A molnupiravir-associated mutational signature in global SARS-CoV-2 genomes.

Molnupiravir, an antiviral medication widely used against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), acts by inducing mutations in the virus genome during replication. Most random mutations are likely to be deleterious to the virus and many will be lethal; thus, molnupiravir-induced elevated mutation rates reduce viral load1,2. However, if some patients treated with molnupiravir do not fully clear the SARS-CoV-2 infections, there could be the potential for onward transmission of molnupiravir-mutated viruses. Here we show that SARS-CoV-2 sequencing databases contain extensive evidence of molnupiravir mutagenesis. Using a systematic approach, we find that a specific class of long phylogenetic branches, distinguished by a high proportion of G-to-A and C-to-T mutations, are found almost exclusively in sequences from 2022, after the introduction of molnupiravir treatment, and in countries and age groups with widespread use of the drug. We identify a mutational spectrum, with preferred nucleotide contexts, from viruses in patients known to have been treated with molnupiravir and show that its signature matches that seen in these long branches, in some cases with onward transmission of molnupiravir-derived lineages. Finally, we analyse treatment records to confirm a direct association between these high G-to-A branches and the use of molnupiravir.

Safety and Biodistribution of Nanoligomers Targeting the SARS-CoV-2 Genome for the Treatment of COVID-19.

As the world braces to enter its fourth year of the coronavirus disease 2019 (COVID-19) pandemic, the need for accessible and effective antiviral therapeutics continues to be felt globally. The recent surge of Omicron variant cases has demonstrated that vaccination and prevention alone cannot quell the spread of highly transmissible variants. A safe and nontoxic therapeutic with an adaptable design to respond to the emergence of new variants is critical for transitioning to the treatment of COVID-19 as an endemic disease. Here, we present a novel compound, called SBCoV202, that specifically and tightly binds the translation initiation site of RNA-dependent RNA polymerase within the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome, inhibiting viral replication. SBCoV202 is a Nanoligomer, a molecule that includes peptide nucleic acid sequences capable of binding viral RNA with single-base-pair specificity to accurately target the viral genome. The compound has been shown to be safe and nontoxic in mice, with favorable biodistribution, and has shown efficacy against SARS-CoV-2 in vitro. Safety and biodistribution were assessed using three separate administration methods, namely, intranasal, intravenous, and intraperitoneal. Safety studies showed the Nanoligomer caused no outward distress, immunogenicity, or organ tissue damage, measured through observation of behavior and body weight, serum levels of cytokines, and histopathology of fixed tissue, respectively. SBCoV202 was evenly biodistributed throughout the body, with most tissues measuring Nanoligomer concentrations well above the compound KD of 3.37 nM. In addition to favorable availability to organs such as the lungs, lymph nodes, liver, and spleen, the compound circulated through the blood and was rapidly cleared through the renal and urinary systems. The favorable biodistribution and lack of immunogenicity and toxicity set Nanoligomers apart from other antisense therapies, while the adaptability of the nucleic acid sequence of Nanoligomers provides a defense against future emergence of drug resistance, making these molecules an attractive potential treatment for COVID-19.

Could a popular antiviral supercharge the pandemic?

Molnupiravir appears to be speeding SARS-CoV-2 evolution.

Characterization of the SARS-CoV-2 ExoN (nsp14ExoN-nsp10) complex: implications for its role in viral genome stability and inhibitor identification.

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14-nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14-nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3'-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14-nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12-nsp7-nsp8 (nsp12-7-8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14-nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.

S-adenosylmethionine-dependent methyltransferase inhibitor DZNep blocks transcription and translation of SARS-CoV-2 genome with a low tendency to select for drug-resistant viral variants.

We report the in vitro antiviral activity of DZNep (3-Deazaneplanocin A; an inhibitor of S-adenosylmethionine-dependent methyltransferase) against SARS-CoV-2, besides demonstrating its protective efficacy against lethal infection of infectious bronchitis virus (IBV, a member of the Coronaviridae family). DZNep treatment resulted in reduced synthesis of SARS-CoV-2 RNA and proteins without affecting other steps of viral life cycle. We demonstrated that deposition of N6-methyl adenosine (m6A) in SARS-CoV-2 RNA in the infected cells recruits heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), an RNA binding protein which serves as a m6A reader. DZNep inhibited the recruitment of hnRNPA1 at m6A-modified SARS-CoV-2 RNA which eventually suppressed the synthesis of the viral genome. In addition, m6A-marked RNA and hnRNPA1 interaction was also shown to regulate early translation to replication switch of SARS-CoV-2 genome. Furthermore, abrogation of methylation by DZNep also resulted in defective synthesis of the 5' cap of viral RNA, thereby resulting in its failure to interact with eIF4E (a cap-binding protein), eventually leading to a decreased synthesis of viral proteins. Most importantly, DZNep-resistant mutants could not be observed upon long-term sequential passage of SARS-CoV-2 in cell culture. In summary, we report the novel role of methylation in the life cycle of SARS-CoV-2 and propose that targeting the methylome using DZNep could be of significant therapeutic value against SARS-CoV-2 infection.

