Stress less: Viral mastery of the RNA G-quadruplex.

RNA G-quadruplexes are dynamically regulated during stress and infection. In this issue of Cell Host & Microbe, Schult et al.1 demonstrate that an RNA G-quadruplex conserved across orthoflaviviruses binds hnRNPH1 to mitigate the host stress response, highlighting the potential of this dynamic proviral RNA structure as a pan-flaviviral target.

Tanomastat exerts multi-targeted inhibitory effects on viral capsid dissociation and RNA replication in human enteroviruses.

BACKGROUND: Global cyclical outbreaks of human enterovirus infections has positioned human enterovirus A71 (EV-A71) as a neurotropic virus of clinical importance. However, there remains a scarcity of internationally approved antivirals and vaccines. METHODS: In pursuit of repurposing drugs for combating human enteroviruses, we employed a comprehensive pharmacophore- and molecular docking-based virtual screen targeting EV-A71 capsid protein VP1-4, 3C protease, and 3D polymerase proteins. Among 15 shortlisted ligand candidates, we dissected the inhibitory mechanism of Tanomastat in cell-based studies and evaluated its in vivo efficacy in an EV-A71-infected murine model. FINDINGS: We demonstrated that Tanomastat exerts dose-dependent inhibition on EV-A71 replication, with comparable efficacy profiles in enterovirus species A, B, C, and D in vitro. Time-course studies suggested that Tanomastat predominantly disrupts early process(es) of the EV-A71 replication cycle. Mechanistically, live virus particle tracking and docking predictions revealed that Tanomastat specifically impedes viral capsid dissociation, potentially via VP1 hydrophobic pocket binding. Bypassing its inhibition on entry stages, we utilized EV-A71 replication-competent, 3Dpol replication-defective, and bicistronic IRES reporter replicons to show that Tanomastat also inhibits viral RNA replication, but not viral IRES translation. We further showed that orally administered Tanomastat achieved 85% protective therapeutic effect and alleviated clinical symptoms in EV-A71-infected neonatal mice. INTERPRETATION: Our study establishes Tanomastat as a broad-spectrum anti-enterovirus candidate with promising pre-clinical efficacy, warranting further testing for potential therapeutic application. FUNDING: MOE Tier 2 grants (MOE-T2EP30221-0005, R571-000-068-592, R571-000-076-515, R571-000-074-733) and A∗STARBiomedical Research Council (BMRC).

DeepIRES: a hybrid deep learning model for accurate identification of internal ribosome entry sites in cellular and viral mRNAs.

The internal ribosome entry site (IRES) is a cis-regulatory element that can initiate translation in a cap-independent manner. It is often related to cellular processes and many diseases. Thus, identifying the IRES is important for understanding its mechanism and finding potential therapeutic strategies for relevant diseases since identifying IRES elements by experimental method is time-consuming and laborious. Many bioinformatics tools have been developed to predict IRES, but all these tools are based on structure similarity or machine learning algorithms. Here, we introduced a deep learning model named DeepIRES for precisely identifying IRES elements in messenger RNA (mRNA) sequences. DeepIRES is a hybrid model incorporating dilated 1D convolutional neural network blocks, bidirectional gated recurrent units, and self-attention module. Tenfold cross-validation results suggest that DeepIRES can capture deeper relationships between sequence features and prediction results than other baseline models. Further comparison on independent test sets illustrates that DeepIRES has superior and robust prediction capability than other existing methods. Moreover, DeepIRES achieves high accuracy in predicting experimental validated IRESs that are collected in recent studies. With the application of a deep learning interpretable analysis, we discover some potential consensus motifs that are related to IRES activities. In summary, DeepIRES is a reliable tool for IRES prediction and gives insights into the mechanism of IRES elements.

[When 2'-O-methylation throws a wrench in HIV-1 reverse transcriptase]. / La 2'-O-méthylation : un grain de sable dans les rouages de la transcriptase inverse du VIH-1.

