Despite the odds: formation of the SARS-CoV-2 methylation complex.

Coronaviruses modify their single-stranded RNA genome with a methylated cap during replication to mimic the eukaryotic mRNAs. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16, while nsp10 acts as a co-factor to both. Additionally, nsp14 carries an exonuclease domain which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins would be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex upon significant allosteric change. This interaction is expected to encourage the formation of mature capped viral mRNA, modulating nsp14's exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.

Feline herpesvirus 1 (FHV-1) enters the cell by receptor-mediated endocytosis.

Feline herpesvirus type 1 (FHV-1) is an enveloped dsDNA virus belonging to the Herpesviridae family and is considered one of the two primary viral etiological factors of feline upper respiratory tract disease. In this study, we investigated the entry of FHV-1 into host cells using two models: the AK-D cell line and primary feline skin fibroblasts (FSFs). We employed confocal microscopy, siRNA silencing, and selective inhibitors of various entry pathways. Our observations revealed that the virus enters cells via pH and dynamin-dependent endocytosis, as the infection was significantly inhibited by NH4Cl, bafilomycin A1, dynasore, and mitmab. Additionally, genistein, nystatin, and filipin treatments, siRNA knock-down of caveolin-1, as well as FHV-1 and caveolin-1 colocalization suggest the involvement of caveolin-mediated endocytosis during the entry process. siRNA knock-down of clathrin heavy chain and analysis of virus particle colocalization with clathrin indicated that clathrin-mediated endocytosis also takes part in the primary cells. This is the first study to systematically examine FHV-1 entry into host cells, and for the first time, we describe FHV-1 replication in AK-D and FSFs. IMPORTANCE Feline herpesvirus 1 (FHV-1) is one of the most prevalent viruses in cats, causing feline viral rhinotracheitis, which is responsible for over half of viral upper respiratory diseases in cats and can lead to ocular lesions resulting in loss of sight. Although the available vaccine reduces the severity of the disease, it does not prevent infection or limit virus shedding. Despite the clinical relevance, the entry mechanisms of FHV-1 have not been thoroughly studied. Considering the limitations of commonly used models based on immortalized cells, we sought to verify our findings using primary feline skin fibroblasts, the natural target for infection in cats.

Novel inhibitors of HSV-1 protease effective in vitro and in vivo.

Herpes simplex virus type 1 (HSV-1) is a widespread human pathogen known to cause infections of diverse severity, ranging from mild ulceration of mucosal and dermal tissues to life-threatening viral encephalitis. In most cases, standard treatment with acyclovir is sufficient to manage the disease progression. However, the emergence of ACV-resistant strains drives the need for new therapeutics and molecular targets. HSV-1 VP24 is a protease indispensable for the assembly of mature virions and, as such, constitutes an interesting target for the therapy. In this study, we present novel compounds, KI207M and EWDI/39/55BF, that block the activity of VP24 protease and consequently inhibit HSV-1 infection in vitro and in vivo. The inhibitors were shown to prevent the egress of viral capsids from the cell nucleus and suppress the cell-to-cell spread of the infection. They were also proven effective against ACV-resistant HSV-1 strains. Considering their low toxicity and high antiviral potency, the novel VP24 inhibitors could provide an alternative for treating ACV-resistant infections or a drug to be used in combined, highly effective therapy.

Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity.

During RNA replication, coronaviruses require proofreading to maintain the integrity of their large genomes. Nsp14 associates with viral polymerase complex to excise the mismatched nucleotides. Aside from the exonuclease activity, nsp14 methyltransferase domain mediates cap methylation, facilitating translation initiation and protecting viral RNA from recognition by the innate immune sensors. The nsp14 exonuclease activity is modulated by a protein co-factor nsp10. While the nsp10/nsp14 complex structure is available, the mechanistic basis for nsp10-mediated modulation remains unclear in the absence of the nsp14 structure. Here, we provide a crystal structure of nsp14 in an apo-form. Comparative analysis of the apo- and nsp10-bound structures explain the modulatory role of the co-factor protein and reveal the allosteric nsp14 control mechanism essential for drug discovery. Further, the flexibility of the N-terminal lid of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp14 structure presented in this study rationalizes the recently proposed idea of nsp14/nsp10/nsp16 ternary complex.

Acriflavine, a clinically approved drug, inhibits SARS-CoV-2 and other betacoronaviruses.

The COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort and available vaccines, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identify acriflavine (ACF) as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.

Cat flu: Broad spectrum polymeric antivirals.

