m6aViewer: software for the detection, analysis, and visualization of N6-methyladenosine peaks from m6A-seq/ME-RIP sequencing data.

Recent methods for transcriptome-wide N6-methyladenosine (m6A) profiling have facilitated investigations into the RNA methylome and established m6A as a dynamic modification that has critical regulatory roles in gene expression and may play a role in human disease. However, bioinformatics resources available for the analysis of m6A sequencing data are still limited. Here, we describe m6aViewer-a cross-platform application for analysis and visualization of m6A peaks from sequencing data. m6aViewer implements a novel m6A peak-calling algorithm that identifies high-confidence methylated residues with more precision than previously described approaches. The application enables data analysis through a graphical user interface, and thus, in contrast to other currently available tools, does not require the user to be skilled in computer programming. m6aViewer and test data can be downloaded here: http://dna2.leeds.ac.uk/m6a.

Hsp70 Isoforms Are Essential for the Formation of Kaposi's Sarcoma-Associated Herpesvirus Replication and Transcription Compartments.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus associated with various AIDS-related malignancies. Like other herpesviruses, multiple processes required for KSHV lytic replication, including viral transcription, viral DNA synthesis and capsid assembly occur in virus-induced intranuclear structures, termed replication and transcription compartments (RTCs). Here we utilised a novel methodology, combining subcellular fractionation and quantitative proteomics, to identify cellular proteins which are recruited to KSHV-induced RTCs and thus play a key role in KSHV lytic replication. We show that several isoforms of the HSP70 chaperone family, Hsc70 and iHsp70, are redistributed from the cytoplasm into the nucleus coinciding with the initial formation of KSHV-induced RTCs. We demonstrate that nuclear chaperone foci are dynamic, initially forming adjacent to newly formed KSHV RTCs, however during later time points the chaperones move within KSHV RTCs and completely co-localise with actively replicating viral DNA. The functional significance of Hsp70 isoforms recruitment into KSHV RTCs was also examined using the specific Hsp70 isoform small molecule inhibitor, VER-155008. Intriguingly, results highlight an essential role of Hsp70 isoforms in the KSHV replication cycle independent of protein stability and maturation. Notably, inhibition of Hsp70 isoforms precluded KSHV RTC formation and RNA polymerase II (RNAPII) relocalisation to the viral genome leading to the abolishment of global KSHV transcription and subsequent viral protein synthesis and DNA replication. These new findings have revealed novel mechanisms that regulate KSHV lytic replication and highlight the potential of HSP70 inhibitors as novel antiviral agents.

NEDDylation is essential for Kaposi's sarcoma-associated herpesvirus latency and lytic reactivation and represents a novel anti-KSHV target.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), which are aggressive malignancies associated with immunocompromised patients. For many non-viral malignancies, therapeutically targeting the ubiquitin proteasome system (UPS) has been successful. Likewise, laboratory studies have demonstrated that inhibition of the UPS might provide a promising avenue for the treatment of KSHV-associated diseases. The largest class of E3 ubiquitin ligases are the cullin-RING ligases (CRLs) that are activated by an additional ubiquitin-like protein, NEDD8. We show that pharmacological inhibition of NEDDylation (using the small molecule inhibitor MLN4924) is cytotoxic to PEL cells by inhibiting NF-κB. We also show that CRL4B is a novel regulator of latency as its inhibition reactivated lytic gene expression. Furthermore, we uncovered a requirement for NEDDylation during the reactivation of the KSHV lytic cycle. Intriguingly, inhibition prevented viral DNA replication but not lytic cycle-associated gene expression, highlighting a novel mechanism that uncouples these two features of KSHV biology. Mechanistically, we show that MLN4924 treatment precluded the recruitment of the viral pre-replication complex to the origin of lytic DNA replication (OriLyt). These new findings have revealed novel mechanisms that regulate KSHV latency and reactivation. Moreover, they demonstrate that inhibition of NEDDylation represents a novel approach for the treatment of KSHV-associated malignancies.

Interactions between KSHV ORF57 and the novel human TREX proteins, CHTOP and CIP29.

The coupling of mRNA processing steps is essential for precise and efficient gene expression. The human transcription/export (hTREX) complex is a highly conserved multi-protein complex responsible for eukaryotic mRNA stability and nuclear export. We have previously shown that the Kaposi's sarcoma-associated open reading frame 57 (ORF57) protein orchestrates the recruitment of the hTREX complex onto viral intronless mRNA, forming a stable and export-competent viral ribonucleoprotein particle (vRNP). Recently, additional cellular proteins, namely CHTOP, CIP29 and POLDIP3 have been proposed as novel hTREX components. Herein, we extend our previous research and provide evidence that ORF57 interacts with CHTOP and CIP29, in contrast to POLDIP3. Moreover, depletion studies show both CHTOP and CIP29 effect ORF57-mediated viral mRNA processing. As such, these results suggest both CHTOP and CIP29 are hTREX components and are recruited to an ORF57-mediated vRNP.

