Dual Myxovirus Screen Identifies a Small-Molecule Agonist of the Host Antiviral Response

As we are confronted with an increasing number of emerging and reemerging viral pathogens, the identification of novel pathogen-specific and broad-spectrum antivirals has become a major developmental objective. Targeting of host factors required for virus replication presents a tangible approach toward obtaining novel hits with a broadened indication range. However, the identification of developable host-directed antiviral candidates remains challenging. We describe a novel screening protocol that interrogates the myxovirus host-pathogen interactome for broad-spectrum drug candidates and simultaneously probes for conventional, pathogen-directed hits. With resource efficiency and pan-myxovirus activity as the central developmental parameters, we explored coscreening against two distinct, independently traceable myxoviruses in a single-well setting. Having identified a pair of unrelated pathogenic myxoviruses (influenza A virus and measles virus) with comparable replication kinetics, we observed unimpaired coreplication of both viruses, generated suitable firefly and Renilla luciferase reporter constructs, respectively, and validated the protocol for up to a 384-well plate format. Combined with an independent counterscreen using a recombinant respiratory syncytial virus luciferase reporter, implementation of the protocol identified candidates with a broadened antimyxovirus profile, in addition to pathogen-specific hits. Mechanistic characterization revealed a newly discovered broad-spectrum lead that does not block viral entry but stimulates effector pathways of the innate cellular antiviral response. In summary, we provide proof of concept for the efficient discovery of broad-spectrum myxovirus inhibitors in parallel to para- and orthomyxovirus-specific hit candidates in a single screening campaign. The newly identified compound provides a basis for the development of a novel broad-spectrum small-molecule antiviral class.     

Chemical Derivatives of a Small Molecule Deubiquitinase Inhibitor Have Antiviral Activity against Several RNA Viruses

Most antiviral treatment options target the invading pathogen and unavoidably encounter loss of efficacy as the pathogen mutates to overcome replication restrictions. A good strategy for circumventing drug resistance, or for pathogens without treatment options, is to target host cell proteins that are utilized by viruses during infection. The small molecule WP1130 is a selective deubiquitinase inhibitor shown previously to successfully reduce replication of noroviruses and some other RNA viruses. In this study, we screened a library of 31 small molecule derivatives of WP1130 to identify compounds that retained the broad-spectrum antiviral activity of the parent compound in vitro but exhibited improved drug-like properties, particularly increased aqueous solubility. Seventeen compounds significantly reduced murine norovirus infection in murine macrophage RAW 264.7 cells, with four causing decreases in viral titers that were similar or slightly better than WP1130 (1.9 to 2.6 log scale). Antiviral activity was observed following pre-treatment and up to 1 hour postinfection in RAW 264.7 cells as well as in primary bone marrow-derived macrophages. Treatment of the human norovirus replicon system cell line with the same four compounds also decreased levels of Norwalk virus RNA. No significant cytotoxicity was observed at the working concentration of 5 µM for all compounds tested. In addition, the WP1130 derivatives maintained their broad-spectrum antiviral activity against other RNA viruses, Sindbis virus, LaCrosse virus, encephalomyocarditis virus, and Tulane virus. Thus, altering structural characteristics of WP1130 can maintain effective broad-spectrum antiviral activity while increasing aqueous solubility.     

Ribosome inactivating proteins from plants inhibiting viruses

Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins, isolated from plants, are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV). Most of the research work related to RIPs has been focused on antiviral activity against HIV; however, the exact mechanism of antiviral activity is still not clear. The mechanism of antiviral activity was thought to follow inactivation of the host cell ribosome, leading to inhibition of viral protein translation and host cell death. Enzymatic activity of RIPs is not limited to depurination of the large rRNA, in addition they can depurinate viral DNA as well as RNA. Recently, Phase I/II clinical trials have demonstrated the potential use of RIPs for treating patients with HIV disease. The aim of this review is to focus on various RIPs from plants associated with anti-HIV activity.     

