Opening of size-selective pores in endosomes during human rhinovirus serotype 2 in vivo uncoating monitored by single-organelle flow analysis

The effect of virus uncoating on endosome integrity during the early steps in viral infection was investigated. Using fluid-phase uptake of 10- and 70-kDa dextrans labeled with a pH-dependent fluorophore (fluorescein isothiocyanate [FITC]) and a pH-independent fluorophore (cyanine 5 [Cy5]), we determined the pHs of labeled compartments in intact HeLa cells by fluorescence-activated cell sorting analysis. Subsequently, the number and pH of fluorescent endosomes in cell homogenates were determined by single-organelle flow analysis. Cointernalization of adenovirus and 70-kDa FITC- and Cy5-labeled dextran (FITC/Cy5-dextran) led to virus-induced endosomal rupture, resulting in the release of the marker from the low-pH environment into the neutral cytosol. Consequently, in the presence of adenovirus, the number of fluorescent endosomes was reduced by 40% compared to that in the control. When human rhinovirus serotype 2 (HRV2) was cointernalized with 10-and 70-kDa FITC/Cy5-dextrans, the 10-kDa dextran was released, whereas the 70-kDa dextran remained within the endosomes, which also maintained their low pH. These data demonstrate that pores are generated in the membrane during HRV2 uncoating and RNA penetration into the cytosol without gross damage of the endosomes; 10-kDa dextran can access the cytosol through these pores. Whereas rhinovirus-mediated pore formation was prevented by the vacuolar ATPase inhibitor bafilomycin A1, adenovirus-mediated endosomal rupture also occurred in the presence of the inhibitor. This finding is in keeping with the low-pH requirement of HRV2 infection; for adenovirus, no pH dependence for endosomal escape was found with this drug.     

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.     

Formation of rhinovirus-soluble ICAM-1 complexes and conformational changes in the virion.

Viral receptors serve both to target viruses to specific cell types and to actively promote the entry of bound virus into cells. Human rhinoviruses (HRVs) can form complexes in vitro with a truncated soluble form of the HRV cell surface receptor, ICAM-1. These complexes appear to be stoichiometric, with approximately 60 ICAM molecules bound per virion or 1 ICAM-1 molecule per icosahedral face of the capsid. The complex can have two fates, either dissociating to yield free virus and free ICAM-1 or uncoating to break down to an 80S empty capsid which has released VP4, viral RNA, and ICAM-1. This uncoating in vitro mimics the uncoating of virus during infection of cells. The stability of the virus-receptor complex is dependent on temperature and the rhinovirus serotype. HRV serotype 14 (HRV14)-ICAM-1 complexes rapidly uncoat, HRV16 forms a stable virus-ICAM complex which does not uncoat detectably at 34 degrees C, and HRV3 has an intermediate phenotype. Rhinovirus can also uncoat after exposure to mildly acidic pH. The sensitivities of individual rhinovirus serotypes to ICAM-1-mediated virus uncoating do not correlate with uncoating promoted by incubation at low pH, suggesting that these two means of virus destabilization occur by different mechanisms. Soluble ICAM-1 and low pH do not act synergistically to promote uncoating. The rate of uncoating does appear to be inversely related to virus affinity for its receptor.     

Uncoating of human rhinovirus serotype 2 from late endosomes.

The internalization pathway and mechanism of uncoating of human rhinovirus serotype 2 (HRV2), a minor-group human rhinovirus, were investigated. Kinetic analysis revealed a late endosomal compartment as the site of capsid modification from D to C antigenicity. The conformational change as well as the infection was prevented by the specific V-ATPase inhibitor bafilomycin A1. A requirement for ATP was also demonstrated with purified endosomes in vitro. Capsid modifications occurred at a pH of 5.5 regardless of whether the virus was entrapped in isolated endosomes or free in solution. These findings suggest that the receptor is not directly involved in the structural modification of HRV2. Viral particles found in purified endosomes of infected cells were mostly devoid of RNA. This supports the hypothesis that uncoating of HRV2 occurs in intact endosomes rather than by a mechanism involving endosomal disruption with subsequent release of the RNA into the cytoplasm.     

