Identification of brevinin-1EMa-derived stapled peptides as broad-spectrum virus entry blockers.

Based on the previously reported 13-residue antibacterial peptide analog, brevinin-1EMa (FLGWLFKVASKVL, peptide B), we attempted to design a novel class of antiviral peptides. For this goal, we synthesized three peptides with different stapling positions (B-2S, B-8S, and B-5S). The most active antiviral peptide with the specific stapling position (B-5S) was further modified in combination with either cysteine (B-5S3C, B-5S7C, and B-5S10C) or hydrophilic amino acid substitution (Bsub and Bsub-5S). Overall, B, B-5S, and Bsub-5S peptides showed superior antiviral activities against enveloped viruses such as retrovirus, lentivirus, hepatitis C virus, and herpes simplex virus with EC50 values of 1-5 µM. Murine norovirus, a non-enveloped virus, was not susceptible to the virucidal actions of these peptides, suggesting the virus membrane disruption as their main antiviral mechanisms of action. We believe that these three novel peptides could serve as promising candidates for further development of membrane-targeting antiviral drugs in the future.

Carrageenans as Broad-Spectrum Microbicides: Current Status and Challenges.

Different kinds of red algae are enriched with chemically diverse carbohydrates. In particular, a group of sulfated polysaccharides, which were isolated from the cell walls of red algae, gained a large amount of attention due to their broad-spectrum antimicrobial activities. Within that group, carrageenans (CGs) were expected to be the first clinically applicable microbicides that could prevent various viral infections due to their superior antiviral potency and desirable safety profiles in subclinical studies. However, their anticipated beneficial effects could not be validated in human studies. To assess the value of a second attempt at pharmacologically developing CGs as a new class of preventive microbicides, all preclinical and clinical development processes of CG-based microbicides need to be thoroughly re-evaluated. In this review, the in vitro toxicities; in vivo safety profiles; and in vitro, ex vivo, and in vivo antiviral activities of CGs are summarized according to the study volume of their target viruses, which include human immunodeficiency virus, herpesviruses, respiratory viruses, human papillomavirus, dengue virus, and other viruses along with a description of their antiviral modes of action and development of antiviral resistance. This evaluation of the strengths and weaknesses of CGs will help provide future research directions that may lead to the successful development of CG-based antimicrobial prophylactics.

Controversial Effects of Vitamin D and Related Genes on Viral Infections, Pathogenesis, and Treatment Outcomes.

Vitamin D (VD) plays an essential role in mineral homeostasis and bone remodeling. A number of different VD-related genes (VDRG) are required for the metabolic activation of VD and the subsequent induction of its target genes. They include a set of genes that encode for VD-binding protein, metabolic enzymes, and the VD receptor. In addition to its well-characterized skeletal function, the immunoregulatory activities of VD and the related polymorphisms of VDRG have been reported and linked to its therapeutic and preventive actions for the control of several viral diseases. However, in regards to their roles in the progression of viral diseases, inconsistent and, in some cases, contradictory results also exist. To resolve this discrepancy, I conducted an extensive literature search by using relevant keywords on the PubMed website. Based on the volume of hit papers related to a certain viral infection, I summarized and compared the effects of VD and VDRG polymorphism on the infection, pathogenesis, and treatment outcomes of clinically important viral diseases. They include viral hepatitis, respiratory viral infections, acquired immunodeficiency syndrome (AIDS), and other viral diseases, which are caused by herpesviruses, dengue virus, rotavirus, and human papillomavirus. This review will provide the most current information on the nutritional and clinical utilization of VD and VDRG in the management of the key viral diseases. This information should be valuable not only to nutritionists but also to clinicians who wish to provide evidence-based recommendations on the use of VD to virally infected patients.

Identification of novel non-nucleoside vinyl-stilbene analogs as potent norovirus replication inhibitors with a potential host-targeting mechanism.

