Highly Efficient SARS-CoV-2 Infection of Human Cardiomyocytes: Spike Protein-Mediated Cell Fusion and Its Inhibition.

Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cardiomyocytes express the angiotensin-converting enzyme 2 (ACE2) receptor but not the transmembrane protease serine 2 (TMPRSS2) that mediates spike protein cleavage in the lungs. Nevertheless, SARS-CoV-2 infection of hiPSC-CMs was prolific; viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CMs, smooth-walled exocytic vesicles contained numerous 65- to 90-nm particles with canonical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand how SARS-CoV-2 spreads in hiPSC-CMs, we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm cell-to-cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin and furin-like proteases abolished cell fusion. A spike mutant with the single amino acid change R682S that disrupts the multibasic furin cleavage motif was fusion inactive. Thus, SARS-CoV-2 replicates efficiently in hiPSC-CMs and furin, and/or furin-like-protease activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform enables target-based drug discovery in cardiac COVID-19. IMPORTANCE Cardiac complications frequently observed in COVID-19 patients are tentatively attributed to systemic inflammation and thrombosis, but viral replication has occasionally been confirmed in cardiac tissue autopsy materials. We developed an in vitro model of SARS-CoV-2 spread in myocardium using induced pluripotent stem cell-derived cardiomyocytes. In these highly differentiated cells, viral transcription levels exceeded those previously documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread, we expressed a fluorescent version of its spike protein that allowed us to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin/furin-like protease cleavage site lost cytopathic function. Of note, the fusion activities of the spike protein of other coronaviruses correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 may cause cardiac damage by fusing cardiomyocytes.

Structural basis of a potent human monoclonal antibody against Zika virus targeting a quaternary epitope.

Zika virus (ZIKV) is a major human pathogen and member of the Flavivirus genus in the Flaviviridae family. In contrast to most other insect-transmitted flaviviruses, ZIKV also can be transmitted sexually and from mother to fetus in humans. During recent outbreaks, ZIKV infections have been linked to microcephaly, congenital disease, and Guillain-Barré syndrome. Neutralizing antibodies have potential as therapeutic agents. We report here a 4-Å-resolution cryo-electron microscopy structure of the ZIKV virion in complex with Fab fragments of the potently neutralizing human monoclonal antibody ZIKV-195. The footprint of the ZIKV-195 Fab fragment expands across two adjacent envelope (E) protein protomers. ZIKV neutralization by this antibody is presumably accomplished by cross-linking the E proteins, which likely prevents formation of E protein trimers required for fusion of the viral and cellular membranes. A single dose of ZIKV-195 administered 5 days after virus inoculation showed marked protection against lethality in a stringent mouse model of infection.

In vitro inhibitory effect of maraviroc on the association of the simian immunodeficiency virus envelope glycoprotein with CCR5.

Asian macaques infected with simian immunodeficiency viruses (SIVs) isolated from African non-human primates develop a disease similar to human AIDS. SIV enters its target cells by binding to CD4 and a coreceptor, typically CCR5. Maraviroc is an entry inhibitor of human immunodeficiency virus type 1 (HIV-1) that prevents the interaction between CCR5 and the surface subunit gp120 of the viral envelope glycoprotein (Env). Thus far, the activity of maraviroc on SIV entry has been poorly studied. Here, we determined in vitro pharmacological parameters of the effect of maraviroc on the SIV Env association with CCR5. Cell-to-cell fusion inhibition assays were used to compare the susceptibility to maraviroc of the SIVsmmPBj Env-CCR5 interaction with that of HIV-1BaL Env. Analysis of dose-response curves and determination of IC50 values demonstrate that increasing concentrations of maraviroc inhibit the membrane fusion activity of SIVsmmPBj Env in a manner and to an extent similar to that of HIV-1BaL Env.

Synthesis of C-2 and C-3 substituted quinolines and their evaluation as anti-HIV-1 agents.

