Antiviral plant-derived natural products to combat RNA viruses: Targets throughout the viral life cycle.

There is a need for new effective antivirals, particularly in response to the development of antiviral drug resistance and emerging RNA viruses such as SARS-CoV-2. Plants are a significant source of structurally diverse bioactive compounds for drug discovery suggesting that plant-derived natural products could be developed as antiviral agents. This article reviews the antiviral activity of plant-derived natural products against RNA viruses, with a focus on compounds targeting specific stages of the viral life cycle. A range of plant extracts and compounds have been identified with antiviral activity, often against multiple virus families suggesting they may be useful as broad-spectrum antiviral agents. The antiviral mechanism of action of many of these phytochemicals is not fully understood and there are limited studies and clinical trials demonstrating their efficacy and toxicity in vivo. Further research is needed to evaluate the therapeutic potential of plant-derived natural products as antiviral agents.

S-farnesylation is essential for antiviral activity of the long ZAP isoform against RNA viruses with diverse replication strategies.

The zinc finger antiviral protein (ZAP) is a broad inhibitor of virus replication. Its best-characterized function is to bind CpG dinucleotides present in viral RNAs and, through the recruitment of TRIM25, KHNYN and other cofactors, target them for degradation or prevent their translation. The long and short isoforms of ZAP (ZAP-L and ZAP-S) have different intracellular localization and it is unclear how this regulates their antiviral activity against viruses with different sites of replication. Using ZAP-sensitive and ZAP-insensitive human immunodeficiency virus type I (HIV-1), which transcribe the viral RNA in the nucleus and assemble virions at the plasma membrane, we show that the catalytically inactive poly-ADP-ribose polymerase (PARP) domain in ZAP-L is essential for CpG-specific viral restriction. Mutation of a crucial cysteine in the C-terminal CaaX box that mediates S-farnesylation and, to a lesser extent, the residues in place of the catalytic site triad within the PARP domain, disrupted the activity of ZAP-L. Addition of the CaaX box to ZAP-S partly restored antiviral activity, explaining why ZAP-S lacks antiviral activity for CpG-enriched HIV-1 despite conservation of the RNA-binding domain. Confocal microscopy confirmed the CaaX motif mediated localization of ZAP-L to vesicular structures and enhanced physical association with intracellular membranes. Importantly, the PARP domain and CaaX box together jointly modulate the interaction between ZAP-L and its cofactors TRIM25 and KHNYN, implying that its proper subcellular localisation is required to establish an antiviral complex. The essential contribution of the PARP domain and CaaX box to ZAP-L antiviral activity was further confirmed by inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, which replicates in double-membrane vesicles derived from the endoplasmic reticulum. Thus, compartmentalization of ZAP-L on intracellular membranes provides an essential effector function in ZAP-L-mediated antiviral activity against divergent viruses with different subcellular replication sites.

Multicellular spatial model of RNA virus replication and interferon responses reveals factors controlling plaque growth dynamics.

Respiratory viruses present major public health challenges, as evidenced by the 1918 Spanish Flu, the 1957 H2N2, 1968 H3N2, and 2009 H1N1 influenza pandemics, and the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Severe RNA virus respiratory infections often correlate with high viral load and excessive inflammation. Understanding the dynamics of the innate immune response and its manifestations at the cell and tissue levels is vital to understanding the mechanisms of immunopathology and to developing strain-independent treatments. Here, we present a novel spatialized multicellular computational model of RNA virus infection and the type-I interferon-mediated antiviral response that it induces within lung epithelial cells. The model is built using the CompuCell3D multicellular simulation environment and is parameterized using data from influenza virus-infected cell cultures. Consistent with experimental observations, it exhibits either linear radial growth of viral plaques or arrested plaque growth depending on the local concentration of type I interferons. The model suggests that modifying the activity of signaling molecules in the JAK/STAT pathway or altering the ratio of the diffusion lengths of interferon and virus in the cell culture could lead to plaque growth arrest. The dependence of plaque growth arrest on diffusion lengths highlights the importance of developing validated spatial models of cytokine signaling and the need for in vitro measurement of these diffusion coefficients. Sensitivity analyses under conditions leading to continuous or arrested plaque growth found that plaque growth is more sensitive to variations of most parameters and more likely to have identifiable model parameters when conditions lead to plaque arrest. This result suggests that cytokine assay measurements may be most informative under conditions leading to arrested plaque growth. The model is easy to extend to include SARS-CoV-2-specific mechanisms or to use as a component in models linking epithelial cell signaling to systemic immune models.

