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.

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.

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.

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.

Exploration of microRNAs as transcriptional regulator in mumps virus infection through computational studies.

Mumps is a common childhood infection caused by the mumps virus (MuV). Aseptic meningitis and encephalitis are usual symptoms of mumps together with orchitis and oophoritis that can arise in males and females, respectively. We have used computational tools: RNA22, miRanda and psRNATarget to predict the microRNA-mRNA binding sites to find the putative microRNAs playing role in the host response to mumps virus infection. Our computational studies indicate that hsa-mir-3155a is most likely involved in mumps infection. This was further investigated by the prediction of binding sites of hsa-mir-3155a to the MuV genome. Additionally, structure prediction using MC-Fold and MC-Sym, respectively has been applied to predict the 3D structures of miRNA and mRNA. The miRNA-mRNA interaction profile between has been confirmed through molecular docking simulation studies. Taken together, the putative miRNA (hsa_miR_6794_5p) has been found to be most likely involved in the regulation of transcriptional activity in the MuV infection.

Role of RNA structural plasticity in modulating HIV-1 genome packaging and translation.

HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5' capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5' polyA element, resulting in formation of an extended 5' polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5' polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs. We identified mutations that stabilize the 5' polyA hairpin structure in 3G RNAs, which promote RNA dimerization and Gag binding without sequestering the 5' cap. None of these 3G transcripts were competitively packaged, confirming that cap exposure is a dominant negative determinant of viral genome packaging. For all RNAs examined, conformations that favored 5' cap exposure were both poorly packaged and more efficiently translated than those that favored 5' cap sequestration. We propose that structural plasticity of 5' polyA and other conserved RNA elements place the 5' leader on a thermodynamic tipping point for low-energetic (~3 kcal/mol) control of global transcript structure and function.

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.

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.

Structural Analysis of the Hepatitis B Virus RNA Encapsidation Signal ε by Selective 2'-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE).

RNA structure is crucial for RNA function, including in viral cis-elements such as the hepatitis B virus (HBV) RNA encapsidation signal ε. Interacting with the viral polymerase ε mediates packaging of the pregenomic (pg) RNA into capsids, initiation of reverse transcription, and it affects the mRNA functions of pgRNA. As free RNA, the 61-nucleotide (nt) ε sequence adopts a bipartite stem-loop structure with a central bulge and an apical loop. Due to stable Watson-Crick base pairing, this was already predicted by early RNA folding programs and confirmed by classical enzymatic and chemical structure probing. A newer, high-resolution probing technique exploits the selective acylation of solvent-accessible 2'-hydroxyls in the RNA backbone by electrophilic compounds such as 2-methylnicotinic acid imidazolide (NAI), followed by mapping of the modified sites by primer extension. This SHAPE principle has meanwhile been extended to numerous applications. Here we provide a basic protocol for NAI-based SHAPE of isolated HBV ε RNA which already provided insights into the impact of mutations, and preliminarily, of polymerase binding on the RNA structural dynamics. While the focus is on NAI modification, we also briefly cover target RNA preparation by in vitro transcription, primer extension using a radiolabeled primer, and analysis of the resulting cDNAs by denaturing polyacrylamide gelelectrophoresis (PAGE). Given the high tolerance of SHAPE chemistry to different conditions, including applicability in live cells, we expect this technique to greatly facilitate deciphering the conformational dynamics underlying the various functions of the ε element, especially in concert with the recently solved three-dimensional structure of the free RNA.

Analysis of N6-Methyladenosine Modification of HBV RNA by Methylated RNA Immunoprecipitation.

Of all the chemical modifications of RNAs, the N6-methyladenosine (m6A) modification is the most prevalent and well-characterized RNA modification that is functionally implicated in a wide range of biological processes. The m6A modification occurs in hepatitis B virus (HBV) RNAs and this modification regulates the HBV life cycle in several ways. Thus, understanding the mechanisms underlying m6A modification of HBV RNAs is crucial in understanding HBV infectious process and associated pathogenesis. Here, we describe the currently utilized method in the detection and characterization of m6A-methylated RNAs during viral infection.

