Structural biology of SARS-CoV-2: open the door for novel therapies.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the causative agent of the pandemic disease COVID-19, which is so far without efficacious treatment. The discovery of therapy reagents for treating COVID-19 are urgently needed, and the structures of the potential drug-target proteins in the viral life cycle are particularly important. SARS-CoV-2, a member of the Orthocoronavirinae subfamily containing the largest RNA genome, encodes 29 proteins including nonstructural, structural and accessory proteins which are involved in viral adsorption, entry and uncoating, nucleic acid replication and transcription, assembly and release, etc. These proteins individually act as a partner of the replication machinery or involved in forming the complexes with host cellular factors to participate in the essential physiological activities. This review summarizes the representative structures and typically potential therapy agents that target SARS-CoV-2 or some critical proteins for viral pathogenesis, providing insights into the mechanisms underlying viral infection, prevention of infection, and treatment. Indeed, these studies open the door for COVID therapies, leading to ways to prevent and treat COVID-19, especially, treatment of the disease caused by the viral variants are imperative.

Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.

Cyclosporine A Inhibits Viral Infection and Release as Well as Cytokine Production in Lung Cells by Three SARS-CoV-2 Variants.

In December 2019, a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) started spreading worldwide causing the coronavirus disease 2019 (COVID-19) pandemic. The hyperactivation of the immune system has been proposed to account for disease severity and death in COVID-19 patients. Despite several approaches having been tested, no therapeutic protocol has been approved. Given that Cyclosporine A (CsA) is well-known to exert a strong antiviral activity on several viral strains and an anti-inflammatory role in different organs with relevant benefits in diverse pathological contexts, we tested its effects on SARS-CoV-2 infection of lung cells. We found that treatment with CsA either before or after infection of CaLu3 cells by three SARS-CoV-2 variants: (i) reduces the expression of both viral RNA and proteins in infected cells; (ii) decreases the number of progeny virions released by infected cells; (iii) dampens the virus-triggered synthesis of cytokines (including IL-6, IL-8, IL1α and TNF-α) that are involved in cytokine storm in patients. Altogether, these data provide a rationale for CsA repositioning for the treatment of severe COVID-19 patients. IMPORTANCE SARS-CoV-2 is the most recently identified member of the betacoronavirus genus responsible for the COVID-19 pandemic. Repurposing of available drugs has been a "quick and dirty" approach to try to reduce mortality and severe symptoms in affected patients initially, and can still represent an undeniable and valuable approach to face COVID-19 as the continuous appearance and rapid diffusion of more "aggressive"/transmissible variants, capable of eluding antibody neutralization, challenges the effectiveness of some anti-SARS-CoV-2 vaccines. Here, we tested a known antiviral and anti-inflammatory drug, Cyclosporine A (CsA), and found that it dampens viral infection and cytokine release from lung cells upon exposure to three different SARS-CoV-2 variants. Knock down of the main intracellular target of CsA, Cyclophilin A, does not phenocopy the drug inhibition of viral infection. Altogether, these findings shed new light on the cellular mechanisms of SARS-CoV-2 infection and provide the rationale for CsA repositioning to treat severe COVID-19 patients.

The Peptide A-3302-B Isolated from a Marine Bacterium Micromonospora sp. Inhibits HSV-2 Infection by Preventing the Viral Egress from Host Cells.

Herpesviruses are highly prevalent in the human population, and frequent reactivations occur throughout life. Despite antiviral drugs against herpetic infections, the increasing appearance of drug-resistant viral strains and their adverse effects prompt the research of novel antiherpetic drugs for treating lesions. Peptides obtained from natural sources have recently become of particular interest for antiviral therapy applications. In this work, we investigated the antiviral activity of the peptide A-3302-B, isolated from a marine bacterium, Micromonospora sp., strain MAG 9-7, against herpes simplex virus type 1, type 2, and human cytomegalovirus. Results showed that the peptide exerted a specific inhibitory activity against HSV-2 with an EC50 value of 14 µM. Specific antiviral assays were performed to investigate the mechanism of action of A-3302-B. We demonstrated that the peptide did not affect the expression of viral proteins, but it inhibited the late events of the HSV-2 replicative cycle. In detail, it reduced the cell-to-cell virus spread and the transmission of the extracellular free virus by preventing the egress of HSV-2 progeny from the infected cells. The dual antiviral and previously reported anti-inflammatory activities of A-3302-B, and its effect against an acyclovir-resistant HSV-2 strain are attractive features for developing a therapeutic to reduce the transmission of HSV-2 infections.