Fullerene Derivatives Prevent Packaging of Viral Genomic RNA into HIV-1 Particles by Binding Nucleocapsid Protein.

Fullerene derivatives with hydrophilic substituents have been shown to exhibit a range of biological activities, including antiviral ones. For a long time, the anti-HIV activity of fullerene derivatives was believed to be due to their binding into the hydrophobic pocket of HIV-1 protease, thereby blocking its activity. Recent work, however, brought new evidence of a novel, protease-independent mechanism of fullerene derivatives' action. We studied in more detail the mechanism of the anti-HIV-1 activity of N,N-dimethyl[70]fulleropyrrolidinium iodide fullerene derivatives. By using a combination of in vitro and cell-based approaches, we showed that these C70 derivatives inhibited neither HIV-1 protease nor HIV-1 maturation. Instead, our data indicate effects of fullerene C70 derivatives on viral genomic RNA packaging and HIV-1 cDNA synthesis during reverse transcription-without impairing reverse transcriptase activity though. Molecularly, this could be explained by a strong binding affinity of these fullerene derivatives to HIV-1 nucleocapsid domain, preventing its proper interaction with viral genomic RNA, thereby blocking reverse transcription and HIV-1 infectivity. Moreover, the fullerene derivatives' oxidative activity and fluorescence quenching, which could be one of the reasons for the inconsistency among reported anti-HIV-1 mechanisms, are discussed herein.

G-Quadruplexes Formation at the Upstream Region of Replication Origin (OriL) of the Pseudorabies Virus: Implications for Antiviral Targets.

Pseudorabies virus (PRV) is the causative agent of Aujeszky's disease, which still causes large economic losses for the swine industry. Therefore, it is urgent to find a new strategy to prevent and control PRV infection. Previous studies have proven that guanine (G)-rich DNA or RNA sequences in some other viruses' genomes have the potential to form G-quadruplex (G4), which serve as promising antivirus targets. In this study, we identified two novel G4-forming sequences, OriL-A and OriL-S, which are located at the upstream origin of replication (OriL) in the PRV genome and conserved across 32 PRV strains. Circular dichroism (CD) spectroscopy and a gel electrophoresis assay showed that the two G-rich sequences can fold into parallel G4 structures in vitro. Moreover, fluorescence resonance energy transfer (FRET) melting and a Taq polymerase stop assay indicated that the G4 ligand PhenDC3 has the capacity to bind and stabilize the G4. Notably, the treatment of PRV-infected cells with G4-stabilizer PhenDC3 significantly inhibited PRV DNA replication in host cells but did not affect PRV's attachment and entry. These results not only expand our knowledge about the G4 characteristics in the PRV genome but also suggest that G4 may serve as an innovative therapeutic target against PRV.

Application of Copper Iodide Nanoparticle-Doped Film and Fabric To Inactivate SARS-CoV-2 via the Virucidal Activity of Cuprous Ions (Cu+).

