Expression and RNA-binding of human zinc-finger antiviral protein

Zinc-finger antiviral protein (ZAP) is a recently isolated host antiviral factor that inhibits the replication of many viruses such as Moloney murine leukemia virus (MLV) and Sindbis virus (SIN) by preventing the accumulation of viral mRNA in the cytoplasm. ZAP comprises four CCCH zinc-finger motifs, the second and fourth of which are responsible for protein activity based on their integrity. Thus far, there have been no reports on whether or not ZAP expressed in Escherichia coli is soluble. Therefore, we expressed N-terminal ZAP (NZAP, 254 amino acids) in E. coli as a fusion protein with several different cleavage sites and protein tags. Cleaved ZAP in soluble form strongly bound to RNA through its four CCCH zinc-finger motifs. Here, we provide evidence indicating that ZAP directly interacted with viral RNA. Each conserved zinc-finger motif of ZAP coordinates a zinc ion using three cysteines and one histidine. These findings suggest that ZAP recruits the cellular RNA degradation machinery for the degradation of viral RNA.     

A Cholesterol Tag at the N Terminus of the Relatively Broad-Spectrum Fusion Inhibitory Peptide Targets an Earlier Stage of Fusion Glycoprotein Activation and Increases the Peptide's Antiviral Potency In Vivo

In previous work, we designed peptides that showed potent inhibition of Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) infections in chicken embryos. In this study, we demonstrate that peptides modified with cholesterol or 3 U of polyethylene glycol (PEG₃) conjugated to the peptides'' N termini showed even more promising antiviral activities when tested in animal models. Both cholesterol- and cholesterol-PEG₃-tagged peptides were able to protect chicken embryos from infection with different serotypes of NDV and IBV when administered 12 h prior to virus inoculation. In comparison, the untagged peptides required intervention closer to the time of viral inoculation to achieve a similar level of protection. Intramuscular injection of cholesterol-tagged peptide at 1.6 mg/kg 1 day before virus infection and then three times at 3-day intervals after viral inoculation protected 70% of the chickens from NDV infection. We further demonstrate that the cholesterol-tagged peptide has an in vivo half-life greater than that of untagged peptides. It also has the potential to cross the blood-brain barrier to enter the avian central nervous system (CNS). Finally, we show that the cholesterol-tagged peptide could play a role before the viral fusion peptide''s insertion into the host cell and thereby target an earlier stage of fusion glycoprotein activation. Our findings are of importance for the further development of antivirals with broad-spectrum protective effects.     

Antiviral effects of statins

Introducing statins as possible widely-available drugs for the treatment of viral infections requires an in depth review of their antiviral properties. Despite some inconsistency, a large body of literature data from experimental and clinical studies suggest that statins may have a role in the treatment of viral infections due to their immunomodulatory properties as well as their ability to inhibit viral replication. In the present review, the role that statins may play while interacting with the immune system during viral infections and the possible inhibitory effects of statins on different stages of virus cell cycle (i.e., from fusion with host cell membranes to extracellular release) and subsequent virus transmission are described. Specifically, cholesterol-dependent and cholesterol-independent mechanisms of the antiviral effects of statins are reported.     

eIF4A,a target of siRNA derived from rice stripe virus,negatively regulates antiviral autophagy by interacting with ATG5 in Nicotiana benthamiana

Autophagy is induced by viral infection and has antiviral functions in plants, but the underlying mechanism is poorly understood. We previously identified a viral small interfering RNA (vsiRNA) derived from rice stripe virus (RSV) RNA4 that contributes to the leaf-twisting and stunting symptoms caused by this virus by targeting the host eukaryotic translation initiation factor 4A (eIF4A) mRNA for silencing. In addition, autophagy plays antiviral roles by degrading RSV p3 protein, a suppressor of RNA silencing. Here, we demonstrate that eIF4A acts as a negative regulator of autophagy in Nicotiana benthamiana. Silencing of NbeIF4A activated autophagy and inhibited RSV infection by facilitating autophagic degradation of p3. Further analysis showed that NbeIF4A interacts with NbATG5 and interferes with its interaction with ATG12. Overexpression of NbeIF4A suppressed NbATG5-activated autophagy. Moreover, expression of vsiRNA-4A, which targets NbeIF4A mRNA for cleavage, induced autophagy by silencing NbeIF4A. Finally, we demonstrate that eIF4A from rice, the natural host of RSV, also interacts with OsATG5 and suppresses OsATG5-activated autophagy, pointing to the conserved function of eIF4A as a negative regulator of antiviral autophagy. Taken together, these results reveal that eIF4A negatively regulates antiviral autophagy by interacting with ATG5 and that its mRNA is recognized by a virus-derived siRNA, resulting in its silencing, which induces autophagy against viral infection.     

