Major Group Of Human Rhinoviruses Research Articles

Vitamin D attenuates rhinovirus-induced expression of intercellular adhesion molecule-1 (ICAM-1) and platelet-activating factor receptor (PAFR) in respiratory epithelial cells

Human rhinoviruses commonly cause upper respiratory infections, which may be complicated by secondary bacterial infection. Vitamin D replacement reduces risk of acute respiratory infections in vitamin D-deficient individuals, but the mechanisms by which such protection is mediated are incompletely understood. We therefore conducted experiments to characterise the influence of the major circulating metabolite 25-hydroxyvitamin D (25[OH]D) and the active metabolite 1,25-dihydroxyvitamin D (1,25[OH]2D) on responses of a respiratory epithelial cell line (A549 cells) to infection with a major group human rhinovirus (RV-16). Pre-treatment of A549 respiratory epithelial cells with a physiological concentration (10−7M) of 25(OH)D induced transient resistance to infection with RV-16 and attenuated RV-16-induced expression of the genes encoding intercellular adhesion molecule 1 (ICAM-1, a cell surface glycoprotein that acts as the cellular receptor for major group rhinoviruses) and platelet-activating factor receptor (PAFR, a G-protein coupled receptor implicated in adhesion of Streptococcus pneumoniae to respiratory epithelial cells). These effects were associated with enhanced expression of the genes encoding the NF-κB inhibitor IκBα and the antimicrobial peptide cathelicidin LL-37. Our findings suggest possible mechanisms by which vitamin D may enhance resistance to rhinovirus infection and reduce risk of secondary bacterial infection in vitamin D-deficient individuals.

Authorised medicinal product Aspecton® Oral Drops containing thyme extract KMTv24497 shows antiviral activity against viruses which cause respiratory infections

The common cold is a respiratory disease often caused by viral pathogens, including a major group of human rhinoviruses (HRV). Currently, there is no specific antiviral approach available against these viruses. Another viral pathogen that causes respiratory diseases is the influenza virus (IV). Even though a few approved drugs are available for antiviral treatment against IV, these are prone to lose their effectiveness due to the rapid emergence of resistant virus variants. Therefore, new effective approaches characterised by a broad antiviral activity and wide availability would be very advantageous. In this study the authors aimed to investigate the antiviral potential of an authorised medicinal product (Aspecton® Oral Drops) containing a specific thyme extract (KMTv24497) against human rhinovirus serotype 1a (HRV1a), as well as against an influenza A virus isolate of the 2009 pandemic (H1N1pdm09) at non-toxic concentrations. To the best of the authors knowledge, they were able to demonstrate, for the first time, the in vitro antiviral activity of a thyme extract containing an authorised medicinal product in non-toxic concentrations against an HRV, as well as against an influenza A virus strain in cell culture. This study represents a first approach to prove a causal therapeutic effect in addition to the already described symptomatic effects for a thyme preparation. Patients with common cold infections may profit from the additional causal therapeutic effects of an expectorant.

Immunization with Live Human Rhinovirus (HRV) 16 Induces Protection in Cotton Rats against HRV14 Infection.

Human rhinoviruses (HRVs) are the main cause of cold-like illnesses, and currently no vaccine or antiviral therapies against HRVs are available to prevent or mitigate HRV infection. There are more than 150 antigenically heterogeneous HRV serotypes, with ∼90 HRVs belonging to major group species A and B. Development of small animal models that are susceptible to infection with major group HRVs would be beneficial for vaccine research. Previously, we showed that the cotton rat (Sigmodon hispidus) is semi-permissive to HRV16 (major group, species HRV-A virus) infection, replicating in the upper and lower respiratory tracts with measurable pathology, mucus production, and expression of inflammatory mediators. Herein, we report that intranasal infection of cotton rats with HRV14 (major group, species HRV-B virus) results in isolation of infectious virus from the nose and lung. Similar to HRV16, intramuscular immunization with live HRV14 induces homologous protection that correlated with high levels of serum neutralizing antibodies. Vaccination and challenge experiments with HRV14 and HRV16 to evaluate the development of cross-protective immunity demonstrate that intramuscular immunization with live HRV16 significantly protects animals against HRV14 challenge. Determination of the immunological mechanisms involved in heterologous protection and further characterization of infection with other major HRV serotypes in the cotton rat could enhance the robustness of the model to define heterotypic relationships between this diverse group of viruses and thereby increase its potential for development of a multi-serotype HRV vaccine.

