Antiviral agent blocks breathing of the common cold virus

A dynamic capsid is critical to the events that shape the viral life cycle; events such as cell attachment, cell entry, and nucleic acid release demand a highly mobile viral surface. Protein mass mapping of the common cold virus, human rhinovirus 14 (HRV14), revealed both viral structural dynamics and the inhibition of such dynamics with an antiviral agent, WIN 52084. Viral capsid digestion fragments resulting from proteolytic time-course experiments provided structural information in good agreement with the HRV14 three-dimensional crystal structure. As expected, initial digestion fragments included peptides from the capsid protein VP1. This observation was expected because VP1 is the most external viral protein. Initial digestion fragments also included peptides belonging to VP4, the most internal capsid protein. The mass spectral results together with x-ray crystallography data provide information consistent with a “breathing” model of the viral capsid. Whereas the crystal structure of HRV14 shows VP4 to be the most internal capsid protein, mass spectral results show VP4 fragments to be among the first digestion fragments observed. Taken together this information demonstrates that VP4 is transiently exposed to the viral surface via viral breathing. Comparative digests of HRV14 in the presence and absence of WIN 52084 revealed a dramatic inhibition of digestion. These results indicate that the binding of the antiviral agent not only causes local conformational changes in the drug binding pocket but actually stabilizes the entire viral capsid against enzymatic degradation. Viral capsid mass mapping provides a fast and sensitive method for probing viral structural dynamics as well as providing a means for investigating antiviral drug efficacy.

Recombinant parvovirus-like particles as an antigen carrier: A novel nonreplicative exogenous antigen to elicit protective antiviral cytotoxic T cells

To develop a strategy that promotes efficient antiviral immunity, hybrid virus-like particles (VLP) were prepared by self-assembly of the modified porcine parvovirus VP2 capsid protein carrying a CD8+ T cell epitope from the lymphocytic choriomeningitis virus nucleoprotein. Immunization of mice with these hybrid pseudoparticles, without adjuvant, induced strong cytotoxic T lymphocyte (CTL) responses against both peptide-coated- or virus-infected-target cells. This CD8+ class I-restricted cytotoxic activity persisted in vivo for at least 9 months. Furthermore, the hybrid parvovirus-like particles were able to induce a complete protection of mice against a lethal lymphocytic choriomeningitis virus infection. To our knowledge, this study represents the first demonstration that hybrid nonreplicative VLP carrying a single viral CTL epitope can induce protection against a viral lethal challenge, in the absence of any adjuvant. These recombinant particles containing a single type of protein are easily produced by the baculovirus expression system and, therefore, represent a promising and safe strategy to induce strong CTL responses for the elimination of virus-infected cells.

Structure of the HIV-1 integrase catalytic domain complexed with an inhibitor: A platform for antiviral drug design

HIV integrase, the enzyme that inserts the viral DNA into the host chromosome, has no mammalian counterpart, making it an attractive target for antiviral drug design. As one of the three enzymes produced by HIV, it can be expected that inhibitors of this enzyme will complement the therapeutic use of HIV protease and reverse transcriptase inhibitors. We have determined the structure of a complex of the HIV-1 integrase core domain with a novel inhibitor, 5ClTEP, 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-propenone, to 2.1-Å resolution. The inhibitor binds centrally in the active site of the integrase and makes a number of close contacts with the protein. Only minor changes in the protein accompany inhibitor binding. This inhibitor complex will provide a platform for structure-based design of an additional class of inhibitors for antiviral therapy.

Analysis of three structurally related antiviral compounds in complex with human rhinovirus 16

Rhinoviruses are a frequent cause of the common cold. A series of antirhinoviral compounds have been developed that bind into a hydrophobic pocket in the viral capsid, stabilizing the capsid and interfering with cell attachment. The structures of a variety of such compounds, complexed with rhinovirus serotypes 14, 16, 1A, and 3, previously have been examined. Three chemically similar compounds, closely related to a drug that is undergoing phase III clinical trials, were chosen to determine the structural impact of the heteroatoms in one of the three rings. The compounds were found to have binding modes that depend on their electronic distribution. In the compound with the lowest efficacy, the terminal ring is displaced by 1 Å and rotated by 180° relative to the structure of the other two. The greater polarity of the terminal ring in one of the three compounds leads to a small displacement of its position relative to the other compounds in the hydrophobic end of the antiviral compound binding pocket to a site where it makes fewer interactions. Its lower efficacy is likely to be the result of the reduced number of interactions. A region of conserved residues has been identified near the entrance to the binding pocket where there is a corresponding conservation of the mode of binding of these compounds to different serotypes. Thus, variations in residues lining the more hydrophobic end of the pocket are primarily responsible for the differences in drug efficacies.