Discovery of Genes that Modulate Flavivirus Replication in an Interferon-Dependent Manner

Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function strategy, we screened an original library of 1156 siRNAs targeting 386 individual curated human genes in stimulated microglial cells infected with Zika virus (ZIKV), an emerging RNA virus that belongs to the flavivirus genus. The screen recovered twenty-one potential host proteins that modulate ZIKV replication in an IFN-dependent manner, including the previously known IFITM3 and LY6E. Further characterization contributed to delineate the spectrum of action of these genes towards other pathogenic RNA viruses, including Hepatitis C virus and SARS-CoV-2. Our data revealed that APOL3 acts as a proviral factor for ZIKV and several other related and unrelated RNA viruses. In addition, we showed that MTA2, a chromatin remodeling factor, possesses potent flavivirus-specific antiviral functions induced by IFN. Our work identified previously unrecognized genes that modulate the replication of RNA viruses in an IFN-dependent manner, opening new perspectives to target weakness points in the life cycle of these viruses.     

Targeting Membrane-Bound Viral RNA Synthesis Reveals Potent Inhibition of Diverse Coronaviruses Including the Middle East Respiratory Syndrome Virus

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS–CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.     

High Content Image-Based Screening of a Protease Inhibitor Library Reveals Compounds Broadly Active against Rift Valley Fever Virus and Other Highly Pathogenic RNA Viruses

High content image-based screening was developed as an approach to test a protease inhibitor small molecule library for antiviral activity against Rift Valley fever virus (RVFV) and to determine their mechanism of action. RVFV is the causative agent of severe disease of humans and animals throughout Africa and the Arabian Peninsula. Of the 849 compounds screened, 34 compounds exhibited ≥50% inhibition against RVFV. All of the hit compounds could be classified into 4 distinct groups based on their unique chemical backbone. Some of the compounds also showed broad antiviral activity against several highly pathogenic RNA viruses including Ebola, Marburg, Venezuela equine encephalitis, and Lassa viruses. Four hit compounds (C795-0925, D011-2120, F694-1532 and G202-0362), which were most active against RVFV and showed broad-spectrum antiviral activity, were selected for further evaluation for their cytotoxicity, dose response profile, and mode of action using classical virological methods and high-content imaging analysis. Time-of-addition assays in RVFV infections suggested that D011-2120 and G202-0362 targeted virus egress, while C795-0925 and F694-1532 inhibited virus replication. We showed that D011-2120 exhibited its antiviral effects by blocking microtubule polymerization, thereby disrupting the Golgi complex and inhibiting viral trafficking to the plasma membrane during virus egress. While G202-0362 also affected virus egress, it appears to do so by a different mechanism, namely by blocking virus budding from the trans Golgi. F694-1532 inhibited viral replication, but also appeared to inhibit overall cellular gene expression. However, G202-0362 and C795-0925 did not alter any of the morphological features that we examined and thus may prove to be good candidates for antiviral drug development. Overall this work demonstrates that high-content image analysis can be used to screen chemical libraries for new antivirals and to determine their mechanism of action and any possible deleterious effects on host cellular biology.     

The interplay between virus infection and the cellular RNA interference machinery

RNA interference (RNAi) plays a pivotal role in the regulation of gene expression to control cell development and differentiation. In plants, insects and nematodes RNAi also functions as an innate defence response against viruses. Similarly, there is accumulating evidence that RNAi functions as an antiviral defence mechanism in mammalian cells. Viruses have evolved highly sophisticated mechanisms for interacting with the host cell machinery, and recent evidence indicates that this also involves RNAi pathways. The cellular RNAi machinery can inhibit virus replication, but viruses may also exploit the RNAi machinery for their own replication. In addition, viruses can encode proteins or RNA molecules that suppress existing RNAi pathways or trigger the silencing of specific host genes. Besides the natural interplay between RNAi and viruses, induced RNAi provides an attractive therapy approach for the fight against human pathogenic viruses. Here, we summarize the latest news on virus-RNAi interactions and RNAi based antiviral therapy.     

Proteomic analysis of virus-host interactions in an infectious context using recombinant viruses