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.     

Major and Minor Receptor Group Human Rhinoviruses Penetrate from Endosomes by Different Mechanisms

Intercellular adhesion molecule 1 and the low-density lipoprotein receptor are used for cell entry by major and minor receptor group human rhinoviruses (HRVs), respectively. Whereas minor-group viruses, exemplified by HRV2, transfer their genomic RNA to the cytoplasm through a pore in the endosomal membrane (E. Prchla, C. Plank, E. Wagner, D. Blaas, and R. Fuchs, J. Cell Biol. 131:111–123, 1995), the mechanism of in vivo uncoating of major-group HRVs has not been elucidated so far. Using free-flow electrophoresis, we performed a comparative analysis of cell entry by HRV2 and the major group rhinovirus HRV14. Here we demonstrate that this technique allows the separation of free viral particles from those associated with early endosomes, late endosomes, and plasma membranes. Upon free-flow electrophoretic separation of microsomes, HRV14 was recovered from endosomes under conditions which prevent uncoating, whereas the proportion of free viral particles increased with time under conditions which promote uncoating. The remaining virus eluted within numerous fractions corresponding to membraneous material, with no clear endosomal peaks being discernible. This suggests that uncoating of HRV14 results in lysis of the endosomal membrane and release of subviral 135S and 80S particles into the cytoplasm.     

Conformational changes,plasma membrane penetration,and infection by human rhinovirus type 2: role of receptors and low pH

Human rhinovirus type 2 (HRV2) is internalized by members of the low-density lipoprotein (LDL) receptor (LDLR) family. It then progresses into late endosomes, where it undergoes conversion from D- to C-antigenicity at pH < 5.6. Upon uncoating, the viral RNA is transferred into the cytoplasm across the endsosomal membrane. However, C-antigenic particles fail to attach to LDLR; this raised the question of whether the virus remains attached to the receptors and is carried to late compartments or rather falls off at the higher pH in early endosomes. We therefore determined the pH dependence of virus-receptor dissociation and virus conversion to C-antigen under conditions preventing endocytosis. (35)S-HRV2 was attached to HeLa cells at 4 degrees C and incubated in buffers of pH 7.4 to 5.0; levels of native virus and C-antigenic particles remaining cell associated or having been released into the medium were determined by immunoprecipitation. At pH 6.0, HRV2 was readily released from plasma membrane receptors in its native form, whereas at pH < or = 5.4, it was entirely converted to C-antigen, which, however, only dissociated from the surface upon prolonged incubation. The antigenic conversion occurred at the same pH regardless of whether HRV2 was free in solution or bound to its receptors. These data suggest that, in vivo, the virus is no longer bound to its receptors when the antigenic conversion and uncoating occur in more acidic late endosomes. When virus was bound to HeLa cells at 4 degrees C, converted into C-antigen by exposure to pH 5.3, and subsequently warmed to 34 degrees C in the presence of bafilomycin (to prevent endosomal uncoating), viral de novo synthesis was detected. This study demonstrates for the first time that a nonenveloped virus such as HRV2 can infect from the plasma membrane when artificially exposed to low pH. This implies that the viral RNA can gain access to the cytoplasm from the plasma membrane.     

Insights into minor group rhinovirus uncoating: the X-ray structure of the HRV2 empty capsid

Upon attachment to their respective receptor, human rhinoviruses (HRVs) are internalized into the host cell via different pathways but undergo similar structural changes. This ultimately results in the delivery of the viral RNA into the cytoplasm for replication. To improve our understanding of the conformational modifications associated with the release of the viral genome, we have determined the X-ray structure at 3.0 Å resolution of the end-stage of HRV2 uncoating, the empty capsid. The structure shows important conformational changes in the capsid protomer. In particular, a hinge movement around the hydrophobic pocket of VP1 allows a coordinated shift of VP2 and VP3. This overall displacement forces a reorganization of the inter-protomer interfaces, resulting in a particle expansion and in the opening of new channels in the capsid core. These new breaches in the capsid, opening one at the base of the canyon and the second at the particle two-fold axes, might act as gates for the externalization of the VP1 N-terminus and the extrusion of the viral RNA, respectively. The structural comparison between native and empty HRV2 particles unveils a number of pH-sensitive amino acid residues, conserved in rhinoviruses, which participate in the structural rearrangements involved in the uncoating process.     