HIV-1 polymerase, commonly known as HIV reverse transcriptase (RT), catalyzes the critical reaction of reverse transcription by synthesizing a double-stranded DNA copy of the viral genomic RNA. During the replication cycle, this synthesized DNA is integrated into the host genome. This entire process is essential for viral replication and is targeted by several antiviral drugs. Numerous studies in biochemistry and structural biology have led to a good understanding of HIV-1 RT functions. However, the discovery of epitranscriptomic marks, such as 2'-O-methylations, on the HIV-1 RNA genome raise the questions about RT's ability to copy RNAs decorated with these biochemical modifications. This review focuses on the importance of RT in the viral cycle, its structure and function and the impact of 2'-O-methylations on its activity and replication regulation, particularly in quiescent cells.

A specific phosphorylation-dependent conformational switch in SARS-CoV-2 nucleocapsid protein inhibits RNA binding.

The nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 encapsidates the viral genome and is essential for viral function. The central disordered domain comprises a serine-arginine-rich (SR) region that is hyperphosphorylated in infected cells. This modification regulates function, although mechanistic details remain unknown. We use nuclear magnetic resonance to follow structural changes occurring during hyperphosphorylation by serine arginine protein kinase 1, glycogen synthase kinase 3, and casein kinase 1, that abolishes interaction with RNA. When eight approximately uniformly distributed sites have been phosphorylated, the SR domain binds the same interface as single-stranded RNA, resulting in complete inhibition of RNA binding. Phosphorylation by protein kinase A does not prevent RNA binding, indicating that the pattern resulting from physiologically relevant kinases is specific for inhibition. Long-range contacts between the RNA binding, linker, and dimerization domains are abrogated, phenomena possibly related to genome packaging and unpackaging. This study provides insight into the recruitment of specific host kinases to regulate viral function.

Transcription start site choice regulates HIV-1 RNA conformation and function.

HIV-1, the causative agent of AIDS, is a retrovirus that packages two copies of unspliced viral RNA as a dimer into newly budding virions. The unspliced viral RNA also serves as an mRNA template for translation of two polyproteins. Recent studies suggest that the fate of the viral RNA (genome or mRNA) is determined at the level of transcription. RNA polymerase II uses heterogeneous transcription start sites to generate major transcripts that differ in only two guanosines at the 5' end. Remarkably, this two-nucleotide difference is sufficient to alter the structure of the 5'-untranslated region and generate two pools of RNA with distinct functions. The presence of both RNA species is needed for optimal viral replication and fitness.

Untangling the pseudoknots of SARS-CoV-2: Insights into structural heterogeneity and plasticity.

Since the onset of the COVID-19 pandemic, one productive area of research has focused on the intricate two- and three-dimensional structures taken on by SARS-CoV-2's RNA genome. These structures control essential viral processes, making them tempting targets for therapeutic intervention. This review focuses on two such structured regions, the frameshift stimulation element (FSE), which controls the translation of viral protein, and the 3' untranslated region (3' UTR), which is thought to regulate genome replication. For the FSE, we discuss its canonical pseudoknot's threaded and unthreaded topologies, as well as the diversity of competing two-dimensional structures formed by local and long-distance base pairing. For the 3' UTR, we review the evidence both for and against the formation of its replication-enabling pseudoknot.

Epitranscriptomic m5C methylation of SARS-CoV-2 RNA regulates viral replication and the virulence of progeny viruses in the new infection.

While the significance of N6-methyladenosine (m6A) in viral regulation has been extensively studied, the functions of 5-methylcytosine (m5C) modification in viral biology remain largely unexplored. In this study, we demonstrate that m5C is more abundant than m6A in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and provide a comprehensive profile of the m5C landscape of SARS-CoV-2 RNA. Knockout of NSUN2 reduces m5C levels in SARS-CoV-2 virion RNA and enhances viral replication. Nsun2 deficiency mice exhibited higher viral burden and more severe lung tissue damages. Combined RNA-Bis-seq and m5C-MeRIP-seq identified the NSUN2-dependent m5C-methylated cytosines across the positive-sense genomic RNA of SARS-CoV-2, and the mutations of these cytosines enhance RNA stability. The progeny SARS-CoV-2 virions from Nsun2 deficiency mice with low levels of m5C modification exhibited a stronger replication ability. Overall, our findings uncover the vital role played by NSUN2-mediated m5C modification during SARS-CoV-2 replication and propose a host antiviral strategy via epitranscriptomic addition of m5C methylation to SARS-CoV-2 RNA.

Potential Transcriptional Enhancers in Coronaviruses: From Infectious Bronchitis Virus to SARS-CoV-2.

