Zika virus (ZIKV) infection remains a worldwide concern, and currently no effective treatments or vaccines are available. Novel therapeutics are an avenue of interest that could probe viral RNA-human protein communication to stop viral replication. One specific RNA structure, G-quadruplexes (G4s), possess various roles in viruses and all domains of life, including transcription and translation regulation and genome stability, and serves as nucleation points for RNA liquid-liquid phase separation. Previous G4 studies on ZIKV using a quadruplex forming G-rich sequences Mapper located a potential G-quadruplex sequence in the 3′ terminal region (TR) and was validated structurally using a 25-mer oligo. It is currently unknown if this structure is conserved and maintained in a large ZIKV RNA transcript and its specific roles in viral replication. Using bioinformatic analysis and biochemical assays, we demonstrate that the ZIKV 3′ TR G4 is conserved across all ZIKV isolates and maintains its structure in a 3′ TR full-length transcript. We further established the G4 formation using pyridostatin and the BG4 G4-recognizing antibody binding assays. Our study also demonstrates that the human DEAD-box helicases, DDX3X_132-607 and DDX17_135-555, bind to the 3′ TR and that DDX17_135-555 unfolds the G4 present in the 3′ TR. These findings provide a path forward in potential therapeutic targeting of DDX3X or DDX17’s binding to the 3′ TR G4 region for novel treatments against ZIKV.

The long non-coding RNAs (lncRNAs) other than rRNA and tRNA were earlier assumed to be ‘junk genomic material’. However, recent advancements in genomics methods have highlighted their roles not only in housekeeping but also in the progression of diseases like cancer as well as viral infections. lncRNAs owing to their length, have both short-range and long-range interactions resulting in complex folded structures that recruit various biomolecules enabling lncRNAs to undertake their various biological functions. Using cell lysate pull-down assays increasing number of lnRNAs-interacting proteins are being identified. These interactions can be further exploited to develop targeted novel therapeutic strategies to inhibit lncRNA–protein interactions. This review attempts to succinctly techniques that can identify and characterize the lnRNAs–protein interactions (i.e. affinity, stoichiometry, and thermodynamics). Furthermore, using other sophisticated biophysical techniques, one can also perform size estimations, and determine low-resolution structures. Since these methods study the biomolecules in solution, large-scale structural observations can be performed in real-time. This review attempts to briefly introduce the readers to biochemical and biophysical techniques, such that they can utilize these methods to obtain a holistic characterization of the biomolecules of interest. Additionally, it should be noted that the use of these methods is not limited to the characterization of the interacting molecules but can also be used to determine the efficacy of the therapeutic molecules to disrupt these interactions.

The dicistrovirus intergenic (IGR) IRES uses the most streamlined translation initiation mechanism: the IRES recruits ribosomes directly without using protein factors and initiates translation from a non-AUG codon. Several subtypes of dicistroviruses IRES have been identified; typically, the IRESs adopt two -to three overlapping pseudoknots with key stem-loop and unpaired regions that interact with specific domains of the ribosomal 40S and 60S subunits to direct translation. We previously predicted an atypical IGR IRES structure and a potential -1 programmed frameshift (-1 FS) signal within the genome of the whitefly Bemisia-associated dicistrovirus 2 (BaDV-2). Here, using bicistronic reporters, we demonstrate that the predicted BaDV-2 -1 FS signal can drive -1 frameshifting in vitro via a slippery sequence and a downstream stem-loop structure that would direct the translation of the viral RNA-dependent RNA polymerase. Moreover, the predicted BaDV-2 IGR can support IRES translation in vitro but does so through a mechanism that is not typical of known factorless dicistrovirus IGR IRES mechanisms. Using deletion and mutational analyses, the BaDV-2 IGR IRES is mapped within a 140-nucleotide element and initiates translation from an AUG codon. Moreover, the IRES does not bind directly to purified ribosomes and is sensitive to eIF2 and eIF4A inhibitors NSC1198983 and hippuristanol, respectively, indicating an IRES-mediated factor-dependent mechanism. Biophysical characterization suggests the BaDV-2 IGR IRES contains several stem-loops; however, mutational analysis suggests a model whereby the IRES is unstructured or adopts distinct conformations for translation initiation. In summary, we have provided evidence of the first -1 FS frameshifting signal and a novel factor-dependent IRES mechanism in this dicistrovirus family, thus highlighting the diversity of viral RNA-structure strategies to direct viral protein synthesis.