Antiviral activities of the soluble extracellular domains of type I interferon receptors

Alternative splicing leads to the expression of multiple isoforms of the subunits (IFNAR1 and IFNAR2) of the type I IFN receptor. Here we describe two transcripts representing extracellular forms of ovine IFNAR1 and show that soluble extracellular forms of both IFNAR2 and IFNAR1, prepared in recombinant form in Escherichia coli, have antiviral (AV) activity in the absence of IFN. Exposure of Madin-Darby bovine kidney cells to the extracellular domain (R2E) of IFNAR2 at concentrations as low as 10 nM afforded complete protection against vesicular stomatitis virus and led to the rapid activation of the transcription factors ISGF3 and GAF. Although R2E can bind IFN (Kd ≈70 nM), activity was observed irrespective of whether or not ligand was present. R2E was inactive on mouse L929 cells but active on L929 cells expressing a membraneanchored, ovine/human chimeric IFNAR2 with an ovine extracellular domain. The data suggest that AV activity is conferred by the ability of soluble R2E to associate with the transfected IFNAR2 subunit rather than resident murine IFNAR1. Soluble extracellular forms of IFNAR1 have lower AV activity than R2E on Madin-Darby bovine kidney cells but are less species-specific and protect wild-type L929 cells as efficiently as the transfected cell line, presumably by interacting with one of the murine receptor subunits.

An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense

Plants contain RNA-dependent RNA polymerase (RdRP) activities that synthesize short cRNAs by using cellular or viral RNAs as templates. During studies of salicylic acid (SA)-induced resistance to viral pathogens, we recently found that the activity of a tobacco RdRP was increased in virus-infected or SA-treated plants. Biologically active SA analogs capable of activating plant defense response also induced the RdRP activity, whereas biologically inactive analogs did not. A tobacco RdRP gene, NtRDRP1, was isolated and found to be induced both by virus infection and by treatment with SA or its biologically active analogs. Tobacco lines deficient in the inducible RDRP activity were obtained by expressing antisense RNA for the NtRDRP1 gene in transgenic plants. When infected by tobacco mosaic virus, these transgenic plants accumulated significantly higher levels of viral RNA and developed more severe disease symptoms than wild-type plants. After infection by a strain of potato virus X that does not spread in wild-type tobacco plants, the transgenic NtRDRP1 antisense plants accumulated virus and developed symptoms not only locally in inoculated leaves but also systemically in upper uninoculated leaves. These results strongly suggest that inducible RdRP activity plays an important role in plant antiviral defense.

Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes

Human rhinoviruses, the most important etiologic agents of the common cold, are messenger-active single-stranded monocistronic RNA viruses that have evolved a highly complex cascade of proteolytic processing events to control viral gene expression and replication. Most maturation cleavages within the precursor polyprotein are mediated by rhinovirus 3C protease (or its immediate precursor, 3CD), a cysteine protease with a trypsin-like polypeptide fold. High-resolution crystal structures of the enzyme from three viral serotypes have been used for the design and elaboration of 3C protease inhibitors representing different structural and chemical classes. Inhibitors having α,β-unsaturated carbonyl groups combined with peptidyl-binding elements specific for 3C protease undergo a Michael reaction mediated by nucleophilic addition of the enzyme’s catalytic Cys-147, resulting in covalent-bond formation and irreversible inactivation of the viral protease. Direct inhibition of 3C proteolytic activity in virally infected cells treated with these compounds can be inferred from dose-dependent accumulations of viral precursor polyproteins as determined by SDS/PAGE analysis of radiolabeled proteins. Cocrystal-structure-assisted optimization of 3C-protease-directed Michael acceptors has yielded molecules having extremely rapid in vitro inactivation of the viral protease, potent antiviral activity against multiple rhinovirus serotypes and low cellular toxicity. Recently, one compound in this series, AG7088, has entered clinical trials.

A mammalian 2-5A system functions as an antiviral pathway in transgenic plants.

Resistance to virus infections in higher vertebrates is mediated in part through catalysis of RNA decay by the, interferon-regulated 2-5A system. A functional 2-5A system requires two enzymes, a 2-5A synthetase that produces 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A) in response to double-stranded RNA, and the 2-5A-dependent RNase L. We have coexpressed these human enzymes in transgenic tobacco plants by using a single plasmid containing the cDNAs for both human RNase L and a low molecular weight form of human 2-5A synthetase under control of different, constitutive promoters. Expression of the human cDNAs in the transgenic plants was demonstrated from Northern blots, by specific enzyme assays, and by immunodetection (for RNase L). Infection of leaves, detached or in planta, of the coexpressing transgenic plants by tobacco mosaic virus, alfalfa [correction of alfafa] mosaic virus, or tobacco etch virus resulted in necrotic lesions. In contrast, leaves expressing 2-5A synthetase or RNase L alone and leaves containing the plasmid vector alone produced typical systemic infections. While alfalfa mosaic virus produced lesions only in the inoculated leaves regardless of the concentration of virus in the inoculum, high, but not low, levels of tobacco etch virus inoculum resulted in escape of virus to uninoculated leaves. Nevertheless, there was a substantial reduction of tobacco etch virus yield as measured by ELISA assay in the coexpressing transgenic plants. These results indicate that expression of a mammalian 2-5A system in plants provides resistance to virus infections.