Norovirus (NV), is the most common cause of acute gastroenteritis worldwide. To date, there is no specific anti-NV drug or vaccine to treat NV infections. In this study, we evaluated the inhibitory effect of different stilbene-based analogs on RNA genome replication of human NV (HNV) using a virus replicon-bearing cell line (HG23). Initial screening of our in-house chemical library against NV led to the identification of a hit containing stilbene scaffold 5 which on initial optimization gave us a vinyl stilbene compound 16c (EC50 = 4.4 µM). Herein we report our structure-activity relationship study of the novel series of vinyl stilbene analogs that inhibits viral RNA genome replication in a human NV-specific manner. Among these newly synthesized compounds, several amide derivatives of vinyl stilbenes exhibited potent anti-NV activity with EC50 values ranging from 1 to 2 µM. A trans-vinyl stilbenoid with an appended substituted piperazine amide (18k), exhibited potent anti-NV activity and also displayed favorable metabolic stability. Compound 18k demonstrated an excellent safety profile, the highest suppressive effect, and was selective for HNV replication via a viral RNA polymerase-independent manner. Its potential host-targeting antiviral mechanism was further supported by specific activation of heat shock factor 1-dependent stress-inducible pathway by 18k. These results suggest that 18k might be a promising lead compound for developing novel NV inhibitors with the novel antiviral mechanism.

Plant-Derived Purification, Chemical Synthesis, and In Vitro/In Vivo Evaluation of a Resveratrol Dimer, Viniferin, as an HCV Replication Inhibitor.

Oligostilbenoid compounds, a group of resveratrol multimers, display several anti-microbial activities through the neutralization of cytotoxic oxidants, and by inhibiting essential host and viral enzymes. In our previous study, we identified a series of oligostilbenoid compounds as potent hepatitis C virus (HCV) replication inhibitors. In particular, vitisin B, a resveratrol tetramer, exhibited the most dramatic anti-HCV activity (EC50 = 6 nM and CC50 > 10 µM) via the disruption of the viral helicase NS3 (IC50 = 3 nM). However, its further development as an HCV drug candidate was halted due to its intrinsic drawbacks, such as poor stability, low water solubility, and restricted in vivo absorption. In order to overcome these limitations, we focused on (+)-ε-viniferin, a resveratrol dimer, as an alternative. We prepared three different versions of (+)-ε-viniferin, including one which was extracted from the grapevine root (EVF) and two which were chemically synthesized with either penta-acetylation (SVF-5Ac) or no acetylation (SVF) using a newly established synthesis method. We confirmed their anti-HCV replication activities and minimal cytotoxicity by using genotype 1b and 2a HCV replicon cells. Their anti-HCV replication action also translated into a significant reduction of viral protein expression. Anti-HCV NS3 helicase activity by EVF was also verified in vitro. Finally, we demonstrated that SVF has improved pharmacokinetic properties over vitisin B. Overall, the favorable antiviral and pharmacokinetic properties of these three versions of viniferin warrant their further study as members of a promising new class of anti-HCV therapeutics.

CRISPR/Cas9-Based Antiviral Strategy: Current Status and the Potential Challenge.

From its unexpected discovery as a bacterial adaptive immune system to its countless applications as one of the most versatile gene-editing tools, the CRISPR/Cas9 system has revolutionized every field of life science. Virology is no exception to this ever-growing list of CRISPR/Cas9-based applications. Direct manipulation of a virus genome by CRISPR/Cas9 has enabled a systematic study of cis-elements and trans-elements encoded in a virus genome. In addition, this virus genome-specific mutagenesis by CRISPR/Cas9 was further funneled into the development of a novel class of antiviral therapy targeting many incurable chronic viral infections. In this review, a general concept on the CRISPR/Cas9-based antiviral strategy will be described first. To understand the current status of the CRISPR/Cas9-based antiviral approach, a series of recently published antiviral studies involving CRISPR/Cas9-mediated control of several clinically-relevant viruses including human immunodeficiency virus, hepatitis B virus, herpesviruses, human papillomavirus, and other viruses will be presented. Lastly, the potential challenge and future prospect for successful clinical translation of this CRISPR/Cas9-based antiviral method will be discussed.

Discovery of Ezrin Expression as a Potential Biomarker for Chemically Induced Ocular Irritation Using Human Corneal Epithelium Cell Line and a Reconstructed Human Cornea-like Epithelium Model.

