The m6A landscape of polyadenylated nuclear (PAN) RNA and its related methylome in the context of KSHV replication.

Polyadenylated nuclear (PAN) RNA is a long noncoding transcript involved in Kaposi's sarcoma-associated herpesvirus (KSHV) lytic reactivation and regulation of cellular and viral gene expression. We have previously shown that PAN RNA has dynamic secondary structure and protein binding profiles that can be influenced by epitranscriptomic modifications. N6-methyladenosine (m6A) is one of the most abundant chemical signatures found in viral RNA genomes and virus-encoded RNAs. Here, we combined antibody-independent next-generation mapping with direct RNA sequencing to address the epitranscriptomic status of PAN RNA in KSHV infected cells. We showed that PAN m6A status is dynamic, reaching the highest number of modifications at the late lytic stages of KSHV infection. Using a newly developed method, termed selenium-modified deoxythymidine triphosphate (SedTTP)-reverse transcription (RT) and ligation assisted PCR analysis of m6A (SLAP), we gained insight into the fraction of modification at identified sites. By applying comprehensive proteomic approaches, we identified writers and erasers that regulate the m6A status of PAN, and readers that can convey PAN m6A phenotypic effects. We verified the temporal and spatial subcellular availability of the methylome components for PAN modification by performing confocal microscopy analysis. Additionally, the RNA biochemical probing (SHAPE-MaP) outlined local and global structural alterations invoked by m6A in the context of full-length PAN RNA. This work represents the first comprehensive overview of the dynamic interplay that takes place between the cellular epitranscriptomic machinery and a specific viral RNA in the context of KSHV infected cells.

Dynamic bulge nucleotides in the KSHV PAN ENE triple helix provide a unique binding platform for small molecule ligands.

Cellular and virus-coded long non-coding (lnc) RNAs support multiple roles related to biological and pathological processes. Several lncRNAs sequester their 3' termini to evade cellular degradation machinery, thereby supporting disease progression. An intramolecular triplex involving the lncRNA 3' terminus, the element for nuclear expression (ENE), stabilizes RNA transcripts and promotes persistent function. Therefore, such ENE triplexes, as presented here in Kaposi's sarcoma-associated herpesvirus (KSHV) polyadenylated nuclear (PAN) lncRNA, represent targets for therapeutic development. Towards identifying novel ligands targeting the PAN ENE triplex, we screened a library of immobilized small molecules and identified several triplex-binding chemotypes, the tightest of which exhibits micromolar binding affinity. Combined biophysical, biochemical, and computational strategies localized ligand binding to a platform created near a dinucleotide bulge at the base of the triplex. Crystal structures of apo (3.3 Å) and ligand-soaked (2.5 Å) ENE triplexes, which include a stabilizing basal duplex, indicate significant local structural rearrangements within this dinucleotide bulge. MD simulations and a modified nucleoside analog interference technique corroborate the role of the bulge and the base of the triplex in ligand binding. Together with recently discovered small molecules that reduce nuclear MALAT1 lncRNA levels by engaging its ENE triplex, our data supports the potential of targeting RNA triplexes with small molecules.

Insights into the secondary and tertiary structure of the Bovine Viral Diarrhea Virus Internal Ribosome Entry Site.

The internal ribosome entry site (IRES) RNA of bovine viral diarrhoea virus (BVDV), an economically significant Pestivirus, is required for the cap-independent translation of viral genomic RNA. Thus, it is essential for viral replication and pathogenesis. We applied a combination of high-throughput biochemical RNA structure probing (SHAPE-MaP) and in silico modelling approaches to gain insight into the secondary and tertiary structures of BVDV IRES RNA. Our study demonstrated that BVDV IRES RNA in solution forms a modular architecture composed of three distinct structural domains (I-III). Two regions within domain III are represented in tertiary interactions to form an H-type pseudoknot. Computational modelling of the pseudoknot motif provided a fine-grained picture of the tertiary structure and local arrangement of helices in the BVDV IRES. Furthermore, comparative genomics and consensus structure predictions revealed that the pseudoknot is evolutionarily conserved among many Pestivirus species. These studies provide detailed insight into the structural arrangement of BVDV IRES RNA H-type pseudoknot and encompassing motifs that likely contribute to the optimal functionality of viral cap-independent translation element.