A null mutation in the gene encoding a type I interferon receptor component eliminates antiproliferative and antiviral responses to interferons alpha and beta and alters macrophage responses.

To examine the in vivo role(s) of type I interferons (IFNs) and to determine the role of a component of the type I IFN receptor (IFNAR1) in mediating responses to these IFNs, we generated mice with a null mutation (-/-) in the IFNAR1 gene. Despite compelling evidence for modulation of cell proliferation and differentiation by type I IFNs, there were no gross signs of abnormal fetal development or morphological changes in adult IFNAR1-/- mice. However, abnormalities of hemopoietic cells were detected in IFNAR1 -/- mice. Elevated levels of myeloid lineage cells were detected in peripheral blood and bone marrow by staining with Mac-1 and Gr-1 antibodies. Furthermore, bone marrow macrophages from IFNAR1 -/- mice showed abnormal responses to colony-stimulating factor 1 and lipopolysaccharide. IFNAR1 -/- mice were highly susceptible to viral infection: viral titers were undetected 24 hr after infection of IFNAR1 +/+ mice but were extremely high in organs of IFNAR1 -/- mice, demonstrating that the type I IFN system is a major acute antiviral defence. In cell lines derived from IFNAR1 -/- mice, there was no signaling in response to IFN-alpha or -beta as measured by induction of 2'-5' oligoadenylate synthetase, antiviral, or antiproliferative responses. Importantly, these studies demonstrate that type I IFNs function in the development and responses of myeloid lineage cells, particularly macrophages, and that the IFNAR1 receptor component is essential for antiproliferative and antiviral responses to IFN-alpha and -beta.

Involvement of the double-stranded-RNA-dependent kinase PKR in interferon expression and interferon-mediated antiviral activity.

The signaling mechanisms responsible for the induced expression of interferon (IFN) genes by viral infection or double-stranded RNA (dsRNA) are not well understood. Here we investigate the role of the interferon-induced dsRNA-dependent protein kinase PKR in the regulation of IFN induction. Biological activities attributed to PKR include regulating protein synthesis, mediating IFN actions, and functioning as a possible tumor suppressor. Since binding of dsRNA is required for its activation, PKR has been considered as a candidate signal transducer for regulating IFN expression. To examine this role of PKR, loss-of-function phenotypes in stable transformants of promonocytic U-937 cells were achieved by two different strategies, overexpression of an antisense PKR transcript or a dominant negative PKR mutant gene. Both types of PKR-deficient cells were more permissive for viral replication than the control U-937 cells. As the result of PKR loss, they also showed impaired induction of IFN-alpha and IFN-beta genes in response to several inducers--specifically, encephalomyocarditis virus, lipopolysaccharide, and phorbol 12-myristate 13-acetate. Interestingly, while IFN-alpha induction by dsRNA was impaired in PKR-deficient cells, IFN-beta induction remained intact. Loss of PKR function also resulted in decreased antiviral activity as elicited by IFN-alpha and, to a greater extent, by IFN-gamma. These results implicate PKR in the regulation of several antiviral activities.

Isolation and characterization of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: identification of residues important for toxicity.

Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the 28S rRNA of eukaryotic ribosomes. PAP has potent antiviral activity against many plant and animal viruses, including human immunodeficiency virus. We describe here development of a positive selection system to isolate PAP mutants with reduced toxicity. In vitro translation in the presence or absence of microsomal membranes shows that PAP is synthesized as a precursor and undergoes at least two different proteolytic processing steps to generate mature PAP. The PAP cDNA was placed under control of the galactose-inducible GAL1 promoter and transformed into Saccharomyces cerevisiae. Induction of PAP expression was lethal to yeast. The PAP expression plasmid was mutagenized and plasmids encoding mutant PAP genes were identified by their failure to kill S. cerevisiae. A number of mutant alleles were sequenced. In one mutant, a point mutation at Glu-177 inactivated enzymatic function in vitro, suggesting that this glutamic acid residue is located at or near the catalytic site. Mutants with either point mutations near the N terminus or a nonsense mutation at residue 237 produced protein that was enzymatically active in vitro, suggesting that the toxicity of PAP is not due solely to enzymatic activity. Toxicity of PAP appears to be a multistep process that involves possibly different domains of the protein.