Lipid Droplets Are Beneficial for Rabies Virus Replication by Facilitating Viral Budding.

Rabies is an old zoonotic disease caused by rabies virus (RABV), but the pathogenic mechanism of RABV is still not completely understood. Lipid droplets (LDs) have been reported to play a role in pathogenesis of several viruses. However, their role in RABV infection remains unclear. Here, we initially found that RABV infection upregulated LD production in multiple cells and mouse brains. After treatment with atorvastatin, a specific inhibitor of LDs, RABV replication in N2a cells decreased. Then we found that RABV infection could upregulate N-myc downstream regulated gene-1 (NDRG1), which in turn enhanced the expression of diacylglycerol acyltransferase 1/2 (DGAT1/2). DGAT1/2 could elevate cellular triglyceride synthesis and ultimately promote intracellular LD formation. Furthermore, we found that RABV-M and RABV-G, which were mainly involved in the viral budding process, could colocalize with LDs, indicating that RABV might utilize LDs as a carrier to facilitate viral budding and eventually increase virus production. Taken together, our study reveals that lipid droplets are beneficial for RABV replication, and their biogenesis is regulated via the NDRG1-DGAT1/2 pathway, which provides novel potential targets for developing anti-RABV drugs. IMPORTANCE Lipid droplets have been proven to play an important role in viral infections, but their role in RABV infection has not yet been elaborated. Here, we find that RABV infection upregulates the generation of LDs by enhancing the expression of N-myc downstream regulated gene-1 (NDRG1). Then NDRG1 elevated cellular triglycerides synthesis by increasing the activity of diacylglycerol acyltransferase 1/2 (DGAT1/2), which promotes the biogenesis of LDs. RABV-M and RABV-G, which are the major proteins involved in viral budding, could utilize LDs as a carrier for transport to cell membrane, resulting in enhanced virus budding. Our findings will extend the knowledge of lipid metabolism in RABV infection and help to explore potential therapeutic targets for RABV.

Hydroxypropyl Methylcellulose-Based Nasal Sprays Effectively Inhibit In Vitro SARS-CoV-2 Infection and Spread.

The ongoing coronavirus disease (COVID-19) pandemic has required a variety of non-medical interventions to limit the transmission of the causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One such option is over-the-counter nasal sprays that aim to block virus entry and transmission within the nasal cavity. In this study, we assessed the ability of three hydroxypropyl methylcellulose (HPMC)-based powder nasal sprays, produced by Nasaleze, to inhibit SARS-CoV-2 infection and release in vitro. Upon application, the HPMC powder forms a gel-like matrix within the nasal cavity-a process we recapitulated in cell culture. We found that virus release from cells previously infected with SARS-CoV-2 was inhibited by the gel matrix product in a dose-dependent manner, with virus levels reduced by >99.99% over a 72 h period at a dose of 6.4 mg/3.5 cm2. We also show that the pre-treatment of cells with product inhibited SARS-CoV-2 infection, independent of the virus variant. The primary mechanism of action appears to be via the formation of a physical, passive barrier. However, the addition of wild garlic provided additional direct antiviral properties in some formulations. We conclude that HPMC-based nasal sprays may offer an additional component to strategies to limit the spread of respiratory viruses, including SARS-CoV-2.

Tomatidine reduces Chikungunya virus progeny release by controlling viral protein expression.

Tomatidine, a natural steroidal alkaloid from unripe green tomatoes has been shown to exhibit many health benefits. We recently provided in vitro evidence that tomatidine reduces the infectivity of Dengue virus (DENV) and Chikungunya virus (CHIKV), two medically important arthropod-borne human infections for which no treatment options are available. We observed a potent antiviral effect with EC50 values of 0.82 µM for DENV-2 and 1.3 µM for CHIKV-LR. In this study, we investigated how tomatidine controls CHIKV infectivity. Using mass spectrometry, we identified that tomatidine induces the expression of p62, CD98, metallothionein and thioredoxin-related transmembrane protein 2 in Huh7 cells. The hits p62 and CD98 were validated, yet subsequent analysis revealed that they are not responsible for the observed antiviral effect. In parallel, we sought to identify at which step of the virus replication cycle tomatidine controls virus infectivity. A strong antiviral effect was seen when in vitro transcribed CHIKV RNA was transfected into Huh7 cells treated with tomatidine, thereby excluding a role for tomatidine during CHIKV cell entry. Subsequent determination of the number of intracellular viral RNA copies and viral protein expression levels during natural infection revealed that tomatidine reduces the RNA copy number and viral protein expression levels in infected cells. Once cells are infected, tomatidine is not able to interfere with active RNA replication yet it can reduce viral protein expression. Collectively, the results delineate that tomatidine controls viral protein expression to exert its antiviral activity. Lastly, sequential passaging of CHIKV in presence of tomatidine did not lead to viral resistance. Collectively, these results further emphasize the potential of tomatidine as an antiviral treatment towards CHIKV infection.