Using SHAPE-MaP to probe small molecule-RNA interactions.

RNA is a regulator and catalyst of many cellular processes. Efforts to therapeutically harness RNA began with the discovery of myriad coding and non-coding RNAs and their versatile modes of action. However, due to its dynamic structure and the polar and repetitive nature of its surface, RNA presents a challenging target for drug design. For an RNA to be druggable, it must contain a motif that assumes a nearly fixed and unique conformation that a small molecule can recognize and bind consistently and with high affinity. Hence, reliable methods for determining the secondary and tertiary structures of RNA, and even the features and occupancy of potential drug binding sites are of utmost importance for the effective design of RNA-based therapeutics. Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) has emerged as such a method, by which RNA secondary structure can be probed at single-nucleotide resolution, under a variety of conditions, and in the presence of RNA-specific small-molecule ligands. In this review, we describe an in-depth protocol for using SHAPE-MaP to characterize RNA-small molecule interactions in cell culture (in cellulo). This method can be applied to transcripts of any size or abundance, and to determine the sites and affinities of small molecule binding, making it an essential and versatile tool for drug discovery.

Towards elucidating the structure, function, and druggability of coronavirus cis-acting RNA motifs / Zglebiajac zawilosci struktur, funkcji i lekowalnosci cis motywów RNA u koronawirusów.

Coronaviruses are the causative agents of mild to severe respiratory and intestinal infections in humans. They are the largest RNA viruses, which genomes and encoded RNAs are known to fold into the highly-order structures that play essential roles in the viral replication and infectivity cycle. The recent outbreaks of new pathogenic coronaviruses steered researchers' attention into the possibility of targeting their RNAs directly with novel RNA-specific drugs and therapeutic strategies. In this manuscript, we highlight the recent biochemical and biophysical methodological advancements that yielded more in-depth insight into the structural and functional composition of coronaviruses cis-acting RNA motifs. We discuss the complexity of these RNA regulatory elements, their intermolecular interactions, post-transcriptional regulation, and their potential as druggable targets. We also indicate the location and function of unstructured and highly-conserved regions in coronaviruses RNA genomes representing viable aims for antisense oligonucleotide or CRISPR-based antiviral strategies.

Unveiling the druggable RNA targets and small molecule therapeutics.

The increasing appreciation for the crucial roles of RNAs in infectious and non-infectious human diseases makes them attractive therapeutic targets. Coding and non-coding RNAs frequently fold into complex conformations which, if effectively targeted, offer opportunities to therapeutically modulate numerous cellular processes, including those linked to undruggable protein targets. Despite the considerable skepticism as to whether RNAs can be targeted with small molecule therapeutics, overwhelming evidence suggests the challenges we are currently facing are not outside the realm of possibility. In this review, we highlight the most recent advances in molecular techniques that have sparked a revolution in understanding the RNA structure-to-function relationship. We bring attention to the application of these modern techniques to identify druggable RNA targets and to assess small molecule binding specificity. Finally, we discuss novel screening methodologies that support RNA drug discovery and present examples of therapeutically valuable RNA targets.

Kaposi's sarcoma-associated herpesvirus polyadenylated nuclear RNA: a structural scaffold for nuclear, cytoplasmic and viral proteins.