ADAR Editing in Viruses: An Evolutionary Force to Reckon with.

Adenosine Deaminases that Act on RNA (ADARs) are RNA editing enzymes that play a dynamic and nuanced role in regulating transcriptome and proteome diversity. This editing can be highly selective, affecting a specific site within a transcript, or nonselective, resulting in hyperediting. ADAR editing is important for regulating neural functions and autoimmunity, and has a key role in the innate immune response to viral infections, where editing can have a range of pro- or antiviral effects and can contribute to viral evolution. Here we examine the role of ADAR editing across a broad range of viral groups. We propose that the effect of ADAR editing on viral replication, whether pro- or antiviral, is better viewed as an axis rather than a binary, and that the specific position of a given virus on this axis is highly dependent on virus- and host-specific factors, and can change over the course of infection. However, more research needs to be devoted to understanding these dynamic factors and how they affect virus-ADAR interactions and viral evolution. Another area that warrants significant attention is the effect of virus-ADAR interactions on host-ADAR interactions, particularly in light of the crucial role of ADAR in regulating neural functions. Answering these questions will be essential to developing our understanding of the relationship between ADAR editing and viral infection. In turn, this will further our understanding of the effects of viruses such as SARS-CoV-2, as well as many others, and thereby influence our approach to treating these deadly diseases.

Povidone-Iodine Attenuates Viral Replication in Ocular Cells: Implications for Ocular Transmission of RNA Viruses.

Several RNA viruses, including SARS-CoV-2, can infect or use the eye as an entry portal to cause ocular or systemic diseases. Povidone-Iodine (PVP-I) is routinely used during ocular surgeries and eye banking as a cost-effective disinfectant due to its broad-spectrum antimicrobial activity, including against viruses. However, whether PVP-I can exert antiviral activities in virus-infected cells remains elusive. In this study, using Zika (ZIKV) and Chikungunya (CHIKV) virus infection of human corneal and retinal pigment epithelial cells, we report antiviral mechanisms of PVP-I. Our data showed that PVP-I, even at the lowest concentration (0.01%), drastically reduced viral replication in corneal and retinal cells without causing cellular toxicity. Antiviral effects of PVP-I against ZIKV and CHIKV were mediated by direct viral inactivation, thus attenuating the ability of the virus to infect host cells. Moreover, one-minute PVP-I exposure of infected ocular cells drastically reduced viral replication and the production of infectious progeny virions. Furthermore, viral-induced (CHIKV) expression of inflammatory genes (TNF-α, IL-6, IL-8, and IL1ß) were markedly reduced in PVP-I treated corneal epithelial cells. Together, our results demonstrate potent antiviral effects of PVP-I against ZIKV and CHIKV infection of ocular cells. Thus, a low dose of PVP-I can be used during tissue harvesting for corneal transplants to prevent potential transmission of RNA viruses via infected cells.

Interferon-Independent Restriction of RNA Virus Entry and Replication by a Class of Damage-Associated Molecular Patterns.