Rottlerin inhibits La Crosse virus-induced encephalitis in mice and blocks release of replicating virus from the Golgi body in neurons.

La Crosse virus (LACV) is a mosquito-borne orthobunyavirus that causes approximately 60 to 80 hospitalized pediatric encephalitis cases in the United States yearly. The primary treatment for most viral encephalitis, including LACV, is palliative care, and specific antiviral therapeutics are needed. We screened the National Center for Advancing Translational Sciences library of 3,833 FDA-approved and bioactive small molecules for the ability to inhibit LACV-induced death in SH-SY5Y neuronal cells. The top three hits from the initial screen were validated by examining their ability to inhibit virus-induced cell death in multiple neuronal cell lines. Rottlerin consistently reduced LACV-induced death by 50% in multiple human and mouse neuronal cell lines with an effective concentration of 0.16-0.69 µg ml-1 depending on cell line. Rottlerin was effective up to 12 hours post-infection in vitro and inhibited virus particle trafficking from the Golgi apparatus to trans-Golgi vesicles. In human inducible pluripotent stem cell-derived cerebral organoids, rottlerin reduced virus production by one log and cell death by 35% compared with dimethyl sulfoxide-treated controls. Administration of rottlerin in mice by intraperitoneal or intracranial routes starting at 3 days post-infection decreased disease development by 30-50%. Furthermore, rottlerin also inhibited virus replication of other pathogenic California serogroup orthobunyaviruses (Jamestown Canyon and Tahyna virus) in neuronal cell lines.

Protease, Growth Factor, and Heparanase-Mediated Syndecan-1 Shedding Leads to Enhanced HSV-1 Egress.

Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPGs) are considered important for the entry of many different viruses. Previously, we demonstrated that heparanase (HPSE), the host enzyme responsible for cleaving HS chains, is upregulated by herpes simplex virus-1 (HSV-1) infection. Higher levels of HPSE accelerate HS removal from the cell surface, facilitating viral release from infected cells. Here, we study the effects of overexpressing HPSE on viral entry, cell-to-cell fusion, plaque formation, and viral egress. We provide new information that higher levels of HPSE reduce syncytial plaque formation while promoting egress and extracellular release of the virions. We also found that transiently enhanced expression of HPSE did not affect HSV-1 entry into host cells or HSV-1-induced cell-to-cell fusion, suggesting that HPSE activation is tightly regulated and facilitates extracellular release of the maturing virions. We demonstrate that an HSPG-shedding agonist, PMA; a protease, thrombin; and a growth factor, EGF as well as bacterially produced recombinant heparinases resulted in enhanced HSV-1 release from HeLa and human corneal epithelial (HCE) cells. Our findings here underscore the significance of syndecan-1 functions in the HSV-1 lifecycle, provide evidence that the shedding of syndecan-1 ectodomain is another way HPSE works to facilitate HSV-1 release, and add new evidence on the significance of various HSPG shedding agonists in HSV-1 release from infected cells.

A Novel Small Molecule Inhibits Hepatitis C Virus Propagation in Cell Culture.