As a result of the novel coronavirus disease 2019 pandemic, strengthening control measures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become an urgent global issue. In addition to antiviral therapy and vaccination strategies, applying available virucidal substances for SARS-CoV-2 inactivation is also a target of research to prevent the spread of infection. Here, we evaluated the SARS-CoV-2 inactivation activity of a copper iodide (CuI) nanoparticle dispersion, which provides Cu+ ions having high virucidal activity, and its mode of actions. In addition, the utility of CuI-doped film and fabric for SARS-CoV-2 inactivation was evaluated. The CuI dispersion exhibited time-dependent rapid virucidal activity. Analyses of the modes of action of CuI performed by Western blotting and real-time reverse transcription-PCR targeting viral proteins and the genome revealed that CuI treatment induced the destruction of these viral components. In this setting, the indirect action of CuI-derived reactive oxygen species contributed to the destruction of viral protein. Moreover, the CuI-doped film and fabric demonstrated rapid inactivation of the SARS-CoV-2 solution in which the viral titer was high. These findings indicated the utility of the CuI-doped film and fabric as anti-SARS-CoV-2 materials for the protection of high-touch environmental surfaces and surgical masks/protective clothes. Throughout this study, we demonstrated the effectiveness of CuI nanoparticles for inactivating SARS-CoV-2 and revealed a part of its virucidal mechanism of action. IMPORTANCE The COVID-19 pandemic has caused an unprecedented number of infections and deaths. As the spread of the disease is rapid and the risk of infection is severe, hand and environmental hygiene may contribute to suppressing contact transmission of SARS-CoV-2. Here, we evaluated the SARS-CoV-2 inactivation activity of CuI nanoparticles, which provide the Cu+ ion as an antiviral agent, and we provided advanced findings of the virucidal mechanisms of action of Cu+. Our results showed that the CuI dispersion, as well as CuI-doped film and fabric, rapidly inactivated SARS-CoV-2 with a high viral titer. We also demonstrated the CuI's virucidal mechanisms of action, specifically the destruction of viral proteins and the genome by CuI treatment. Protein destruction largely depended on CuI-derived reactive oxygen species. This study provides novel information about the utility and mechanisms of action of promising virucidal material against SARS-CoV-2.

Rhinovirus Inhibitors: Including a New Target, the Viral RNA.

Rhinoviruses (RVs) are the main cause of recurrent infections with rather mild symptoms characteristic of the common cold. Nevertheless, RVs give rise to enormous numbers of absences from work and school and may become life-threatening in particular settings. Vaccination is jeopardised by the large number of serotypes eliciting only poorly cross-neutralising antibodies. Conversely, antivirals developed over the years failed FDA approval because of a low efficacy and/or side effects. RV species A, B, and C are now included in the fifteen species of the genus Enteroviruses based upon the high similarity of their genome sequences. As a result of their comparably low pathogenicity, RVs have become a handy model for other, more dangerous members of this genus, e.g., poliovirus and enterovirus 71. We provide a short overview of viral proteins that are considered potential drug targets and their corresponding drug candidates. We briefly mention more recently identified cellular enzymes whose inhibition impacts on RVs and comment novel approaches to interfere with infection via aggregation, virus trapping, or preventing viral access to the cell receptor. Finally, we devote a large part of this article to adding the viral RNA genome to the list of potential drug targets by dwelling on its structure, folding, and the still debated way of its exit from the capsid. Finally, we discuss the recent finding that G-quadruplex stabilising compounds impact on RNA egress possibly via obfuscating the unravelling of stable secondary structural elements.

Severe Acute Respiratory Syndrome Coronavirus-2 Inactivation Activity of the Polyphenol-Rich Tea Leaf Extract with Concentrated Theaflavins and Other Virucidal Catechins.

Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is producing a large number of infections and deaths globally, the development of supportive and auxiliary treatments is attracting increasing attention. Here, we evaluated SARS-CoV-2-inactivation activity of the polyphenol-rich tea leaf extract TY-1 containing concentrated theaflavins and other virucidal catechins. The TY-1 was mixed with SARS-CoV-2 solution, and its virucidal activity was evaluated. To evaluate the inhibition activity of TY-1 in SARS-CoV-2 infection, TY-1 was co-added with SARS-CoV-2 into cell culture media. After 1 h of incubation, the cell culture medium was replaced, and the cells were further incubated in the absence of TY-1. The viral titers were then evaluated. To evaluate the impacts of TY-1 on viral proteins and genome, TY-1-treated SARS-CoV-2 structural proteins and viral RNA were analyzed using western blotting and real-time RT-PCR, respectively. TY-1 showed time- and concentration-dependent virucidal activity. TY-1 inhibited SARS-CoV-2 infection of cells. The results of western blotting and real-time RT-PCR suggested that TY-1 induced structural change in the S2 subunit of the S protein and viral genome destruction, respectively. Our findings provided basic insights in vitro into the possible value of TY-1 as a virucidal agent, which could enhance the current SARS-CoV-2 control measures.

Identification and Structural Aspects of G-Quadruplex-Forming Sequences from the Influenza A Virus Genome.

