overlapping reading frames, and complex splicing patterns. Direct RNA sequencing (direct RNA-seq) using nanopore arrays offers an exciting alternative whereby individual polyadenylated RNAs are sequenced directly, without the recoding and amplification biases inherent to other sequencing methodologies. Here we use direct RNA-seq to profile the herpes simplex virus type 1 (HSV-1) transcriptome during productive infection of primary cells. We show how direct RNA-seq data can be used to define transcription initiation and RNA cleavage sites associated with all polyadenylated viral RNAs and demonstrate that low level read-through transcription produces a novel class of chimeric HSV-1 transcripts, including a functional mRNA encoding a fusion of the viral E3 ubiquitin ligase ICP0 and viral membrane glycoprotein L. Thus, direct RNA-seq offers a powerful method to characterize the changing transcriptional landscape of viruses with complex genomes.
PMID: 30765700
ISSN: 2041-1723
CID: 3656412
Going the distance: optimizing RNA-Seq strategies for transcriptomic analysis of complex viral genomes
Depledge, Daniel P; Mohr, Ian; Wilson, Angus C
Transcriptome profiling has become routine in studies of many biological processes. However, favored approaches such as short-read Illumina RNA sequencing are giving way to long-read sequencing platforms better suited to interrogating the complex transcriptomes typical of many RNA and DNA viruses. Here, we provide a guide - tailored to molecular virologists - to the ins-and-outs of viral transcriptome sequencing and discuss the strengths and weaknesses of the major RNA sequencing technologies as tools to analyze the abundance and diversity of viral transcripts made during infection.
PMID: 30305358
ISSN: 1098-5514
CID: 3335052
Shared ancestry of herpes simplex virus 1 strain Patton with recent clinical isolates from Asia and with strain KOS63
Pourchet, Aldo; Copin, Richard; Mulvey, Matthew C; Shopsin, Bo; Mohr, Ian; Wilson, Angus C
Herpes simplex virus 1 (HSV-1) is a widespread pathogen that persists for life, replicating in surface tissues and establishing latency in peripheral ganglia. Increasingly, molecular studies of latency use cultured neuron models developed using recombinant viruses such as HSV-1 GFP-US11, a derivative of strain Patton expressing green fluorescent protein (GFP) fused to the viral US11 protein. Visible fluorescence follows viral DNA replication, providing a real time indicator of productive infection and reactivation. Patton was isolated in Houston, Texas, prior to 1973, and distributed to many laboratories. Although used extensively, the genomic structure and phylogenetic relationship to other strains is poorly known. We report that wild type Patton and the GFP-US11 recombinant contain the full complement of HSV-1 genes and differ within the unique regions at only eight nucleotides, changing only two amino acids. Although isolated in North America, Patton is most closely related to Asian viruses, including KOS63.
PMCID:5653468
PMID: 28957690
ISSN: 1096-0341
CID: 2717522
Viral Ubiquitin Ligase Stimulates Selective Host MicroRNA Expression by Targeting ZEB Transcriptional Repressors
Lutz, Gabriel; Jurak, Igor; Kim, Eui Tae; Kim, Ju Youn; Hackenberg, Michael; Leader, Andrew; Stoller, Michelle L; Fekete, Donna M; Weitzman, Matthew D; Coen, Donald M; Wilson, Angus C
Infection with herpes simplex virus-1 (HSV-1) brings numerous changes in cellular gene expression. Levels of most host mRNAs are reduced, limiting synthesis of host proteins, especially those involved in antiviral defenses. The impact of HSV-1 on host microRNAs (miRNAs), an extensive network of short non-coding RNAs that regulate mRNA stability/translation, remains largely unexplored. Here we show that transcription of the miR-183 cluster (miR-183, miR-96, and miR-182) is selectively induced by HSV-1 during productive infection of primary fibroblasts and neurons. ICP0, a viral E3 ubiquitin ligase expressed as an immediate-early protein, is both necessary and sufficient for this induction. Nuclear exclusion of ICP0 or removal of the RING (really interesting new gene) finger domain that is required for E3 ligase activity prevents induction. ICP0 promotes the degradation of numerous host proteins and for the most part, the downstream consequences are unknown. Induction of the miR-183 cluster can be mimicked by depletion of host transcriptional repressors zinc finger E-box binding homeobox 1 (ZEB1)/-crystallin enhancer binding factor 1 (deltaEF1) and zinc finger E-box binding homeobox 2 (ZEB2)/Smad-interacting protein 1 (SIP1), which we establish as new substrates for ICP0-mediated degradation. Thus, HSV-1 selectively stimulates expression of the miR-183 cluster by ICP0-mediated degradation of ZEB transcriptional repressors.
PMCID:5580467
PMID: 28783105
ISSN: 1999-4915
CID: 2663802
Modeling HSV-1 Latency in Human Embryonic Stem Cell-Derived Neurons
Pourchet, Aldo; Modrek, Aram S; Placantonakis, Dimitris G; Mohr, Ian; Wilson, Angus C
Herpes simplex virus 1 (HSV-1) uses latency in peripheral ganglia to persist in its human host, however, recurrent reactivation from this reservoir can cause debilitating and potentially life-threatening disease. Most studies of latency use live-animal infection models, but these are complex, multilayered systems and can be difficult to manipulate. Infection of cultured primary neurons provides a powerful alternative, yielding important insights into host signaling pathways controlling latency. However, small animal models do not recapitulate all aspects of HSV-1 infection in humans and are limited in terms of the available molecular tools. To address this, we have developed a latency model based on human neurons differentiated in culture from an NIH-approved embryonic stem cell line. The resulting neurons are highly permissive for replication of wild-type HSV-1, but establish a non-productive infection state resembling latency when infected at low viral doses in the presence of the antivirals acyclovir and interferon-alpha. In this state, viral replication and expression of a late viral gene marker are not detected but there is an accumulation of the viral latency-associated transcript (LAT) RNA. After a six-day establishment period, antivirals can be removed and the infected cultures maintained for several weeks. Subsequent treatment with sodium butyrate induces reactivation and production of new infectious virus. Human neurons derived from stem cells provide the appropriate species context to study this exclusively human virus with the potential for more extensive manipulation of the progenitors and access to a wide range of preexisting molecular tools.