Chicken and duck myotubes are highly susceptible and permissive to influenza virus infection.

UNLABELLED: Skeletal muscle, at 30 to 40% of body mass, is the most abundant soft tissue in the body. Besides its primary function in movement and posture, skeletal muscle is a significant innate immune organ with the capacity to produce cytokines and chemokines and respond to proinflammatory cytokines. Little is known about the role of skeletal muscle during systemic influenza A virus infection in any host and particularly avian species. Here we used primary chicken and duck multinucleated myotubes to examine their susceptibility and innate immune response to influenza virus infections. Both chicken and duck myotubes expressed avian and human sialic acid receptors and were readily susceptible to low-pathogenicity (H2N3 A/mallard duck/England/7277/06) and high-pathogenicity (H5N1 A/turkey/England/50-92/91 and H5N1 A/turkey/Turkey/1/05) avian and human H1N1 (A/USSR/77) influenza viruses. Both avian host species produced comparable levels of progeny H5N1 A/turkey/Turkey/1/05 virus. Notably, the rapid accumulation of viral nucleoprotein and matrix (M) gene RNA in chicken and duck myotubes was accompanied by extensive cytopathic damage with marked myotube apoptosis (widespread microscopic blebs, caspase 3/7 activation, and annexin V binding at the plasma membrane). Infected chicken myotubes produced significantly higher levels of proinflammatory cytokines than did the corresponding duck cells. Additionally, in chicken myotubes infected with H5N1 viruses, the induction of interferon beta (IFN-ß) and IFN-inducible genes, including the melanoma differentiation-associated protein 5 (MDA-5) gene, was relatively weak compared to infection with the corresponding H2N3 virus. Our findings highlight that avian skeletal muscle fibers are capable of productive influenza virus replication and are a potential tissue source of infection. IMPORTANCE: Infection with high-pathogenicity H5N1 viruses in ducks is often asymptomatic, and skeletal muscle from such birds could be a source of infection of humans and animals. Little is known about the ability of influenza A viruses to replicate in avian skeletal muscle fibers. We show here that cultured chicken and duck myotubes were highly susceptible to infection with both low- and high-pathogenicity avian influenza viruses. Infected myotubes of both avian species displayed rapid virus accumulation, apoptosis, and extensive cellular damage. Our results indicate that avian skeletal muscle fibers of chicken and duck could be significant contributors to progeny production of highly pathogenic H5N1 viruses.

Elucidation of the Epitranscriptomic RNA Modification Landscape of Chikungunya Virus.

The genomes of positive-sense (+) single-stranded RNA (ssRNA) viruses are believed to be subjected to a wide range of RNA modifications. In this study, we focused on the chikungunya virus (CHIKV) as a model (+) ssRNA virus to study the landscape of viral RNA modification in infected human cells. Among the 32 distinct RNA modifications analysed by mass spectrometry, inosine was found enriched in the genomic CHIKV RNA. However, orthogonal validation by Illumina RNA-seq analyses did not identify any inosine modification along the CHIKV RNA genome. Moreover, CHIKV infection did not alter the expression of ADAR1 isoforms, the enzymes that catalyse the adenosine to inosine conversion. Together, this study highlights the importance of a multidisciplinary approach to assess the presence of RNA modifications in viral RNA genomes.

N6-methyladenosine modification is not a general trait of viral RNA genomes.

Despite the nuclear localization of the m6A machinery, the genomes of multiple exclusively-cytoplasmic RNA viruses, such as chikungunya (CHIKV) and dengue (DENV), are reported to be extensively m6A-modified. However, these findings are mostly based on m6A-Seq, an antibody-dependent technique with a high rate of false positives. Here, we address the presence of m6A in CHIKV and DENV RNAs. For this, we combine m6A-Seq and the antibody-independent SELECT and nanopore direct RNA sequencing techniques with functional, molecular, and mutagenesis studies. Following this comprehensive analysis, we find no evidence of m6A modification in CHIKV or DENV transcripts. Furthermore, depletion of key components of the host m6A machinery does not affect CHIKV or DENV infection. Moreover, CHIKV or DENV infection has no effect on the m6A machinery's localization. Our results challenge the prevailing notion that m6A modification is a general feature of cytoplasmic RNA viruses and underscore the importance of validating RNA modifications with orthogonal approaches.

