Minding the message: tactics controlling RNA decay, modification, and translation in virus-infected cells.

With their categorical requirement for host ribosomes to translate mRNA, viruses provide a wealth of genetically tractable models to investigate how gene expression is remodeled post-transcriptionally by infection-triggered biological stress. By co-opting and subverting cellular pathways that control mRNA decay, modification, and translation, the global landscape of post-transcriptional processes is swiftly reshaped by virus-encoded factors. Concurrent host cell-intrinsic countermeasures likewise conscript post-transcriptional strategies to mobilize critical innate immune defenses. Here we review strategies and mechanisms that control mRNA decay, modification, and translation in animal virus-infected cells. Besides settling infection outcomes, post-transcriptional gene regulation in virus-infected cells epitomizes fundamental physiological stress responses in health and disease.

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.

RNA m6 A modification enzymes shape innate responses to DNA by regulating interferon ß.

Modification of mRNA by N6-adenosine methylation (m6A) on internal bases influences gene expression in eukaryotes. How the dynamic genome-wide landscape of m6A-modified mRNAs impacts virus infection and host immune responses remains poorly understood. Here, we show that type I interferon (IFN) production triggered by dsDNA or human cytomegalovirus (HCMV) is controlled by the cellular m6A methyltrasferase subunit METTL14 and ALKBH5 demethylase. While METTL14 depletion reduced virus reproduction and stimulated dsDNA- or HCMV-induced IFNB1 mRNA accumulation, ALKBH5 depletion had the opposite effect. Depleting METTL14 increased both nascent IFNB1 mRNA production and stability in response to dsDNA. In contrast, ALKBH5 depletion reduced nascent IFNB1 mRNA production without detectably influencing IFN1B mRNA decay. Genome-wide transcriptome profiling following ALKBH5 depletion identified differentially expressed genes regulating antiviral immune responses, while METTL14 depletion altered pathways impacting metabolic reprogramming, stress responses, and aging. Finally, we determined that IFNB1 mRNA was m6A-modified within both the coding sequence and the 3' untranslated region (UTR). This establishes that the host m6A modification machinery controls IFNß production triggered by HCMV or dsDNA. Moreover, it demonstrates that responses to nonmicrobial dsDNA in uninfected cells, which shape host immunity and contribute to autoimmune disease, are regulated by enzymes controlling m6A epitranscriptomic changes.

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.

CCR4-NOT differentially controls host versus virus poly(a)-tail length and regulates HCMV infection.

Unlike most RNA and DNA viruses that broadly stimulate mRNA decay and interfere with host gene expression, human cytomegalovirus (HCMV) extensively remodels the host translatome without producing an mRNA decay enzyme. By performing a targeted loss-of-function screen in primary human fibroblasts, we here identify the host CCR4-NOT deadenylase complex members CNOT1 and CNOT3 as unexpected pro-viral host factors that selectively regulate HCMV reproduction. We find that the scaffold subunit CNOT1 is specifically required for late viral gene expression and genome-wide host responses in CCR4-NOT-disrupted cells. By profiling poly(A)-tail lengths of individual HCMV and host mRNAs using nanopore direct RNA sequencing, we reveal poly(A)-tails of viral messages to be markedly longer than those of cellular mRNAs and significantly less sensitive to CCR4-NOT disruption. Our data establish that mRNA deadenylation by host CCR4-NOT is critical for productive HCMV replication and define a new mechanism whereby herpesvirus infection subverts cellular mRNA metabolism to remodel the gene expression landscape of the infected cell. Moreover, we expose an unanticipated host factor with potential to become a therapeutic anti-HCMV target.

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.

Vaccinia virus D10 has broad decapping activity that is regulated by mRNA splicing.

The mRNA 5' cap structure serves both to protect transcripts from degradation and promote their translation. Cap removal is thus an integral component of mRNA turnover that is carried out by cellular decapping enzymes, whose activity is tightly regulated and coupled to other stages of the mRNA decay pathway. The poxvirus vaccinia virus (VACV) encodes its own decapping enzymes, D9 and D10, that act on cellular and viral mRNA, but may be regulated differently than their cellular counterparts. Here, we evaluated the targeting potential of these viral enzymes using RNA sequencing from cells infected with wild-type and decapping mutant versions of VACV as well as in uninfected cells expressing D10. We found that D9 and D10 target an overlapping subset of viral transcripts but that D10 plays a dominant role in depleting the vast majority of human transcripts, although not in an indiscriminate manner. Unexpectedly, the splicing architecture of a gene influences how robustly its corresponding transcript is targeted by D10, as transcripts derived from intronless genes are less susceptible to enzymatic decapping by D10. As all VACV genes are intronless, preferential decapping of transcripts from intron-containing genes provides an unanticipated mechanism for the virus to disproportionately deplete host transcripts and remodel the infected cell transcriptome.