Mammalian cells detect microbial molecules known as pathogen-associated molecular patterns (PAMPs) as indicators of potential infection. Upon PAMP detection, diverse defensive responses are induced by the host, including those that promote inflammation and cell-intrinsic antimicrobial activities. Host-encoded molecules released from dying or damaged cells, known as damage-associated molecular patterns (DAMPs), also induce defensive responses. Both DAMPs and PAMPs are recognized for their inflammatory potential, but only the latter are well established to stimulate cell-intrinsic host defense. Here, we report a class of DAMPs that engender an antiviral state in human epithelial cells. These DAMPs include oxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine), PGPC (1-palmitoyl-2-glutaryl phosphatidylcholine), and POVPC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphatidylcholine], oxidized lipids that are naturally released from dead or dying cells. Exposing cells to these DAMPs prior to vesicular stomatitis virus (VSV) infection limits viral replication. Mechanistically, these DAMPs prevent viral entry, thereby limiting the percentage of cells that are productively infected and consequently restricting viral load. We found that the antiviral actions of oxidized lipids are distinct from those mediated by the PAMP Poly I:C, in that the former induces a more rapid antiviral response without the induction of the interferon response. These data support a model whereby interferon-independent defensive activities can be induced by DAMPs, which may limit viral replication before PAMP-mediated interferon responses are induced. This antiviral activity may impact viruses that disrupt interferon responses in the oxygenated environment of the lung, such as influenza virus and SARS-CoV-2.IMPORTANCE In this work, we explored how a class of oxidized lipids, spontaneously created during tissue damage and unprogrammed cell lysis, block the earliest events in RNA virus infection in the human epithelium. This gives us novel insight into the ways that we view infection models, unveiling a built-in mechanism to slow viral growth that neither engages the interferon response nor is subject to known viral antagonism. These oxidized phospholipids act prior to infection, allowing time for other, better-known innate immune mechanisms to take effect. This discovery broadens our understanding of host defenses, introducing a soluble factor that alters the cellular environment to protect from RNA virus infection.

SARS-CoV-2: lessons from both the history of medicine and from the biological behavior of other well-known viruses.

SARS-CoV-2 is the etiological agent of the current pandemic worldwide and its associated disease COVID-19. In this review, we have analyzed SARS-CoV-2 characteristics and those ones of other well-known RNA viruses viz. HIV, HCV and Influenza viruses, collecting their historical data, clinical manifestations and pathogenetic mechanisms. The aim of the work is obtaining useful insights and lessons for a better understanding of SARS-CoV-2. These pathogens present a distinct mode of transmission, as SARS-CoV-2 and Influenza viruses are airborne, whereas HIV and HCV are bloodborne. However, these viruses exhibit some potential similar clinical manifestations and pathogenetic mechanisms and their understanding may contribute to establishing preventive measures and new therapies against SARS-CoV-2.

Role of Heat Shock Proteins (HSP70 and HSP90) in Viral Infection.

Heat shock proteins (HSPs) are a large group of chaperones found in most eukaryotes and bacteria. They are responsible for the correct protein folding, protection of the cell against stressors, presenting immune and inflammatory cytokines; furthermore, they are important factors in regulating cell differentiation, survival and death. Although the biological function of HSPs is to maintain cell homeostasis, some of them can be used by viruses both to fold their proteins and increase the chances of survival in unfavorable host conditions. Folding viral proteins as well as replicating many different viruses are carried out by, among others, proteins from the HSP70 and HSP90 families. In some cases, the HSP70 family proteins directly interact with viral polymerase to enhance viral replication or they can facilitate the formation of a viral replication complex and/or maintain the stability of complex proteins. It is known that HSP90 is important for the expression of viral genes at both the transcriptional and the translational levels. Both of these HSPs can form a complex with HSP90 and, consequently, facilitate the entry of the virus into the cell. Current studies have shown the biological significance of HSPs in the course of infection SARS-CoV-2. A comprehensive understanding of chaperone use during viral infection will provide new insight into viral replication mechanisms and therapeutic potential. The aim of this study is to describe the molecular basis of HSP70 and HSP90 participation in some viral infections and the potential use of these proteins in antiviral therapy.

An isoform of Dicer protects mammalian stem cells against multiple RNA viruses.