Hepatitis C virus (HCV) can cause acute and chronic infection that is associated with considerable liver-related morbidity and mortality. In recent years, there has been a shift in the treatment paradigm with the discovery and approval of agents that target specific proteins vital for viral replication. We employed a cell culture-adapted strain of HCV and human hepatoma-derived cells lines to test the effects of our novel small-molecule compound (AO13) on HCV. Virus inhibition was tested by analyzing RNA replication, protein expression, and virus production in virus-infected cells treated with AO13. Treatment with AO13 inhibited virus spread in cell culture and showed a 100-fold reduction in the levels of infectious virus production. AO13 significantly reduced the level of viral RNA contained within cell culture fluids and reduced the cellular levels of HCV core protein, suggesting that the compound might act on a late step in the viral life cycle. Finally, we observed that AO13 did not affect the release of infectious virus from infected cells. Docking studies and molecular dynamics analyses suggested that AO13 might target the NS5B RNA polymerase, however, real-time RT-PCR analyses of cellular levels of HCV RNA showed only an ∼2-fold reduction in viral RNA levels in the presence of AO13. Taken together, this study revealed that AO13 showed consistent, but low-level antiviral effect against HCV, although the mechanism of action remains unclear. IMPORTANCE The discovery of curative antiviral drugs for a chronic disease such as HCV infection has encouraged drug discovery in the context of other viruses for which no curative drugs currently exist. Since we currently face a novel virus that has caused a pandemic, the need for new antiviral agents is more apparent than ever. We describe here a novel compound that shows a modest antiviral effect against HCV that could serve as a lead compound for future drug development against other important viruses such as SARS-CoV-2.

Inhibitors of Protein Glycosylation Are Active against the Coronavirus Severe Acute Respiratory Syndrome Coronavirus SARS-CoV-2.

Repurposing clinically available drugs to treat the new coronavirus disease 2019 (COVID-19) is an urgent need in the course of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2) pandemic, as very few treatment options are available. The iminosugar Miglustat is a well-characterized drug for the treatment of rare genetic lysosome storage diseases, such as Gaucher and Niemann-Pick type C, and has also been described to be active against a variety of enveloped viruses. The activity of Miglustat is here demonstrated in the micromolar range for SARS-CoV-2 in vitro. The drug acts at the post-entry level and leads to a marked decrease of viral proteins and release of infectious viruses. The mechanism resides in the inhibitory activity toward α-glucosidases that are involved in the early stages of glycoprotein N-linked oligosaccharide processing in the endoplasmic reticulum, leading to a marked decrease of the viral Spike protein. Indeed, the antiviral potential of protein glycosylation inhibitors against SARS-CoV-2 is further highlighted by the low-micromolar activity of the investigational drug Celgosivir. These data point to a relevant role of this approach for the treatment of COVID-19.

Compounds Identified from Marine Mangrove Plant (Avicennia alba) as Potential Antiviral Drug Candidates Against WDSV, an In-Silico Approach.

Walleye dermal sarcoma virus (WDSV) is a type of retrovirus, which affects most of the adult walleye fishes during the spawning time. The virus causes multiple epithelial tumors on the fish's skin and fins that are liable for more than 50% of the mortality rate of fish around the world. Till now, no effective antiviral drug or vaccine candidates have been developed that can block the progression of the disease caused by the pathogen. It was found that the 582-amino-acid (aa) residues long internal structural gag polyprotein of the virus plays an important role in virus budding and virion maturation outside of the cell. Inhibition of the protein can block the budding and virion maturation process and can be developed as an antiviral drug candidate against the virus. Therefore, the study aimed to identify potential natural antiviral drug candidates from the tropical mangrove marine plant Avicennia alba, which will be able to block the budding and virion maturation process by inhibiting the activity of the gag protein of the virus. Initially, a homology modeling approach was applied to identify the 3D structure, followed by refinement and validation of the protein. The refined protein structures were then utilized for molecular docking simulation. Eleven phytochemical compounds have been isolated from the marine plant and docked against the virus gag polyprotein. Three compounds, namely Friedlein (CID244297), Phytosterols (CID12303662), and 1-Triacontanol (CID68972) have been selected based on their docking score -8.5 kcal/mol, -8.0 kcal/mol and -7.9 kcal/mol, respectively, and were evaluated through ADME (Absorption, Distribution, Metabolism and Excretion), and toxicity properties. Finally, molecular dynamics (MD) simulation was applied to confirm the binding stability of the protein-ligands complex structure. The ADME and toxicity analysis reveal the efficacy and non-toxic properties of the compounds, where MD simulation confirmed the binding stability of the selected three compounds with the targeted protein. This computational study revealed the virtuous value of the selected three compounds against the targeted gag polyprotein and will be effective and promising antiviral candidates against the pathogen in a significant and worthwhile manner. Although in vitro and in vivo study is required for further evaluation of the compounds against the targeted protein.