Translational control of the activation of transcription factor NF-κB and production of type I interferon by phosphorylation of the translation factor eIF4E.

Type I interferon is an integral component of the antiviral response, and its production is tightly controlled at the levels of transcription and translation. The eukaryotic translation-initiation factor eIF4E is a rate-limiting factor whose activity is regulated by phosphorylation of Ser209. Here we found that mice and fibroblasts in which eIF4E cannot be phosphorylated were less susceptible to virus infection. More production of type I interferon, resulting from less translation of Nfkbia mRNA (which encodes the inhibitor IκBα), largely explained this phenotype. The lower abundance of IκBα resulted in enhanced activity of the transcription factor NF-κB, which promoted the production of interferon-ß (IFN-ß). Thus, regulated phosphorylation of eIF4E has a key role in antiviral host defense by selectively controlling the translation of an mRNA that encodes a critical suppressor of the innate antiviral response.

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.

Preventing translational inhibition from ribosomal protein insufficiency by a herpes simplex virus-encoded ribosome-associated protein.

In addition to being required for protein synthesis, ribosomes and ribosomal proteins (RPs) also regulate messenger RNA translation in uninfected and virus-infected cells. By individually depleting 85 RPs using RNA interference, we found that overall protein synthesis in uninfected primary fibroblasts was more sensitive to RP depletion than those infected with herpes simplex virus-1 (HSV-1). Although representative RP depletion (uL3, uS4, uL5) inhibited protein synthesis in cells infected with two different DNA viruses (human cytomegalovirus, vaccinia virus), HSV-1-infected cell protein synthesis unexpectedly endured and required a single virus-encoded gene product, VP22. During individual RP insufficiency, VP22-expressing HSV-1 replicated better than a VP22-deficient variant. Furthermore, VP22 promotes polysome accumulation in virus-infected cells when uL3 or ribosome availability is limiting and cosediments with initiating and elongating ribosomes in infected and uninfected cells. This identifies VP22 as a virus-encoded, ribosome-associated protein that compensates for RP insufficiency to support viral protein synthesis and replication. Moreover, it reveals an unanticipated class of virus-encoded, ribosome-associated effectors that reduce the dependence of protein synthesis upon host RPs and broadly support translation during physiological stress such as infection.

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.

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.

Co-opting the Fanconi anemia genomic stability pathway enables herpesvirus DNA synthesis and productive growth.

DNA damage associated with viral DNA synthesis can result in double-strand breaks that threaten genome integrity and must be repaired. Here, we establish that the cellular Fanconi anemia (FA) genomic stability pathway is exploited by herpes simplex virus 1 (HSV-1) to promote viral DNA synthesis and enable its productive growth. Potent FA pathway activation in HSV-1-infected cells resulted in monoubiquitination of FA effector proteins FANCI and FANCD2 (FANCI-D2) and required the viral DNA polymerase. FANCD2 relocalized to viral replication compartments, and FANCI-D2 interacted with a multisubunit complex containing the virus-encoded single-stranded DNA-binding protein ICP8. Significantly, whereas HSV-1 productive growth was impaired in monoubiquitination-defective FA cells, this restriction was partially surmounted by antagonizing the DNA-dependent protein kinase (DNA-PK), a critical enzyme required for nonhomologous end-joining (NHEJ). This identifies the FA-pathway as a cellular factor required for herpesvirus productive growth and suggests that FA-mediated suppression of NHEJ is a fundamental step in the viral life cycle.

Inhibition of ULK1 and Beclin1 by an α-herpesvirus Akt-like Ser/Thr kinase limits autophagy to stimulate virus replication.

Autophagy is a powerful host defense that restricts herpes simplex virus-1 (HSV-1) pathogenesis in neurons. As a countermeasure, the viral ICP34.5 polypeptide, which is exclusively encoded by HSV, antagonizes autophagy in part through binding Beclin1. However, whether autophagy is a cell-type-specific antiviral defense or broadly restricts HSV-1 reproduction in nonneuronal cells is unknown. Here, we establish that autophagy limits HSV-1 productive growth in nonneuronal cells and is repressed by the Us3 gene product. Phosphorylation of the autophagy regulators ULK1 and Beclin1 in virus-infected cells was dependent upon the HSV-1 Us3 Ser/Thr kinase. Furthermore, Beclin1 was unexpectedly identified as a direct Us3 kinase substrate. Although disabling autophagy did not impact replication of an ICP34.5-deficient virus in primary human fibroblasts, depleting Beclin1 and ULK1 partially rescued Us3-deficient HSV-1 replication. This shows that autophagy restricts HSV-1 reproduction in a cell-intrinsic manner in nonneuronal cells and is suppressed by multiple, independent viral functions targeting Beclin1 and ULK1. Moreover, it defines a surprising role regulating autophagy for the Us3 kinase, which unlike ICP34.5 is widely encoded by alpha-herpesvirus subfamily members.