Numerous studies have attempted to develop a new in vitro eye irritation test (EIT). To obtain more reliable results from EIT, potential new biomarkers that reflect eye irritation by chemicals must be identified. We investigated candidate biomarkers for eye irritation, using a proteomics approach. Sodium lauryl sulfate (SLS) or benzalkonium chloride (BAC) was applied on a reconstructed human cornea-like epithelium model, MCTT HCE, and corneal protein expression was examined by two-dimensional gel electrophoresis. We found that ezrin (EZR) was significantly upregulated by SLS or BAC. In addition, upregulation of EZR in immortalized human corneal cells treated with SLS or BAC was confirmed by quantitative reverse transcription-PCR and western blot analysis. Furthermore, other well-known eye irritants such as cetylpyridinium bromide, Triton X-100, cyclohexanol, ethanol, 2-methyl-1-pentanol, and sodium hydroxide significantly increased EZR expression in immortalized human corneal cells. Induction of EZR promoter activity in irritant-treated human corneal cells was confirmed by a luciferase gene reporter assay. In conclusion, EZR expression may be a potential biomarker for detecting eye irritation, which may substantially improve the performance of in vitro EIT.

Establishment of Immortalized Primary Human Foreskin Keratinocytes and Their Application to Toxicity Assessment and Three Dimensional Skin Culture Construction.

In spite of frequent usage of primary human foreskin keratinocytes (HFKs) in the study of skin biology, senescence-induced blockage of in vitro proliferation has been a big hurdle for their effective utilization. In order to overcome this passage limitation, we first isolated ten HFK lines from circumcision patients and successfully immortalized four of them via a retroviral transduction of high-risk human papillomavirus (HPV) E6 and E7 oncogenes. We confirmed expression of a keratinocyte marker protein, keratin 14 and two viral oncoproteins in these immortalized HFKs. We also observed their robust responsiveness to various exogenous stimuli, which was evidenced by increased mRNA expression of epithelial differentiation markers and pro-inflammatory genes in response to three reactive chemicals. In addition, their applicability to cytotoxicity assessment turned out to be comparable to that of HaCaT cells. Finally, we confirmed their differentiation capacity by construction of well-stratified three dimensional skin cultures. These newly established immortalized HFKs will be valuable tools not only for generation of in vitro skin disease models but also for prediction of potential toxicities of various cosmetic chemicals.

US28, a Virally-Encoded GPCR as an Antiviral Target for Human Cytomegalovirus Infection.

Viruses continue to evolve a new strategy to take advantage of every aspect of host cells in order to maximize their survival. Due to their central roles in transducing a variety of transmembrane signals, GPCRs seem to be a prime target for viruses to pirate for their own use. Incorporation of GPCR functionality into the genome of herpesviruses has been demonstrated to be essential for pathogenesis of many herpesviruses-induced diseases. Here, we introduce US28 of human cytomegalovirus (HCMV) as the beststudied example of virally-encoded GPCRs to manipulate host GPCR signaling. In this review, we wish to summarize a number of US28-related topics including its regulation of host signaling pathways, its constitutive internalization, its structural and functional analysis, its roles in HCMV biology and pathogenesis, its proliferative activities and role in oncogenesis, and pharmacological modulation of its biological activities. This review will aid in our understanding of how pathogenic viruses usurp the host GPCR signaling for successful viral infection. This kind of knowledge will enable us to build a better strategy to control viral infection by normalizing the virally-dysregulated host GPCR signaling.

Mangosteen xanthones suppress hepatitis C virus genome replication.