Coronaviruses constitute a global threat to human and animal health. It is essential to investigate the long-distance RNA-RNA interactions that approximate remote regulatory elements in strategies, including genome circularization, discontinuous transcription, and transcriptional enhancers, aimed at the rapid replication of their large genomes, pathogenicity, and immune evasion. Based on the primary sequences and modeled RNA-RNA interactions of two experimentally defined coronaviral enhancers, we detected via an in silico primary and secondary structural analysis potential enhancers in various coronaviruses, from the phylogenetically ancient avian infectious bronchitis virus (IBV) to the recently emerged SARS-CoV-2. These potential enhancers possess a core duplex-forming region that could transition between closed and open states, as molecular switches directed by viral or host factors. The duplex open state would pair with remote sequences in the viral genome and modulate the expression of downstream crucial genes involved in viral replication and host immune evasion. Consistently, variations in the predicted IBV enhancer region or its distant targets coincide with cases of viral attenuation, possibly driven by decreased open reading frame (ORF)3a immune evasion protein expression. If validated experimentally, the annotated enhancer sequences could inform structural prediction tools and antiviral interventions.

Insights into the structure of RNPs from segmented negative-sense RNA viruses.

The genome of segmented negative-sense single-stranded RNA viruses, such as influenza virus and bunyaviruses, is coated by viral nucleoproteins (NPs), forming a ribonucleoprotein (RNP). In this issue of Structure, Dick et al.1 expand our knowledge on the RNPs of these viruses by solving the structures of Thogoto virus NP and RNP.

RNA replication-independent, DNA linearization-dependent expression of reporter genes from a SARS-CoV-2 replicon-encoding DNA in human cells.

Replicons, derived from RNA viruses, are genetic constructs retaining essential viral enzyme genes while lacking key structural protein genes. Upon introduction into cells, the genes carried by the replicon RNA are expressed, and the RNA self-replicates, yet viral particle production does not take place. Typically, RNA replicons are transcribed in vitro and are then electroporated in cells. However, it would be advantageous for the replicon to be generated in cells following DNA transfection instead of RNA. In this study, a bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon under control of a T7 promoter was transfected into HEK293T cells engineered to functionally express the T7 RNA polymerase (T7 RNAP). Upon transfection of the BAC DNA, we observed low, but reproducible expression of reporter proteins GFP and luciferase carried by this replicon. Expression of the reporter proteins required linearization of the BAC DNA prior to transfection. Moreover, expression occurred independently of T7 RNAP. Gene expression was also insensitive to remdesivir treatment, suggesting that it did not involve self-replication of replicon RNA. Similar results were obtained in highly SARS-CoV-2 infection-permissive Calu-3 cells. Strikingly, prior expression of the SARS-CoV-2 N protein boosted expression from transfected SARS-CoV-2 RNA replicon but not from the replicon BAC DNA. In conclusion, transfection of a large DNA encoding a coronaviral replicon led to reproducible replicon gene expression through an unidentified mechanism. These findings highlight a novel pathway toward replicon gene expression from transfected replicon cDNA, offering valuable insights for the development of methods for DNA-based RNA replicon applications.

Structures of influenza A and B replication complexes give insight into avian to human host adaptation and reveal a role of ANP32 as an electrostatic chaperone for the apo-polymerase.

Replication of influenza viral RNA depends on at least two viral polymerases, a parental replicase and an encapsidase, and cellular factor ANP32. ANP32 comprises an LRR domain and a long C-terminal low complexity acidic region (LCAR). Here we present evidence suggesting that ANP32 is recruited to the replication complex as an electrostatic chaperone that stabilises the encapsidase moiety within apo-polymerase symmetric dimers that are distinct for influenza A and B polymerases. The ANP32 bound encapsidase, then forms the asymmetric replication complex with the replicase, which is embedded in a parental ribonucleoprotein particle (RNP). Cryo-EM structures reveal the architecture of the influenza A and B replication complexes and the likely trajectory of the nascent RNA product into the encapsidase. The cryo-EM map of the FluB replication complex shows extra density attributable to the ANP32 LCAR wrapping around and stabilising the apo-encapsidase conformation. These structures give new insight into the various mutations that adapt avian strain polymerases to use the distinct ANP32 in mammalian cells.

