WTAP Targets the METTL3 m6A-Methyltransferase Complex to Cytoplasmic Hepatitis C Virus RNA to Regulate Infection.

Modification of the hepatitis C virus (HCV) positive-strand RNA genome by N6-methyladenosine (m6A) regulates the viral life cycle. This life cycle takes place solely in the cytoplasm, while m6A addition on cellular mRNA takes place in the nucleus. Thus, the mechanisms by which m6A is deposited on the viral RNA have been unclear. In this work, we find that m6A modification of HCV RNA by the m6A-methyltransferase proteins methyltransferase-like 3 and 14 (METTL3 and METTL14) is regulated by Wilms' tumor 1-associating protein (WTAP). WTAP, a predominantly nuclear protein, is an essential member of the cellular mRNA m6A-methyltransferase complex and known to target METTL3 to mRNA. We found that HCV infection induces localization of WTAP to the cytoplasm. Importantly, we found that WTAP is required for both METTL3 interaction with HCV RNA and m6A modification across the viral RNA genome. Further, we found that WTAP, like METTL3 and METTL14, negatively regulates the production of infectious HCV virions, a process that we have previously shown is regulated by m6A. Excitingly, WTAP regulation of both HCV RNA m6A modification and virion production was independent of its ability to localize to the nucleus. Together, these results reveal that WTAP is critical for HCV RNA m6A modification by METTL3 and METTL14 in the cytoplasm. IMPORTANCE Positive-strand RNA viruses such as HCV represent a significant global health burden. Previous work has described that HCV RNA contains the RNA modification m6A and how this modification regulates viral infection. Yet, how this modification is targeted to HCV RNA has remained unclear due to the incompatibility of the nuclear cellular processes that drive m6A modification with the cytoplasmic HCV life cycle. In this study, we present evidence for how m6A modification is targeted to HCV RNA in the cytoplasm by a mechanism in which WTAP recruits the m6A-methyltransferase METTL3 to HCV RNA. This targeting strategy for m6A modification of cytoplasmic RNA viruses is likely relevant for other m6A-modified positive-strand RNA viruses with cytoplasmic life cycles such as enterovirus 71 and SARS-CoV-2 and provides an exciting new target for potential antiviral therapies.

WTAP targets the METTL3 m 6 A-methyltransferase complex to cytoplasmic hepatitis C virus RNA to regulate infection.

Modification of the hepatitis C virus (HCV) positive-strand RNA genome by N6-methyladenosine (m 6 A) regulates the viral lifecycle. This lifecycle takes place solely in the cytoplasm, while m 6 A addition on cellular mRNA takes place in the nucleus. Thus, the mechanisms by which m 6 A is deposited on the viral RNA have been unclear. In this work, we find that m 6 A modification of HCV RNA by the m 6 A-methyltransferase proteins METTL3 and METTL14 is regulated by WTAP. WTAP, a predominantly nuclear protein, is an essential member of the cellular mRNA m 6 A-methyltransferase complex and known to target METTL3 to mRNA. We found that HCV infection induces localization of WTAP to the cytoplasm. Importantly, we found that WTAP is required for both METTL3 interaction with HCV RNA and for m 6 A modification across the viral RNA genome. Further, we found that WTAP, like METTL3 and METTL14, negatively regulates the production of infectious HCV virions, a process that we have previously shown is regulated by m 6 A. Excitingly, WTAP regulation of both HCV RNA m 6 A modification and virion production were independent of its ability to localize to the nucleus. Together, these results reveal that WTAP is critical for HCV RNA m 6 A modification by METTL3 and METTL14 in the cytoplasm. IMPORTANCE: Positive-strand RNA viruses such as HCV represent a significant global health burden. Previous work has described how HCV RNA contains the RNA modification m 6 A and how this modification regulates viral infection. Yet, how this modification is targeted to HCV RNA has remained unclear due to the incompatibility of the nuclear cellular processes that drive m 6 A modification with the cytoplasmic HCV lifecycle. In this study, we present evidence for how m 6 A modification is targeted to HCV RNA in the cytoplasm by a mechanism in which WTAP recruits the m 6 A-methyltransferase METTL3 to HCV RNA. This targeting strategy for m 6 A modification of cytoplasmic RNA viruses is likely relevant for other m 6 A-modified positive-strand RNA viruses with cytoplasmic lifecycles such as enterovirus 71 and SARS-CoV-2 and provides an exciting new target for potential antiviral therapies.

FTO Suppresses STAT3 Activation and Modulates Proinflammatory Interferon-Stimulated Gene Expression.

Signaling initiated by type I interferon (IFN) results in the induction of hundreds of IFN-stimulated genes (ISGs). The type I IFN response is important for antiviral restriction, but aberrant activation of this response can lead to inflammation and autoimmunity. Regulation of this response is incompletely understood. We previously reported that the mRNA modification m6A and its deposition enzymes, METTL3 and METTL14 (METTL3/14), promote the type I IFN response by directly modifying the mRNA of a subset of ISGs to enhance their translation. Here, we determined the role of the RNA demethylase fat mass and obesity-associated protein (FTO) in the type I IFN response. FTO, which can remove either m6A or cap-adjacent m6Am RNA modifications, has previously been associated with obesity and body mass index, type 2 diabetes, cardiovascular disease, and inflammation. We found that FTO suppresses the transcription of a distinct set of ISGs, including many known pro-inflammatory genes, and that this regulation requires its catalytic activity but is not through the actions of FTO on m6Am. Interestingly, depletion of FTO led to activation of the transcription factor STAT3, whose role in the type I IFN response is not well understood. This activation of STAT3 increased the expression of a subset of ISGs. Importantly, this increased ISG induction resulting from FTO depletion was partially ablated by depletion of STAT3. Together, these results reveal that FTO negatively regulates STAT3-mediated signaling that induces proinflammatory ISGs during the IFN response, highlighting an important role for FTO in suppression of inflammatory genes.

