Antibody-mediated neutralization of human rhinovirus 14 explored by means of cryoelectron microscopy and X-ray crystallography of virus-Fab complexes.

The structures of three different human rhinovirus 14 (HRV14)-Fab complexes have been explored with X-ray crystallography and cryoelectron microscopy procedures. All three antibodies bind to the NIm-IA site of HRV14, which is the beta-B-beta-C loop of the viral capsid protein VP1. Two antibodies, Fab17-IA (Fab17) and Fab12-IA (Fab12), bind bivalently to the virion surface and strongly neutralize viral infectivity whereas Fab1-IA (Fab1) strongly aggregates and weakly neutralizes virions.

WIN 52035-dependent human rhinovirus 16: assembly deficiency caused by mutations near the canyon surface.

Three drug-dependent mutants of human rhinovirus 16 (HRV16) were characterized by sequence analyses of spontaneous mutant isolates and were genetically reconstructed from a parental cDNA plasmid. These mutants formed plaques in the presence but not in the absence of the selecting antiviral drug, WIN 52035, which binds to the capsid of wild-type virus and inhibits its attachment to the host cell. The drug-dependent phenotype of each mutant was caused by a single amino acid substitution in the VP1 coat protein.

The refined structure of human rhinovirus 16 at 2.15 A resolution: implications for the viral life cycle.

Background: Rhinoviruses belong to the picornavirus family and are small, icosahedral, non-enveloped viruses containing one positive RNA strand. Human rhinovirus 16 (HRV16) belongs to the major receptor group of rhinoviruses, for which the cellular receptor is intercellular adhesion molecule-1 (ICAM-1). In many rhinoviruses, one of the viral coat proteins (VP1) contains a hydrophobic pocket which is occupied by a fatty acid-like molecule, or so-called 'pocket factor'.

Complete replication of an animal virus and maintenance of expression vectors derived from it in Saccharomyces cerevisiae.

Here we describe the first instances to our knowledge of animal virus genome replication, and of de novo synthesis of infectious virions by a nonendogenous virus, in the yeast Saccharomyces cerevisiae, whose versatile genetics offers significant advantages for studying viral replication and virus-host interactions. Flock house virus (FHV) is the most extensively studied member of the Nodaviridae family of (+) strand RNA animal viruses. Transfection of yeast with FHV genomic RNA induced viral RNA replication, transcription, and assembly of infectious virions.

Structural studies on human rhinovirus 14 drug-resistant compensation mutants.

Structures have been determined of three human rhinovirus 14 (HRV14) compensation mutants that have resistance to the antiviral capsid binding compounds WIN 52035 and WIN 52084. In addition, the structure of HRV14 is reported, with a site-directed mutation at residue 1219 in VP1. A spontaneous mutation occurs at the same site in one of the compensation mutants.

WIN51711-resistant mutants of poliovirus type 3: capsid residues important in uncoating functions.

Capsid-binding drugs that inhibit the first stage of picornaviral uncoating were used to select drug-resistant mutants of the Sabin strain of poliovirus type 3. Such mutants provide information about parts of the capsid that are important for functions blocked by the drugs, and also about pathways to drug resistance. Amino-acid substitutions allowing virus to produce progeny in the presence of drug were mapped to 13 different residues occupying three distinct locations: (I) the canyon base; (II) the lining of the drug-binding pocket; and (III) the base of the protomer.

Complete sequence of the RNA genome of human rhinovirus 16, a clinically useful common cold virus belonging to the ICAM-1 receptor group.

We report here the complete nucleotide sequence and predicted polyprotein sequence of HeLa cell-adapted human rhinovirus 16 (HRV16). This virus is more suitable than human rhinovirus 14 (HRV14) for clinical studies, and its growth and physical properties are favorable for biochemical and crystallographic analysis. The complete message-sense RNA genome of HRV16 is composed of 7124 bases, not including the poly(A) tail.

Distribution of drug resistance mutations in type 3 poliovirus identifies three regions involved in uncoating functions.

We have previously described the use of an uncoating inhibitor, WIN 51711, to select drug-resistant mutants of the Sabin strain of poliovirus type 3. Two-thirds of the mutants proved to be dependent on the drug for plaque formation because of extreme thermolability (A. G.

Structure determination of an Fab fragment that neutralizes human rhinovirus 14 and analysis of the Fab-virus complex.

The crystal structure of Fab17-IA, an antigen-binding fragment from a murine immunoglobulin that neutralizes human rhinovirus 14 (HRV14), has been solved to 2.7 A resolution. Fab17-IA crystallized into three different space groups depending upon the method used to purify the intact antibody.

Production and crystallization of virus-like particles assembled in a heterologous protein expression system.

It is of considerable interest to separate the processes of viral infectivity and virion assembly. Until recently this has only been possible with viruses that could be disassembled and reassembled in vitro. Even in these cases it was difficult to establish the authenticity of reassembled capsid protein because of possible irreversible damage that may have occurred to the protein during disassembly.

Reconstitution of Flock House provirions: a model system for studying structure and assembly.

Assembly of Flock House virus in infected Drosophila cells proceeds through an intermediate, the provirion, which lacks infectivity until the coat precursor protein, alpha, undergoes a spontaneous "maturation" cleavage (A. Schneemann, W. Zhong, T.

Capsid assembly in a family of animal viruses primes an autoproteolytic maturation that depends on a single aspartic acid residue.

