G-quadruplex in the TMV Genome Regulates Viral Proliferation and Acts as Antiviral Target of Photodynamic Therapy.

Plant viruses seriously disrupt crop growth and development, and classic protein-targeted antiviral drugs could not provide complete protection against them. It is urgent to develop antiviral compounds with novel targets. Photodynamic therapy shows potential in controlling agricultural pests, but nonselective damage from reactive oxygen species (ROS) unexpectedly affects healthy tissues. A G-quadruplex (G4)-forming sequence in the tobacco mosaic virus (TMV) genome was identified to interfere the RNA replication in vitro, and affect the proliferation of TMV in tobacco. N-methyl mesoporphyrin IX stabilizing the G4 structure exhibited inhibition against viral proliferation, which was comparable to the inhibition effect of ribavirin. This indicated that G4 could work as an antiviral target. The large conjugate planes shared by G4 ligands and photosensitizers (PSs) remind us that the PSs could work as antiviral agents by targeting G4 in the genome of TMV. Chlorin e6 (Ce6) was identified to stabilize the G4 structure in the dark and selectively cleave the G4 sequence by producing ROS upon LED-light irradiation, leading to 92.2% inhibition against TMV in vivo, which is higher than that of commercial ningnanmycin. The inhibition of Ce6 was lost against the mutant variants lacking the G4-forming sequence. These findings indicated that the G-quadruplex in the TMV genome worked as an important structural element regulating viral proliferation, and could act as the antiviral target of photodynamic therapy.

Unfolding of an RNA G-quadruplex motif in the negative strand genome of porcine reproductive and respiratory syndrome virus by host and viral helicases to promote viral replication.

G-quadruplex (G4) is a unique secondary structure formed by guanine-rich nucleic acid sequences. Growing studies reported that the genomes of some viruses harbor G4 structures associated with viral replication, opening up a new field to dissect viral infection. Porcine reproductive and respiratory syndrome virus (PRRSV), a representative member of Arteriviridae, is an economically significant pathogen that has devastated the swine industry worldwide for over 30 years. In this study, we identified a highly conserved G-rich sequence with parallel-type G4 structure (named PRRSV-G4) in the negative strand genome RNA of PRRSV. Pyridostatin (PDS), a well-known G4-binding ligand, stabilized the PRRSV-G4 structure and inhibited viral replication. By screening the proteins interacting with PRRSV-G4 in PRRSV-infected cells and single-molecule magnetic tweezers analysis, we found that two helicases, host DDX18 and viral nsp10, interact with and efficiently unwound the PRRSV-G4 structure, thereby facilitating viral replication. Using a PRRSV reverse genetics system, we confirmed that recombinant PRRSV with a G4-disruptive mutation exhibited resistance to PDS treatment, thereby displaying higher replication than wild-type PRRSV. Collectively, these results demonstrate that the PRRSV-G4 structure plays a crucial regulatory role in viral replication, and targeting this structure represents a promising strategy for antiviral therapies.

An overview of single-molecule techniques and applications in the study of nucleic acid structure and function.

Nucleic acids are an indispensable component in all known life forms. The biological processes are regulated by Nucleic acids, which associate to form special high-order structures. since the high-level structures of nucleic acids are related to gene expression in cancer cells or viruses, it is very likely to become a potential drug target. Traditional biochemical methods are limited to distinguish the conformational distribution and dynamic transition process of single nucleic acid structure. The ligands based on the intermediate and transition states between different conformations are not designed by traditional biochemical methods. The single-molecule techniques enable real-time observation of the individual nucleic acid behavior due to its high resolution. Here, we introduce the application of single-molecule techniques in the study of small molecules to recognize nucleic acid structures, such as single-molecule FRET, magnetic tweezers, optical tweezers and atomic force microscopy. At the same time, we also introduce the specific advantages of single-molecule technology compared with traditional biochemical methods and some problems arisen in current research.

BMPQ-1 binds selectively to (3+1) hybrid topologies in human telomeric G-quadruplex multimers.

A single G-quadruplex forming sequence from the human telomere can adopt six distinct topologies that are inter-convertible under physiological conditions. This presents challenges to design ligands that show selectivity and specificity towards a particular conformation. Additional complexity is introduced in differentiating multimeric G-quadruplexes over monomeric species, which would be able to form in the single-stranded 3' ends of telomeres. A few ligands have been reported that bind to dimeric quadruplexes, but their preclinical pharmacological evaluation is limited. Using multidisciplinary approaches, we identified a novel quinoline core ligand, BMPQ-1, which bound to human telomeric G-quadruplex multimers over monomeric G-quadruplexes with high selectivity, and induced the formation of G-quadruplex DNA along with the related DNA damage response at the telomere. BMPQ-1 reduced tumor cell proliferation with an IC50 of ∼1.0 µM and decreased tumor growth rate in mouse by half. Biophysical analysis using smFRET identified a mixture of multiple conformations coexisting for dimeric G-quadruplexes in solution. Here, we showed that the titration of BMPQ-1 shifted the conformational ensemble of multimeric G-quadruplexes towards (3+1) hybrid-2 topology, which became more pronounced as further G-quadruplex units are added.