RNA viruses exhibit small-sized genomes encoding few proteins, but still establish complex networks of interactions with host cell components to achieve replication and spreading. Ideally, these virus-host protein interactions should be mapped directly in infected cell culture, but such a high standard is often difficult to reach when using conventional approaches. We thus developed a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect physical binding partners during infection. As a proof of concept, we engineered a recombinant measles virus (MV) expressing one of its virulence factors, the MV-V protein, with a One-STrEP amino-terminal tag. This allowed virus-host protein complex analysis directly from infected cells by combining modified tandem affinity chromatography and mass spectrometry analysis. Using this approach, we established a prosperous list of 245 cellular proteins interacting either directly or indirectly with MV-V, and including four of the nine already known partners of this viral factor. These interactions were highly specific of MV-V because they were not recovered when the nucleoprotein MV-N, instead of MV-V, was tagged. Besides key components of the antiviral response, cellular proteins from mitochondria, ribosomes, endoplasmic reticulum, protein phosphatase 2A, and histone deacetylase complex were identified for the first time as prominent targets of MV-V and the critical role of the later protein family in MV replication was addressed. Most interestingly, MV-V showed some preferential attachment to essential proteins in the human interactome network, as assessed by centrality and interconnectivity measures. Furthermore, the list of MV-V interactors also showed a massive enrichment for well-known targets of other viruses. Altogether, this clearly supports our approach based on reverse genetics of viruses combined with high-throughput proteomics to probe the interaction network that viruses establish in infected cells.     

Virucidal activity of a scorpion venom peptide variant mucroporin-M1 against measles, SARS-CoV and influenza H5N1 viruses

Outbreaks of SARS-CoV, influenza A (H5N1, H1N1) and measles viruses in recent years have raised serious concerns about the measures available to control emerging and re-emerging infectious viral diseases. Effective antiviral agents are lacking that specifically target RNA viruses such as measles, SARS-CoV and influenza H5N1 viruses, and available vaccinations have demonstrated variable efficacy. Therefore, the development of novel antiviral agents is needed to close the vaccination gap and silence outbreaks. We previously indentified mucroporin, a cationic host defense peptide from scorpion venom, which can effectively inhibit standard bacteria. The optimized mucroporin-M1 can inhibit gram-positive bacteria at low concentrations and antibiotic-resistant pathogens. In this investigation, we further tested mucroporin and the optimized mucroporin-M1 for their antiviral activity. Surprisingly, we found that the antiviral activities of mucroporin-M1 against measles, SARS-CoV and influenza H5N1 viruses were notably increased with an EC₅₀ of 7.15 μg/ml (3.52 μM) and a CC₅₀ of 70.46 μg/ml (34.70 μM) against measles virus, an EC₅₀ of 14.46 μg/ml (7.12 μM) against SARS-CoV and an EC₅₀ of 2.10 μg/ml (1.03 μM) against H5N1, while the original peptide mucroporin showed no antiviral activity against any of these three viruses. The inhibition model could be via a direct interaction with the virus envelope, thereby decreasing the infectivity of virus. This report provides evidence that host defense peptides from scorpion venom can be modified for antiviral activity by rational design and represents a practical approach for developing broad-spectrum antiviral agents, especially against RNA viruses.     

Enhancement of replication of RNA viruses by ADAR1 via RNA editing and inhibition of RNA-activated protein kinase

Adenosine deaminase acting on RNA 1 (ADAR1) is a double-stranded RNA binding protein and RNA-editing enzyme that modifies cellular and viral RNAs, including coding and noncoding RNAs. This interferon (IFN)-induced protein was expected to have an antiviral role, but recent studies have demonstrated that it promotes the replication of many RNA viruses. The data from these experiments show that ADAR1 directly enhances replication of hepatitis delta virus, human immunodeficiency virus type 1, vesicular stomatitis virus, and measles virus. The proviral activity of ADAR1 occurs through two mechanisms: RNA editing and inhibition of RNA-activated protein kinase (PKR). While these pathways have been found independently, the two mechanisms can act in concert to increase viral replication and contribute to viral pathogenesis. This novel type of proviral regulation by an IFN-induced protein, combined with some antiviral effects of hyperediting, sheds new light on the importance of ADAR1 during viral infection and transforms our overall understanding of the innate immune response.     

Viral Subversion of Nucleocytoplasmic Trafficking

Trafficking of proteins and RNA into and out of the nucleus occurs through the nuclear pore complex (NPC). Because of its critical function in many cellular processes, the NPC and transport factors are common targets of several viruses that disrupt key constituents of the machinery to facilitate viral replication. Many viruses such as poliovirus and severe acute respiratory syndrome (SARS) virus inhibit protein import into the nucleus, whereas viruses such as influenza A virus target and disrupt host mRNA nuclear export. Current evidence indicates that these viruses may employ such strategies to avert the host immune response. Conversely, many viruses co‐opt nucleocytoplasmic trafficking to facilitate transport of viral RNAs. As viral proteins interact with key regulators of the host nuclear transport machinery, viruses have served as invaluable tools of discovery that led to the identification of novel constituents of nuclear transport pathways. This review explores the importance of nucleocytoplasmic trafficking to viral pathogenesis as these studies revealed new antiviral therapeutic strategies and exposed previously unknown cellular mechanisms. Further understanding of nuclear transport pathways will determine whether such therapeutics will be useful treatments for important human pathogens.      

