Molecular dynamics simulations of human rhinovirus and an antiviral compound

The human rhinovirus 14 (HRV14) protomer, with or without the antiviral compound WIN 52084s, was simulated using molecular dynamics and rotational symmetry boundary conditions to model the effect of the entire icosahedral capsid. The protein asymmetrical unit, comprising four capsid proteins (VP1, VP2, VP3, and VP4) and two calcium ions, was solvated both on the exterior and the interior to fill the inside of the capsid. The stability of the simulations of this large system (~800 residues and 6,650 water molecules) is comparable to more conventional globular protein simulations. The influence of the antiviral compound on compressibility and positional fluctuations is reported. The compressibility, estimated from the density fluctuations in the region of the binding pocket, was found to be greater with WIN 52084s bound than without the drug, substantiating previous computations on reduced viral systems. An increase in compressibility correlates with an entropically more favorable system. In contrast to the increase in density fluctuations and compressibility, the positional fluctuations decreased dramatically for the external loops of VP1 and the N-terminus of VP3 when WIN 52084s is bound. Most of these VP1 and VP3 loops are found near the fivefold axis, a region whose mobility was not considered in reduced systems, but can be observed with this simulation of the full viral protomer. Altered loop flexibility is consistent with changes in proteolytic sensitivity observed experimentally. Moreover, decreased flexibility in these intraprotomeric loops is noteworthy since the externalization of VP4, part of VP1, and RNA during the uncoating process is thought to involve areas near the fivefold axis. Both the decrease in positional fluctuations at the fivefold axis and the increase in compressibility near the WIN pocket are discussed in relationship to the antiviral activity of stabilizing the virus against uncoating.     

Genetic and molecular analyses of spontaneous mutants of human rhinovirus 14 that are resistant to an antiviral compound

Spontaneous mutants of human rhinovirus 14 resistant to WIN 52084, an antiviral compound that inhibits attachment to cells, were isolated by selecting plaques that developed when wild-type virus was plated in the presence of high (2 micrograms/ml) or low (0.1 to 0.4 micrograms/ml) concentrations of the compound. Two classes of drug resistance were observed: a high-resistance (HR) class with a frequency of about 4 x 10(-5), and a low-resistance (LR) class with a 10- to 30-fold-higher frequency. The RNA genomes of 56 HR mutants and 13 LR mutants were sequenced in regions encoding the drug-binding site. The HR mutations mapped to only 2 of the 16 amino acid residues that form the walls of the drug-binding pocket. The side chains of these two residues point directly into the pocket and were invariably replaced by bulkier groups. These findings, and patterns of resistance to related WIN compounds, support the concept that HR mutations may hinder the entry or seating of drug within the binding pocket. In contrast, all of the LR mutations mapped to portions of the polypeptide chain near the canyon floor that move when the drug is inserted. Because several LR mutations partially reverse the attachment-inhibiting effect of WIN compounds, these mutants provide useful tools for studying the regions of the capsid structure involved in attachment. This paper shows that the method of escape mutant analysis, previously used to identify antibody binding sites on human rhinovirus 14, is also applicable to analysis of antiviral drug activity.     

The antiviral compound enviroxime targets the 3A coding region of rhinovirus and poliovirus.

Enviroxime is an antiviral compound that inhibits the replication of rhinoviruses and enteroviruses. We have explored the mechanism of action of enviroxime by using poliovirus type 1 and human rhinovirus type 14 as model systems. By varying the time of drug addition to virus-infected cells, we determined that enviroxime could be added several hours postinfection without significant loss of inhibition. This suggested that the drug targeted a step involved in RNA replication or protein processing. To identify this target, we mapped 23 independent mutations in mutants that could multiply in the presence of 1 microgram of enviroxime per ml. Each of these mutants contained a single nucleotide substitution that altered one amino acid in the 3A coding region. Using oligonucleotide-directed mutagenesis of cDNA clones, we have confirmed that these single-amino-acid substitutions are sufficient to confer the resistance phenotype. In addition, we conducted two experiments to support the hypothesis that enviroxime inhibits a 3A function. First, we determined by dot blot analysis of RNA from poliovirus-infected cells that enviroxime preferentially inhibits synthesis of the viral plus strand. Second, we demonstrated that enviroxime inhibits the initiation of plus-strand RNA synthesis as measured by the addition of [32P]uridine to 3AB in poliovirus crude replication complexes. To our knowledge, this is the first evidence that 3A can be targeted by antiviral drugs. We anticipate that enviroxime will be a useful tool for investigating the natural function of the 3A protein.     