Prazoles Targeting Tsg101 Inhibit Release of Epstein-Barr Virus following Reactivation from Latency.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus responsible for several diseases, including cancers of lymphoid and epithelial cells. EBV cancers typically exhibit viral latency; however, the production and release of EBV through its lytic phase are essential for cancer development. Antiviral agents that specifically target EBV production do not currently exist. Previously, we reported that the proton pump inhibitor tenatoprazole, which blocks the interaction of ubiquitin with the ESCRT-1 factor Tsg101, inhibits production of several enveloped viruses, including EBV. Here, we show that three structurally distinct prazoles impair mature particle formation postreactivation and identify the impact on stages of replication. The prazoles did not impair expression of lytic genes representative of the different kinetic classes but interfered with capsid maturation in the nucleus as well as virion transport from the nucleus. Replacement of endogenous Tsg101 with a mutant Tsg101 refractory to prazole-mediated inhibition rescued EBV release. These findings directly implicate Tsg101 in EBV nuclear egress and identify prazoles as potential therapeutic candidates for conditions that rely on EBV replication, such as chronic active EBV infection and posttransplant lymphoproliferative disorders. IMPORTANCE Production of virions is necessary for the ubiquitous Epstein-Barr virus (EBV) to persist in humans and can set the stage for development of EBV cancers in at-risk individuals. In our attempts to identify inhibitors of the EBV lytic phase, we previously found that a prazole proton pump inhibitor, known to block the interaction of ubiquitin with the ESCRT-1 factor Tsg101, blocks production of EBV. We now find that three structurally distinct prazoles impair maturation of EBV capsids and virion transport from the nucleus and, by interfering with Tsg101, prevent EBV release from lytically active cells. Our findings not only implicate Tsg101 in EBV production but also identify widely used prazoles as candidates to prevent development of posttransplant EBV lymphomas.

Properties of Oligomeric Interaction of the Cytomegalovirus Core Nuclear Egress Complex (NEC) and Its Sensitivity to an NEC Inhibitory Small Molecule.

Herpesviral nuclear egress is a regulated process shared by all family members, ensuring the efficient cytoplasmic release of viral capsids. In the case of human cytomegalovirus (HCMV), the core of the nuclear egress complex (NEC) consists of the pUL50-pUL53 heterodimer that builds hexameric lattices for capsid binding and multicomponent interaction, including NEC-associated host factors. A characteristic feature of NEC interaction is the N-terminal hook structure of pUL53 that binds to an alpha-helical groove of pUL50, thus termed as hook-into-groove interaction. This central regulatory element is essential for viral replication and shows structural-functional conservation, which has been postulated as a next-generation target of antiviral strategies. However, a solid validation of this concept has been missing. In the present study, we focused on the properties of oligomeric HCMV core NEC interaction and the antiviral activity of specifically targeted prototype inhibitors. Our data suggest the following: (i) transiently expressed, variably tagged versions of HCMV NEC proteins exert hook-into-groove complexes, putatively in oligomeric assemblies that are distinguishable from heterodimers, as shown by in vitro assembly and coimmunoprecipitation approaches; (ii) this postulated oligomeric binding pattern was further supported by the use of a pUL50::pUL53 fusion construct also showing a pronounced multi-interaction potency; (iii) using confocal imaging cellular NEC-associated proteins were found partly colocalized with the tagged core NECs; (iv) a small inhibitory molecule, recently identified by an in vitro binding inhibition assay, was likewise active in blocking pUL50-pUL53 oligomeric assembly and in exerting antiviral activity in HCMV-infected fibroblasts. In summary, the findings refine the previous concept of HCMV core NEC formation and nominate this drug-accessible complex as a validated antiviral drug target.

Triterpenoid-Mediated Inhibition of Virus-Host Interaction: Is Now the Time for Discovering Viral Entry/Release Inhibitors from Nature?

Fatal infectious diseases caused by HIV-1, influenza A virus, Ebola virus, and currently pandemic coronavirus highlight the great need for the discovery of antiviral agents in mechanisms different from current viral replication-targeted approaches. Given the critical role of virus-host interactions in the viral life cycle, the development of entry or shedding inhibitors may expand the current repertoire of antiviral agents; the combination of antireplication inhibitors and entry or shedding inhibitors would create a multifaceted drug cocktail with a tandem antiviral mechanism. Therefore, we provide critical information about triterpenoids as potential antiviral agents targeting entry and release, focusing specifically on the emerging aspect of triterpenoid-mediated inhibition of a variety of virus-host membrane fusion mechanisms via a trimer-of-hairpin motif. These properties of triterpenoids supply their host an evolutionary advantage for chemical defense and may protect against an increasingly diverse array of viruses infecting mammals, providing a direction for antiviral drug discovery.