Hepatitis C virus (HCV) is a hepatotropic single-stranded RNA virus. HCV infection is causally linked with development of liver cirrhosis and hepatocellular carcinoma. Enhanced production of reactive oxygen species by HCV has been implicated to play an important role in HCV-induced pathogenesis. Mangosteen has been widely used as a traditional medicine as well as a dietary supplement ,thanks to its powerful anti-oxidant effect. In the present study, we demonstrated that the ethanol extract from mangosteen fruit peels (MG-EtOH) is able to block HCV genome replication using HCV genotype 1b Bart79I subgenomic (EC50 5.1 µg/mL) and genotype 2a J6/JFH-1 infectious replicon systems (EC50 3.8 µg/mL). We found that inhibition of HCV replication by MG-EtOH led to subsequent down-regulation of expression of HCV proteins. Interestingly, MG-EtOH exhibited a modest inhibitory effect on in vitro RNA polymerase activity of NS5B. Among a number of xanthones compounds identified within this MG-EtOH, we discovered α-MG (EC50 6.3 µM) and γ-MG (EC50 2.7 µM) as two major single molecules responsible for suppression of HCV replication. This finding will provide a valuable molecular basis to further develop mangosteen as an important dietary supplement to combat HCV-induced liver diseases.

Roles of the PDZ domain-binding motif of the human papillomavirus type 16 E6 on the immortalization and differentiation of primary human foreskin keratinocytes.

A number of PDZ domain-containing proteins have been identified as binding partners for the oncoprotein E6 of the high-risk type human papillomaviruses (HPVs). These include hDlg, hScrib, MAGI1, MAGI2, and MAGI3, MUPP1, 14-3-3zeta, Na/H exchange regulatory factor 1, PTPN13, TIP-2/GIPC, Tip-1, and PATJ. The PDZ domain-binding motif (-X-T-X-V) at the carboxy terminus of E6 is essential for targeting PDZ proteins for proteasomal degradation. However, contribution of degradation of PDZ proteins by E6 to HPV-induced oncogenesis is still controversial. In order to clarify potential roles of molecular interactions between high-risk HPV E6 and one of best characterized PDZ proteins, hDlg in HPV-induced transformation, we used a retroviral infection system to overexpress HPV16 E7 gene alone or together with either HPV16 E6 wild type or E6 mutant gene lacking the PDZ domain-binding motif and investigated the effect of mutating the PDZ domain-binding motif of E6 on the immortalization and differentiation of human foreskin keratinocytes (HFKs) by the high-risk type HPV E6 and E7. Although the PDZ domain-binding motif of E6 was found to be required for the efficient growth of HFKs, it was not necessary for the E6 and E7-induced immortalization of HFKs. Furthermore, the overexpression of E6 and E7 neither induced degradation nor altered cellular localization of hDlg in undifferentiated or differentiated HFKs. These data indicate that the PDZ domain-binding motif of E6 contributes to the efficient cellular growth through mechanisms other than degradation and changes in the subcellular localizations of hDlg.

Daclatasvir: potential role in hepatitis C.

Chronic hepatitis C virus (HCV) infection is responsible for the development of liver cirrhosis and hepatocellular carcinoma. It has been a tremendous burden on global health care systems. With the advent of a number of new direct-acting and host-targeting antiviral agents, current interferon-α- and ribavirin-based HCV therapy has started to move towards an interferon-sparing or even interferon-free strategy. In this regard, a recently identified NS5A inhibitor, daclatasvir, showed a great promise in clinical trials as another new class of direct-acting anti-HCV therapeutics, with a distinct mechanism of action. In this review, a variety of preclinical as well as clinical proof-of-concept studies of daclatasvir, including the studies of its discovery, mechanism of action, viral resistance, and host polymorphism profiles are reviewed. In addition, a role of daclatasvir in the future therapy for HCV patients is discussed briefly.

Interaction of hepatitis C virus core protein with janus kinase is required for efficient production of infectious viruses.

Chronic hepatitis C virus (HCV) infection is responsible for the development of liver cirrhosis and hepatocellular carcinoma. HCV core protein plays not only a structural role in the virion morphogenesis by encapsidating a virus RNA genome but also a non-structural role in HCV-induced pathogenesis by blocking innate immunity. Especially, it has been shown to regulate JAK-STAT signaling pathway through its direct interaction with Janus kinase (JAK) via its proline-rich JAK-binding motif ((79)PGYPWP(84)). However, little is known about the physiological significance of this HCV core-JAK association in the context of the virus life cycle. In order to gain an insight, a mutant HCV genome (J6/JFH1-79A82A) was constructed to express the mutant core with a defective JAK-binding motif ((79)AGYAWP(84)) using an HCV genotype 2a infectious clone (J6/JFH1). When this mutant HCV genome was introduced into hepatocarcinoma cells, it was found to be severely impaired in its ability to produce infectious viruses in spite of its robust RNA genome replication. Taken together, all these results suggest an essential requirement of HCV core-JAK protein interaction for efficient production of infectious viruses and the potential of using core-JAK blockers as a new anti-HCV therapy.