Influenza A virus (IAV) causes seasonal epidemics and sporadic pandemics, therefore is an important research subject for scientists around the world. Despite the high variability of its genome, the structure of viral RNA (vRNA) possesses features that remain constant between strains and are biologically important for virus replication. Therefore, conserved structural motifs of vRNA can represent a novel therapeutic target. Here, we focused on the presence of G-rich sequences within the influenza A/California/07/2009(H1N1) genome and their ability to form RNA G-quadruplex structures (G4s). We identified 12 potential quadruplex-forming sequences (PQS) and determined their conservation among the IAV strains using bioinformatics tools. Then we examined the propensity of PQS to fold into G4s by various biophysical methods. Our results revealed that six PQS oligomers could form RNA G-quadruplexes. However, three of them were confirmed to adopt G4 structures by all utilized methods. Moreover, we showed that these PQS motifs are present within segments encoding polymerase complex proteins indicating their possible role in the virus biology.

In Vitro and In Silico Anti-Arboviral Activities of Dihalogenated Phenolic Derivates of L-Tyrosine.

Despite the serious public health problem represented by the diseases caused by dengue (DENV), Zika (ZIKV) and chikungunya (CHIKV) viruses, there are still no specific licensed antivirals available for their treatment. Here, we examined the potential anti-arbovirus activity of ten di-halogenated compounds derived from L-tyrosine with modifications in amine and carboxyl groups. The activity of compounds on VERO cell line infection and the possible mechanism of action of the most promising compounds were evaluated. Finally, molecular docking between the compounds and viral and cellular proteins was evaluated in silico with Autodock Vina®, and the molecular dynamic with Gromacs®. Only two compounds (TDC-2M-ME and TDB-2M-ME) inhibited both ZIKV and CHIKV. Within the possible mechanism, in CHIKV, the two compounds decreased the number of genome copies and in the pre-treatment strategy the infectious viral particles. In the ZIKV model, only TDB-2M-ME inhibited the viral protein and demonstrate a virucidal effect. Moreover, in the U937 cell line infected with CHIKV, both compounds inhibited the viral protein and TDB-2M-ME inhibited the viral genome too. Finally, the in silico results showed a favorable binding energy between the compounds and the helicases of both viral models, the NSP3 of CHIKV and cellular proteins DDC and ß2 adrenoreceptor.

Unconventional viral gene expression mechanisms as therapeutic targets.

Unlike the human genome that comprises mostly noncoding and regulatory sequences, viruses have evolved under the constraints of maintaining a small genome size while expanding the efficiency of their coding and regulatory sequences. As a result, viruses use strategies of transcription and translation in which one or more of the steps in the conventional gene-protein production line are altered. These alternative strategies of viral gene expression (also known as gene recoding) can be uniquely brought about by dedicated viral enzymes or by co-opting host factors (known as host dependencies). Targeting these unique enzymatic activities and host factors exposes vulnerabilities of a virus and provides a paradigm for the design of novel antiviral therapies. In this Review, we describe the types and mechanisms of unconventional gene and protein expression in viruses, and provide a perspective on how future basic mechanistic work could inform translational efforts that are aimed at viral eradication.

Supramolecular Cylinders Target Bulge Structures in the 5' UTR of the RNA Genome of SARS-CoV-2 and Inhibit Viral Replication*.

The untranslated regions (UTRs) of viral genomes contain a variety of conserved yet dynamic structures crucial for viral replication, providing drug targets for the development of broad spectrum anti-virals. We combine in vitro RNA analysis with molecular dynamics simulations to build the first 3D models of the structure and dynamics of key regions of the 5' UTR of the SARS-CoV-2 genome. Furthermore, we determine the binding of metallo-supramolecular helicates (cylinders) to this RNA structure. These nano-size agents are uniquely able to thread through RNA junctions and we identify their binding to a 3-base bulge and the central cross 4-way junction located in stem loop 5. Finally, we show these RNA-binding cylinders suppress SARS-CoV-2 replication, highlighting their potential as novel anti-viral agents.

A novel cell culture system modeling the SARS-CoV-2 life cycle.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the global pandemic of COVID-19. SARS-CoV-2 is classified as a biosafety level-3 (BSL-3) agent, impeding the basic research into its biology and the development of effective antivirals. Here, we developed a biosafety level-2 (BSL-2) cell culture system for production of transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). This trVLP expresses a reporter gene (GFP) replacing viral nucleocapsid gene (N), which is required for viral genome packaging and virion assembly (SARS-CoV-2 GFP/ΔN trVLP). The complete viral life cycle can be achieved and exclusively confined in the cells ectopically expressing SARS-CoV or SARS-CoV-2 N proteins, but not MERS-CoV N. Genetic recombination of N supplied in trans into viral genome was not detected, as evidenced by sequence analysis after one-month serial passages in the N-expressing cells. Moreover, intein-mediated protein trans-splicing approach was utilized to split the viral N gene into two independent vectors, and the ligated viral N protein could function in trans to recapitulate entire viral life cycle, further securing the biosafety of this cell culture model. Based on this BSL-2 SARS-CoV-2 cell culture model, we developed a 96-well format high throughput screening for antivirals discovery. We identified salinomycin, tubeimoside I, monensin sodium, lycorine chloride and nigericin sodium as potent antivirals against SARS-CoV-2 infection. Collectively, we developed a convenient and efficient SARS-CoV-2 reverse genetics tool to dissect the virus life cycle under a BSL-2 condition. This powerful tool should accelerate our understanding of SARS-CoV-2 biology and its antiviral development.