Mammalian innate resistance to highly pathogenic avian influenza H5N1 virus infection is mediated through reduced proinflammation and infectious virus release.

Respiratory epithelial cells and macrophages are the key innate immune cells that play an important role in the pathogenesis of influenza A virus infection. We found that these two cell types from both human and pig showed comparable susceptibilities to initial infection with a highly pathogenic avian influenza (HPAI) H5N1 virus (A/turkey/Turkey/1/05) and a moderately pathogenic human influenza H1N1 virus (A/USSR/77), but there were contrasting differences in host innate immune responses. Human cells mounted vigorous cytokine (tumor necrosis factor alpha [TNF-α] and interleukin-6 [IL-6]) and chemokine (CXCL9, CXCL10, and CXCL11) responses to H5N1 virus infection. However, pig epithelial cells and macrophages showed weak or no TNF-α and chemokine induction with the same infections. The apparent lack of a strong proinflammatory response, corroborated by the absence of TNF-α induction in H5N1 virus-challenged pigs, coincided with greater cell death and the reduced release of infectious virus from infected pig epithelial cells. Suppressor of cytokine signaling 3 (SOCS3), a protein suppressor of the JAK-STAT pathway, was constitutively highly expressed and transcriptionally upregulated in H5N1 virus-infected pig epithelial cells and macrophages, in contrast to the corresponding human cells. The overexpression of SOCS3 in infected human macrophages dampened TNF-α induction. In summary, we found that the reported low susceptibility of pigs to contemporary Eurasian HPAI H5N1 virus infections coincides at the level of innate immunity of respiratory epithelial cells and macrophages with a reduced output of viable virus and an attenuated proinflammatory response, possibly mediated in part by SOCS3, which could serve as a target in the treatment or prevention of virus-induced hypercytokinemia, as observed for humans.

m6A Regulates the Stability of Cellular Transcripts Required for Efficient KSHV Lytic Replication.

The epitranscriptomic modification N6-methyladenosine (m6A) is a ubiquitous feature of the mammalian transcriptome. It modulates mRNA fate and dynamics to exert regulatory control over numerous cellular processes and disease pathways, including viral infection. Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation from the latent phase leads to the redistribution of m6A topology upon both viral and cellular mRNAs within infected cells. Here we investigate the role of m6A in cellular transcripts upregulated during KSHV lytic replication. Our results show that m6A is crucial for the stability of the GPRC5A mRNA, whose expression is induced by the KSHV latent-lytic switch master regulator, the replication and transcription activator (RTA) protein. Moreover, we demonstrate that GPRC5A is essential for efficient KSHV lytic replication by directly regulating NFκB signalling. Overall, this work highlights the central importance of m6A in modulating cellular gene expression to influence viral infection.

A simplified but robust method for the isolation of avian and mammalian muscle satellite cells.

BACKGROUND: Current methods of isolation of muscle satellite cells from different animal species are highly variable making inter-species comparisons problematic. This variation mainly stems from the use of different proteolytic enzymes to release the satellite cells from the muscle tissue (sometimes a single enzyme is used but often a combination of enzymes is preferred) and the different extracellular matrix proteins used to coat culture ware. In addition, isolation of satellite cells is frequently laborious and sometimes may require pre-plating of the cell preparation on uncoated flasks or Percoll centrifugation to remove contaminating fibroblasts. The methodology employed to isolate and culture satellite cells in vitro can critically determine the fusion of myoblasts into multi-nucleated myotubes. These terminally differentiated myotubes resemble mature myofibres in the muscle tissue in vivo, therefore optimal fusion is a keystone of in vitro muscle culture. Hence, a simple method of muscle satellite cell isolation and culture of different vertebrate species that can result in a high fusion rate is highly desirable. RESULTS: We demonstrate here a relatively simple and rapid method of isolating highly enriched muscle satellite cells from different avian and mammalian species. In brief, muscle tissue was mechanically dissociated, digested with a single enzyme (pronase), triturated with a 10-ml pipette, filtered and directly plated onto collagen coated flasks. Following this method and after optimization of the cell culture conditions, excellent fusion rates were achieved in the duck, chicken, horse and cow (with more than 50% cell fusion), and to a lesser extent pig, pointing to pronase as a highly suitable enzyme to release satellite cells from muscle tissue. CONCLUSIONS: Our simplified method presents a quick and simple alternative to isolating highly enriched muscle satellite cell cultures which can subsequently rapidly differentiate into well developed primary myotubes. The use of the same isolation protocol allows better inter-species comparisons of muscle satellite cells. Of all the farm animal species investigated, harvested chicken muscle cells showed the highest percentage of muscle satellite cells, and equine muscle cells presented the highest fusion index, an impressive ≈ 77%. Porcine cells displayed the lowest amount of satellite cells but still achieved a modest fusion rate of ≈ 41%.