Kaposi's sarcoma-associated herpes virus (KSHV) polyadenylated nuclear (PAN) RNA facilitates lytic infection, modulating the cellular immune response by interacting with viral and cellular proteins and DNA. Although a number nucleoprotein interactions involving PAN have been implicated, our understanding of binding partners and PAN RNA binding motifs remains incomplete. Herein, we used SHAPE-mutational profiling (SHAPE-MaP) to probe PAN in its nuclear, cytoplasmic or viral environments or following cell/virion lysis and removal of proteins. We thus characterized and put into context discrete RNA structural elements, including the cis-acting Mta responsive element and expression and nuclear retention element (1,2). By comparing mutational profiles in different biological contexts, we identified sites on PAN either protected from chemical modification by protein binding or characterized by a loss of structure. While some protein binding sites were selectively localized, others were occupied in all three biological contexts. Individual binding sites of select KSHV gene products on PAN RNA were also identified in in vitro experiments. This work constitutes the most extensive structural characterization of a viral lncRNA and interactions with its protein partners in discrete biological contexts, providing a broad framework for understanding the roles of PAN RNA in KSHV infection.

The "Topological Train Ride" of a viral long non-coding RNA.

As the notion of small molecule targeting of regulatory viral and cellular RNAs gathers momentum, understanding their structure, and variations thereof, in the appropriate biological context will play a critical role. This is especially true of the ∼1100-nt polyadenylated nuclear (PAN) long non-coding (lnc) RNA of Kaposi's sarcoma herpesvirus (KSHV), whose interaction with viral and cellular proteins is central to lytic infection. Nuclear accumulation of PAN RNA is mediated via a unique triple helical structure at its 3' terminus (within the expression and nuclear retention element, or ENE) which protects it from deadenylation-dependent decay. Additionally, significant levels of PAN RNA have been reported in both the cytoplasm of KSHV-infected cells and in budding virions, leading us to consider which viral and host proteins might associate with, or dissociate from, this lncRNA during its "journey" through the cell. By combining the power of SHAPE-mutational profiling (SHAPE-MaP) with large scale virus culture facilities of the National Cancer Institute, Frederick MD, Sztuba-Solinska et al. have provide the first detailed description of KSHV PAN nucleoprotein complexes in multiple biological contexts, complementing this by mapping sites of recombinant KSHV proteins on an in vitro-synthesized, polyadenylated counterpart.

A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8.

Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA-RNA and RNA-protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. Selective 2'-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3' stem-loop (nucleotides 1868-1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication.

Expression of Alternatively Spliced Human T-Cell Leukemia Virus Type 1 mRNAs Is Influenced by Mitosis and by a Novel cis-Acting Regulatory Sequence.

UNLABELLED: Human T-cell leukemia virus type 1 (HTLV-1) expression depends on the concerted action of Tax, which drives transcription of the viral genome, and Rex, which favors expression of incompletely spliced mRNAs and determines a 2-phase temporal pattern of viral expression. In the present study, we investigated the Rex dependence of the complete set of alternatively spliced HTLV-1 mRNAs. Analyses of cells transfected with Rex-wild-type and Rex-knockout HTLV-1 molecular clones using splice site-specific quantitative reverse transcription (qRT)-PCR revealed that mRNAs encoding the p30Tof, p13, and p12/8 proteins were Rex dependent, while the p21rex mRNA was Rex independent. These findings provide a rational explanation for the intermediate-late temporal pattern of expression of the p30tof, p13, and p12/8 mRNAs described in previous studies. All the Rex-dependent mRNAs contained a 75-nucleotide intronic region that increased the nuclear retention and degradation of a reporter mRNA in the absence of other viral sequences. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis revealed that this sequence formed a stable hairpin structure. Cell cycle synchronization experiments indicated that mitosis partially bypasses the requirement for Rex to export Rex-dependent HTLV-1 transcripts. These findings indicate a link between the cycling properties of the host cell and the temporal pattern of viral expression/latency that might influence the ability of the virus to spread and evade the immune system. IMPORTANCE: HTLV-1 is a complex retrovirus that causes two distinct pathologies termed adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-1-associated myelopathy in about 5% of infected individuals. Expression of the virus depends on the concerted action of Tax, which drives transcription of the viral genome, and Rex, which favors expression of incompletely spliced mRNAs and determines a 2-phase temporal pattern of virus expression. The findings reported in this study revealed a novel cis-acting regulatory element and indicated that mitosis partially bypasses the requirement for Rex to export Rex-dependent HTLV-1 transcripts. Our results add a layer of complexity to the mechanisms controlling the expression of alternatively spliced HTLV-1 mRNAs and suggest a link between the cycling properties of the host cell and the temporal pattern of viral expression/latency that might influence the ability of the virus to spread and evade the immune system.