Pronounced therapeutic potential of oligonucleotides fixed on inorganic nanoparticles against highly pathogenic H5N1 influenza A virus in vivo.

This study describes the effective attack of oligonucleotides on the viral genome of highly pathogenic H5N1 influenza A virus (IAV) in vivo using for the first time the new delivery system consisting of biocompatible low-toxic titanium dioxide nanoparticles and immobilized polylysine-containing oligonucleotides with the native (ODN) and partially modified (ODNm) internucleotide bonds. Intraperitoneal injection of the TiO2•PL-ODN nanocomposite provided 65-70% survival of mice, while intraperitoneal or oral administration of TiO2•PL-ODNm was somewhat more efficient (~80% survival). The virus titer in the lung was reduced by two-three orders of magnitude. The nanocomposites are nontoxic to mice under the used conditions. TiO2 nanoparticles, unbound ODN, and the nanocomposite bearing the random oligonucleotide showed an insignificant protective effect, which indicates the ability of targeted oligonucleotides delivered in mice in the nanocomposites to site-specifically interact with complementary RNAs. The protection of oligonucleotides in nanocomposites by TiO2 nanoparticles and partial modification of the internucleotide bonds provides a continued presence of oligonucleotides in the body for the effective and specific action on the viral RNA. The proposed oligonucleotide delivery system can claim not only to effectively inhibit IAV genes but also to turn off other genes responsible for diseases caused by nucleic acids.

The SARS-CoV-2-Inactivating Activity of Hydroxytyrosol-Rich Aqueous Olive Pulp Extract (HIDROX®) and Its Use as a Virucidal Cream for Topical Application.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally. Although measures to control SARS-CoV-2, namely, vaccination, medication, and chemical disinfectants are being investigated, there is an increase in the demand for auxiliary antiviral approaches using natural compounds. Here we have focused on hydroxytyrosol (HT)-rich aqueous olive pulp extract (HIDROX®) and evaluated its SARS-CoV-2-inactivating activity in vitro. We showed that the HIDROX solution exhibits time- and concentration-dependent SARS-CoV-2-inactivating activities, and that HIDROX has more potent virucidal activity than pure HT. The evaluation of the mechanism of action suggested that both HIDROX and HT induced structural changes in SARS-CoV-2, which changed the molecular weight of the spike proteins. Even though the spike protein is highly glycosylated, this change was induced regardless of the glycosylation status. In addition, HIDROX or HT treatment disrupted the viral genome. Moreover, the HIDROX-containing cream applied on film showed time- and concentration-dependent SARS-CoV-2-inactivating activities. Thus, the HIDROX-containing cream can be applied topically as an antiviral hand cream. Our findings suggest that HIDROX contributes to improving SARS-CoV-2 control measures.

SARS-CoV-2 evolution during treatment of chronic infection.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.

Human Papillomavirus G-Rich Regions as Potential Antiviral Drug Targets.

Herein, we report, for the first time, the screening of several ligands in terms of their ability to bind and stabilize G-quadruplexes (G4) found in seven human Papillomavirus (HPV) genomes. Using a variety of biophysical assays, HPV G-quadruplexes were shown to possess a high degree of structural polymorphism upon ligand binding, which may have an impact on transcription, replication, and viral protein production. A sequence found in high-risk HPV16 genotype folds into multiple non-canonical DNA structures; it was converted into a major G4 conformation upon interaction with a well-characterized highly selective G4 ligand, PhenDC3, which may have an impact on the viral infection. Likewise, HPV57 and 58, which fold into multiple G4 structures, were found to form single stable complexes in the presence of two other G4 ligands, C8 and pyridostatin, respectively. In addition, one of the selected compounds, the acridine derivative C8, demonstrated a significant antiviral effect in HPV18-infected organotypic raft cultures. Altogether, these results indicate that targeting HPV G4s may be an alternative route for the development of novel antiviral therapies.