👤 Darren L Gemmill

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. Show less

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. Show less

DEF-3(g16/NY-LU-12) encodes a novel RNA binding protein isolated by positional cloning from an SCLC homozygous deletion region in 3p21.3 and, in parallel, as a differentially expressed gene during myelopoiesis from FDCPmix-A4 cells. DEF-3(g16/NY-LU-12) is ubiquitously expressed during mouse embryogenesis and in adult organs while human hematopoietic tissues showed differential expression. The mouse and human proteins are highly conserved containing two RNA recognition motifs (RRMs) and other domains associated with RNA binding and protein-protein interactions. A database search identified related proteins in human, rat, C. elegans and S. pombe including the 3p21.3 co-deleted gene, LUCA15. Recombinant proteins containing the RRMs of DEF-3(g16/NY-LU-12) and LUCA15 specifically bound poly(G) RNA homopolymers in vitro. These RRMs also show similarity to those of the Hu protein family. Since anti-Hu RRM domain antibodies are associated with an anti-tumor effect and paraneoplastic encephalomyelitis, we tested sera from Hu syndrome patients with the RRMs of DEF-3(g16/NY-LU-12) and LUCA15. These were non-reactive. Thus, DEF-3(g16/NY-LU-12) and LUCA15 represent members of a novel family of RNA binding proteins with similar expression patterns and in vitro RNA binding characteristics. They are co-deleted in some lung cancers and immunologically distinct from the Hu proteins. Show less