From A to m6A: The Emerging Viral Epitranscriptome.

There are over 100 different chemical RNA modifications, collectively known as the epitranscriptome. N6-methyladenosine (m6A) is the most commonly found internal RNA modification in cellular mRNAs where it plays important roles in the regulation of the mRNA structure, stability, translation and nuclear export. This modification is also found in viral RNA genomes and in viral mRNAs derived from both RNA and DNA viruses. A growing body of evidence indicates that m6A modifications play important roles in regulating viral replication by interacting with the cellular m6A machinery. In this review, we will exhaustively detail the current knowledge on m6A modification, with an emphasis on its function in virus biology.

m6A: Widespread regulatory control in virus replication.

N6-methyladenosine (m6A) is a highly pervasive and dynamic modification found on eukaryotic RNA. Despite the failure to comprehend the true regulatory potential of this epitranscriptomic mark for decades, our knowledge of m6A has rapidly expanded in recent years. The modification has now been functionally linked to all stages of mRNA metabolism and demonstrated to regulate a variety of biological processes. Furthermore, m6A has been identified on transcripts encoded by a wide range of viruses. Studies to investigate m6A function in viral-host interactions have highlighted distinct roles indicating widespread regulatory control over viral life cycles. As a result, unveiling the true influence of m6A modification could revolutionise our comprehension of the regulatory mechanisms controlling viral replication. This article is part of a Special Issue entitled: mRNA modifications in gene expression control edited by Dr. Soller Matthias and Dr. Fray Rupert.

The Tudor SND1 protein is an m6A RNA reader essential for replication of Kaposi's sarcoma-associated herpesvirus.

N6-methyladenosine (m6A) is the most abundant internal RNA modification of cellular mRNAs. m6A is recognised by YTH domain-containing proteins, which selectively bind to m6A-decorated RNAs regulating their turnover and translation. Using an m6A-modified hairpin present in the Kaposi's sarcoma associated herpesvirus (KSHV) ORF50 RNA, we identified seven members from the 'Royal family' as putative m6A readers, including SND1. RIP-seq and eCLIP analysis characterised the SND1 binding profile transcriptome-wide, revealing SND1 as an m6A reader. We further demonstrate that the m6A modification of the ORF50 RNA is critical for SND1 binding, which in turn stabilises the ORF50 transcript. Importantly, SND1 depletion leads to inhibition of KSHV early gene expression showing that SND1 is essential for KSHV lytic replication. This work demonstrates that members of the 'Royal family' have m6A-reading ability, greatly increasing their epigenetic functions beyond protein methylation.

Utilising proteomic approaches to understand oncogenic human herpesviruses (Review).

The γ-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are successful pathogens, each infecting a large proportion of the human population. These viruses persist for the life of the host and may each contribute to a number of malignancies, for which there are currently no cures. Large-scale proteomic-based approaches provide an excellent means of increasing the collective understanding of the proteomes of these complex viruses and elucidating their numerous interactions within the infected host cell. These large-scale studies are important for the identification of the intricacies of viral infection and the development of novel therapeutics against these two important pathogens.

Kaposi's sarcoma-associated herpesvirus ORF57 protein: exploiting all stages of viral mRNA processing.

Nuclear mRNA export is a highly complex and regulated process in cells. Cellular transcripts must undergo successful maturation processes, including splicing, 5'-, and 3'-end processing, which are essential for assembly of an export competent ribonucleoprotein particle. Many viruses replicate in the nucleus of the host cell and require cellular mRNA export factors to efficiently export viral transcripts. However, some viral mRNAs undergo aberrant mRNA processing, thus prompting the viruses to express their own specific mRNA export proteins to facilitate efficient export of viral transcripts and allowing translation in the cytoplasm. This review will focus on the Kaposi's sarcoma-associated herpesvirus ORF57 protein, a multifunctional protein involved in all stages of viral mRNA processing and that is essential for virus replication. Using the example of ORF57, we will describe cellular bulk mRNA export pathways and highlight their distinct features, before exploring how the virus has evolved to exploit these mechanisms.