Targeting the m6A RNA modification pathway blocks SARS-CoV-2 and HCoV-OC43 replication.

N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human ß-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m6A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m6A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m6A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m6A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m6A-modified and host m6A pathway components control ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.

TOP2ß-Dependent Nuclear DNA Damage Shapes Extracellular Growth Factor Responses via Dynamic AKT Phosphorylation to Control Virus Latency.

The mTOR pathway integrates both extracellular and intracellular signals and serves as a central regulator of cell metabolism, growth, survival, and stress responses. Neurotropic viruses, such as herpes simplex virus-1 (HSV-1), also rely on cellular AKT-mTORC1 signaling to achieve viral latency. Here, we define a novel genotoxic response whereby spatially separated signals initiated by extracellular neurotrophic factors and nuclear DNA damage are integrated by the AKT-mTORC1 pathway. We demonstrate that endogenous DNA double-strand breaks (DSBs) mediated by Topoisomerase 2ß-DNA cleavage complex (TOP2ßcc) intermediates are required to achieve AKT-mTORC1 signaling and maintain HSV-1 latency in neurons. Suppression of host DNA-repair pathways that remove TOP2ßcc trigger HSV-1 reactivation. Moreover, perturbation of AKT phosphorylation dynamics by downregulating the PHLPP1 phosphatase led to AKT mis-localization and disruption of DSB-induced HSV-1 reactivation. Thus, the cellular genome integrity and environmental inputs are consolidated and co-opted by a latent virus to balance lifelong infection with transmission.

Reanalysis of single-cell RNA sequencing data does not support herpes simplex virus 1 latency in non-neuronal ganglionic cells in mice.

Most individuals are latently infected with herpes simplex virus type 1 (HSV-1), and it is well-established that HSV-1 establishes latency in sensory neurons of peripheral ganglia. However, it was recently proposed that latent HSV-1 is also present in immune cells recovered from the ganglia of experimentally infected mice. Here, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data that formed the basis for that conclusion. Unexpectedly, off-target priming in 3' scRNA-Seq experiments enabled the detection of non-polyadenylated HSV-1 latency-associated transcript (LAT) intronic RNAs. However, LAT reads were near-exclusively detected in mixed populations of cells undergoing cell death. Specific loss of HSV-1 LAT and neuronal transcripts during quality control filtering indicated widespread destruction of neurons, supporting the presence of contaminating cell-free RNA in other cells following tissue processing. In conclusion, the reported detection of latent HSV-1 in non-neuronal cells is best explained using compromised scRNA-Seq datasets.IMPORTANCEMost people are infected with herpes simplex virus type 1 (HSV-1) during their life. Once infected, the virus generally remains in a latent (silent) state, hiding within the neurons of peripheral ganglia. Periodic reactivation (reawakening) of the virus may cause fresh diseases such as cold sores. A recent study using single-cell RNA sequencing (scRNA-Seq) proposed that HSV-1 can also establish latency in the immune cells of mice, challenging existing dogma. We reanalyzed the data from that study and identified several flaws in the methodologies and analyses performed that invalidate the published conclusions. Specifically, we showed that the methodologies used resulted in widespread destruction of neurons which resulted in the presence of contaminants that confound the data analysis. We thus conclude that there remains little to no evidence for HSV-1 latency in immune cells.

Herpes Simplex Virus-1 ICP27 Nuclear Export Signal Mutants Exhibit Cell Type-Dependent Deficits in Replication and ICP4 Expression.

Herpes simplex virus type-1 (HSV-1) protein ICP27 is an essential immediate early (IE) protein that promotes the expression of viral early (E) and late (L) genes via multiple mechanisms. Our understanding of this complex regulatory protein has been greatly enhanced by the characterization of HSV-1 mutants bearing engineered alterations in the ICP27 gene. However, much of this analysis has been performed in interferon-deficient Vero monkey cells. Here, we assessed the replication of a panel of ICP27 mutants in several other cell types. Our analysis shows that mutants lacking ICP27's amino (N)-terminal nuclear export signal (NES) display a striking cell type-dependent growth phenotype, i.e., they grow semi-permissively in Vero and some other cells but are tightly blocked for replication in primary human fibroblasts and multiple human cell lines. This tight growth defect correlates with a failure of these mutants to replicate viral DNA. We also report that HSV-1 NES mutants are deficient in expressing the IE protein ICP4 at early times postinfection. Analysis of viral RNA levels suggests that this phenotype is due, at least in part, to a defect in the export of ICP4 mRNA to the cytoplasm. In combination, our results (i) show that ICP27's NES is critically important for HSV-1 replication in many human cells, and (ii) suggest that ICP27 plays a heretofore unappreciated role in the expression of ICP4. IMPORTANCE HSV-1 IE proteins drive productive HSV-1 replication. The major paradigm of IE gene induction, developed over many years, involves the parallel activation of the five IE genes by the viral tegument protein VP16, which recruits the host RNA polymerase II (RNAP II) to the IE gene promoters. Here, we provide evidence that ICP27 can enhance ICP4 expression early in infection. Because ICP4 is required for transcription of viral E and L genes, this finding may be relevant to understanding how HSV-1 enters and exits the latent state in neurons.