In mammals, early resistance to viruses relies on interferons, which protect differentiated cells but not stem cells from viral replication. Many other organisms rely instead on RNA interference (RNAi) mediated by a specialized Dicer protein that cleaves viral double-stranded RNA. Whether RNAi also contributes to mammalian antiviral immunity remains controversial. We identified an isoform of Dicer, named antiviral Dicer (aviD), that protects tissue stem cells from RNA viruses-including Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-by dicing viral double-stranded RNA to orchestrate antiviral RNAi. Our work sheds light on the molecular regulation of antiviral RNAi in mammalian innate immunity, in which different cell-intrinsic antiviral pathways can be tailored to the differentiation status of cells.

Applications of Deep Mutational Scanning in Virology.

Several recently developed high-throughput techniques have changed the field of molecular virology. For example, proteomics studies reveal complete interactomes of a viral protein, genome-wide CRISPR knockout and activation screens probe the importance of every single human gene in aiding or fighting a virus, and ChIP-seq experiments reveal genome-wide epigenetic changes in response to infection. Deep mutational scanning is a relatively novel form of protein science which allows the in-depth functional analysis of every nucleotide within a viral gene or genome, revealing regions of importance, flexibility, and mutational potential. In this review, we discuss the application of this technique to RNA viruses including members of the Flaviviridae family, Influenza A Virus and Severe Acute Respiratory Syndrome Coronavirus 2. We also briefly discuss the reverse genetics systems which allow for analysis of viral replication cycles, next-generation sequencing technologies and the bioinformatics tools that facilitate this research.

NPC1-regulated dynamic of clathrin-coated pits is essential for viral entry.

Viruses utilize cellular lipids and manipulate host lipid metabolism to ensure their replication and spread. Therefore, the identification of lipids and metabolic pathways that are suitable targets for antiviral development is crucial. Using a library of compounds targeting host lipid metabolic factors and testing them for their ability to block pseudorabies virus (PRV) and vesicular stomatitis virus (VSV) infection, we found that U18666A, a specific inhibitor of Niemann-Pick C1 (NPC1), is highly potent in suppressing the entry of diverse viruses including pseudotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). NPC1 deficiency markedly attenuates viral growth by decreasing cholesterol abundance in the plasma membrane, thereby inhibiting the dynamics of clathrin-coated pits (CCPs), which are indispensable for clathrin-mediated endocytosis. Significantly, exogenous cholesterol can complement the dynamics of CCPs, leading to efficient viral entry and infectivity. Administration of U18666A improves the survival and pathology of PRV- and influenza A virus-infected mice. Thus, our studies demonstrate a unique mechanism by which NPC1 inhibition achieves broad antiviral activity, indicating a potential new therapeutic strategy against SARS-CoV-2, as well as other emerging viruses.

Multiscale Electron Microscopy for the Study of Viral Replication Organelles.

During infection with positive-strand RNA viruses, viral RNA synthesis associates with modified intracellular membranes that form unique and captivating structures in the cytoplasm of the infected cell. These viral replication organelles (ROs) play a key role in the replicative cycle of important human pathogens like coronaviruses, enteroviruses, or flaviviruses. From their discovery to date, progress in our understanding of viral ROs has closely followed new developments in electron microscopy (EM). This review gives a chronological account of this progress and an introduction to the different EM techniques that enabled it. With an ample repertoire of imaging modalities, EM is nowadays a versatile technique that provides structural and functional information at a wide range of scales. Together with well-established approaches like electron tomography or labeling methods, we examine more recent developments, such as volume scanning electron microscopy (SEM) and in situ cryotomography, which are only beginning to be applied to the study of viral ROs. We also highlight the first cryotomography analyses of viral ROs, which have led to the discovery of macromolecular complexes that may serve as RO channels that control the export of newly-made viral RNA. These studies are key first steps towards elucidating the macromolecular complexity of viral ROs.

iPSC screening for drug repurposing identifies anti-RNA virus agents modulating host cell susceptibility.