A hepatitis C virus NS4B inhibitor suppresses viral genome replication by disrupting NS4B's dimerization/multimerization as well as its interaction with NS5A.

Chronic hepatitis C virus (HCV) infection is responsible for severe liver diseases including liver cirrhosis and hepatocellular carcinoma. An HCV non-structural protein 4B (NS4B) plays an essential role in viral RNA genome replication by building multi-vesicular structures around endoplasmic reticulum membranes. Especially, the second amphipathic helix of NS4B (NS4B-AH2) was shown to be essential for this process. By screening compounds against a membrane-aggregating activity of NS4B-AH2, several anti-HCV replication small molecules targeting NS4B-AH2 were discovered. However, little is known about detailed molecular mechanism of action for these NS4B-AH2 inhibitors. In this report, we provide evidences that NS4B-AH2 is required for NS4B's dimerization/multimerization, its proper subcellular localization, as well as its interaction with NS5A. More importantly, one of NS4B-AH2 inhibitors called "anguizole" was found to be able to disrupt all of these NS4B-AH2-mediated biological functions of NS4B. This newly elucidated mechanism of action will enable us not only to better understand a central role of NS4B-AH2 in HCV life cycle but also to develop a more safe and effective new class of NS4B-AH2 inhibitors of HCV replication in the future.

Discovery of hepatitis C virus NS5A inhibitors as a new class of anti-HCV therapy.

Chronic hepatitis C virus (HCV) infection is responsible for development of liver cirrhosis and hepatocellular carcinoma. In addition to PEGylated interferon-α, ribavirin, and HCV NS3 protease inhibitors, recently identified HCV NS5A inhibitors such as BMS-790052 showed a great promise in clinical trials as another new class of direct-acting anti-HCV therapeutics with a distinct mechanism of action. This clinical proof-of-concept study with NS5A inhibitors demonstrated that small molecules targeting a viral protein without any known enzymatic activity can also have profound antiviral effects. In conclusion, NS5A inhibitors will serve as a valuable component of future therapy for HCV patients.

Structural linkage between ligand discrimination and receptor activation by type I interferons.

Type I Interferons (IFNs) are important cytokines for innate immunity against viruses and cancer. Sixteen human type I IFN variants signal through the same cell-surface receptors, IFNAR1 and IFNAR2, yet they can evoke markedly different physiological effects. The crystal structures of two human type I IFN ternary signaling complexes containing IFNα2 and IFNω reveal recognition modes and heterotrimeric architectures that are unique among the cytokine receptor superfamily but conserved between different type I IFNs. Receptor-ligand cross-reactivity is enabled by conserved receptor-ligand "anchor points" interspersed among ligand-specific interactions that "tune" the relative IFN-binding affinities, in an apparent extracellular "ligand proofreading" mechanism that modulates biological activity. Functional differences between IFNs are linked to their respective receptor recognition chemistries, in concert with a ligand-induced conformational change in IFNAR1, that collectively control signal initiation and complex stability, ultimately regulating differential STAT phosphorylation profiles, receptor internalization rates, and downstream gene expression patterns.

Binding dynamics of hepatitis C virus' NS5A amphipathic peptide to cell and model membranes.