Novel Drosophila Viruses Encode Host-Specific Suppressors of RNAi

The ongoing conflict between viruses and their hosts can drive the co-evolution between host immune genes and viral suppressors of immunity. It has been suggested that an evolutionary ‘arms race’ may occur between rapidly evolving components of the antiviral RNAi pathway of Drosophila and viral genes that antagonize it. We have recently shown that viral protein 1 (VP1) of Drosophila melanogaster Nora virus (DmelNV) suppresses Argonaute-2 (AGO2)-mediated target RNA cleavage (slicer activity) to antagonize antiviral RNAi. Here we show that viral AGO2 antagonists of divergent Nora-like viruses can have host specific activities. We have identified novel Nora-like viruses in wild-caught populations of D. immigrans (DimmNV) and D. subobscura (DsubNV) that are 36% and 26% divergent from DmelNV at the amino acid level. We show that DimmNV and DsubNV VP1 are unable to suppress RNAi in D. melanogaster S2 cells, whereas DmelNV VP1 potently suppresses RNAi in this host species. Moreover, we show that the RNAi suppressor activity of DimmNV VP1 is restricted to its natural host species, D. immigrans. Specifically, we find that DimmNV VP1 interacts with D. immigrans AGO2, but not with D. melanogaster AGO2, and that it suppresses slicer activity in embryo lysates from D. immigrans, but not in lysates from D. melanogaster. This species-specific interaction is reflected in the ability of DimmNV VP1 to enhance RNA production by a recombinant Sindbis virus in a host-specific manner. Our results emphasize the importance of analyzing viral RNAi suppressor activity in the relevant host species. We suggest that rapid co-evolution between RNA viruses and their hosts may result in host species-specific activities of RNAi suppressor proteins, and therefore that viral RNAi suppressors could be host-specificity factors.     

Modeling Innate Antiviral Immunity in Physiological Context

An effective innate antiviral response is critical for the mitigation of severe disease and host survival following infection. In vivo, the innate antiviral response is triggered by cells that detect the invading pathogen and then communicate through autocrine and paracrine signaling to stimulate the expression of genes that inhibit viral replication, curtail cell proliferation, or modulate the immune response. In other words, the innate antiviral response is complex and dynamic. Notably, in the laboratory, culturing viruses and assaying viral life cycles frequently utilizes cells that are derived from tissues other than those that support viral replication during natural infection, while the study of viral pathogenesis often employs animal models. In recapitulating the human antiviral response, it is important to consider that variation in the expression and function of innate immune sensors and antiviral effectors exists across species, cell types, and cell differentiation states, as well as when cells are placed in different contexts. Thus, to gain novel insight into the dynamics of the host response and how specific sensors and effectors impact infection kinetics by a particular virus, the model system must be selected carefully. In this review, we briefly introduce key signaling pathways involved in the innate antiviral response and highlight how these differ between systems. We then review the application of tissue-engineered or 3D models for studying the antiviral response, and suggest how these in vitro culture systems could be further utilized to assay physiologically-relevant host responses and reveal novel insight into virus-host interactions.     

Defective Viral Genomes: Critical Danger Signals of Viral Infections

Viruses efficiently block the host antiviral response in order to replicate and spread before host intervention. The mechanism initiating antiviral immunity during stealth viral replication is unknown, but recent data demonstrate that defective viral genomes generated at peak virus replication are critical for this process in vivo. This article summarizes the supporting evidence and highlights gaps in our understanding of the mechanisms and impact of immunostimulatory defective viral genomes generated during natural infections.     