Role of human rhinovirus in triggering human airway epithelial-mesenchymal transition

BackgroundStructural changes in the airways, collectively referred to as airway remodeling, are a characteristic feature of asthma, and are now known to begin in early life. Human rhinovirus (HRV)-induced wheezing illnesses during early life are a potential inciting stimulus for remodeling. Increased deposition of matrix proteins causes thickening of the lamina reticularis, which is a well-recognized component of airway remodeling. Increased matrix protein deposition is believed to be due to the presence of increased numbers of activated mesenchymal cells (fibroblasts/myofibroblasts) in the subepithelial region of asthmatic airways. The origin of these increased mesenchymal cells is not clear, but one potential contributor is the process of epithelial-mesenchymal transition (EMT). We hypothesized that HRV infection may help to induce EMT.MethodsWe used the BEAS-2B human bronchial epithelial cells line, which uniformly expresses the major group HRV receptor, to examine the effects of stimulation with HRV alone, transforming growth factor-β1 (TGF-β1), alone, and the combination, on induction of changes consistent with EMT. Western blotting was used to examine expression of epithelial and mesenchymal phenotypic marker proteins and selected signaling molecules. Cell morphology was also examined.ResultsIn this study, we show that two different strains of HRV, which use two different cellular receptors, are each capable of triggering phenotypic changes consistent with EMT. Moreover, both HRV serotypes synergistically induced changes consistent with EMT when used in the presence of TGF-β1. Morphological changes were also most pronounced with the combination of HRV and TGF-β1. Viral replication was not essential for phenotypic changes. The synergistic interactions between HRV and TGF-β1 were mediated, at least in part, via activation of mitogen activated protein kinase pathways, and via induction of the transcription factor SLUG.ConclusionsThese data support a role for HRV in the induction of EMT, which may contribute to matrix protein deposition and thickening of the lamina reticularis in airways of patients with asthma.

Haemophilus influenzae increases the susceptibility and inflammatory response of airway epithelial cells to viral infections.

Nontypeable Haemophilus influenzae (NTHI), a common colonizer of lungs of patients with chronic obstructive pulmonary disease (COPD), can enhance expression of the cellular receptor intercellular adhesion molecule 1 (ICAM-1), which in turn can be used by major group human rhinoviruses (HRVs) for attachment. Here, we evaluated the effect of NTHI-induced up-regulation of ICAM-1 on viral replication and inflammatory responses toward different respiratory viruses. Therefore, human bronchial epithelial cells were pretreated with heat-inactivated NTHI (hi-NTHI) and subsequently infected with either HRV16 (major group), HRV1B (minor group), or respiratory syncytial virus (RSV). Pretreatment with hi-NTHI significantly up-regulated ICAM-1 in BEAS-2B cells and primary bronchial epithelial cells. Concomitantly, release of infectious HRV16 particles was increased in cells pretreated with hi-NTHI. Pretreatment with hi-NTHI also caused a significant increase in HRV16 RNA, whereas replication of HRV1B and RSV were increased to a far lesser extent and only at later time points. Interestingly, release of IL-6 and IL-8 after RSV, but not HRV, infection was synergistically increased in hi-NTHI-pretreated BEAS-2B cells. In summary, exposure to hi-NTHI significantly enhanced sensitivity toward HRV16 but not HRV1B or RSV, probably through ICAM-1 up-regulation. Furthermore, hi-NTHI pretreatment may enhance the inflammatory response to RSV infection, suggesting that preexisting bacterial infections might exaggerate inflammation during secondary viral infection.

An Anti-Human ICAM-1 Antibody Inhibits Rhinovirus-Induced Exacerbations of Lung Inflammation

Human rhinoviruses (HRV) cause the majority of common colds and acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Effective therapies are urgently needed, but no licensed treatments or vaccines currently exist. Of the 100 identified serotypes, ∼90% bind domain 1 of human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor, making this an attractive target for development of therapies; however, ICAM-1 domain 1 is also required for host defence and regulation of cell trafficking, principally via its major ligand LFA-1. Using a mouse anti-human ICAM-1 antibody (14C11) that specifically binds domain 1 of human ICAM-1, we show that 14C11 administered topically or systemically prevented entry of two major groups of rhinoviruses, HRV16 and HRV14, and reduced cellular inflammation, pro-inflammatory cytokine induction and virus load in vivo. 14C11 also reduced cellular inflammation and Th2 cytokine/chemokine production in a model of major group HRV-induced asthma exacerbation. Interestingly, 14C11 did not prevent cell adhesion via human ICAM-1/LFA-1 interactions in vitro, suggesting the epitope targeted by 14C11 was specific for viral entry. Thus a human ICAM-1 domain-1-specific antibody can prevent major group HRV entry and induction of airway inflammation in vivo.