PMCID:5488658
PMID: 28594343
ISSN: 2076-0817
CID: 2590522
Immune Escape via a Transient Gene Expression Program Enables Productive Replication of a Latent Pathogen
Linderman, Jessica A; Kobayashi, Mariko; Rayannavar, Vinayak; Fak, John J; Darnell, Robert B; Chao, Moses V; Wilson, Angus C; Mohr, Ian
How type I and II interferons prevent periodic reemergence of latent pathogens in tissues of diverse cell types remains unknown. Using homogeneous neuron cultures latently infected with herpes simplex virus 1, we show that extrinsic type I or II interferon acts directly on neurons to induce unique gene expression signatures and inhibit the reactivation-specific burst of viral genome-wide transcription called phase I. Surprisingly, interferons suppressed reactivation only during a limited period early in phase I preceding productive virus growth. Sensitivity to type II interferon was selectively lost if viral ICP0, which normally accumulates later in phase I, was expressed before reactivation. Thus, interferons suppress reactivation by preventing initial expression of latent genomes but are ineffective once phase I viral proteins accumulate, limiting interferon action. This demonstrates that inducible reactivation from latency is only transiently sensitive to interferon. Moreover, it illustrates how latent pathogens escape host immune control to periodically replicate by rapidly deploying an interferon-resistant state.
PMCID:5340258
PMID: 28147283
ISSN: 2211-1247
CID: 2424422
Restarting Lytic Gene Transcription at the Onset of Herpes Simplex Virus Reactivation
Cliffe, Anna R; Wilson, Angus C
Herpes simplex virus type (HSV) establishes a latent reservoir in neurons of human peripheral nerves. In this quiescent state the viral genome persists as a circular, histone-associated episome and transcription of viral lytic-cycle genes is largely suppressed through epigenetic processes. Periodically latent virus undergoes reactivation whereby lytic genes are activated and viral replication occurs. In this GEM we review recent evidence that mechanisms governing the initial transcription of lytic genes are distinct from those of de novo infection and directly link reactivation to neuronal stress response pathways. We also discuss evidence that lytic cycle gene expression can be uncoupled from the full reactivation program, arguing for a less sharply bimodal definition of latency.
PMCID:5215350
PMID: 27807236
ISSN: 1098-5514
CID: 2296982
Expression of Herpes Simplex Virus 1 MicroRNAs in Cell Culture Models of Quiescent and Latent Infection
Jurak, Igor; Hackenberg, Michael; Kim, Ju Youn; Pesola, Jean M; Everett, Roger D; Preston, Chris M; Wilson, Angus C; Coen, Donald M
To facilitate studies of herpes simplex virus 1 latency, cell culture models of quiescent or latent infection have been developed. Using deep sequencing, we analyzed the expression of viral microRNAs (miRNAs) in two models employing human fibroblasts and one using rat neurons. In all cases, the expression patterns differed from that in productively infected cells, with the rat neuron pattern most closely resembling that found in latently infected human or mouse ganglia in vivo.
PMCID:3911564
PMID: 24307587
ISSN: 0022-538x
CID: 836682
Using homogeneous primary neuron cultures to study fundamental aspects of HSV-1 latency and reactivation
Kim, Ju Youn; Shiflett, Lora A; Linderman, Jessica A; Mohr, Ian; Wilson, Angus C
We describe a primary neuronal culture system suitable for molecular characterization of herpes simplex virus type 1 (HSV-1) infection, latency, and reactivation. While several alternative models are available, including infections of live animal and explanted ganglia, these are complicated by the presence of multiple cell types, including immune cells, and difficulties in manipulating the neuronal environment. The highly pure neuron culture system described here can be readily manipulated and is ideal for molecular studies that focus exclusively on the relationship between the virus and host neuron, the fundamental unit of latency. As such it allows for detailed investigations of both viral and neuronal factors involved in the establishment and maintenance of HSV-1 latency and in viral reactivation induced by defined stimuli.
PMID: 24671683
ISSN: 1064-3745
CID: 970092
A cultured affair: HSV latency and reactivation in neurons
Wilson, Angus C; Mohr, Ian
After replicating in surface epithelia, herpes simplex virus type-1 (HSV-1) enters the axonal terminals of peripheral neurons. The viral genome translocates to the nucleus, where it establishes a specialized infection known as latency, re-emerging periodically to seed new infections. Studies using cultured neuron models that faithfully recapitulate the molecular hallmarks of latency and reactivation defined in live animal models have provided fresh insight into the control of latency and connections to neuronal physiology. With this comes a growing appreciation for how the life cycles of HSV-1 and other herpesviruses are governed by key host pathways controlling metabolic homeostasis and cell identity.
PMCID:3989139
PMID: 22963857
ISSN: 0966-842x
CID: 182432