The Phlebovirus Ribonucleoprotein: An Overview.

In negative strand RNA viruses, ribonucleoproteins, not naked RNA, constitute the template used by the large protein endowed with polymerase activity for replicating and transcribing the viral genome. Here we give an overview of the structures and functions of the ribonucleoprotein from phleboviruses. The nucleocapsid monomer, which constitutes the basic structural unit, possesses a flexible arm allowing for a conformational switch between a closed monomeric state and the formation of a polymeric filamentous structure competent for viral RNA binding and encapsidation in the open state of N. The modes of N-N oligomerization as well as interactions with vRNA are described. Finally, recent advances in tomography open exciting perspectives for a more complete understanding of N-L interactions and the design of specific antiviral compounds.

Characterization of RVFV Nucleocapsid Protein Binding Sites on RNA by iCLIP-seq.

The nucleocapsid protein (N) in Rift Valley fever virus is an RNA-binding protein that functions in viral transcription, replication, and packaging. In this chapter, the method for studying protein-RNA interactions in context of viral infection using individual nucleotide resolution, cross-linking, immunoprecipitation, and sequencing (iCLIP-seq) is explained. The method is useful for identifying the interactions between both host and viral RNAs with N and can identify RNA motifs that interact with the protein of interest.

Nucleotide Resolution Mapping of Rift Valley Fever Virus Nucleoprotein-Genome RNA Interactions.

Rift Valley fever virus (RVFV; genus Phlebovirus, family Phenuiviridae, order Bunyavirales) is a mosquito-borne zoonotic pathogen endemic in Africa. Its negative-stranded genomic RNA (vRNA) is divided into three segments termed L, M, and S. Both vRNAs and antigenomic cRNAs are encapsidated by viral nucleoprotein (N) to form nucleocapsids, which constitute the template for genome transcription and replication. Based on a number of electron microscopy and structural studies, the viral RNAs of negative-strand RNA viruses, including phleboviruses, are commonly considered to be entirely and uniformly covered by N protein. However, high resolution data supporting this notion was missing to date.Here, we describe a method how to globally map all N-RNA interactions of RVFV by using iCLIP (individual-nucleotide resolution UV cross-linking and immunoprecipitation). The protocol is based on covalent cross-linking of direct protein-RNA interactions by UV irradiation. Following sample lysis, a selective isolation of N in complex with its RNA targets is achieved by immunoprecipitation. Then, N-RNA complexes are separated by SDS-PAGE, and after membrane transfer, RNA is isolated and subjected to library preparation and high-throughput sequencing. We explain how the standard iCLIP protocol can be adapted to RVFV N-RNA interaction studies. The protocol describes mapping of all N interactions with the vRNAs and cRNAs derived either from RVFV particles or from infected cells.

Bacillaceae serine proteases and Streptomyces epsilon-poly-L-lysine synergistically inactivate Caliciviridae by inhibiting RNA genome release.

Human norovirus (HuNoV) is an enteric infectious pathogen belonging to the Caliciviridae family that causes occasional epidemics. Circulating alcohol-tolerant viral particles that are readily transmitted via food-borne routes significantly contribute to the global burden of HuNoV-induced gastroenteritis. Moreover, contact with enzymes secreted by other microorganisms in the environment can impact the infectivity of viruses. Hence, understanding the circulation dynamics of Caliciviridae is critical to mitigating epidemics. Accordingly, in this study, we screened whether environmentally abundant secretase components, particularly proteases, affect Caliciviridae infectivity. Results showed that combining Bacillaceae serine proteases with epsilon-poly-L-lysine (EPL) produced by Streptomyces-a natural antimicrobial-elicited anti-Caliciviridae properties, including against the epidemic HuNoV GII.4_Sydney_2012 strain. In vitro and in vivo biochemical and virological analyses revealed that EPL has two unique synergistic viral inactivation functions. First, it maintains an optimal pH to promote viral surface conformational changes to the protease-sensitive structure. Subsequently, it inhibits viral RNA genome release via partial protease digestion at the P2 and S domains in the VP1 capsid. This study provides new insights regarding the high-dimensional environmental interactions between bacteria and Caliciviridae, while promoting the development of protease-based anti-viral disinfectants.