Development of a Homogeneous High-Throughput Screening Assay for Biological Inhibitors of Human Rhinovirus Infection

Infection with human rhinovirus (HRV) is thought to result in acute respiratory exacerbations of chronic obstructive pulmonary disorder (COPD). Consequently, prevention of HRV infection may provide therapeutic benefit to these patients. As all major group HRV serotypes infect cells via an interaction between viral coat proteins and intercellular adhesion molecule–1 (ICAM-1), it is likely that inhibitors of this interaction would prevent or reduce infections. Our objective was to use phage display technology in conjunction with naive human antibody libraries to identify anti–ICAM-1 antibodies capable of functional blockade of HRV infection. Key to success was the development of a robust, functionally relevant high-throughput screen (HTS) compatible with the specific challenges of antibody screening. In this article, we describe the development of a novel homogeneous time-resolved fluorescence (HTRF) assay based on the inhibition of soluble ICAM-1 binding to live HRV16. We describe the implementation of the method in an antibody screening campaign and demonstrate the biological relevance of the assay by confirming the activity of resultant antibodies in a cell-based in vitro HRV infection assay.

Entry of a heparan sulphate-binding HRV8 variant strictly depends on dynamin but not on clathrin, caveolin, and flotillin

The major group human rhinovirus type 8 can enter cells via heparan sulphate. When internalized into ICAM-1 negative rhabdomyosarcoma (RD) cells, HRV8 accumulated in the cells but caused CPE only after 3days when used at high MOI. Adaptation by three blind passages alternating between RD and HeLa cells resulted in variant HRV8v with decreased stability at acidic pH allowing for productive infection in the absence of ICAM-1. HRV8v produced CPE at 10 times lower MOI within 1day. Confocal fluorescence microscopy colocalization and the use of pharmacological and dominant negative inhibitors revealed that viral uptake is clathrin, caveolin, and flotillin independent. However, it is blocked by dynasore, amiloride, and EIPA. Furthermore, HRV8v induced FITC-dextran uptake and colocalized with this fluid phase marker. Except for the complete inhibition by dynasore, the entry pathway of HRV8v via HS is similar to that of HRV14 in RD cells that overexpress ICAM-1.

Uncoating of human rhinoviruses

Human rhinoviruses (HRVs) are a major cause of the common cold. The more than one hundred serotypes, divided into species HRV-A and HRV-B, either bind intercellular adhesion molecule 1 (major group viruses) or members of the low-density lipoprotein receptor (minor group viruses) for cell entry. Some major group HRVs can also access the host cell via heparan sulphate proteoglycans. The cell attachment protein(s) of the recently discovered phylogenetic clade HRV-C is unknown. The respective receptors direct virus uptake via clathrin-dependent or independent endocytosis or via macropinocytosis. Triggered by ICAM-1 and/or the low pH environment in endosomes the virions undergo conformational alterations giving rise to hydrophobic subviral particles. These are handed over from the receptors to the endosomal membrane. According to the current view, the RNA genome is released through an opening at one of the fivefold axes of the icosahedral capsid and crosses the membrane through a pore presumably formed by viral proteins. Alternatively, the membrane may be ruptured allowing subviral particles and RNA to enter the cytosol. Whether a channel is formed or the membrane is disrupted most probably depends on the respective HRV receptor.