Maturation of noninfectious nodavirus provirions occurs by autoproteolytic cleavage of most of the 180 copies of the alpha-protein that make up the icosahedral capsid. This maturation, which is much slower than viral assembly, produces an infectious particle that is more stable than the provirion and makes viral uncoating thermodynamically distinct from assembly, allowing assembly and (a time-delayed) uncoating to occur under similar conditions. The results of structural, computational, and molecular genetic studies suggest that maturation depends both on intrasubunit strain, produced during assembly, and on a critical aspartic acid residue.

Flock house virus: a simple model for studying persistent infection in cultured Drosophila cells.

Flock house virus (FHV), isolated from twenty Drosophila melanogaster cell lines, persistently infected with the virus, were examined during successive serial passages by plaque assay and sequence analysis. No phenotypic or genotypic changes in the virus were observed during the establishment of persistent infection, suggesting that it was a cellular modification that led to the first step in establishing the persistent state. Once this state was initiated, the virus was relieved of the need for a functional coat protein to propagate itself and mutations began to accumulate selectively in RNA2, the gene for the coat protein.

Use of drug-resistance mutants to identify functional regions in picornavirus capsid proteins.

The WIN drugs and similar hydrophobic compounds that insert into the capsid of picornaviruses have been shown to block viral uncoating. In some of the human rhinoviruses they also block attachment of virus to cells. Spontaneously occurring drug-resistant mutants of human rhinovirus 14 and poliovirus type 3 were selected for their ability to make plaques in the presence of the selecting drug.

The structure of human rhinovirus 16.

Background: Rhinoviruses and the homologous polioviruses have hydrophobic pockets below their receptor-binding sites, which often contain unidentified electron density ('pocket factors'). Certain antiviral compounds also bind in the pocket, displacing the pocket factor and inhibiting uncoating. However, human rhinovirus (HRV)14, which belongs to the major group of rhinoviruses that use intercellular adhesion molecule-1 (ICAM-1) as a receptor, has an empty pocket.

Crystallization of viruslike particles assembled from flock house virus coat protein expressed in a baculovirus system.

Flock house virus coat protein expressed in a baculovirus system spontaneously assembles into viruslike particles, which undergo an autocatalytic postassembly cleavage equivalent to that of the native virus. Mutations of the asparagine at the Asn/Ala cleavage site result in assembly of provirion-like particles that are cleavage defective. Crystals of the mutant provirions have been grown, and they diffract X rays beyond 3.

Use of recombinant baculoviruses in synthesis of morphologically distinct viruslike particles of flock house virus, a nodavirus.

Flock house virus (FHV) is a small icosahedral insect virus of the family Nodaviridae. Its genome consists of two messenger-sense RNA molecules, both of which are encapsidated in the same particle. RNA1 (3.

Flock house virus: down-regulation of subgenomic RNA3 synthesis does not involve coat protein and is targeted to synthesis of its positive strand.

Flock house virus is a small insect virus with a bipartite RNA genome consisting of RNA1 and RNA2. RNA3 is a subgenomic element encoded by RNA1, the genomic segment required for viral RNA synthesis (T. M.

WIN 52035-2 inhibits both attachment and eclipse of human rhinovirus 14.

WIN compounds inhibit attachment of human rhinovirus 14 by binding to a hydrophobic pocket within the capsid and inducing conformational changes in the canyon floor, the region that binds the cellular receptor. To study the basis of drug resistance, we isolated and characterized a family of human rhinovirus 14 mutants resistant to WIN 52035-2. Thermostabilization data and single-cycle growth curves provided evidence for two classes of resistant mutants.

Role of maturation cleavage in infectivity of picornaviruses: activation of an infectosome.

Maturation of picornaviruses involves assembly of a "provirion," which undergoes an autocatalytic cleavage of VP0 to VP2 plus VP4. RNA transcripts from a cDNA clone of human rhinovirus 14 mutated at asparagine 68, one of the residues in the maturation cleavage site, generated normal yields of 150S particles which were noninfectious in the plaque assay because they were unable to initiate a second cycle of infection. These cleavage-defective provirions were otherwise indistinguishable from mature virions in sedimentation coefficient, binding affinity to monoclonal antibodies against neutralization sites IA, II, and III, attachment to HeLa cell receptors, and rate of cell-mediated conformational changes to form 125S A-particles and 80S empty capsids.

WIN 51711-dependent mutants of poliovirus type 3: evidence that virions decay after release from cells unless drug is present.

Twenty-two spontaneous mutants of the Sabin strain of poliovirus type 3 were selected for drug resistance by plating on HeLa cell monolayers in the presence of WIN 51711, an uncoating inhibitor. When replated in the presence and absence of drug, two classes of mutants were observed; mutants displayed either a drug-dependent or a non-drug-dependent phenotype, in the proportion 14:8. Non-drug-dependent mutants plaqued with equal efficiency in the presence or absence of drug.

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.

Evidence that the packaging signal for nodaviral RNA2 is a bulged stem-loop.

Flock house virus is an insect virus belonging to the family Nodaviridae; members of this family are characterized by a small bipartite positive-stranded RNA genome. The larger genomic segment, RNA1, encodes viral replication proteins, whereas the smaller one, RNA2, encodes coat protein. Both RNAs are packaged in a single particle.

Maturation cleavage required for infectivity of a nodavirus.

Nodaviral morphogenesis involves formation of labile precursor particles, called provirions, which mature by autocatalytic cleavage of the 407-residue coat precursor protein between asparagine residue 363 and alanine residue 364. It has previously been demonstrated that maturation results in increased physicochemical stability of the virion. We show here that cleavage of coat protein in purified provirions of Flock House virus was accompanied by a five- to eightfold increase in specific infectivity.