Sensing of an HIV-1-Derived Single-Stranded RNA-Oligonucleotide Induces Arginase 1-Mediated Tolerance.

Small synthetic oligodeoxynucleotides (ODNs) can mimic microbial nucleic acids by interacting with receptor systems and promoting immunostimulatory activities. Nevertheless, some ODNs can act differently on the plasmacytoid dendritic cell (pDC) subset, shaping their immunoregulatory properties and rendering them suitable immunotherapeutic tools in several clinical settings for treating overwhelming immune responses. We designed HIV-1-derived, DNA- and RNA-based oligonucleotides (gag, pol, and U5 regions) and assessed their activity in conferring a tolerogenic phenotype to pDCs in skin test experiments. RNA-but not DNA-oligonucleotides are capable of inducing tolerogenic features in pDCs. Interestingly, sensing the HIV-1-derived single-stranded RNA-gag oligonucleotide (RNA-gag) requires both TLR3 and TLR7 and the engagement of the TRIF adaptor molecule. Moreover, the induction of a suppressive phenotype in pDCs by RNA-gag is contingent upon the induction and activation of the immunosuppressive enzyme Arginase 1. Thus, our data suggest that sensing of the synthetic RNA-gag oligonucleotide in pDCs can induce a suppressive phenotype in pDCs, a property rendering RNA-gag a potential tool for therapeutic strategies in allergies and autoimmune diseases.

May I Help You with Your Coat? HIV-1 Capsid Uncoating and Reverse Transcription.

The human immunodeficiency virus type 1 (HIV-1) capsid is a protein core formed by multiple copies of the viral capsid (CA) protein. Inside the capsid, HIV-1 harbours all the viral components required for replication, including the genomic RNA and viral enzymes reverse transcriptase (RT) and integrase (IN). Upon infection, the RT transforms the genomic RNA into a double-stranded DNA molecule that is subsequently integrated into the host chromosome by IN. For this to happen, the viral capsid must open and release the viral DNA, in a process known as uncoating. Capsid plays a key role during the initial stages of HIV-1 replication; therefore, its stability is intimately related to infection efficiency, and untimely uncoating results in reverse transcription defects. How and where uncoating takes place and its relationship with reverse transcription is not fully understood, but the recent development of novel biochemical and cellular approaches has provided unprecedented detail on these processes. In this review, we present the latest findings on the intricate link between capsid stability, reverse transcription and uncoating, the different models proposed over the years for capsid uncoating, and the role played by other cellular factors on these processes.

Inhibition of SARS-CoV-2 Replication by Self-Assembled siRNA Nanoparticles Targeting Multiple Highly Conserved Viral Sequences.

Coronavirus infectious disease 2019 (COVID-19), caused by severe acute respiratory virus type 2 (SARS-CoV-2), has caused a global public health crisis. As an RNA virus, the high gene mutability of SARS-CoV-2 poses significant challenges to the development of broad-spectrum vaccines and antiviral therapeutics. There remains a lack of specific therapeutics directly targeting SARS-CoV-2. With the ability to efficiently inhibit the expression of target genes in a sequence-specific way, small interfering RNA (siRNA) therapy has exhibited significant potential in antiviral and other disease treatments. In this work, we presented a highly effective self-assembled siRNA nanoparticle targeting multiple highly conserved regions of SARS-CoV-2. The siRNA sequences targeting viral conserved regions were first screened and evaluated by their thermodynamic features, off-target effects, and secondary structure toxicities. RNA motifs including siRNA sequences were then designed and self-assembled into siRNA nanoparticles. These siRNA nanoparticles demonstrated remarkable uniformity and stability and efficiently entered cells directly through cellular endocytic pathways. Moreover, these nanoparticles effectively inhibited the replication of SARS-CoV-2, exhibiting a superior inhibitory effect compared to free siRNA. These results demonstrated that these self-assembled siRNA nanoparticles targeting highly conserved regions of SARS-CoV-2 represent highly effective antiviral candidates for the treatment of infections, and are promisingly effective against current and future viral variants.