Human pathogenic RNA viruses are threats to public health because they are prone to escaping the human immune system through mutations of genomic RNA, thereby causing local outbreaks and global pandemics of emerging or re-emerging viral diseases. While specific therapeutics and vaccines are being developed, a broad-spectrum therapeutic agent for RNA viruses would be beneficial for targeting newly emerging and mutated RNA viruses. In this study, we conducted a screen of repurposed drugs using Sendai virus (an RNA virus of the family Paramyxoviridae), with human-induced pluripotent stem cells (iPSCs) to explore existing drugs that may present anti-RNA viral activity. Selected hit compounds were evaluated for their efficacy against two important human pathogens: Ebola virus (EBOV) using Huh7 cells and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using Vero E6 cells. Selective estrogen receptor modulators (SERMs), including raloxifene, exhibited antiviral activities against EBOV and SARS-CoV-2. Pioglitazone, a PPARγ agonist, also exhibited antiviral activities against SARS-CoV-2, and both raloxifene and pioglitazone presented a synergistic antiviral effect. Finally, we demonstrated that SERMs blocked entry steps of SARS-CoV-2 into host cells. These findings suggest that the identified FDA-approved drugs can modulate host cell susceptibility against RNA viruses.

Ivermectin also inhibits the replication of bovine respiratory viruses (BRSV, BPIV-3, BoHV-1, BCoV and BVDV) in vitro.

Bovine respiratory disease (BRD) complex is an important viral infection that causes huge economic losses in cattle herds worldwide. However, there is no directly effective antiviral drug application against respiratory viral pathogens; generally, the metaphylactic antibacterial drug applications are used for BRD. Ivermectin (IVM) is currently used as a broad-spectrum anti-parasitic agent both for veterinary and human medicine on some occasions. Moreover, since it is identified as an inhibitor for importin α/ß-mediated nuclear localization signal (NLS), IVM is also reported to have antiviral potential against several RNA and DNA viruses. Since therapeutic use of IVM in COVID-19 cases has recently been postulated, the potential antiviral activity of IVM against bovine respiratory viruses including BRSV, BPIV-3, BoHV-1, BCoV and BVDV are evaluated in this study. For these purposes, virus titration assay was used to evaluate titers in viral harvest from infected cells treated with non-cytotoxic IVM concentrations (1, 2.5 and 5 µM) and compared to titers from non-treated infected cells. This study indicated that IVM inhibits the replication of BCoV, BVDV, BRSV, BPIV-3 and BoHV-1 in a dose-dependent manner in vitro as well as number of extracellular infectious virions. In addition, it was demonstrated that IVM has no clear effect on the attachment and penetration steps of the replication of the studied viruses. Finally, this study shows for the first time that IVM can inhibit infection of BRD-related viral agents namely BCoV, BPIV-3, BVDV, BRSV and BoHV-1 at the concentrations of 2.5 and 5 µM. Consequently, IVM, which is licensed for antiparasitic indications, also deserves to be evaluated as a broad-spectrum antiviral in BRD cases caused by viral pathogens.

Natural Products with Potential to Treat RNA Virus Pathogens Including SARS-CoV-2.

Three families of RNA viruses, the Coronaviridae, Flaviviridae, and Filoviridae, collectively have great potential to cause epidemic disease in human populations. The current SARS-CoV-2 (Coronaviridae) responsible for the COVID-19 pandemic underscores the lack of effective medications currently available to treat these classes of viral pathogens. Similarly, the Flaviviridae, which includes such viruses as Dengue, West Nile, and Zika, and the Filoviridae, with the Ebola-type viruses, as examples, all lack effective therapeutics. In this review, we present fundamental information concerning the biology of these three virus families, including their genomic makeup, mode of infection of human cells, and key proteins that may offer targeted therapies. Further, we present the natural products and their derivatives that have documented activities to these viral and host proteins, offering hope for future mechanism-based antiviral therapeutics. By arranging these potential protein targets and their natural product inhibitors by target type across these three families of virus, new insights are developed, and crossover treatment strategies are suggested. Hence, natural products, as is the case for other therapeutic areas, continue to be a promising source of structurally diverse new anti-RNA virus therapeutics.

mTOR inhibition in COVID-19: A commentary and review of efficacy in RNA viruses.