How RNA modifications regulate the antiviral response.

Induction of the antiviral innate immune response is highly regulated at the RNA level, particularly by RNA modifications. Recent discoveries have revealed how RNA modifications play key roles in cellular surveillance of nucleic acids and in controlling gene expression in response to viral infection. These modifications have emerged as being essential for a functional antiviral response and maintaining cellular homeostasis. In this review, we will highlight these and other discoveries that describe how the antiviral response is controlled by modifications to both viral and cellular RNA, focusing on how mRNA cap modifications, N6-methyladenosine, and RNA editing all contribute to coordinating an efficient response that properly controls viral infection.

Flipping the script: viral capitalization of RNA modifications.

RNA encoded by RNA viruses is highly regulated so that it can function in multiple roles during the viral life cycle. These roles include serving as the mRNA template for translation or the genetic material for replication as well as being packaged into progeny virions. RNA modifications provide an emerging regulatory dimension to the RNA of viruses. Modification of the viral RNA can increase the functional genomic capacity of the RNA viruses without the need to encode and translate additional genes. Further, RNA modifications can facilitate interactions with host or viral RNA-binding proteins that promote replication or can prevent interactions with antiviral RNA-binding proteins. The mechanisms by which RNA viruses facilitate modification of their RNA are diverse. In this review, we discuss some of these mechanisms, including exploring the unknown mechanism by which the RNA of viruses that replicate in the cytoplasm could acquire the RNA modification N6-methyladenosine.

Pervasive tertiary structure in the dengue virus RNA genome.

RNA virus genomes are efficient and compact carriers of biological information, encoding information required for replication both in their primary sequences and in higher-order RNA structures. However, the ubiquity of RNA elements with higher-order folds-in which helices pack together to form complex 3D structures-and the extent to which these elements affect viral fitness are largely unknown. Here we used single-molecule correlated chemical probing to define secondary and tertiary structures across the RNA genome of dengue virus serotype 2 (DENV2). Higher-order RNA structures are pervasive and involve more than one-third of nucleotides in the DENV2 genomic RNA. These 3D structures promote a compact overall architecture and contribute to viral fitness. Disrupting RNA regions with higher-order structures leads to stable, nonreverting mutants and could guide the development of vaccines based on attenuated RNA viruses. The existence of extensive regions of functional RNA elements with tertiary folds in viral RNAs, and likely many other messenger and noncoding RNAs, means that there are significant regions with pocket-containing surfaces that may serve as novel RNA-directed drug targets.

Engineered Oncolytic Poliovirus PVSRIPO Subverts MDA5-Dependent Innate Immune Responses in Cancer Cells.

We are pursuing cancer immunotherapy with a neuro-attenuated recombinant poliovirus, PVSRIPO. PVSRIPO is the live attenuated type 1 (Sabin) poliovirus vaccine carrying a heterologous internal ribosomal entry site (IRES) of human rhinovirus type 2 (HRV2). Intratumoral infusion of PVSRIPO is showing promise in the therapy of recurrent WHO grade IV malignant glioma (glioblastoma), a notoriously treatment-refractory cancer with dismal prognosis. PVSRIPO exhibits profound cytotoxicity in infected neoplastic cells expressing the poliovirus receptor CD155. In addition, it elicits intriguing persistent translation and replication, giving rise to sustained type I interferon (IFN)-dominant proinflammatory stimulation of antigen-presenting cells. A key determinant of the inflammatory footprint generated by neoplastic cell infection and its role in shaping the adaptive response after PVSRIPO tumor infection is the virus's inherent relationship to the host's innate antiviral response. In this report, we define subversion of innate host immunity by PVSRIPO, enabling productive viral translation and cytopathogenicity with extremely low multiplicities of infection in the presence of an active innate antiviral IFN response.IMPORTANCE Engaging innate antiviral responses is considered key for instigating tumor-antigen-specific antitumor immunity with cancer immunotherapy approaches. However, they are a double-edged sword for attempts to enlist viruses in such approaches. In addition to their role in the transition from innate to adaptive immunity, innate antiviral IFN responses may intercept the viral life cycle in cancerous cells, prevent viral cytopathogenicity, and restrict viral spread. This has been shown to reduce overall antitumor efficacy of several proposed oncolytic virus prototypes, presumably by limiting direct cell killing and the ensuing inflammatory profile within the infected tumor. In this report, we outline how an unusual recalcitrance of polioviruses toward innate antiviral responses permits viral cytotoxicity and propagation in neoplastic cells, combined with engaging active innate antiviral IFN responses.

A CRISPR Activation Screen Identifies a Pan-avian Influenza Virus Inhibitory Host Factor.

Influenza A virus (IAV) is a pathogen that poses significant risks to human health. It is therefore critical to develop strategies to prevent influenza disease. Many loss-of-function screens have been performed to identify the host proteins required for viral infection. However, there has been no systematic screen to identify the host factors that, when overexpressed, are sufficient to prevent infection. In this study, we used CRISPR/dCas9 activation technology to perform a genome-wide overexpression screen to identify IAV restriction factors. The major hit from our screen, B4GALNT2, showed inhibitory activity against influenza viruses with an α2,3-linked sialic acid receptor preference. B4GALNT2 overexpression prevented the infection of every avian influenza virus strain tested, including the H5, H9, and H7 subtypes, which have previously caused disease in humans. Thus, we have used CRISPR/dCas9 activation technology to identify a factor that can abolish infection by avian influenza viruses.