VP4 protein from human rhinovirus 14 is released by pressure and locked in the capsid by the antiviral compound WIN

Rhinoviruses are the major causative agents of the common cold in humans. Here, we studied the stability of human rhinovirus type 14 (HRV14) under conditions of high hydrostatic pressure, low temperature, and urea in the absence and presence of an antiviral drug. Capsid dissociation and changes in the protein conformation were monitored by fluorescence spectroscopy, light scattering, circular dichroism, gel filtration chromatography, mass spectrometry and infectivity assays. The data show that high pressure induces the dissociation of HRV14 and that this process is inhibited by WIN 52084. MALDI-TOF mass spectrometry experiments demonstrate that VP4, the most internal viral protein, is released from the capsid by pressure treatment. This release of VP4 is concomitant with loss of infectivity. Our studies also show that at least one antiviral effect of the WIN drugs involves the locking of VP4 inside the capsid by blocking the dynamics associated with cell attachment.     

Structure of human rhinovirus complexed with Fab fragments from a neutralizing antibody.

We have determined the structure of a human rhinovirus (HRV)-Fab complex by using cryoelectron microscopy and image reconstruction techniques. This is the first view of an intact human virus complexed with a monoclonal Fab (Fab17-IA) for which both atomic structures are known. The surface area on HRV type 14 (HRV14) in contact with Fab17-IA was approximately 500 A2 (5 nm2), which is much larger than the area that constitutes the NIm-IA epitope (on viral protein VP1) defined by natural escape mutants. From modeling studies and electrostatic potential calculations, charged residues outside the neutralizing immunogenic site IA (NIm-IA) were also predicted to be involved in antibody recognition. These predictions were confirmed by site-specific mutations and analysis of the Fab17-IA-HRV14 complex, along with knowledge of the crystallographic structures of HRV14 and Fab17-IA. The bound Fab17-IA reaches across a surface depression (the canyon) and meets a related Fab at the nearest icosahedral twofold axis. By adjusting the elbow angles of the bound Fab fragments from 162 degrees to 198 degrees, an intact antibody molecule can be easily modeled. This, along with aggregation and binding stoichiometry results, supports the earlier proposal that this antibody binds bivalently to the surface of HRV14 across icosahedral twofold axes. One prediction of this model, that the intact canyon-spanning immunoglobulin G molecule would block attachment of the virus to HeLa cells, was confirmed experimentally.     

Structural studies of two rhinovirus serotypes complexed with fragments of their cellular receptor

Two human rhinovirus serotypes complexed with two- and five-domain soluble fragments of the cellular receptor, intercellular adhesion molecule-1, have been investigated by X-ray crystallographic analyses of the individual components and by cryo-electron microscopy of the complexes. The three-dimensional image reconstructions provide a molecular envelope within which the crystal structures of the viruses and the receptor fragments can be positioned with accuracy. The N-terminal domain of the receptor binds to the rhinovirus 'canyon' surrounding the icosahedral 5-fold axes. Fitting of molecular models into the image reconstruction density identified the residues on the virus that interact with those on the receptor surface, demonstrating complementarity of the electrostatic patterns for the tip of the N-terminal receptor domain and the floor of the canyon. The complexes seen in the image reconstructions probably represent the first stage of a multistep binding process. A mechanism is proposed for the subsequent viral uncoating process.     

Human rhinovirus HRV14 uncoats from early endosomes in the presence of bafilomycin

Various studies have provided evidence for the existence of spontaneously active cytosolic species of protein phosphatase 1, but these enzymes have never been purified and characterized. We have used chromatography on microcystin-Sepharose and Resource Q to purify cytosolic protein phosphatases from rat liver. Two of the isolated enzymes were identified by Western analysis and peptide sequencing as complexes of the catalytic subunit of protein phosphatase 1 and either the inhibitor NIPP1 or the myosin-binding subunit MYPT1, which reportedly is not present in chicken liver. In contrast, PCR cloning revealed the expression of two MYPT1 splice variants in rat liver.     