A Novel Antiviral Target Structure Involved in the RNA Binding,Dimerization, and Nuclear Export Functions of the Influenza A Virus Nucleoprotein

Developing antiviral therapies for influenza A virus (IAV) infection is an ongoing process because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. The ideal strategy is to develop drugs that target well-conserved, functionally restricted, and unique surface structures without affecting host cell function. We recently identified the antiviral compound, RK424, by screening a library of 50,000 compounds using cell-based infection assays. RK424 showed potent antiviral activity against many different subtypes of IAV in vitro and partially protected mice from a lethal dose of A/WSN/1933 (H1N1) virus in vivo. Here, we show that RK424 inhibits viral ribonucleoprotein complex (vRNP) activity, causing the viral nucleoprotein (NP) to accumulate in the cell nucleus. In silico docking analysis revealed that RK424 bound to a small pocket in the viral NP. This pocket was surrounded by three functionally important domains: the RNA binding groove, the NP dimer interface, and nuclear export signal (NES) 3, indicating that it may be involved in the RNA binding, oligomerization, and nuclear export functions of NP. The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424. Surface plasmon resonance (SPR) and pull-down assays showed that RK424 inhibited both the NP-RNA and NP-NP interactions, whereas size exclusion chromatography showed that RK424 disrupted viral RNA-induced NP oligomerization. In addition, in vitro nuclear export assays confirmed that RK424 inhibited nuclear export of NP. The amino acid residues comprising the NP pocket play a crucial role in viral replication and are highly conserved in more than 7,000 NP sequences from avian, human, and swine influenza viruses. Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules. Taken together, these results describe a promising new approach to developing influenza virus drugs that target a novel pocket structure within NP.     

Viral macrodomains: a structural and evolutionary assessment of the pharmacological potential

Viral macrodomains possess the ability to counteract host ADP-ribosylation, a post-translational modification implicated in the creation of an antiviral environment via immune response regulation. This brought them into focus as promising therapeutic targets, albeit the close homology to some of the human macrodomains raised concerns regarding potential cross-reactivity and adverse effects for the host. Here, we evaluate the structure and function of the macrodomain of SARS-CoV-2, the causative agent of COVID-19. We show that it can antagonize ADP-ribosylation by PARP14, a cellular (ADP-ribosyl)transferase necessary for the restriction of coronaviral infections. Furthermore, our structural studies together with ligand modelling revealed the structural basis for poly(ADP-ribose) binding and hydrolysis, an emerging new aspect of viral macrodomain biology. These new insights were used in an extensive evolutionary analysis aimed at evaluating the druggability of viral macrodomains not only from the Coronaviridae but also Togaviridae and Iridoviridae genera (causing diseases such as Chikungunya and infectious spleen and kidney necrosis virus disease, respectively). We found that they contain conserved features, distinct from their human counterparts, which may be exploited during drug design.     

Measles Virus Fusion Machinery Activated by Sialic Acid Binding Globular Domain

Paramyxoviruses, including the human pathogen measles virus (MV) and the avian Newcastle disease virus (NDV), enter host cells through fusion of the viral envelope with the target cell membrane. This fusion is driven by the concerted action of two viral envelope glycoproteins: the receptor binding protein and the fusion protein (F). The MV receptor binding protein (hemagglutinin [H]) attaches to proteinaceous receptors on host cells, while the receptor binding protein of NDV (hemagglutinin-neuraminidase [HN]) interacts with sialic acid-containing receptors. The receptor-bound HN/H triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. The mechanism of fusion activation has been proposed to be different for sialic acid-binding viruses and proteinaceous receptor-binding viruses. We report that a chimeric protein containing the NDV HN receptor binding region and the MV H stalk domain can activate MV F to fuse, suggesting that the signal to the stalk of a protein-binding receptor binding molecule can be transmitted from a sialic acid binding domain. By engineering the NDV HN globular domain to interact with a proteinaceous receptor, the fusion activation signal was preserved. Our findings are consistent with a unified mechanism of fusion activation, at least for the Paramyxovirinae subfamily, in which the receptor binding domains of the receptor binding proteins are interchangeable and the stalk determines the specificity of F activation.     

Efficient Herpes Simplex Virus 1 Replication Requires Cellular ATR Pathway Proteins

Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that replicates in the nucleus of the host cell and is known to interact with several components of the cellular DNA-damage-signaling machinery. We have previously reported that the DNA damage response kinase, ATR, is specifically inactivated in HSV-1-infected cells. On the other hand, we have also shown that ATR and its scaffolding protein, ATRIP, are recruited to viral replication compartments, where they play beneficial roles during HSV-1 replication. In order to better understand this apparent discrepancy, we tested the hypothesis that some of the components of the ATR pathway may exert an antiviral effect on infection. In fact, we learned that all 10 of the canonical ATR pathway proteins are stable in HSV-infected cells and are recruited to viral replication compartments; furthermore, short hairpin RNA (shRNA) knockdown shows that several, including ATRIP, RPA70, TopBP1, Claspin, and CINP, are required for efficient HSV-1 replication. We also determined that activation of the ATR kinase prior to infection did not affect virus yield but did result in reduced levels of recombination between coinfecting viruses. Together, these data suggest that ATR pathway proteins are not antiviral per se but that activation of ATR signaling may have negative consequences during viral replication, such as inhibiting recombination.     