In vitro reconstitution reveals membrane clustering and RNA recruitment by the enteroviral AAA+ ATPase 2C.

Enteroviruses are a vast genus of positive-sense RNA viruses that cause diseases ranging from common cold to poliomyelitis and viral myocarditis. They encode a membrane-bound AAA+ ATPase, 2C, that has been suggested to serve several roles in virus replication, e.g. as an RNA helicase and capsid assembly factor. Here, we report the reconstitution of full-length, poliovirus 2C's association with membranes. We show that the N-terminal membrane-binding domain of 2C contains a conserved glycine, which is suggested by structure predictions to divide the domain into two amphipathic helix regions, which we name AH1 and AH2. AH2 is the main mediator of 2C oligomerization, and is necessary and sufficient for its membrane binding. AH1 is the main mediator of a novel function of 2C: clustering of membranes. Cryo-electron tomography reveal that several 2C copies mediate this function by localizing to vesicle-vesicle interfaces. 2C-mediated clustering is partially outcompeted by RNA, suggesting a way by which 2C can switch from an early role in coalescing replication organelles and lipid droplets, to a later role where 2C assists RNA replication and particle assembly. 2C is sufficient to recruit RNA to membranes, with a preference for double-stranded RNA (the replicating form of the viral genome). Finally, the in vitro reconstitution revealed that full-length, membrane-bound 2C has ATPase activity and ATP-independent, single-strand ribonuclease activity, but no detectable helicase activity. Together, this study suggests novel roles for 2C in membrane clustering, RNA membrane recruitment and cleavage, and calls into question a role of 2C as an RNA helicase. The reconstitution of functional, 2C-decorated vesicles provides a platform for further biochemical studies into this protein and its roles in enterovirus replication.

Poly(rC)-Binding Protein 2 Does Not Directly Participate in HCV Translation or Replication, but Rather Modulates Genome Packaging.

The hepatitis C virus (HCV) co-opts many cellular factors-including proteins and microRNAs-to complete its life cycle. A cellular RNA-binding protein, poly(rC)-binding protein 2 (PCBP2), was previously shown to bind to the hepatitis C virus (HCV) genome; however, its precise role in the viral life cycle remained unclear. Herein, using the HCV cell culture (HCVcc) system and assays that isolate each step of the viral life cycle, we found that PCBP2 does not have a direct role in viral entry, translation, genome stability, or HCV RNA replication. Rather, our data suggest that PCBP2 depletion only impacts viral RNAs that can undergo genome packaging. Taken together, our data suggest that endogenous PCBP2 modulates the early steps of genome packaging, and therefore only has an indirect effect on viral translation and RNA replication, likely by increasing the translating/replicating pool of viral RNAs to the detriment of virion assembly.

Molecular Bases and Specificity behind the Activation of the Immune System OAS/RNAse L Pathway by Viral RNA.

The first line of defense against invading pathogens usually relies on innate immune systems. In this context, the recognition of exogenous RNA structures is primordial to fight, notably, against RNA viruses. One of the most efficient immune response pathways is based on the sensing of RNA double helical motifs by the oligoadenylate synthase (OAS) proteins, which in turn triggers the activity of RNase L and, thus, cleaves cellular and viral RNA. In this contribution, by using long-range molecular dynamics simulations, complemented with enhanced sampling techniques, we elucidate the structural features leading to the activation of OAS by interaction with a model double-strand RNA oligomer mimicking a viral RNA. We characterize the allosteric regulation induced by the nucleic acid leading to the population of the active form of the protein. Furthermore, we also identify the free energy profile connected to the active vs. inactive conformational transitions in the presence and absence of RNA. Finally, the role of two RNA mutations, identified as able to downregulate OAS activation, in shaping the protein/nucleic acid interface and the conformational landscape of OAS is also analyzed.

Phase Separation of SARS-CoV-2 Nucleocapsid Protein with TDP-43 Is Dependent on C-Terminus Domains.