Human Rhinovirus 14 Enters Rhabdomyosarcoma Cells Expressing ICAM-1 by a Clathrin-, Caveolin-, and Flotillin-Independent Pathway

Intercellular adhesion molecule 1 (ICAM-1) mediates binding and entry of major group human rhinoviruses (HRVs). Whereas the entry pathway of minor group HRVs has been studied in detail and is comparatively well understood, the pathway taken by major group HRVs is largely unknown. Use of immunofluorescence microscopy, colocalization with specific endocytic markers, dominant negative mutants, and pharmacological inhibitors allowed us to demonstrate that the major group virus HRV14 enters rhabdomyosarcoma cells transfected to express human ICAM-1 in a clathrin-, caveolin-, and flotillin-independent manner. Electron microscopy revealed that many virions accumulated in long tubular structures, easily distinguishable from clathrin-coated pits and caveolae. Virus entry was strongly sensitive to the Na(+)/H(+) ion exchange inhibitor amiloride and moderately sensitive to cytochalasin D. Thus, cellular uptake of HRV14 occurs via a pathway exhibiting some, but not all, characteristics of macropinocytosis and is similar to that recently described for adenovirus 3 entry via alpha(v) integrin/CD46 in HeLa cells.

Low pH-Triggered Beta-Propeller Switch of the Low-Density Lipoprotein Receptor Assists Rhinovirus Infection

Minor group human rhinoviruses (HRVs) bind three members of the low-density lipoprotein receptor (LDLR) family: LDLR proper, very-LDLR (VLDLR) and LDLR-related protein (LRP). Whereas ICAM-1, the receptor of major group HRVs actively contributes to viral uncoating, LDLRs are rather considered passive vehicles for cargo delivery to the low-pH environment of endosomes. Since the Tyr-Trp-Thr-Asp beta-propeller domain of LDLR has been shown to be involved in the dissociation of bound LDL via intramolecular competition at low pH, we studied whether it also plays a role in HRV infection. Human cell lines deficient in LDLR family proteins are not available. Therefore, we used CHO-ldla7 cells that lack endogenous LDLR. These were stably transfected to express either wild-type (wt) human LDLR or a mutant with a deletion of the beta-propeller. When HRV2 was attached to the propeller-negative LDLR, a lower pH was required for conversion to subviral particles than when attached to wt LDLR. This indicates that high-avidity receptor binding maintains the virus in its native conformation. HRV2 internalization directed the mutant LDLR but not wt LDLR to lysosomes, resulting in reduced plasma membrane expression of propeller-negative LDLR. Infection assays using a CHO-adapted HRV2 variant showed a delay in intracellular viral conversion and de novo viral synthesis in cells expressing the truncated LDLR. Our data indicate that the beta-propeller attenuates the virus-stabilizing effect of LDLR binding and thereby facilitates RNA release from endosomes, resulting in the enhancement of infection. This is a nice example of a virus exploiting high-avidity multimodule receptor binding with an intrinsic release mechanism.

Distinguishing Molecular Features and Clinical Characteristics of a Putative New Rhinovirus Species, Human Rhinovirus C (HRV C)

BackgroundHuman rhinoviruses (HRVs) are the most frequently detected pathogens in acute respiratory tract infections (ARTIs) and yet little is known about the prevalence, recurrence, structure and clinical impact of individual members. During 2007, the complete coding sequences of six previously unknown and highly divergent HRV strains were reported. To catalogue the molecular and clinical features distinguishing the divergent HRV strains, we undertook, for the first time, in silico analyses of all available polyprotein sequences and performed retrospective reviews of the medical records of cases in which variants of the prototype strain, HRV-QPM, had been detected.Methodology/Principle FindingsGenomic analyses revealed that the six divergent strains, residing within a clade we previously called HRV A2, had the shortest polyprotein of all picornaviruses investigated. Structure-based amino acid alignments identified conserved motifs shared among members of the genus Rhinovirus as well as substantive deletions and insertions unique to the divergent strains. Deletions mostly affected regions encoding proteins traditionally involved in antigenicity and serving as HRV and HEV receptor footprints. Because the HRV A2 strains cannot yet be cultured, we created homology models of predicted HRV-QPM structural proteins. In silico comparisons confirmed that HRV-QPM was most closely related to the major group HRVs. HRV-QPM was most frequently detected in infants with expiratory wheezing or persistent cough who had been admitted to hospital and required supplemental oxygen. It was the only virus detected in 65% of positive individuals. These observations contributed to an objective clinical impact ranging from mild to severe.ConclusionsThe divergent strains did not meet classification requirements for any existing species of the genus Rhinovirus or Enterovirus. HRV A2 strains should be partitioned into at least one new species, putatively called Human rhinovirus C, populated by members detected with high frequency, from individuals with respiratory symptoms requiring hospital admission.