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.     

Tumor Progression Locus 2 Promotes Induction of IFNλ, Interferon Stimulated Genes and Antigen-Specific CD8+ T Cell Responses and Protects against Influenza Virus

Mitogen-activated protein kinase (MAP) cascades are important in antiviral immunity through their regulation of interferon (IFN) production as well as virus replication. Although the serine-threonine MAP kinase tumor progression locus 2 (Tpl2/MAP3K8) has been implicated as a key regulator of Type I (IFNα/β) and Type II (IFNγ) IFNs, remarkably little is known about how Tpl2 might contribute to host defense against viruses. Herein, we investigated the role of Tpl2 in antiviral immune responses against influenza virus. We demonstrate that Tpl2 is an integral component of multiple virus sensing pathways, differentially regulating the induction of IFNα/β and IFNλ in a cell-type specific manner. Although Tpl2 is important in the regulation of both IFNα/β and IFNλ, only IFNλ required Tpl2 for its induction during influenza virus infection both in vitro and in vivo. Further studies revealed an unanticipated function for Tpl2 in transducing Type I IFN signals and promoting expression of interferon-stimulated genes (ISGs). Importantly, Tpl2 signaling in nonhematopoietic cells is necessary to limit early virus replication. In addition to early innate alterations, impaired expansion of virus-specific CD8⁺ T cells accompanied delayed viral clearance in Tpl2^-/- mice at late time points. Consistent with its critical role in facilitating both innate and adaptive antiviral responses, Tpl2 is required for restricting morbidity and mortality associated with influenza virus infection. Collectively, these findings establish an essential role for Tpl2 in antiviral host defense mechanisms.     

Actinobacillus pleuropneumoniae Possesses an Antiviral Activity against Porcine Reproductive and Respiratory Syndrome Virus

Pigs are often colonized by more than one bacterial and/or viral species during respiratory tract infections. This phenomenon is known as the porcine respiratory disease complex (PRDC). Actinobacillus pleuropneumoniae (App) and porcine reproductive and respiratory syndrome virus (PRRSV) are pathogens that are frequently involved in PRDC. The main objective of this project was to study the in vitro interactions between these two pathogens and the host cells in the context of mixed infections. To fulfill this objective, PRRSV permissive cell lines such as MARC-145, SJPL, and porcine alveolar macrophages (PAM) were used. A pre-infection with PRRSV was performed at 0.5 multiplicity of infection (MOI) followed by an infection with App at 10 MOI. Bacterial adherence and cell death were compared. Results showed that PRRSV pre-infection did not affect bacterial adherence to the cells. PRRSV and App co-infection produced an additive cytotoxicity effect. Interestingly, a pre-infection of SJPL and PAM cells with App blocked completely PRRSV infection. Incubation of SJPL and PAM cells with an App cell-free culture supernatant is also sufficient to significantly block PRRSV infection. This antiviral activity is not due to LPS but rather by small molecular weight, heat-resistant App metabolites (<1 kDa). The antiviral activity was also observed in SJPL cells infected with swine influenza virus but to a much lower extent compared to PRRSV. More importantly, the PRRSV antiviral activity of App was also seen with PAM, the cells targeted by the virus in vivo during infection in pigs. The antiviral activity might be due, at least in part, to the production of interferon γ. The use of in vitro experimental models to study viral and bacterial co-infections will lead to a better understanding of the interactions between pathogens and their host cells, and could allow the development of novel prophylactic and therapeutic tools.     

The Cytoplasmic Location of Chicken Mx Is Not the Determining Factor for Its Lack of Antiviral Activity

Background

Chicken Mx belongs to the Mx family of interferon-induced dynamin-like GTPases, which in some species possess potent antiviral properties. Conflicting data exist for the antiviral capability of chicken Mx. Reports of anti-influenza activity of alleles encoding an Asn631 polymorphism have not been supported by subsequent studies. The normal cytoplasmic localisation of chicken Mx may influence its antiviral capacity. Here we report further studies to determine the antiviral potential of chicken Mx against Newcastle disease virus (NDV), an economically important cytoplasmic RNA virus of chickens, and Thogoto virus, an orthomyxovirus known to be exquisitely sensitive to the cytoplasmic MxA protein from humans. We also report the consequences of re-locating chicken Mx to the nucleus.