Structural complexity of Dengue virus untranslated regions: cis-acting RNA motifs and pseudoknot interactions modulating functionality of the viral genome.

The Dengue virus (DENV) genome contains multiple cis-acting elements required for translation and replication. Previous studies indicated that a 719-nt subgenomic minigenome (DENV-MINI) is an efficient template for translation and (-) strand RNA synthesis in vitro. We performed a detailed structural analysis of DENV-MINI RNA, combining chemical acylation techniques, Pb(2+) ion-induced hydrolysis and site-directed mutagenesis. Our results highlight protein-independent 5'-3' terminal interactions involving hybridization between recognized cis-acting motifs. Probing analyses identified tandem dumbbell structures (DBs) within the 3' terminus spaced by single-stranded regions, internal loops and hairpins with embedded GNRA-like motifs. Analysis of conserved motifs and top loops (TLs) of these dumbbells, and their proposed interactions with downstream pseudoknot (PK) regions, predicted an H-type pseudoknot involving TL1 of the 5' DB and the complementary region, PK2. As disrupting the TL1/PK2 interaction, via 'flipping' mutations of PK2, previously attenuated DENV replication, this pseudoknot may participate in regulation of RNA synthesis. Computer modeling implied that this motif might function as autonomous structural/regulatory element. In addition, our studies targeting elements of the 3' DB and its complementary region PK1 indicated that communication between 5'-3' terminal regions strongly depends on structure and sequence composition of the 5' cyclization region.

The HIV-2 Rev-response element: determining secondary structure and defining folding intermediates.

Interaction between the viral protein Rev and the RNA motifs known as Rev response elements (RREs) is required for transport of unspliced and partially spliced human immunodeficiency virus (HIV)-1 and HIV-2 RNAs from the nucleus to the cytoplasm during the later stages of virus replication. A more detailed understanding of these nucleoprotein complexes and the host factors with which they interact should accelerate the development of new antiviral drugs targeting cis-acting RNA regulatory signals. In this communication, the secondary structures of the HIV-2 RRE and two RNA folding precursors have been identified using the SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical probing methodology together with a novel mathematical approach for determining the secondary structures of RNA conformers present in a mixture. A complementary chemical probing technique was also used to support these secondary structure models, to confirm that the RRE2 RNA undergoes a folding transition and to obtain information about the relative positioning of RRE2 substructures in three dimensions. Our analysis collectively suggests that the HIV-2 RRE undergoes two conformational transitions before assuming the energetically most favorable conformer. The 3D models for the HIV-2 RRE and folding intermediates are also presented, wherein the Rev-binding stem-loops (IIB and I) are located coaxially in the former, which is in agreement with previous models for HIV-1 Rev-RRE binding.

Identification of biologically active, HIV TAR RNA-binding small molecules using small molecule microarrays.

Identifying small molecules that selectively bind to structured RNA motifs remains an important challenge in developing potent and specific therapeutics. Most strategies to find RNA-binding molecules have identified highly charged compounds or aminoglycosides that commonly have modest selectivity. Here we demonstrate a strategy to screen a large unbiased library of druglike small molecules in a microarray format against an RNA target. This approach has enabled the identification of a novel chemotype that selectively targets the HIV transactivation response (TAR) RNA hairpin in a manner not dependent on cationic charge. Thienopyridine 4 binds to and stabilizes the TAR hairpin with a Kd of 2.4 µM. Structure-activity relationships demonstrate that this compound achieves activity through hydrophobic and aromatic substituents on a heterocyclic core, rather than cationic groups typically required. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis was performed on a 365-nucleotide sequence derived from the 5' untranslated region (UTR) of the HIV-1 genome to determine global structural changes in the presence of the molecule. Importantly, the interaction of compound 4 can be mapped to the TAR hairpin without broadly disrupting any other structured elements of the 5' UTR. Cell-based anti-HIV assays indicated that 4 inhibits HIV-induced cytopathicity in T lymphocytes with an EC50 of 28 µM, while cytotoxicity was not observed at concentrations approaching 1 mM.