Human Rhinovirus Type 54 Infection via Heparan Sulfate Is Less Efficient and Strictly Dependent on Low Endosomal pH

K-type major-group human rhinoviruses (HRVs) (including HRV54) share a prominent lysine residue in the HI surface loop of VP1 with all minor-group HRVs. Despite the presence of this residue, they cannot use members of the low-density lipoprotein receptor family for productive infection. Reexamining all K-type viruses for receptor usage, we noticed that HRV54 is able to replicate in RD cells that lack the major-group receptor intercellular adhesion molecule 1 (ICAM-1). By using receptor blocking assays, inhibition of sulfation, enzymatic digestion, and proteoglycan-deficient cell lines, we show here that wild-type HRV54, without any adaptation, uses heparan sulfate (HS) proteoglycan as an alternate receptor. However, infection via HS is less efficient than infection via ICAM-1. Moreover, HRV54 has an acid lability profile similar to that of the minor-group virus HRV2. In ICAM-1-deficient cells its replication is completely blocked by the H(+)-ATPase inhibitor bafilomycin A1, whereas in ICAM-1-expressing cells it replicates in the presence of the drug. Thus, use of a "noncatalytic" receptor requires the virus to be highly unstable at low pH.

Human Rhinoviruses Inhibit the Accessory Function of Dendritic Cells by Inducing Sialoadhesin and B7-H1 Expression

Dendritic cells (DC) are professional APCs with an unmatched ability to interact with and activate T cells. There is accumulating evidence that DC not only efficiently stimulate T cell activation but also regulate T cell responses. However, little is known about cell surface structures on DC involved in the regulation of T cell responses. We demonstrate that human rhinoviruses (HRV) can efficiently inhibit the accessory function of DC through induction of inhibitory cell surface receptors. We observed that treatment of DC with HRV14 (R-DC), a member of the major group HRV family, diminished their T cell stimulatory capacity and induced a promiscuous and deep anergic state in cocultured T cells despite high levels of MHC molecules as well as costimulatory molecules, e.g., B7-1 (CD80) and B7-2 (CD86), and independent of inhibitory soluble factors such as IL-10. In contrast, expression of inhibitory B7-H1 molecules was up-regulated and R-DC de novo expressed sialoadhesin (Sn). Most importantly, blocking of B7-H1 and Sn on R-DC with specific mAbs against both receptors reverted the inhibitory phenotype. Thus, inhibitory signals delivered from R-DC to T cells via B7-H1 and Sn were critical for the induction of anergy. These observations suggest that an altered accessory molecule repertoire on DC upon interaction with HRV down-modulates adaptive immune responses during the viral infection.

The Crystal Structure of Coxsackievirus A21 and Its Interaction with ICAM-1

CVA21 and polioviruses both belong to the Enterovirus genus in the family of Picornaviridae, whereas rhinoviruses form a distinct picornavirus genus. Nevertheless, CVA21 and the major group of human rhinoviruses recognize intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor, whereas polioviruses use poliovirus receptor. The crystal structure of CVA21 has been determined to 3.2 A resolution. Its structure has greater similarity to poliovirus structures than to other known picornavirus structures. Cryo-electron microscopy (cryo-EM) was used to determine an 8.0 A resolution structure of CVA21 complexed with an ICAM-1 variant, ICAM-1(Kilifi). The cryo-EM map was fitted with the crystal structures of ICAM-1 and CVA21. Significant differences in the structure of CVA21 with respect to the poliovirus structures account for the inability of ICAM-1 to bind polioviruses. The interface between CVA21 and ICAM-1 has shape and electrostatic complementarity with many residues being conserved among those CVAs that bind ICAM-1.