A universal dual mechanism immunotherapy for the treatment of influenza virus infections.

Seasonal influenza epidemics lead to 3-5 million severe infections and 290,000-650,000 annual global deaths. With deaths from the 1918 influenza pandemic estimated at >50,000,000 and future pandemics anticipated, the need for a potent influenza treatment is critical. In this study, we design and synthesize a bifunctional small molecule by conjugating the neuraminidase inhibitor, zanamivir, with the highly immunogenic hapten, dinitrophenyl (DNP), which specifically targets the surface of free virus and viral-infected cells. We show that this leads to simultaneous inhibition of virus release, and immune-mediated elimination of both free virus and virus-infected cells. Intranasal or intraperitoneal administration of a single dose of drug to mice infected with 100x MLD50 virus is shown to eradicate advanced infections from representative strains of both influenza A and B viruses. Since treatments of severe infections remain effective up to three days post lethal inoculation, our approach may successfully treat infections refractory to current therapies.

A Novel Mechanism Underlying Antiviral Activity of an Influenza Virus M2-Specific Antibody.

Protective immunity against influenza A viruses (IAVs) generally depends on antibodies to the major envelope glycoprotein, hemagglutinin (HA), whose antigenicity is distinctive among IAV subtypes. On the other hand, the matrix 2 (M2) protein is antigenically highly conserved and has been studied as an attractive vaccine antigen to confer cross-protective immunity against multiple subtypes of IAVs. However, antiviral mechanisms of M2-specific antibodies are not fully understood. Here, we report the molecular basis of antiviral activity of an M2-specific monoclonal antibody (MAb), rM2ss23. We first found that rM2ss23 inhibited A/Aichi/2/1968 (H3N2) (Aichi) but not A/PR/8/1934 (H1N1) (PR8) replication. rM2ss23 altered the cell surface distribution of M2, likely by cross-linking the molecules, and interfered with the colocalization of HA and M2, resulting in reduced budding of progeny viruses. However, these effects were not observed for another strain, PR8, despite the binding capacity of rM2ss23 to PR8 M2. Interestingly, HA was also involved in the resistance of PR8 to rM2ss23. We also found that two amino acid residues at positions 54 and 57 in the M2 cytoplasmic tail were critical for the insensitivity of PR8 to rM2ss2. These findings suggest that the disruption of the M2-HA colocalization on infected cells and subsequent reduction of virus budding is one of the principal mechanisms of antiviral activity of M2-specific antibodies and that anti-M2 antibody-sensitive and -resistant IAVs have different properties in the interaction between M2 and HA.IMPORTANCE Although the IAV HA is the major target of neutralizing antibodies, most of the antibodies are HA subtype specific, restricting the potential of HA-based vaccines. On the contrary, the IAV M2 protein has been studied as a vaccine antigen to confer cross-protective immunity against IAVs with multiple HA subtypes, since M2 is antigenically conserved. Although a number of studies highlight the protective role of anti-HA neutralizing and nonneutralizing antibodies, precise information on the molecular mechanism of action of M2-specific antibodies is still obscure. In this study, we found that an anti-M2 antibody interfered with the HA-M2 association, which is important for efficient budding of progeny virus particles from infected cells. The antiviral activity was IAV strain dependent despite the similar binding capacity of the antibody to M2, and, interestingly, HA was involved in susceptibility to the antibody. Our data provide a novel mechanism underlying antiviral activity of M2-specific antibodies.

Clustered Lysine Residues of the Canine Distemper Virus Matrix Protein Regulate Membrane Association and Budding Activity.