Cracking the Code: Enhancing Molecular Tools for Progress in Nanobiotechnology.

Nature continually refines its processes for optimal efficiency, especially within biological systems. This article explores the collaborative efforts of researchers worldwide, aiming to mimic nature's efficiency by developing smarter and more effective nanoscale technologies and biomaterials. Recent advancements highlight progress and prospects in leveraging engineered nucleic acids and proteins for specific tasks, drawing inspiration from natural functions. The focus is developing improved methods for characterizing, understanding, and reprogramming these materials to perform user-defined functions, including personalized therapeutics, targeted drug delivery approaches, engineered scaffolds, and reconfigurable nanodevices. Contributions from academia, government agencies, biotech, and medical settings offer diverse perspectives, promising a comprehensive approach to broad nanobiotechnology objectives. Encompassing topics from mRNA vaccine design to programmable protein-based nanocomputing agents, this work provides insightful perspectives on the trajectory of nanobiotechnology toward a future of enhanced biomimicry and technological innovation.

Mutations in the antiviral RNAi defense pathway modify Brome mosaic virus RNA recombinant profiles.

RNA interference (RNAi) mechanism targets viral RNA for degradation. To test whether RNAi gene products contributed to viral RNA recombination, a series of Arabidopsis thaliana RNAi-defective mutants were infected with Brome mosaic virus (BMV) RNAs that have been engineered to support crossovers within the RNA3 segment. Single-cross RNA3-RNA1, RNA3-RNA2, and RNA3-RNA3 recombinants accumulated in both the wild-type (wt) and all knock-out lines at comparable frequencies. However, a reduced accumulation of novel 3' mosaic RNA3 recombinants was observed in ago1, dcl2, dcl4, and rdr6 lines but not in wt Col-0 or the dcl3 line. A BMV replicase mutant accumulated a low level of RNA3-RNA1 single-cross recombinants in Col-0 plants while, in a dcl2 dcl4 double mutant, the formation of both RNA3-RNA1 and mosaic recombinants was at a low level. A control infection in the cpr5-2 mutant, a more susceptible BMV Arabidopsis host, generated similar-to-Col-0 profiles of both single-cross and mosaic recombinants, indicating that recombinant profiles were, to some extent, independent of a viral replication rate. Also, the relative growth experiments revealed similar selection pressure for recombinants among the host lines. Thus, the altered recombinant RNA profiles have originated at the level of recombinant formation rather than because of altered selection. In conclusion, the viral replicase and the host RNAi gene products contribute in distinct ways to BMV RNA recombination. Our studies reveal that the antiviral RNAi mechanisms are utilized by plant RNA viruses to increase their variability, reminiscent of phenomena previously demonstrated in fungi.

Label-Free Detection of Human Coronaviruses in Infected Cells Using Enhanced Darkfield Hyperspectral Microscopy (EDHM).

Human coronaviruses (HCoV) are causative agents of mild to severe intestinal and respiratory infections in humans. In the last 15 years, we have witnessed the emergence of three zoonotic, highly pathogenic HCoVs. Thus, early and accurate detection of these viral pathogens is essential for preventing transmission and providing timely treatment and monitoring of drug resistance. Herein, we applied enhanced darkfield hyperspectral microscopy (EDHM), a novel non-invasive, label-free diagnostic tool, to rapidly and accurately identify two strains of HCoVs, i.e., OC43 and 229E. The EDHM technology allows collecting the optical image with spectral and spatial details in a single measurement without direct contact between the specimen and the sensor. Thus, it can directly map spectral signatures specific for a given viral strain in a complex biological milieu. Our study demonstrated distinct spectral patterns for HCoV-OC43 and HCoV-229E virions in the solution, serving as distinguishable parameters for their differentiation. Furthermore, spectral signatures obtained for both HCoV strains in the infected cells displayed a considerable peak wavelength shift compared to the uninfected cell, indicating that the EDHM is applicable to detect HCoV infection in mammalian cells.