A plenty of natural products and synthetic derivatives containing quinoline moiety have been reported to possess various pharmacological activities. Quinolines such as 2-styrylquinolines and 8-hydroxyquinolines are extensively studied for their anti-HIV-1 activity and found to act mainly through HIV-1 integrase enzyme inhibition. In continuation of our efforts to search for newer anti-HIV-1 molecules, thirty-one quinoline derivatives with different linkers to ancillary phenyl ring were synthesized and evaluated for in vitro anti-HIV-1 activity using TZM-bl assays. Compound 31 showed higher activity in TZM-bl cell line against HIV-1VB59 and HIV-1UG070 cell associated virus (IC50 3.35 ±â€¯0.87 and 2.57 ±â€¯0.71 µM) as compared to other derivatives. Compound 31 was further tested against cell free virus HIV-1VB59 and HIV-1UG070 (IC50 1.27 ±â€¯0.31 and 2.88 ±â€¯1.79 µM, TI 42.20 and 18.61, respectively). This lead molecule also showed good activity in viral entry inhibition assay and cell fusion assay defining its mode of action. The activity of compound 31 was confirmed by testing against HIV-1VB51 in activated peripheral blood mononuclear cells (PBMCs). Binding interactions of 31 were compared with known entry inhibitors.

Cryo-EM structures elucidate neutralizing mechanisms of anti-chikungunya human monoclonal antibodies with therapeutic activity.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe acute and chronic disease in humans. Although highly inhibitory murine and human monoclonal antibodies (mAbs) have been generated, the structural basis of their neutralizing activity remains poorly characterized. Here, we determined the cryo-EM structures of chikungunya virus-like particles complexed with antibody fragments (Fab) of two highly protective human mAbs, 4J21 and 5M16, that block virus fusion with host membranes. Both mAbs bind primarily to sites within the A and B domains, as well as to the B domain's ß-ribbon connector of the viral glycoprotein E2. The footprints of these antibodies on the viral surface were consistent with results from loss-of-binding studies using an alanine scanning mutagenesis-based epitope mapping approach. The Fab fragments stabilized the position of the B domain relative to the virus, particularly for the complex with 5M16. This finding is consistent with a mechanism of neutralization in which anti-CHIKV mAbs that bridge the A and B domains impede movement of the B domain away from the underlying fusion loop on the E1 glycoprotein and therefore block the requisite pH-dependent fusion of viral and host membranes.

Enhanced potency of bivalent small molecule gp41 inhibitors.

Low molecular weight peptidomimetic inhibitors with hydrophobic pocket binding properties and moderate fusion inhibitory activity against HIV-1 gp41-mediated cell fusion were elaborated by increasing the available surface area for interacting with the heptad repeat-1 (HR1) coiled coil on gp41. Two types of modifications were tested: 1) increasing the overall hydrophobicity of the molecules with an extension that could interact in the HR1 groove, and 2) forming symmetrical dimers with two peptidomimetic motifs that could potentially interact simultaneously in two hydrophobic pockets on the HR1 trimer. The latter approach was more successful, yielding 40-60times improved potency against HIV fusion over the monomers. Biophysical characterization, including equilibrium binding studies by fluorescence and kinetic analysis by Surface Plasmon Resonance, revealed that inhibitor potency was better correlated to off-rates than to binding affinity. Binding and kinetic data could be fit to a model of bidentate interaction of dimers with the HR1 trimer as an explanation for the slow off-rate, albeit with minimal cooperativity due to the highly flexible ligand structures. The strong cooperativity observed in fusion inhibitory activity of the dimers implied accentuated potency due to the transient nature of the targeted intermediate. Optimization of monomer, dimer or higher order structures has the potential to lead to highly potent non-peptide fusion inhibitors by targeting multiple hydrophobic pockets.

Super short membrane-active lipopeptides inhibiting the entry of influenza A virus.

Influenza A viruses (IAV) are significant pathogens that result in millions of human infections and impose a substantial health and economic burdens worldwide. Due to the limited anti-influenza A therapeutics available and the emergence of drug resistant viral strains, it is imperative to develop potent anti-IAV agents with different mode of action. In this study, by applying a pseudovirus based screening approach, two super short membrane-active lipopeptides of C12-KKWK and C12-OOWO were identified as effective anti-IAV agents with IC50 value of 7.30±1.57 and 8.48±0.74 mg/L against A/Puerto Rico/8/34 strain, and 6.14±1.45 and 7.22±0.67 mg/L against A/Aichi/2/68 strain, respectively. The mechanism study indicated that the anti-IAV activity of these peptides would result from the inhibition of virus entry by interacting with HA2 subunit of hemagglutinin (HA). Thus, these peptides may have potentials as lead peptides for the development of new anti-IAV therapeutics to block the entry of virus into host cells.

Broadly Applicable, Virus-Free Dual Reporter Assay to Identify Compounds Interfering with Membrane Fusion: Performance for HSV-1 and SARS-CoV-2.