Repression of eEF2K transcription by NF-κB tunes translation elongation to inflammation and dsDNA-sensing.

Gene expression is rapidly remodeled by infection and inflammation in part via transcription factor NF-κB activation and regulated protein synthesis. While protein synthesis is largely controlled by mRNA translation initiation, whether cellular translation elongation factors are responsive to inflammation and infection remains poorly understood. Here, we reveal a surprising mechanism whereby NF-κB restricts phosphorylation of the critical translation elongation factor eEF2, which catalyzes the protein synthesis translocation step. Upon exposure to NF-κB-activating stimuli, including TNFα, human cytomegalovirus infection, or double-stranded DNA, eEF2 phosphorylation on Thr56, which slows elongation to limit protein synthesis, and the overall abundance of eEF2 kinase (eEF2K) are reduced. Significantly, this reflected a p65 NF-κB subunit-dependent reduction in eEF2K pre-mRNA, indicating that NF-κB activation represses eEF2K transcription to decrease eEF2K protein levels. Finally, we demonstrate that reducing eEF2K abundance regulates protein synthesis in response to a bacterial toxin that inactivates eEF2. This establishes that NF-κB activation by diverse physiological effectors controls eEF2 activity via a transcriptional repression mechanism that reduces eEF2K polypeptide abundance to preclude eEF2 phosphorylation, thereby stimulating translation elongation and protein synthesis. Moreover, it illustrates how nuclear transcription regulation shapes translation elongation factor activity and exposes how eEF2 is integrated into innate immune response networks orchestrated by NF-κB.

Coupling 40S ribosome recruitment to modification of a cap-binding initiation factor by eIF3 subunit e.

40S ribosomes are loaded onto capped mRNAs via the multisubunit translation initiation factors eIF3 and eIF4F. While eIF4E is the eIF4F cap recognition component, the eIF4G subunit associates with 40S-bound eIF3. How this intricate process is coordinated remains poorly understood. Here, we identify an eIF3 subunit that regulates eIF4F modification and show that eIF3e is required for inducible eIF4E phosphorylation. Significantly, recruitment of the eIF4E kinase Mnk1 (MAPK signal-integrating kinase 1) to eIF4F depended on eIF3e, and eIF3e was sufficient to promote Mnk1-binding to eIF4G. This establishes a mechanism by which 40S ribosome loading imparts a phosphorylation mark on the cap-binding eIF4F complex that regulates selective mRNA translation and is synchronized by a specific eIF3 subunit.

A new role for the cellular PABP repressor Paip2 as an innate restriction factor capable of limiting productive cytomegalovirus replication.

The capacity of polyadenylate-binding protein PABPC1 (PABP1) to stimulate translation is regulated by its repressor, Paip2. Paradoxically, while PABP accumulation promotes human cytomegalovirus (HCMV) protein synthesis, we show that this is accompanied by an analogous increase in the abundance of Paip2 and EDD1, an E3 ubiquitin ligase that destabilizes Paip2. Coordinate control of PABP1, Paip2, and EDD1 required the virus-encoded UL38 mTORC1 activator and resulted in augmented Paip2 synthesis, stability, and association with PABP1. Paip2 synthesis also increased following serum stimulation of uninfected normal fibroblasts, suggesting that this coregulation may play a role in how uninfected cells respond to stress. Significantly, Paip2 accumulation was dependent on PABP accrual, as preventing PABP1 accumulation suppressed viral replication and inhibited the corresponding Paip2 increase. Furthermore, depleting Paip2 restored the ability of infected cells to assemble the translation initiation factor eIF4F, promoting viral protein synthesis and replication without increasing PABP1. This establishes a new role for the cellular PABP1 inhibitor Paip2 as an innate defense that restricts viral protein synthesis and replication. Moreover, it illustrates how a stress-induced rise in PABP1 triggered by virus infection can counter and surpass a corresponding increase in Paip2 abundance and stability.

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.