Novel viral splicing events and open reading frames revealed by long-read direct RNA sequencing of adenovirus transcripts.

Adenovirus is a common human pathogen that relies on host cell processes for transcription and processing of viral RNA and protein production. Although adenoviral promoters, splice junctions, and polyadenylation sites have been characterized using low-throughput biochemical techniques or short read cDNA-based sequencing, these technologies do not fully capture the complexity of the adenoviral transcriptome. By combining Illumina short-read and nanopore long-read direct RNA sequencing approaches, we mapped transcription start sites and RNA cleavage and polyadenylation sites across the adenovirus genome. In addition to confirming the known canonical viral early and late RNA cassettes, our analysis of splice junctions within long RNA reads revealed an additional 35 novel viral transcripts that meet stringent criteria for expression. These RNAs include fourteen new splice junctions which lead to expression of canonical open reading frames (ORFs), six novel ORF-containing transcripts, and 15 transcripts encoding for messages that could alter protein functions through truncation or fusion of canonical ORFs. In addition, we detect RNAs that bypass canonical cleavage sites and generate potential chimeric proteins by linking distinct gene transcription units. Among these chimeric proteins we detected an evolutionarily conserved protein containing the N-terminus of E4orf6 fused to the downstream DBP/E2A ORF. Loss of this novel protein, E4orf6/DBP, was associated with aberrant viral replication center morphology and poor viral spread. Our work highlights how long-read sequencing technologies combined with mass spectrometry can reveal further complexity within viral transcriptomes and resulting proteomes.

Single-cell transcriptomics identifies Gadd45b as a regulator of herpesvirus-reactivating neurons.

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for dissecting the complexity of normal and diseased tissues, enabling characterization of cell diversity and heterogeneous phenotypic states in unprecedented detail. However, this technology has been underutilized for exploring the interactions between the host cell and viral pathogens in latently infected cells. Herein, we use scRNA-seq and single-molecule sensitivity fluorescent in situ hybridization (smFISH) technologies to investigate host single-cell transcriptome changes upon the reactivation of a human neurotropic virus, herpes simplex virus-1 (HSV-1). We identify the stress sensor growth arrest and DNA damage-inducible 45 beta (Gadd45b) as a critical antiviral host factor that regulates HSV-1 reactivation events in a subpopulation of latently infected primary neurons. We show that distinct subcellular localization of Gadd45b correlates with the viral late gene expression program, as well as the expression of the viral transcription factor, ICP4. We propose that a hallmark of a "successful" or "aborted" HSV-1 reactivation state in primary neurons is determined by a unique subcellular localization signature of the stress sensor Gadd45b.

Widespread remodeling of the m6A RNA-modification landscape by a viral regulator of RNA processing and export.

N6-methyladenosine (m6A) is the most abundant internal messenger RNA (mRNA) modification, contributing to the processing, stability, and function of methylated RNAs. Methylation occurs in the nucleus during pre-mRNA synthesis and requires a core methyltransferase complex consisting of METTL3, METTL14, and WTAP. During herpes simplex virus (HSV-1) infection, cellular gene expression is profoundly suppressed, allowing the virus to monopolize the host transcription and translation apparatus and antagonize antiviral responses. The extent to which HSV-1 uses or manipulates the m6A pathway is not known. Here, we show that, in primary fibroblasts, HSV-1 orchestrates a striking redistribution of the nuclear m6A machinery that progresses through the infection cycle. METTL3 and METTL14 are dispersed into the cytoplasm, whereas WTAP remains nuclear. Other regulatory subunits of the methyltransferase complex, along with the nuclear m6A-modified RNA binding protein YTHDC1 and nuclear demethylase ALKBH5, are similarly redistributed. These changes require ICP27, a viral regulator of host mRNA processing that mediates the nucleocytoplasmic export of viral late mRNAs. Viral gene expression is initially reduced by small interfering RNA (siRNA)-mediated inactivation of the m6A methyltransferase but becomes less impacted as the infection advances. Redistribution of the nuclear m6A machinery is accompanied by a wide-scale reduction in the installation of m6A and other RNA modifications on both host and viral mRNAs. These results reveal a far-reaching mechanism by which HSV-1 subverts host gene expression to favor viral replication.