The International Society of RNA Nanotechnology and Nanomedicine (ISRNN): The Present and Future of the Burgeoning Field.

The International Society of RNA Nanotechnology and Nanomedicine (ISRNN) hosts an annual meeting series focused on presenting the latest research achievements involving RNA-based therapeutics and strategies, aiming to expand their current biomedical applications while overcoming the remaining challenges of the burgeoning field of RNA nanotechnology. The most recent online meeting hosted a series of engaging talks and discussions from an international cohort of leading nanotechnologists that focused on RNA modifications and modulation, dynamic RNA structures, overcoming delivery limitations using a variety of innovative platforms and approaches, and addressing the newly explored potential for immunomodulation with programmable nucleic acid nanoparticles. In this Nano Focus, we summarize the main discussion points, conclusions, and future directions identified during this two-day webinar as well as more recent advances to highlight and to accelerate this exciting field.

The Structure-To-Function Relationships of Gammaherpesvirus-Encoded Long Non-Coding RNAs and Their Contributions to Viral Pathogenesis.

Advances in next-generation sequencing have facilitated the discovery of a multitude of long non-coding RNAs (lncRNAs) with pleiotropic functions in cellular processes, disease, and viral pathogenesis. It came as no surprise when viruses were also revealed to transcribe their own lncRNAs. Among them, gammaherpesviruses, one of the three subfamilies of the Herpesviridae, code their largest number. These structurally and functionally intricate non-coding (nc) transcripts modulate cellular and viral gene expression to maintain viral latency or prompt lytic reactivation. These lncRNAs allow for the virus to escape cytosolic surveillance, sequester, and re-localize essential cellular factors and modulate the cell cycle and proliferation. Some viral lncRNAs act as "messenger molecules", transferring information about viral infection to neighboring cells. This broad range of lncRNA functions is achieved through lncRNA structure-mediated interactions with effector molecules of viral and host origin, including other RNAs, proteins and DNAs. In this review, we discuss examples of gammaherpesvirus-encoded lncRNAs, emphasize their unique structural attributes, and link them to viral life cycle, pathogenesis, and disease progression. We will address their potential as novel targets for drug discovery and propose future directions to explore lncRNA structure and function relationship.

Polymorphisms in KSHV-encoded microRNA sequences affect levels of mature viral microRNA in Kaposi Sarcoma lesions.

Background: We previously reported Kaposi sarcoma-associated herpesvirus (KSHV) microRNA sequence variants in clinical samples correlated with increased risk of multicentric Castleman's disease (MCD). We then demonstrated that microRNAs with variant sequence have different maturation and mature microRNA expression in vitro. Here, we illustrate the association between microRNA sequence and changes in mature microRNA levels within Kaposi sarcoma (KS) lesions. Methods: KSHV microRNA sequences were determined from 20 KS lesions and 4 control skin biopsies from individuals evaluated for KS. Levels of mature KSHV microRNAs were measured with 21 custom small RNA qRT-PCR assays using RNA RNU6B as endogenous control. Results: The levels of 13 KSHV-encoded microRNAs were elevated in KS lesions compared to control biopsies. MicroRNA 9-5p was strongly down regulated in South African vs. US biopsies. Low levels of K12-9-5p were associated with single nucleotide polymorphisms (SNPs) in miR-K12-9-5p, 4-5p, 5-3p, 7-3p and pri-miR-K12-3. One SNP in pri-miR-K12-3 resulted in down regulation of miR-K12-6-3p, 8-3p, 10-3p, 12-5p and the upregulation of 5-5p, illustrating sequence variants outside pre-microRNAs were also associated with changes in mature microRNA levels. Conclusions: The levels of mature KSHV-encoded microRNAs in KS lesions correlate with sequence variation reflecting changes in secondary and tertiary RNA structure.