Mapping and characterization of G-quadruplexes in monkeypox genomes.

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.

Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus covalently closed circular DNA.

Approximately 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and are at increased risk of developing cirrhosis and hepatocellular carcinoma. The HBV genome persists as covalently closed circular DNA (cccDNA), which serves as the template for all HBV mRNA transcripts. Current nucleos(t)ide analogs used to treat HBV do not directly target the HBV cccDNA genome and thus cannot eradicate HBV infection. Here, we report the discovery of a unique G-quadruplex structure in the pre-core promoter region of the HBV genome that is conserved among nearly all genotypes. This region is central to critical steps in the viral life cycle, including the generation of pregenomic RNA, synthesis of core and polymerase proteins, and genome encapsidation; thus, an increased understanding of the HBV pre-core region may lead to the identification of novel anti-HBV cccDNA targets. We utilized biophysical methods (circular dichroism and small-angle X-ray scattering) to characterize the HBV G-quadruplex and the effect of three distinct G to A mutants. We also used microscale thermophoresis to quantify the binding affinity of G-quadruplex and its mutants with a known quadruplex-binding protein (DHX36). To investigate the physiological relevance of HBV G-quadruplex, we employed assays using DHX36 to pull-down cccDNA and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids by monitoring the levels of genomic DNA, pregenomic RNA, and antigens. Further evaluation of this critical host-protein interaction site in the HBV cccDNA genome may facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.

Human DDX3X Unwinds Japanese Encephalitis and Zika Viral 5' Terminal Regions.

Flavivirus genus includes many deadly viruses such as the Japanese encephalitis virus (JEV) and Zika virus (ZIKV). The 5' terminal regions (TR) of flaviviruses interact with human proteins and such interactions are critical for viral replication. One of the human proteins identified to interact with the 5' TR of JEV is the DEAD-box helicase, DDX3X. In this study, we in vitro transcribed the 5' TR of JEV and demonstrated its direct interaction with recombinant DDX3X (Kd of 1.66 ± 0.21 µM) using microscale thermophoresis (MST). Due to the proposed structural similarities of 5' and 3' TRs of flaviviruses, we investigated if the ZIKV 5' TR could also interact with human DDX3X. Our MST studies suggested that DDX3X recognizes ZIKV 5' TR with a Kd of 7.05 ± 0.75 µM. Next, we performed helicase assays that suggested that the binding of DDX3X leads to the unwinding of JEV and ZIKV 5' TRs. Overall, our data indicate, for the first time, that DDX3X can directly bind and unwind in vitro transcribed flaviviral TRs. In summary, our work indicates that DDX3X could be further explored as a therapeutic target to inhibit Flaviviral replication.

Analytical ultracentrifuge: an ideal tool for characterization of non-coding RNAs.

Analytical ultracentrifugation (AUC) has emerged as a robust and reliable technique for biomolecular characterization with extraordinary sensitivity. AUC is widely used to study purity, conformational changes, biomolecular interactions, and stoichiometry. Furthermore, AUC is used to determine the molecular weight of biomolecules such as proteins, carbohydrates, and DNA and RNA. Due to the multifaceted role(s) of non-coding RNAs from viruses, prokaryotes, and eukaryotes, research aimed at understanding the structure-function relationships of non-coding RNAs is rapidly increasing. However, due to their large size, flexibility, complicated secondary structures, and conformations, structural studies of non-coding RNAs are challenging. In this review, we are summarizing the application of AUC to evaluate the homogeneity, interactions, and conformational changes of non-coding RNAs from adenovirus as well as from Murray Valley, Powassan, and West Nile viruses. We also discuss the application of AUC to characterize eukaryotic long non-coding RNAs, Xist, and HOTAIR. These examples highlight the significant role AUC can play in facilitating the structural determination of non-coding RNAs and their complexes.