The SARS-CoV-2 nucleocapsid protein (N protein) is critical in viral replication by undergoing liquid-liquid phase separation to seed the formation of a ribonucleoprotein (RNP) complex to drive viral genomic RNA (gRNA) translation and in suppressing both stress granules and processing bodies, which is postulated to increase uncoated gRNA availability. The N protein can also form biomolecular condensates with a broad range of host endogenous proteins including RNA binding proteins (RBPs). Amongst these RBPs are proteins that are associated with pathological, neuronal, and glial cytoplasmic inclusions across several adult-onset neurodegenerative disorders, including TAR DNA binding protein 43 kDa (TDP-43) which forms pathological inclusions in over 95% of amyotrophic lateral sclerosis cases. In this study, we demonstrate that the N protein can form biomolecular condensates with TDP-43 and that this is dependent on the N protein C-terminus domain (N-CTD) and the intrinsically disordered C-terminus domain of TDP-43. This process is markedly accelerated in the presence of RNA. In silico modeling suggests that the biomolecular condensate that forms in the presence of RNA is composed of an N protein quadriplex in which the intrinsically disordered TDP-43 C terminus domain is incorporated.

Epstein-Barr virus noncoding RNA EBER1 promotes the expression of a ribosomal protein paralog to boost oxidative phosphorylation.

Epstein-Barr virus (EBV) is a highly successful pathogen that infects ~95% of the adult population and is associated with diverse cancers and autoimmune diseases. The most abundant viral factor in latently infected cells is not a protein but a noncoding RNA called EBV-encoded RNA 1 (EBER1). Even though EBER1 is highly abundant and was discovered over forty years ago, the function of EBER1 has remained elusive. EBER1 interacts with the ribosomal protein L22, which normally suppresses the expression of its paralog L22-like 1 (L22L1). Here we show that when L22 binds EBER1, it cannot suppress L22L1, resulting in L22L1 being expressed and incorporated into ribosomes. We further show that L22L1-containing ribosomes preferentially translate mRNAs involved in the oxidative phosphorylation pathway. Moreover, upregulation of L22L1 is indispensable for growth transformation and immortalization of resting B cells upon EBV infection. Taken together, our results suggest that the function of EBER1 is to modulate host gene expression at the translational level, thus bypassing the need for dysregulating host gene transcription.

Endogenous ZAP affects Zika virus RNA interactome.

One of the most recent advances in the analysis of viral RNA-cellular protein interactions is the Comprehensive Identification of RNA-binding Proteins by Mass Spectrometry (ChIRP-MS). Here, we used ChIRP-MS in mock-infected and Zika-infected wild-type cells and cells knockout for the zinc finger CCCH-type antiviral protein 1 (ZAP). We characterized 'ZAP-independent' and 'ZAP-dependent' cellular protein interactomes associated with flavivirus RNA and found that ZAP affects cellular proteins associated with Zika virus RNA. The ZAP-dependent interactome identified with ChIRP-MS provides potential ZAP co-factors for antiviral activity against Zika virus and possibly other viruses. Identifying the full spectrum of ZAP co-factors and mechanisms of how they act will be critical to understanding the ZAP antiviral system and may contribute to the development of antivirals.

Regulation viral RNA transcription and replication by higher-order RNA structures within the nsp1 coding region of MERS coronavirus.

Coronavirus (CoV) possesses numerous functional cis-acting elements in its positive-strand genomic RNA. Although most of these RNA structures participate in viral replication, the functions of RNA structures in the genomic RNA of CoV in viral replication remain unclear. In this study, we investigated the functions of the higher-order RNA stem-loop (SL) structures SL5B, SL5C, and SL5D in the ORF1a coding region of Middle East respiratory syndrome coronavirus (MERS-CoV) in viral replication. Our approach, using reverse genetics of a bacterial artificial chromosome system, revealed that SL5B and SL5C play essential roles in the discontinuous transcription of MERS-CoV. In silico analyses predicted that SL5C interacts with a bulged stem-loop (BSL) in the 3' untranslated region, suggesting that the RNA structure of SL5C is important for viral RNA transcription. Conversely, SL5D did not affect transcription, but mediated the synthesis of positive-strand genomic RNA. Additionally, the RNA secondary structure of SL5 in the revertant virus of the SL5D mutant was similar to that of the wild-type, indicating that the RNA structure of SL5D can finely tune RNA replication in MERS-CoV. Our data indicate novel regulatory mechanisms of viral RNA transcription and replication by higher-order RNA structures in the MERS-CoV genomic RNA.