Membrane fusion constitutes an essential step in the replication cycle of numerous viral pathogens, hence it represents an important druggable target. In the present study, we established a virus-free, stable reporter fusion inhibition assay (SRFIA) specifically designed to identify compounds interfering with virus-induced membrane fusion. The dual reporter assay is based on two stable Vero cell lines harboring the third-generation tetracycline (Tet3G) transactivator and a bicistronic reporter gene cassette under the control of the tetracycline responsive element (TRE3G), respectively. Cell-cell fusion by the transient transfection of viral fusogens in the presence of doxycycline results in the expression of the reporter enzyme secreted alkaline phosphatase (SEAP) and the fluorescent nuclear localization marker EYFPNuc. A constitutively expressed, secreted form of nanoluciferase (secNLuc) functioned as the internal control. The performance of the SRFIA was tested for the quantification of SARS-CoV-2- and HSV-1-induced cell-cell fusion, respectively, showing high sensitivity and specificity, as well as the reliable identification of known fusion inhibitors. Parallel quantification of secNLuc enabled the detection of cytotoxic compounds or insufficient transfection efficacy. In conclusion, the SRFIA reported here is well suited for high-throughput screening for new antiviral agents and essentially will be applicable to all viral fusogens causing cell-cell fusion in Vero cells.

Ginkgolic Acid Inhibits Herpes Simplex Virus Type 1 Skin Infection and Prevents Zosteriform Spread in Mice.

Herpes simplex virus type 1 (HSV-1) causes a lifelong latent infection with an estimated global prevalence of 66%. Primary and recurrent HSV infections are characterized by a tingling sensation, followed by an eruption of vesicles, which can cause painful erosions. Commonly used antiviral drugs against HSV infection are nucleoside analogues including acyclovir (ACV), famciclovir, and valacyclovir. Although these nucleoside analogues reduce morbidity and mortality in immunocompetent individuals, ACV-resistant HSV strains (ACVR-HSV) have been isolated from immunocompromised patients. Thus, ACVR-HSV infection poses a critical emerging public health concern. Recently, we reported that ginkgolic acid (GA) inhibits HSV-1 by disrupting viral structure, blocking fusion, and inhibiting viral protein synthesis. Additionally, we showed GA affords a broad spectrum of fusion inhibition of all three classes of fusion proteins, including those of HIV, Ebola, influenza A and Epstein Barr viruses. Here we report GA's antiviral activity against HSV-1 skin infection in BALB/cJ mice. GA-treated mice demonstrated a significantly reduced mortality rate and decreased infection scores compared to controls treated with dimethylsulfoxide (DMSO)-vehicle. Furthermore, GA efficiently inhibited ACVR-HSV-1 strain 17+ in vitro and in vivo. Since GA's mechanism of action includes virucidal activity and fusion inhibition, it is expected to work alone or synergistically with other anti-viral drugs, and we anticipate it to be effective against additional cutaneous and potentially systemic viral infections.

"Fusion and binding inhibition" key target for HIV-1 treatment and pre-exposure prophylaxis: targets, drug delivery and nanotechnology approaches.

More than 35 million people are living with HIV worldwide with approximately 2.3 million new infections per year. Cascade of events (cell entry, virus replication, assembly and release of newly formed virions) is involved in the HIV-1 transmission process. Every single step offers a potential therapeutic strategy to halt this progression and HIV fusion into the human host cell is one such stage. Controlling the initial event of HIV-1 transmission is the best way to control its dissemination especially when prophylaxis is concerned. Action is required either on the HIV's or host's cell surface which is logically more rational when compared with other intracellular acting moieties. Aim of this manuscript is to detail the significance and current strategies to halt this initial step, thus blocking the entry of HIV-1 for further infection. Both HIV-1 and the possible host cell's receptors/co-receptors are under focus while specifying the targets available for inhibiting this fusion. Current and under investigation moieties are categorized based on their versatile mechanisms. Advanced drug delivery and nanotechnology approaches present a key tool to exploit the therapeutic potential in a boosted way. Current drug delivery and the impact of nanotechnology in potentiating this strategy are detailed.

Anti-herpes simplex virus effect of aristolochic acids in vitro and in vivo / 中国药理学通报

Aim To investigate the inhibition effects and mechanisms of aristolochic acids(AAs)against herpes simplex virus(HSV)in vitro and in vivo. Methods The cytopathic effect(CPE), plaque assay, indirect immunofluorescence and others were used to explore the anti-HSV effects and mechanisms of aristolochic acid in Vero cells, and the in vivo anti-HSV activity of AAs was evaluated using HSV-1 infected BALB/c mouse model. Results The IC