In this commentary, we shed light on the role of the mammalian target of rapamycin (mTOR) pathway in viral infections. The mTOR pathway has been demonstrated to be modulated in numerous RNA viruses. Frequently, inhibiting mTOR results in suppression of virus growth and replication. Recent evidence points towards modulation of mTOR in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We discuss the current literature on mTOR in SARS-CoV-2 and highlight evidence in support of a role for mTOR inhibitors in the treatment of coronavirus disease 2019.

Novel Broad-Spectrum Antiviral Inhibitors Targeting Host Factors Essential for Replication of Pathogenic RNA Viruses.

Recent RNA virus outbreaks such as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ebola virus (EBOV) have caused worldwide health emergencies highlighting the urgent need for new antiviral strategies. Targeting host cell pathways supporting viral replication is an attractive approach for development of antiviral compounds, especially with new, unexplored viruses where knowledge of virus biology is limited. Here, we present a strategy to identify host-targeted small molecule inhibitors using an image-based phenotypic antiviral screening assay followed by extensive target identification efforts revealing altered cellular pathways upon antiviral compound treatment. The newly discovered antiviral compounds showed broad-range antiviral activity against pathogenic RNA viruses such as SARS-CoV-2, EBOV and Crimean-Congo hemorrhagic fever virus (CCHFV). Target identification of the antiviral compounds by thermal protein profiling revealed major effects on proteostasis pathways and disturbance in interactions between cellular HSP70 complex and viral proteins, illustrating the supportive role of HSP70 on many RNA viruses across virus families. Collectively, this strategy identifies new small molecule inhibitors with broad antiviral activity against pathogenic RNA viruses, but also uncovers novel virus biology urgently needed for design of new antiviral therapies.

Novel viruses detected in bats in the Republic of Korea.

Bats are natural reservoirs for potential zoonotic viruses. In this study, next-generation sequencing was performed to obtain entire genome sequences of picornavirus from a picornavirus-positive bat feces sample (16BF77) and to explore novel viruses in a pooled bat sample (16BP) from samples collected in South Korea, 2016. Fourteen mammalian viral sequences were identified from 16BF77 and 29 from 16BP, and verified by RT-PCR. The most abundant virus in 16BF77 was picornavirus. Highly variable picornavirus sequences encoding 3Dpol were classified into genera Kobuvirus, Shanbavirus, and an unassigned group within the family Picornaviridae. Amino acid differences between these partial 3Dpol sequences were ≥ 65.7%. Results showed that one bat was co-infected by picornaviruses of more than two genera. Retrovirus, coronavirus, and rotavirus A sequences also were found in the BP sample. The retrovirus and coronavirus genomes were identified in nine and eight bats, respectively. Korean bat retroviruses and coronavirus demonstrated strong genetic relationships with a Chinese bat retrovirus (RfRV) and coronavirus (HKU5-1), respectively. A co-infection was identified in one bat with a retrovirus and a coronavirus. Our results indicate that Korean bats were multiply infected by several mammal viruses.

Interferons and viruses induce a novel truncated ACE2 isoform and not the full-length SARS-CoV-2 receptor.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19, utilizes angiotensin-converting enzyme 2 (ACE2) for entry into target cells. ACE2 has been proposed as an interferon-stimulated gene (ISG). Thus, interferon-induced variability in ACE2 expression levels could be important for susceptibility to COVID-19 or its outcomes. Here, we report the discovery of a novel, transcriptionally independent truncated isoform of ACE2, which we designate as deltaACE2 (dACE2). We demonstrate that dACE2, but not ACE2, is an ISG. In The Cancer Genome Atlas, the expression of dACE2 was enriched in squamous tumors of the respiratory, gastrointestinal and urogenital tracts. In vitro, dACE2, which lacks 356 amino-terminal amino acids, was non-functional in binding the SARS-CoV-2 spike protein and as a carboxypeptidase. Our results suggest that the ISG-type induction of dACE2 in IFN-high conditions created by treatments, an inflammatory tumor microenvironment or viral co-infections is unlikely to increase the cellular entry of SARS-CoV-2 and promote infection.