Membrane association of the hepatitis C virus NS5A protein is required for viral replication. This association is dependent on an N-terminal amphipathic helix (AH) within NS5A and is restricted to a subset of host cell intracellular membranes. The mechanism underlying this specificity is not known, but it may suggest a novel strategy for developing specific antiviral therapy. Here we have probed the mechanistic details of NS5A AH-mediated binding to both cell-derived and model membranes by use of biochemical membrane flotation and quartz crystal microbalance (QCM) with dissipation. With both assays, we observed AH-mediated binding to model lipid bilayers. When cell-derived membranes were coated on the quartz nanosensor, however, significantly more binding was detected, and the QCM-derived kinetic measurements suggested the existence of an interacting receptor in the target membranes. Biochemical flotation assays performed with trypsin-treated cell-derived membranes exhibited reduced AH-mediated membrane binding, while membrane binding of control cytochrome b5 remained unaffected. Similarly, trypsin treatment of the nanosensor coated with cellular membranes abolished AH peptide binding to the cellular membranes but did not affect the binding of a control lipid-binding peptide. These results therefore suggest that a protein plays a critical role in mediating and stabilizing the binding of NS5A's AH to its target membrane. These results also demonstrate the successful development of a new nanosensor technology ideal both for studying the interaction between a protein and its target membrane and for developing inhibitors of that interaction.

Analysis of the roles of E6 binding to E6TP1 and nuclear localization in the human papillomavirus type 31 life cycle.

The E6 oncoproteins of high-risk human papillomaviruses provide important functions not only for malignant transformation but also in the productive viral life cycle. E6 proteins have been shown to bind to a number of cellular factors, but only a limited number of analyses have investigated the effects of these interactions on the viral life cycle. In this study, we investigated the consequences of HPV 31 E6 binding to E6TP1, a putative Rap1 GAP protein. HPV 16 E6 has been shown to bind as well as induce the rapid turnover of E6TP1, and similar effects were observed with HPV 31 E6. Mutation of amino acid 128 in HPV 31 E6 was found to abrogate the ability to bind and degrade E6TP1 but did not alter binding to another alpha-helical domain protein, E6AP. When HPV 31 genomes containing mutations at amino acid 128 were transfected into human keratinocytes, the viral DNAs were not stably maintained as episomes indicating the importance of this residue for pathogenesis. Many E6 binding partners including E6TP1 are cytoplasmic proteins, but E6 has been also reported to be localized to the nucleus. We therefore investigated the importance of E6 localization to the nucleus in the viral life cycle. Using a fusion of E6 to Green Fluorescent Protein, we mapped one component of the nuclear localization sequences to residues 121 to 124 of HPV 31 E6. Mutation of these residues in the context of the HPV 31 genome abrogated the ability for episomes to be stably maintained and impaired the ability to extend the life span of cells. These studies identify two activities of HPV 31 E6 that are important for its function in the viral life cycle and for extension of cell life span.

Role of the PDZ domain-binding motif of the oncoprotein E6 in the pathogenesis of human papillomavirus type 31.

A number of PDZ domain-containing proteins have been identified as binding partners for the oncoprotein E6 of the high-risk type human papillomaviruses (HPVs). These include hDlg, hScrib, MAGI-1, MAGI-2, MAGI-3, and MUPP1. The PDZ domain-binding motif (-X-T-X-V) at the carboxy terminus of E6 is essential for targeting PDZ proteins for proteasomal degradation. The presence of this motif only in the high-risk HPVs suggests its possible role in HPV-induced oncogenesis. To investigate the role of the PDZ domain-binding motif of E6 in the HPV life cycle, two mutant HPV31 genomes were constructed: E6ValDelta, with a deletion of the last amino acid residue of E6 (valine), and E6ETQVDelta, with a deletion of the entire PDZ domain-binding motif of E6 (ETQV). Three human foreskin keratinocyte (HFK) cell lines were established which maintained transfected wild-type HPV31 or either of two mutant genomes. Cells containing either of two mutant genomes were significantly retarded in their growth rates and reduced in their viral copy numbers compared to those transfected with wild-type genomes. Western analysis did not reveal any significant changes in the levels of PDZ proteins following stable transfection of any HPV31 genomes into HFKs. Although the E6ETQVDelta-transfected HFKs exhibited a pattern of morphological differentiation that appeared different from the HPV31 wild-type-transfected HFKs in organotypic raft cultures, immunohistochemical analysis failed to identify substantial changes in the differentiation-dependent membrane localization of hDlg proteins. These results suggest that binding of E6 to PDZ proteins modulates the early viral functions such as proliferation and maintenance of the viral copy number in undifferentiated cells.