The canine distemper virus (CDV) matrix (M) protein is multifunctional; it orchestrates viral assembly and budding, drives the formation of virus-like particles (VLPs), regulates viral RNA synthesis, and may support additional functions. CDV M may assemble into dimers, where each protomer is constituted by N-terminal and C-terminal domains (NTD and CTD, respectively). Here, to investigate whether electrostatic interactions between CDV M and the plasma membrane (PM) may contribute to budding activity, selected surface-exposed positively charged lysine residues, which are located within a large basic patch of CTD, were replaced by amino acids with selected properties. We found that some M mutants harboring amino acids with neutral and positive charge (methionine and arginine, respectively) maintained full functionality, including proper interaction and localization with the PM as well as intact VLP and progeny virus production as demonstrated by employing a cell exit-complementation system. Conversely, while the overall structural integrity remained mostly unaltered, most of the nonconservative M variants (carrying a glutamic acid; negatively charged) exhibited a cytosolic phenotype secondary to the lack of interaction with the PM. Consequently, such M variants were entirely defective in VLP production and viral particle formation. Furthermore, the proteasome inhibitor bortezomib significantly reduced wild-type M-mediated VLP production. Nevertheless, in the absence of the compound, all engineered M lysine variants exhibited unaffected ubiquitination profiles, consistent with other residues likely involved in this functionally essential posttranslational modification. Altogether, our data identified multiple surface-exposed lysine residues located within a basic patch of CDV M-CTD, critically contributing to PM association and ensuing membrane budding activity.IMPORTANCE Although vaccines against some morbilliviruses exist, infections still occur, which can result in dramatic brain disease or fatal outcome. Postexposure prophylaxis with antivirals would support global vaccination campaigns. Unfortunately, there is no efficient antiviral drug currently approved. The matrix (M) protein of morbilliviruses coordinates viral assembly and egress through interaction with multiple cellular and viral components. However, molecular mechanisms supporting these functions remain poorly understood, which preclude the rationale design of inhibitors. Here, to investigate potential interactions between canine distemper virus (CDV) M and the plasma membrane (PM), we combined structure-guided mutagenesis of selected surface-exposed lysine residues with biochemical, cellular, and virological assays. We identified several lysines clustering in a basic patch microdomain of the CDV M C-terminal domain, which contributed to PM association and budding activity. Our findings provide novel mechanistic information of how morbilliviruses assemble and egress from infected cells, thereby delivering bases for future antiviral drug development.

Anti-Chikungunya Virus Monoclonal Antibody That Inhibits Viral Fusion and Release.

Chikungunya fever, a mosquito-borne disease manifested by fever, rash, myalgia, and arthralgia, is caused by chikungunya virus (CHIKV), which belongs to the genus Alphavirus of the family Togaviridae Anti-CHIKV IgG from convalescent patients is known to directly neutralize CHIKV, and the state of immunity lasts throughout life. Here, we examined the epitope of a neutralizing mouse monoclonal antibody against CHIKV, CHE19, which inhibits viral fusion and release. In silico docking analysis showed that the epitope of CHE19 was localized in the viral E2 envelope and consisted of two separate segments, an N-linker and a ß-ribbon connector, and that its bound Fab fragment on E2 overlapped the position that the E3 glycoprotein originally occupied. We showed that CHIKV-E2 is lost during the viral internalization and that CHE19 inhibits the elimination of CHIKV-E2. These findings suggested that CHE19 stabilizes the E2-E1 heterodimer instead of E3 and inhibits the protrusion of the E1 fusion loop and subsequent membrane fusion. In addition, the antigen-bound Fab fragment configuration showed that CHE19 connects to the CHIKV spikes existing on the two individual virions, leading us to conclude that the CHE19-CHIKV complex was responsible for the large virus aggregations. In our subsequent filtration experiments, large viral aggregations by CHE19 were trapped by a 0.45-µm filter. This virion-connecting characteristic of CHE19 could explain the inhibition of viral release from infected cells by the tethering effect of the virion itself. These findings provide clues toward the development of effective prophylactic and therapeutic monoclonal antibodies against the Alphavirus infection.IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Neither a specific antiviral drug nor a commercial vaccine for CHIKV infection are available. Here, we show a detailed model of the docking between the envelope glycoprotein of CHIKV and our unique anti-CHIKV-neutralizing monoclonal antibody (CHE19), which inhibits CHIKV membrane fusion and virion release from CHIKV-infected cells. Homology modeling of the neutralizing antibody CHE19 and protein-protein docking analysis of the CHIKV envelope glycoprotein and CHE19 suggested that CHE19 inhibits the viral membrane fusion by stabilizing the E2-E1 heterodimer and inhibits virion release by facilitating the formation of virus aggregation due to the connecting virions, and these predictions were confirmed by experiments. Sequence information of CHE19 and the CHIKV envelope glycoprotein and their docking model will contribute to future development of an effective prophylactic and therapeutic agent.