DLK-Dependent Biphasic Reactivation of Herpes Simplex Virus Latency Established in the Absence of Antivirals.

Understanding the molecular mechanisms of herpes simplex virus 1 (HSV-1) latent infection and reactivation in neurons requires the use of in vitro model systems. Establishing a quiescent infection in cultured neurons is problematic, as any infectious virus released can superinfect the cultures. Previous studies have used the viral DNA replication inhibitor acyclovir to prevent superinfection and promote latency establishment. Data from these previous models have shown that reactivation is biphasic, with an initial phase I expression of all classes of lytic genes, which occurs independently of histone demethylase activity and viral DNA replication but is dependent on the cell stress protein DLK. Here, we describe a new model system using HSV-1 Stayput-GFP, a reporter virus that is defective for cell-to-cell spread and establishes latent infections without the need for acyclovir. The establishment of a latent state requires a longer time frame than previous models using DNA replication inhibitors. This results in a decreased ability of the virus to reactivate using established inducers, and as such, a combination of reactivation triggers is required. Using this system, we demonstrate that biphasic reactivation occurs even when latency is established in the absence of acyclovir. Importantly, phase I lytic gene expression still occurs in a histone demethylase and viral DNA replication-independent manner and requires DLK activity. These data demonstrate that the two waves of viral gene expression following HSV-1 reactivation are independent of secondary infection and not unique to systems that require acyclovir to promote latency establishment. IMPORTANCE Herpes simplex virus-1 (HSV-1) enters a latent infection in neurons and periodically reactivates. Reactivation manifests as a variety of clinical symptoms. Studying latency and reactivation in vitro is invaluable, allowing the molecular mechanisms behind both processes to be targeted by therapeutics that reduce the clinical consequences. Here, we describe a novel in vitro model system using a cell-to-cell spread-defective HSV-1, known as Stayput-GFP, which allows for the study of latency and reactivation at the single neuron level. We anticipate this new model system will be an incredibly valuable tool for studying the establishment and reactivation of HSV-1 latent infection in vitro. Using this model, we find that initial reactivation events are dependent on cellular stress kinase DLK but independent of histone demethylase activity and viral DNA replication. Our data therefore further validate the essential role of DLK in mediating a wave of lytic gene expression unique to reactivation.

DRUMMER-rapid detection of RNA modifications through comparative nanopore sequencing.

MOTIVATION: The chemical modification of ribonucleotides regulates the structure, stability and interactions of RNAs. Profiling of these modifications using short-read (Illumina) sequencing techniques provides high sensitivity but low-to-medium resolution i.e. modifications cannot be assigned to specific transcript isoforms in regions of sequence overlap. An alternative strategy uses current fluctuations in nanopore-based long read direct RNA sequencing (DRS) to infer the location and identity of nucleotides that differ between two experimental conditions. While highly sensitive, these signal-level analyses require high-quality transcriptome annotations and thus are best suited to the study of model organisms. By contrast, the detection of RNA modifications in microbial organisms which typically have no or low-quality annotations requires an alternative strategy. Here, we demonstrate that signal fluctuations directly influence error rates during base-calling and thus provides an alternative approach for identifying modified nucleotides. RESULTS: DRUMMER (Detection of Ribonucleic acid Modifications Manifested in Error Rates) (i) utilizes a range of statistical tests and background noise correction to identify modified nucleotides with high confidence, (ii) operates with similar sensitivity to signal-level analysis approaches and (iii) correlates very well with orthogonal approaches. Using well-characterized DRS datasets supported by independent meRIP-Seq and miCLIP-Seq datasets we demonstrate that DRUMMER operates with high sensitivity and specificity. AVAILABILITY AND IMPLEMENTATION: DRUMMER is written in Python 3 and is available as open source in the GitHub repository: https://github.com/DepledgeLab/DRUMMER. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Going the Distance: Optimizing RNA-Seq Strategies for Transcriptomic Analysis of Complex Viral Genomes.