Evolution of RNA Viruses: Reasons for the Existence of Separate Plus, Minus, and Double-Strand Replication Strategies.

Plus, minus, and double-strand RNA viruses are all found in nature. We use computational models to study the relative success of these strategies. We consider translation, replication, and virion assembly inside one cell, and transmission of virions between cells. For viruses which do not incorporate a polymerase in the capsid, transmission of only plus strands is the default strategy because virions containing minus strands are not infectious. Packaging only plus strands has a significant advantage if the number of RNA strands produced per cell is larger than the number of capsids. In this case, by not packaging minus strands, the virus produces more plus-strand virions. Therefore, plus-strand viruses are selected at low multiplicity of infection. However, at high multiplicity of infection, it is preferable to package both strands because the additional minus virions produced are helpful when there are multiple infections per cell. The fact that plus-strand viruses are widespread while viruses that package both strands are not seen in nature suggests that RNA strands are indeed produced in excess over capsids, and that the multiplicity of infection is not sufficiently high to favor the production of both kinds of virions. For double-strand viruses, we show that it is advantageous to produce only plus strands from the double strand within the cell, as is observed in real viruses. The reason for the success of minus-strand viruses is more puzzling initially. For viruses that incorporate a polymerase in the virion, minus virions are infectious. However, this is not sufficient to explain the success of minus-strand viruses, because in this case, viruses that package both strands outcompete those that package only minus or only plus. Real minus-strand viruses make use of replicable strands that are coated by a nucleoprotein, and separate translatable plus strands that are uncoated. Here we show that when there are distinct replicable and translatable strands, minus-strand viruses are selected.

Functional Analysis of GRSF1 in the Nuclear Export and Translation of Influenza A Virus mRNAs.

Influenza A viruses (IAV) utilize host proteins throughout their life cycle to infect and replicate in their hosts. We previously showed that host adaptive mutations in avian IAV PA help recruit host protein G-Rich RNA Sequence Binding Factor 1 (GRSF1) to the nucleoprotein (NP) 5' untranslated region (UTR), leading to the enhanced nuclear export and translation of NP mRNA. In this study, we evaluated the impact of GRSF1 in the viral life cycle. We rescued and characterized a 2009 pH1N1 virus with a mutated GRSF1 binding site in the 5' UTR of NP mRNA. Mutant viral growth was attenuated relative to pH1N1 wild-type (WT) in mammalian cells. We observed a specific reduction in the NP protein production and cytosolic accumulation of NP mRNAs, indicating a critical role of GRSF1 in the nuclear export of IAV NP mRNAs. Further, in vitro-transcribed mutated NP mRNA was translated less efficiently than WT NP mRNA in transfected cells. Together, these findings show that GRSF1 binding is important for both mRNA nuclear export and translation and affects overall IAV growth. Enhanced association of GRSF1 to NP mRNA by PA mutations leads to rapid virus growth, which could be a key process of mammalian host adaptation of IAV.

Identifying small-molecules binding sites in RNA conformational ensembles with SHAMAN.

The rational targeting of RNA with small molecules is hampered by our still limited understanding of RNA structural and dynamic properties. Most in silico tools for binding site identification rely on static structures and therefore cannot face the challenges posed by the dynamic nature of RNA molecules. Here, we present SHAMAN, a computational technique to identify potential small-molecule binding sites in RNA structural ensembles. SHAMAN enables exploring the conformational landscape of RNA with atomistic molecular dynamics simulations and at the same time identifying RNA pockets in an efficient way with the aid of probes and enhanced-sampling techniques. In our benchmark composed of large, structured riboswitches as well as small, flexible viral RNAs, SHAMAN successfully identifies all the experimentally resolved pockets and ranks them among the most favorite probe hotspots. Overall, SHAMAN sets a solid foundation for future drug design efforts targeting RNA with small molecules, effectively addressing the long-standing challenges in the field.