The Role of p38 MAPK in Rhinovirus-Induced Monocyte Chemoattractant Protein-1 Production by Monocytic-Lineage Cells

Viral respiratory infections are a major cause of asthma exacerbations and can contribute to the pathogenesis of asthma. Major group human rhinovirus enters cells by binding to the cell surface molecule ICAM-1 that is present on epithelial and monocytic lineage cells. The focus of the resulting viral infection is in bronchial epithelia. However, previous studies of the cytokine dysregulation that follows rhinovirus infection have implicated monocytic lineage cells in establishing the inflammatory environment even though productive infection is not a result. We have determined that human alveolar macrophages and human peripheral blood monocytes release MCP-1 upon exposure to human rhinovirus 16 (HRV16). Indeed, we have found p38 MAPK activation in human alveolar macrophages within 15 min of exposure to HRV16, and this activation lasts up to 1 h. The targets of p38 MAPK activation include transcriptional activators of the MCP-1 promoter. The transcription factor ATF-2, a p38 MAPK substrate, is phosphorylated 45 min after HRV16 exposure. Furthermore, IkappaBalpha, the inhibitor of the transcription factor NF-kappaB, is degraded. Prevention of HRV16 binding was effective in blocking p38 MAPK activation, ATF-2 phosphorylation, and MCP-1 release. This is the first report of a relationship between HRV16 exposure, MCP-1 release and monocytic-lineage cells suggesting that MCP-1 plays a role in establishing the inflammatory microenvironment initiated in the human airway upon exposure to rhinovirus.

The Minor Receptor Group of Human Rhinovirus (HRV) Includes HRV23 and HRV25, but the Presence of a Lysine in the VP1 HI Loop Is Not Sufficient for Receptor Binding

Like all 10 minor receptor group human rhinoviruses (HRVs), HRV23 and HRV25, previously classified as major group viruses, are neutralized by maltose binding protein (MBP)-V33333 (a soluble recombinant concatemer of five copies of repeat 3 of the very-low-density lipoprotein receptor fused to MBP), bind to low-density lipoprotein receptor in virus overlay blots, and replicate in intercellular adhesion molecule 1 (ICAM-1)-negative COS-7 cells. From phylogenetic analysis of capsid protein VP1-coding sequences, they are also known to cluster together with other minor group strains. Therefore, they belong to the minor group; there are now 12 minor group and 87 major group HRV serotypes. Sequence comparison of the VP1 capsid proteins of all HRVs revealed that the lysine in the HI loop, strictly conserved in the 12 minor group HRVs, is also present in 9 major group serotypes that are neutralized by soluble ICAM-1. Despite the presence of this lysine, they are not neutralized by MBP-V33333 and fail to replicate in COS-7 cells and in HeLa cells in the presence of an ICAM-1-blocking antibody. These nine serotypes are therefore "true" major group viruses.

Human Rhinovirus Type 89 Variants Use Heparan Sulfate Proteoglycan for Cell Attachment

We have previously isolated mutants of the major-group human rhinovirus type 89 that grow in cells deficient in intercellular adhesion molecule 1 (ICAM-1), the receptor used by the wild-type virus for cell entry [A. Reischl, M. Reithmayer, G. Winsauer, R. Moser, I. Goesler, and D. Blaas., J. Virol. 75:9312-9319, 2001]. We now demonstrate that one of these variants utilizes heparan sulfate proteoglycan (HSPG) as a cellular receptor. Adaptation to ICAM-1-deficient cells not only resulted in the newly acquired receptor specificity but also rendered the virus less stable at low pH and at elevated temperatures. This instability might compensate for the absence of the uncoating activity of ICAM-1. Whereas wild-type virus infection via ICAM-1 proceeded in the presence of the vesicular H(+)-ATPase inhibitor bafilomycin A1, infection by the mutant via HSPG was prevented by the drug. This suggests that the low pH prevailing in endosomal compartments is required for uncoating in the absence of the catalytic activity of ICAM-1.

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.

Receptor priming of major group human rhinoviruses for uncoating and entry at mild low-pH environments.

Receptor priming of low-pH-triggered virus entry has been described for an enveloped virus (15). Here we show with major group human rhinoviruses (HRV) and its intercellular adhesion molecule-1 receptor that nonenveloped viruses follow this novel cell entry principle. In vitro the receptor primed HRV for efficient uncoating at mild low pH (5.5 to 6.0). Agents preventing endosomal acidification reduced or blocked rhinovirus cell infection, while nocodazole had no effect on infection of any serotype tested. The entry inhibitory effect of lysosomotropic agents was overcome by exposing cell-internalized HRV to mild low pH (5.5 to 6.0). We therefore conclude that receptor priming of major group HRV must occur in vivo as well. Cooperation of a cellular receptor and low pH in virus uncoating will polarize the exit of the genome to the receptor-bound, membrane-proximal region of the virus particle during acidification of endosomes. This process must be required for efficient penetration of the cellular membrane by viruses.