Feline herpesvirus type 1 (FHV-1) and feline calicivirus (FCV) are considered as main causes of feline upper respiratory tract disease and the most common clinical manifestations include rhinotracheitis, conjunctivitis, and nasal/facial ulcerations. While the primary infection is relatively mild, secondary infections pose a threat to young or immunocompromised cats and may result in a fatal outcome. In this study, we made an effort to evaluate antiviral potency of poly(sodium 4-styrenesulfonates) (PSSNa) as potent FHV-1 and FCV inhibitors for topical use. Mechanistic studies showed that PSSNa exhibits a different mechanism of action depending on target species. While PSSNa acts directly on FHV-1 particles blocking their interaction with the host's cell and preventing the infection, the antiviral potency against FCV is based on inhibition at late stages of the viral replication cycle. Altogether, PSSNa polymers are promising drug candidates to be used in the treatment and prevention of the viral upper respiratory tract disease (URTD), regardless of the cause.

Highly Effective and Safe Polymeric Inhibitors of Herpes Simplex Virus in Vitro and in Vivo.

A series of poly(ethylene glycol)-block-poly(3-(methacryloylamino)propyl trimethylammonium chloride) (PEG-b-PMAPTAC) water-soluble block copolymers consisting of PEG and PMPTAC were obtained by reversible addition-fragmentation chain-transfer (RAFT) polymerization and demonstrated to function as highly effective herpes simplex virus type 1 (HSV-1) inhibitors as shown by in vitro tests (Vero E6 cells) and in vivo experiments (mouse model). Half-maximal inhibitory concentration (IC50) values were determined by quantitative polymerase chain reaction to be 0.36 ± 0.08 µg/mL for the most effective polymer PEG45-b-PMAPTAC52 and 0.84 ± 1.24 µg/mL for the less effective one, PEG45-b-PMAPTAC74. The study performed on the mouse model showed that the polymers protect mice from lethal infection. The polymers are not toxic to the primary human skin fibroblast cells up to the concentration of 100 µg/mL and to the Vero E6 cells up to 500 µg/mL. No systemic or topical toxicity was observed in vivo, even with mice treated with concentrated formulation (100 mg/mL). The mechanistic studies indicated that polymers interacted with the cell and blocked the formation of the entry/fusion complex. Physicochemical and biological properties of PEGx-b-PMAPTACy make them promising drug candidates.

Small-molecule inhibitors of PD-1/PD-L1 immune checkpoint alleviate the PD-L1-induced exhaustion of T-cells.

Antibodies targeting the PD-1/PD-L1 immune checkpoint achieved spectacular success in anticancer therapy in the recent years. In contrast, no small molecules with cellular activity have been reported so far. Here we provide evidence that small molecules are capable of alleviating the PD-1/PD-L1 immune checkpoint-mediated exhaustion of Jurkat T-lymphocytes. The two optimized small-molecule inhibitors of the PD-1/PD-L1 interaction, BMS-1001 and BMS-1166, developed by Bristol-Myers Squibb, bind to human PD-L1 and block its interaction with PD-1, when tested on isolated proteins. The compounds present low toxicity towards tested cell lines and block the interaction of soluble PD-L1 with the cell surface-expressed PD-1. As a result, BMS-1001 and BMS-1166 alleviate the inhibitory effect of the soluble PD-L1 on the T-cell receptor-mediated activation of T-lymphocytes. Moreover, the compounds were effective in attenuating the inhibitory effect of the cell surface-associated PD-L1. We also determined the X-ray structures of the complexes of BMS-1001 and BMS-1166 with PD-L1, which revealed features that may be responsible for increased potency of the compounds compared to their predecessors. Further development may lead to the design of an anticancer therapy based on the orally delivered immune checkpoint inhibition.

Inhibition of Herpes Simplex Viruses by Cationic Dextran Derivatives.

Human herpesviruses are among the most prevalent pathogens and currently there are no drugs available that could cure diseases induced by them. The most widely utilized antiherpes drugs, acyclovir and its derivatives, have serious limitations, such as low bioavailability and severe side effects. The current paper reports on the synthesis and characterization of cationic dextran derivatives (DEXxDSy) of various molecular weights and various degrees of substitution with ammonium groups, which were tested as antiherpes agents. DEXxDSy showed high effectiveness against HSV-1 and HSV-2 viruses, as found using a variety of techniques. Importantly, no toxicity was observed for these compounds in the range of active concentrations, demonstrating their potential as antivirals. The mechanism of action of DEXxDSy was assessed. We hypothesize that they may limit virus transmission, as extensive examination showed that they hamper the interaction between the virus and the cellular attachment receptor.

Novel peptidyl α-aminoalkylphosphonates as inhibitors of hepatitis C virus NS3/4A protease.

Herein, we describe the synthesis and application of novel phosphonic inhibitors designed to target the NS3/4A protease, which is crucial for the life cycle of hepatitis C virus. We examined the inhibitory potency of our synthesized compounds against two genotypes (1a and 1b) of NS3/4A protease and four mutant strains of HCV. The most potent inhibitors displayed k2/KI values of 79 850 M-1s-1 and 60 850 M-1s-1 against genotype 1a and 1b protease, respectively. Further in vitro evaluation of the most potent inhibitors revealed that vastly reduced HCV replication. Cellular toxicity, plasma stability, reactivity with selected human proteases as well the stability of inhibitor-protease complex and its intracellular availability are also discussed.