Transcriptome profiling has become routine in studies of many biological processes. However, the 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 the viral transcripts made during infection.

2019 Colorado Alphaherpesvirus Latency Society symposium.

Meeting Report on the 9th Annual Symposium of the Colorado Alphaherpesvirus Latency Society (CALS) held on May 8-11, 2019, in Vail, CO.

Control of viral latency in neurons by axonal mTOR signaling and the 4E-BP translation repressor.

Latent herpes simplex virus-1 (HSV1) genomes in peripheral nerve ganglia periodically reactivate, initiating a gene expression program required for productive replication. Whether molecular cues detected by axons can be relayed to cell bodies and harnessed to regulate latent genome expression in neuronal nuclei is unknown. Using a neuron culture model, we found that inhibiting mTOR, depleting its regulatory subunit raptor, or inducing hypoxia all trigger reactivation. While persistent mTORC1 activation suppressed reactivation, a mutant 4E-BP (eIF4E-binding protein) translational repressor unresponsive to mTORC1 stimulated reactivation. Finally, inhibiting mTOR in axons induced reactivation. Thus, local changes in axonal mTOR signaling that control translation regulate latent HSV1 genomes in a spatially segregated compartment.

Restarting Lytic Gene Transcription at the Onset of Herpes Simplex Virus Reactivation.

Herpes simplex virus (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.

Expression of herpes simplex virus 1 microRNAs in cell culture models of quiescent and latent infection.

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.

Transient reversal of episome silencing precedes VP16-dependent transcription during reactivation of latent HSV-1 in neurons.

Herpes simplex virus type-1 (HSV-1) establishes latency in peripheral neurons, creating a permanent source of recurrent infections. The latent genome is assembled into chromatin and lytic cycle genes are silenced. Processes that orchestrate reentry into productive replication (reactivation) remain poorly understood. We have used latently infected cultures of primary superior cervical ganglion (SCG) sympathetic neurons to profile viral gene expression following a defined reactivation stimulus. Lytic genes are transcribed in two distinct phases, differing in their reliance on protein synthesis, viral DNA replication and the essential initiator protein VP16. The first phase does not require viral proteins and has the appearance of a transient, widespread de-repression of the previously silent lytic genes. This allows synthesis of viral regulatory proteins including VP16, which accumulate in the cytoplasm of the host neuron. During the second phase, VP16 and its cellular cofactor HCF-1, which is also predominantly cytoplasmic, concentrate in the nucleus where they assemble an activator complex on viral promoters. The transactivation function supplied by VP16 promotes increased viral lytic gene transcription leading to the onset of genome amplification and the production of infectious viral particles. Thus regulated localization of de novo synthesized VP16 is likely to be a critical determinant of HSV-1 reactivation in sympathetic neurons.

Nanopore guided annotation of transcriptome architectures.

Nanopore direct RNA sequencing (DRS) enables the capture and full-length sequencing of native RNAs, without recoding or amplification bias. Resulting data sets may be interrogated to define the identity and location of chemically modified ribonucleotides, as well as the length of poly(A) tails, on individual RNA molecules. The success of these analyses is highly dependent on the provision of high-resolution transcriptome annotations in combination with workflows that minimize misalignments and other analysis artifacts. Existing software solutions for generating high-resolution transcriptome annotations are poorly suited to small gene-dense genomes of viruses due to the challenge of identifying distinct transcript isoforms where alternative splicing and overlapping RNAs are prevalent. To resolve this, we identified key characteristics of DRS data sets that inform resulting read alignments and developed the nanopore guided annotation of transcriptome architectures (NAGATA) software package (https://github.com/DepledgeLab/NAGATA). We demonstrate, using a combination of synthetic and original DRS data sets derived from adenoviruses, herpesviruses, coronaviruses, and human cells, that NAGATA outperforms existing transcriptome annotation software and yields a consistently high level of precision and recall when reconstructing both gene sparse and gene-dense transcriptomes. Finally, we apply NAGATA to generate the first high-resolution transcriptome annotation of the neglected pathogen human adenovirus type F41 (HAdV-41) for which we identify 77 distinct transcripts encoding at least 23 different proteins. IMPORTANCE: The transcriptome of an organism denotes the full repertoire of encoded RNAs that may be expressed. This is critical to understanding the biology of an organism and for accurate transcriptomic and epitranscriptomic-based analyses. Annotating transcriptomes remains a complex task, particularly in small gene-dense organisms such as viruses which maximize their coding capacity through overlapping RNAs. To resolve this, we have developed a new software nanopore guided annotation of transcriptome architectures (NAGATA) which utilizes nanopore direct RNA sequencing (DRS) datasets to rapidly produce high-resolution transcriptome annotations for diverse viruses and other organisms.