Monkeypox virus (MPXV) is a double‐stranded DNA virus from the family Poxviridae, which is endemic in West and Central Africa. Various human outbreaks occurred in the 1980s, resulting from a cessation of smallpox vaccination. Recently, MPXV cases have reemerged in non‐endemic nations, and the 2022 outbreak has been declared a public health emergency. Treatment optionsare limited, and many countries lack the infrastructure to provide symptomatic treatments. The development of cost‐effective antivirals could ease severe health outcomes. G‐quadruplexes have been a target of interest in treating viral infections with different chemicals. In the present work, a genomic‐scale mapping of different MPXV isolates highlighted two conserved putative quadruplex‐forming sequences MPXV‐exclusive in 590 isolates. Subsequently, we assessed the G‐quadruplex formation using circular dichroism spectroscopy and solution small‐angle X‐ray scattering. Furthermore, biochemical assays indicated the ability of MPXV quadruplexes to be recognized by two specific G4‐binding partners—Thioflavin T and DHX36. Additionally, our work also suggests that a quadruplex binding small‐molecule with previously reported antiviral activity, TMPyP4, interacts with MPXV G‐quadruplexes with nanomolar affinity in the presence and absence of DHX36. Finally, cell biology experiments suggests that TMPyP4 treatment substantially reduced gene expression of MPXV proteins. In summary, our work provides insights into the G‐quadruplexes from the MPXV genome that can be further exploited to develop therapeutics.

Zika virus (ZIKV) infection causes significant human disease that, with no approved treatment or vaccine, constitutes a major public health concern. Its life cycle entirely relies on the cytoplasmic fate of the viral RNA genome (vRNA) through a fine-tuned equilibrium between vRNA translation, replication, and packaging into new virions, all within virus-induced replication organelles (vROs). In this study, with an RNA interference (RNAi) mini-screening and subsequent functional characterization, we have identified insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) as a new host dependency factor that regulates vRNA synthesis. In infected cells, IGF2BP2 associates with viral NS5 polymerase and redistributes to the perinuclear viral replication compartment. Combined fluorescence in situ hybridization-based confocal imaging, in vitro binding assays, and immunoprecipitation coupled to RT-qPCR showed that IGF2BP2 directly interacts with ZIKV vRNA 3’ nontranslated region. Using ZIKV sub-genomic replicons and a replication-independent vRO induction system, we demonstrated that IGF2BP2 knockdown impairs de novo vRO biogenesis and, consistently, vRNA synthesis. Finally, the analysis of immunopurified IGF2BP2 complex using quantitative mass spectrometry and RT-qPCR revealed that ZIKV infection alters the protein and RNA interactomes of IGF2BP2. Altogether, our data support that ZIKV hijacks and remodels the IGF2BP2 ribonucleoprotein complex to regulate vRO biogenesis and vRNA neosynthesis.

To achieve a virological cure for hepatitis B virus (HBV), innovative strategies are required to target the covalently closed circular DNA (cccDNA) genome. Guanine‐quadruplexes (G4s) are a secondary structure that can be adopted by DNA and play a significant role in regulating viral replication, transcription, and translation. Antibody‐based probes and small molecules have been developed to study the role of G4s in the context of the human genome, but none have been specifically made to target G4s in viral infection. Herein, we describe the development of a humanized single‐domain antibody (S10) that can target a G4 located in the PreCore (PreC) promoter of the HBV cccDNA genome. MicroScale Thermophoresis demonstrated that S10 has a strong nanomolar affinity to the PreC G4 in its quadruplex form and a structural electron density envelope of the complex was determined using Small‐Angle X‐ray Scattering. Lentiviral transduction of S10 into HepG2‐NTCP cells shows nuclear localization, and chromatin immunoprecipitation coupled with next‐generation sequencing demonstrated that S10 can bind to the HBV PreC G4 present on the cccDNA. This research validates the existence of a G4 in HBV cccDNA and demonstrates that this DNA secondary structure can be targeted with high structural and sequence specificity using S10.