Cathelicidins display conserved direct antiviral activity towards rhinovirus

Human rhinoviruses (HRVs) are the most common cause of viral respiratory tract infections, and are associated with significant morbidity and mortality in immunocompromised individuals and patients with pre-existing pulmonary conditions. The therapeutic options available are extremely limited and therefore novel therapeutics for HRV infections are of significant interest. Cathelicidins have been shown to have potent antiviral activity against a range of pathogens and are known to be key immunomodulatory mediators during infection. We therefore assessed the antiviral potential of cathelicidins from humans and other mammalian species against HRV, together with the potential for the human cathelicidin to modulate apoptotic pathways and alter cell viability during HRV infection. We demonstrate that LL-37, the porcine cathelicidin Protegrin-1, and the ovine cathelicidin SMAP-29 display potent antiviral activity towards HRV and that this activity is visible when either the virus is exposed to the peptides prior to cell infection or after cells have been infected. We further demonstrate that, in contrast to established findings with bacterial infection models, LL-37 does not induce apoptosis or necrosis in HRV-infected lung epithelial cells at physiological or superphysiological concentrations, but does reduce the metabolic activity of infected cells compared to uninfected cells treated with similar peptide concentrations. Collectively, the findings from this study demonstrate that the mechanism of action of cathelicidins against rhinovirus is by directly affecting the virus and we propose that the delivery of exogenous cathelicidins, or novel synthetic analogues, represent an exciting and novel therapeutic strategy for rhinovirus infection.     

Cytoplasmic DNA synthesis in rhinovirus type 14-inoculated KB cells.

Rhinovirus type 14 (RV14) incuced a transient statistically significant stimulation in synthesis of DNA which appeared between 0 and 3 h post-inoculation in the cytoplasm of high density monolayer cultures of KB cells. Newly synthesized DNA was measured by incorporation of [3H] thymidine into acid-insoluble DNAase-sensitive material and the cytoplasmic location established by cell fractionation and electron microscope radioautographic methods. A minimum of 10 plaque-forming units per cell of RV14 was required to stimulate DNA synthesis which did not occur above 34.5 degrees C, a temperature optimal for virus replication. Cytoplasmic DNA taken from RV14-infected or control cells could be differentiated from the bulk of cell (nuclear) DNA by several criteria, including: (1) RV14 induction of synthesis; (2) lower buoyant density and greater heterogeneity in CsCl and ethidium bromide/CsCl gradients; and (3) a different kinetic complexity upon reannealing. The Cot 1/2 value of cytoplasmic DNA, calclated as 50--100 from reassociation profiles, was about 10-fold less complex than the Cot 1/2 value of nuclear DNA (800-1000). These data rule out the possibility that cytoplasmic DNA arises by random breakage of nuclear DNA during cell disruption and extraction and are compatible with the hypothesis that inoculation of KB cells with RV14 results in stimulation of synthesis of a specific class of cell DNA which is detected in the cytoplasm.     

Structural analysis of human rhinovirus complexed with ICAM-1 reveals the dynamics of receptor-mediated virus uncoating

Intercellular adhesion molecule 1 (ICAM-1) functions as the cellular receptor for the major group of human rhinoviruses, being not only the target of viral attachment but also the mediator of viral uncoating. The configurations of HRV3-ICAM-1 complexes prepared both at 4 degrees C and physiological temperature (37 degrees C) were analyzed by cryoelectron microscopy and image reconstruction. The particle diameters of two complexes (with and without RNA) representing uncoating intermediates generated at 37 degrees C were each 4% larger than that of those prepared at 4 degrees C. The larger virus particle arose by an expansive movement of the capsid pentamers along the fivefold axis, which loosens interprotomer contacts, particularly at the canyon region where the ICAM-1 receptor bound. Particle expansion required receptor binding and preceded the egress of the viral RNA. These observations suggest that receptor-mediated uncoating could be a consequence of restrained capsid motion, where the bound receptors maintain the viral capsid in an expanded open state for subsequent genome release.     

Crystal structure of a human rhinovirus neutralizing antibody complexed with a peptide derived from viral capsid protein VP2.

The three-dimensional structure of the complex between the Fab fragment of an anti-human rhinovirus neutralizing antibody (8F5) and a cross-reactive synthetic peptide from the viral capsid protein VP2 has been determined at 2.5 A resolution by crystallographic methods. The refinement is presently at an R factor of 0.18 and the antigen-binding site and viral peptide are well defined. The peptide antigen adopts a compact fold by two tight turns and interacts through hydrogen bonds, some with ionic character, and van der Waals contacts with antibody residues from the six hypervariable loops as well as several framework amino acids. The conformation adopted by the peptide is closely related to the corresponding region of the viral protein VP2 on the surface of human rhinovirus 1A whose three-dimensional structure is known. Implications for the cross-reactivity between peptides and the viral capsid are discussed. The peptide-antibody interactions, together with the analysis of mutant viruses that escape neutralization by 8F5 suggest two different mechanisms for viral escape. The comparison between the complexed and uncomplexed antibody structures shows important conformational rearrangements, especially in the hypervariable loops of the heavy chain. Thus, it constitutes a clear example of the 'induced fit' molecular recognition mechanism.     