Nanopore Guided Annotation of Transcriptome Architectures.

High-resolution annotations of transcriptomes from all domains of life are essential for many sequencing-based RNA analyses, including Nanopore direct RNA sequencing (DRS), which would otherwise be hindered by misalignments and other analysis artefacts. DRS allows the capture and full-length sequencing of native RNAs, without recoding or amplification bias, and resulting data may be interrogated to define the identity and location of chemically modified ribonucleotides, as well as the length of poly(A) tails on individual RNA molecules. Existing software solutions for generating high-resolution transcriptome annotations are poorly suited to small gene dense organisms such as viruses due to the challenge of identifying distinct transcript isoforms where alternative splicing and overlapping RNAs are prevalent. To resolve this, we identified key characteristics of DRS datasets and developed a novel approach to transcriptome. We demonstrate, using a combination of synthetic and original datasets, that our novel approach yields a high level of precision and recall when reconstructing both gene sparse and gene dense transcriptomes from DRS datasets. We further apply this approach to generate a new high resolution transcriptome annotation of the neglected pathogen human adenovirus type F 41 for which we identify 77 distinct transcripts encoding at least 23 different proteins.

Cooperation between viral interferon regulatory factor 4 and RTA to activate a subset of Kaposi's sarcoma-associated herpesvirus lytic promoters.

The four Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded interferon (IFN) regulatory factor homologues (vIRF1 to vIRF4) are used to counter innate immune defenses and suppress p53. The vIRF genes are arranged in tandem but differ in function and expression. In KSHV-infected effusion lymphoma lines, K10.5/vIRF3 and K11/vIRF2 mRNAs are readily detected during latency, whereas K9/vIRF1 and K10/vIRF4 mRNAs are upregulated during reactivation. Here we show that the K10/vIRF4 promoter responds to the lytic switch protein RTA in KSHV-infected cells but is essentially unresponsive in uninfected cells. Coexpression of RTA with vIRF4 is sufficient to restore regulation, a property not shared by other vIRFs. The K9/vIRF1 promoter behaves similarly, and production of infectious virus is enhanced by the presence of vIRF4. Synergy requires the DNA-binding domain (DBD) and C-terminal IRF homology regions of vIRF4. Mutations of arginine residues within the putative DNA recognition helix of vIRF4 or the invariant cysteines of the adjacent CxxC motif abolish cooperation with RTA, in the latter case by preventing self-association. The oligomerization and transactivation functions of RTA are also essential for synergy. The K10/vIRF4 promoter contains two transcription start sites (TSSs), and a 105-bp fragment containing the proximal promoter is responsive to vIRF4/RTA. Binding of a cellular factor(s) to this fragment is altered when both viral proteins are present, suggesting a possible mechanism for transcriptional synergy. Reliance on coregulators encoded by either the host or viral genome provides an elegant strategy for expanding the regulatory potential of a master regulator, such as RTA.

Reanalysis of single-cell RNA sequencing data does not support herpes simplex virus 1 latency in non-neuronal ganglionic cells in mice.

Most individuals are latently infected with herpes simplex virus type 1 (HSV-1) and it is well-established that HSV-1 establishes latency in sensory neurons of peripheral ganglia. However, it was recently proposed that latent virus is also present in immune cells recovered from ganglia in a mouse model used for studying latency. Here, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data that formed the basis for this conclusion. Unexpectedly, off-target priming in 3' scRNA-Seq experiments enabled the detection of non-polyadenylated HSV-1 latency-associated transcript (LAT) intronic RNAs. However, LAT reads were nearexclusively detected in a mixed population of cells undergoing cell death. Specific loss of HSV1 LAT and neuronal transcripts during quality control filtering indicated widespread destruction of neurons, supporting the presence of contaminating cell-free RNA in other cells following tissue processing. In conclusion, the reported detection of latent HSV-1 in non-neuronal cells is best explained by inaccuracies in the data analyses.