Interaction of the innate immune system with positive-strand RNA virus replication organelles

The potential health risks associated with (re-)emerging positive-strand RNA (+RNA) viruses emphasizes the need for understanding host-pathogen interactions for these viruses. The innate immune system forms the first line of defense against pathogenic organisms like these and is responsible for detecting pathogen-associated molecular patterns (PAMPs). Viral RNA is a potent inducer of antiviral innate immune signaling, provoking an antiviral state by directing expression of interferons (IFNs) and pro-inflammatory cytokines. However, +RNA viruses developed various methods to avoid detection and downstream signaling, including isolation of viral RNA replication in membranous viral replication organelles (ROs). These structures therefore play a central role in infection, and consequently, loss of RO integrity might simultaneously result in impaired viral replication and enhanced antiviral signaling. This review summarizes the first indications that the innate immune system indeed has tools to disrupt viral ROs and other non- or aberrant-self membrane structures, and may do this by marking these membranes with proteins such as microtubule-associated protein 1A/1B-light chain 3 (LC3) and ubiquitin, resulting in the recruitment of IFN-inducible GTPases. Further studies should evaluate whether this process forms a general effector mechanism in +RNA virus infection, thereby creating the opportunity for development of novel antiviral therapies.     

Broad action of Hsp90 as a host chaperone required for viral replication

Viruses are intracellular pathogens responsible for a vast number of human diseases. Due to their small genome size, viruses rely primarily on the biosynthetic apparatus of the host for their replication. Recent work has shown that the molecular chaperone Hsp90 is nearly universally required for viral protein homeostasis. As observed for many endogenous cellular proteins, numerous different viral proteins have been shown to require Hsp90 for their folding, assembly, and maturation. Importantly, the unique characteristics of viral replication cause viruses to be hypersensitive to Hsp90 inhibition, thus providing a novel therapeutic avenue for the development of broad-spectrum antiviral drugs. The major developments in this emerging field are hereby discussed. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

Host cellular signaling induced by influenza virus

A wide range of host cellular signal transduction pathways can be stimulated by influenza virus infection. Some of these signal transduction pathways induce the host cell’s innate immune response against influenza virus, while others are essential for efficient influenza virus replication. This review examines the cellular signaling induced by influenza virus infection in host cells, including host pattern recognition receptor (PRR)-related signaling, protein kinase C (PKC), Raf/MEK/ERK and phosphatidylinositol- 3-kinase (PI3K)/Akt signaling, and the corresponding effects on the host cell and/or virus, such as recognition of virus by the host cell, viral absorption and entry, viral ribonucleoprotein (vRNP) export, translation control of cellular and viral proteins, and virus-induced cell apoptosis. Research into influenza virus-induced cell signaling promotes a clearer understanding of influenza virus-host interactions and assists in the identification of novel antiviral targets and antiviral strategies.     

The intricate interplay between RNA viruses and NF-κB

RNA viruses have rapidly evolving genomes which often allow cross-species transmission and frequently generate new virus variants with altered pathogenic properties. Therefore infections by RNA viruses are a major threat to human health. The infected host cell detects trace amounts of viral RNA and the last years have revealed common principles in the biochemical mechanisms leading to signal amplification that is required for mounting of a powerful antiviral response. Components of the RNA sensing and signaling machinery such as RIG-I-like proteins, MAVS and the inflammasome inducibly form large oligomers or even fibers that exhibit hallmarks of prions. Following a nucleation event triggered by detection of viral RNA, these energetically favorable and irreversible polymerization events trigger signaling cascades leading to the induction of antiviral and inflammatory responses, mediated by interferon and NF-κB pathways. Viruses have evolved sophisticated strategies to manipulate these host cell signaling pathways in order to ensure their replication. We will discuss at the examples of influenza and HTLV-1 viruses how a fascinating diversity of biochemical mechanisms is employed by viral proteins to control the NF-κB pathway at all levels.