Similar interactions of the poliovirus and rhinovirus 3D polymerases with the 3' untranslated region of rhinovirus 14

We showed previously that a human rhinovirus 14 (HRV14) 3' untranslated region (3' UTR) on a poliovirus genome was able to replicate with nearly wild-type kinetics (J. B. Rohll, D. H. Moon, D. J. Evans, and J. W. Almond, J. Virol 69:7835-7844, 1995). This enabled the HRV14 single 3' UTR stem-loop structure to be studied in combination with a sensitive reporter system, poliovirus FLC/REP, in which the capsid coding region is replaced by an in-frame chloramphemicol acetyltransferase (CAT) gene. Using such a construct, we identified a mutant (designated mut4), in which the structure and stability of the stem were predicted to be maintained, that replicated very poorly as determined by its level of CAT activity. The effect of this mutant 3' UTR on replication has been further investigated by transferring it onto the full-length cDNAs of both poliovirus type 3 (PV3) and HRV14. Virus was recovered with a parental plaque phenotype at a low frequency, indicating the acquisition of compensating changes, which sequence analysis revealed were, in both poliovirus- and rhinovirus-derived viruses, located in the active-site cleft of 3D polymerase and involved the substitution of Asn18 for Tyr. These results provide further evidence of a specific interaction between the 3' UTR of picornaviruses and the viral polymerase and also indicate similar interactions of the 3' UTR of rhinovirus with both poliovirus and rhinovirus polymerases.     

Evidence for at least two dominant neutralization antigens on human rhinovirus 14.

A collection of 28 mutants of human rhinovirus 14, selected for resistance to 10 individual neutralizing monoclonal antibodies, was used to identify two major neutralization antigens, N-Ag I and N-Ag II. Isoelectric analysis showed that all 16 of the N-Ag I mutants analyzed were charge altered in VP1;8 of 12 N-Ag II mutants were altered in VP3. These results suggest that N-Ag I resides on VP1, whereas N-Ag II lies on VP3. The frequency of charge alterations was much higher than predicted by the genetic code, suggesting that charged amino acids on the antigenic sites play an important role in interaction with neutralizing antibody. Antibodies against N-Ag I and N-Ag II neutralize with widely different efficiencies.     

Capsid coding sequence is required for efficient replication of human rhinovirus 14 RNA.

Mechanisms by which the plus-sense RNA genomes of picornaviruses are replicated remain poorly defined, but existing models do not suggest a role for sequences encoding the capsid proteins. However, candidate RNA replicons (delta P1 beta gal and delta P1Luc), representing the sequence of human rhinovirus 14 virus (HRV-14) with reporter protein sequences (beta-galactosidase or luciferase, respectively) replacing most of the P1 capsid-coding region, failed to replicate in transfected H1-HeLa cells despite efficient primary cleavage of the polyprotein. To determine which P1 sequences might be required for RNA replication, HRV-14 mutants in which segments of the P1 region were removed to frame from the genome were constructed. Mutants with deletions involving the 5'proximal 1,489 nucleotides of the P1 region replicated efficiently, while those with deletions involving the 3' 1,079 nucleotides did not. Reintroduction of the 3' P1 sequence into the nonreplicating delta P1Luc construct resulted in a new candidate replicon, delta P1Luc/VP3, which replicated well and expressed luciferase efficiently. Capsid proteins provided in trans by helper virus failed to rescue the nonreplicating delta P1Luc genome but were able to package the larger-than-genome-length delta P1Luc/VP3 replicon. Thus, a 3'-distal P1 capsid-coding sequence has a previously unrecognized cis-active function related to replication of HRV-14 RNA.     

Properties and origins of infectious rhinovirus type 14 particles of different buoyant densities.