Methodology/Principal Findings

Chicken Mx was tested in virus infection assays using NDV. Neither the Asn631 nor Ser631 Mx alleles (when transfected into 293T cells) showed inhibition of virus-directed gene expression when the cells were subsequently infected with NDV. Human MxA however did show significant inhibition of NDV-directed gene expression. Chicken Mx failed to inhibit a Thogoto virus (THOV) minireplicon system in which the cytoplasmic human MxA protein showed potent and specific inhibition. Relocalisation of chicken Mx to the nucleus was achieved by inserting the Simian Virus 40 large T antigen nuclear localisation sequence (SV40 NLS) at the N-terminus of chicken Mx. Nuclear re-localised chicken Mx did not inhibit influenza (A/PR/8/34) gene expression during virus infection in cell culture or influenza polymerase activity in A/PR/8/34 or A/Turkey/50-92/91 minireplicon systems.

Conclusions/Significance

The chicken Mx protein (Asn631) lacks inhibitory effects against THOV and NDV, and is unable to suppress influenza replication when artificially re-localised to the cell nucleus. Thus, the natural cytoplasmic localisation of the chicken Mx protein does not account for its lack of antiviral activity.     

MCPIP1 ribonuclease exhibits broad-spectrum antiviral effects through viral RNA binding and degradation

Monocyte chemoattractant protein 1-induced protein 1 (MCPIP1), belonging to the MCPIP family with highly conserved CCCH-type zinc finger and Nedd4-BP1, YacP Nuclease domains, has been implicated in negative regulation of the cellular inflammatory responses. In this report, we demonstrate for the first time that this RNA-binding nuclease also targets viral RNA and possesses potent antiviral activities. Overexpression of the human MCPIP1, but not MCPIP2, MCPIP3 or MCPIP4, inhibited Japanese encephalitis virus (JEV) and dengue virus (DEN) replication. The functional analysis of MCPIP1 revealed that the activities of RNase, RNA binding and oligomerization, but not deubiqutinase, are required for its antiviral potential. Furthermore, infection of other positive-sense RNA viruses, such as sindbis virus and encephalomyocarditis virus, and negative-sense RNA virus, such as influenza virus, as well as DNA virus, such as adenovirus, can also be blocked by MCPIP1. Moreover, the endogenous MCPIP1 gene expression was induced by JEV and DEN infection, and knockdown of MCPIP1 expression enhanced the replication of JEV and DEN in human cells. Thus, MCPIP1 can act as a host innate defense via RNase activity for targeting and degrading viral RNA.     

Influenza A virus nucleoprotein induces apoptosis in human airway epithelial cells: implications of a novel interaction between nucleoprotein and host protein Clusterin

Apoptosis induction is an antiviral host response, however, influenza A virus (IAV) infection promotes host cell death. The nucleoprotein (NP) of IAV is known to contribute to viral pathogenesis, but its role in virus-induced host cell death was hitherto unknown. We observed that NP contributes to IAV infection induced cell death and heterologous expression of NP alone can induce apoptosis in human airway epithelial cells. The apoptotic effect of IAV NP was significant when compared with other known proapoptotic proteins of IAV. The cell death induced by IAV NP was executed through the intrinsic apoptosis pathway. We screened host cellular factors for those that may be targeted by NP for inducing apoptosis and identified human antiapoptotic protein Clusterin (CLU) as a novel interacting partner. The interaction between IAV NP and CLU was highly conserved and mediated through β-chain of the CLU protein. Also CLU was found to interact specifically with IAV NP and not with any other known apoptosis modulatory protein of IAV. CLU prevents induction of the intrinsic apoptosis pathway by binding to Bax and inhibiting its movement into the mitochondria. We found that the expression of IAV NP reduced the association between CLU and Bax in mammalian cells. Further, we observed that CLU overexpression attenuated NP-induced cell death and had a negative effect on IAV replication. Collectively, these findings indicate a new function for IAV NP in inducing host cell death and suggest a role for the host antiapoptotic protein CLU in this process.