Isopycnic centrifugation of rhinovirus type 14 (RV14), purified from infected HeLa or KB cell cultures, into CsCl gradients resolved two bands of infectious virus particles with buoyant density values of 1.409 +/- 0.007 (H virus) and 1.386 +/- 0.004 (L virus) g/ml. Only H virus was detected by incorporation of radiolabeled uridine into viral RNA, and H virus accounted for the majority of infectivity in gradients. H and L virus could not be differentiated by plaque morphology, extent of neutralization by RV14-specific antiserum, or particle size. Electron microscope studies showed that most L-virus particles were associated with an amorphous material. Treatment of L virus with proteolytic enzymes or rebanding L virus in CsCl gradients resulted in recovery of the majority of infectivity as H virus. Virus purified from cell-free fluids from infected HeLa or KB cell cultures banded only as H virus. HeLa cell cultures challenged with purified H virus and harvested at 3 h postinoculation for virus purification yielded only infectious H virus. Both H and L viruses were detected in cell cultures that had been challenged with purified H virus and harvested at 12 h postinoculation. The data suggest that H virus represents progeny virus, whereas L virus represents sequestered infectious virus particles which become associated with an amorphous material and do not enter into viral replicative processes.     

The complete nucleotide sequence of a common cold virus: human rhinovirus 14.

The complete nucleotide sequence of the single-stranded RNA genome of human rhinovirus 14, one of the causative agents of the common cold, has been determined from cDNA cloned in E. coli. The genome is typical of the picornaviridae family, comprising a 5' non-coding region of 624 nucleotides, a long open reading frame of 6537 nucleotides (90.8% of the genome) and a 3' non-coding region of 47 nucleotides. Comparison of the nucleotide sequence and the predicted amino acid sequence with those of the polioviruses reveals a surprising degree of homology which may allow recognition of regions of antigenic importance and prediction of the virus polyprotein cleavage sites. The results presented here imply a closer genetic relationship between the rhinovirus and enterovirus genera than previously suspected.     

Cryoelectron microscopy analysis of the structural changes associated with human rhinovirus type 14 uncoating

Release of the human rhinovirus (HRV) genome into the cytoplasm of the cell involves a concerted structural modification of the viral capsid. The intracellular adhesion molecule 1 (ICAM-1) cellular receptor of the major-group HRVs and the low-density lipoprotein (LDL) receptor of the minor-group HRVs have different nonoverlapping binding sites. While ICAM-1 binding catalyzes uncoating, LDL receptor binding does not. Uncoating of minor-group HRVs is initiated by the low pH of late endosomes. We have studied the conformational changes concomitant with uncoating in the major-group HRV14 and compared them with previous results for the minor-group HRV2. The structure of empty HRV14 was determined by cryoelectron microscopy, and the atomic structure of native HRV14 was used to examine the conformational changes of the capsid and its constituent viral proteins. For both HRV2 and HRV14, the transformation from full to empty capsid involves an overall 4% expansion and an iris type of movement of viral protein VP1 to open up a 10-A-diameter channel on the fivefold axis to allow exit of the RNA genome. The beta-cylinders formed by the N termini of the VP3 molecules inside the capsid on the fivefold axis all open up in HRV2, but we propose that only one opens up in HRV14. The release of VP4 is less efficient in HRV14 than in HRV2, and the N termini of VP1 may exit at different points. The N-terminal loop of VP2 is modified in both viruses, probably to detach the RNA, but it bends only inwards in HRV2.     

Chemical disinfection to interrupt transfer of rhinovirus type 14 from environmental surfaces to hands.

Rhinoviruses can survive on environmental surfaces for several hours under ambient conditions. Hands can readily become contaminated after contact with such surfaces, and self-inoculation may lead to infection. Whereas hand washing is crucial in preventing the spread of rhinovirus colds, proper disinfection of environmental surfaces may further reduce rhinovirus transmission. In this study, the capacities of Lysol Disinfectant Spray (0.1% o-phenylphenol and 79% ethanol), a domestic bleach (6% sodium hypochlorite diluted to give 800 ppm of free chlorine), a quaternary ammonium-based product (7.05% quaternary ammonium diluted 1:128 in tap water), and a phenol-based product (14.7% phenol diluted 1:256 in tap water) were compared in interrupting the transfer of rhinovirus type 14 from stainless steel disks to fingerpads of human volunteers upon a 10-s contact at a pressure of 1 kg/cm2. Ten microliters of the virus, suspended in bovine mucin (5 mg/ml), was placed on each disk, and the inoculum was dried under ambient conditions; the input number on each disk ranged from 0.5 x 10(5) to 2.1 x 10(6) PFU. The dried virus was exposed to 20 microliters of the test disinfectant. The Lysol spray was able to reduce virus infectivity by > 99.99% after a contact of either 1 or 10 min, and no detectable virus was transferred to fingerpads from Lysol-treated disks. The bleach (800 ppm of free chlorine) reduced the virus titer by 99.7% after a contact time of 10 min, and again no virus was transferred from the disks treated with it.(ABSTRACT TRUNCATED AT 250 WORDS)