Elevated endosomal pH in HeLa cells overexpressing mutant dynamin can affect infection by pH-sensitive viruses

Many viruses gain access to the cell via the endosomal route and require low endosomal pH for infectivity. The GTPase dynamin is essential for clathrin-dependent endocytosis, and in HeLa cells overexpressing the nonfunctional dynamin^K44A mutant the formation of clathrin-coated vesicles is halted. HRV2, a human minor group rhinovirus, is internalized by members of the low-density lipoprotein receptor family in a clathrin-independent manner. The low endosomal pH then leads to conversion of the capsid to C-antigen, which is required for release (uncoating) and transfer of the viral RNA into the cytosol and de novo synthesis of infectious virus. We here demonstrate that overexpression of dynamin^K44A reduces this antigenic conversion and results in diminished viral synthesis. In contrast, lysosomal degradation is unaffected. The kinetics of the formation of C-antigen in vitro and in vivo suggest that the pH in endosomes is elevated by about 0.4 units upon overexpression of dynamin^K44A. As a consequence, HRV2 uncoating is diminished early after internalization but attains control levels upon prolonged internalization. Thus, overexpression of dynamin^K44A, in addition to trafficking defects, results in an elevated endosomal pH and thereby affects virus infection and most likely endosomal sorting and processing.     

Rhinovirus-mediated endosomal release of transfection complexes.

Endocytosis is an efficient method for transfer of genes into mammalian cells. Incorporation of adenovirus particles into gene transfer complexes greatly enhances gene delivery, probably by the release of endocytosed DNA into the cytoplasm. We report here that two different serotypes of human rhinovirus (HRV), HRV2 and HRV14, are also able to enhance receptor-mediated gene transfer. The effect of several compounds known to inhibit viral infection on HRV2- and HRV14-enhanced transfection was examined. WIN I(s) and WIN IV, two compounds which inhibit viral uncoating, had different effects on HRV2- and HRV14-enhanced gene transfer to NIH 3T3 cells. While HRV14-enhanced gene transfer was severely reduced in the presence of these compounds, virtually no effects were observed when HRV2 was used. The use of antiviral compounds thus allowed transfection of human cells, which are normally lysed rapidly upon infection with HRV. Viral activity could be mimicked by using a peptide derived from the N terminus of VP1 of HRV2. This peptide possesses pH-dependent membrane-disrupting activity and enhances gene transfer to NIH 3T3 and HeLa cells.     

Kinesin-1-mediated capsid disassembly and disruption of the nuclear pore complex promote virus infection

Many viruses deliver their genomes into the host cell nucleus for replication. However, the size restrictions of the nuclear pore complex (NPC), which regulates the passage of proteins, nucleic acids, and solutes through the nuclear envelope, require virus capsid uncoating before viral DNA can access the nucleus. We report a microtubule motor kinesin-1-mediated and NPC-supported mechanism of adenovirus uncoating. The capsid binds to the NPC filament protein Nup214 and kinesin-1 light-chain Klc1/2. The nucleoporin Nup358, which is bound to Nup214/Nup88, interacts with the kinesin-1 heavy-chain Kif5c to indirectly link the capsid to the kinesin motor. Kinesin-1 disrupts capsids docked at Nup214, which compromises the NPC and dislocates nucleoporins and capsid fragments into the cytoplasm. NPC disruption increases nuclear envelope permeability as indicated by the nuclear influx of large cytoplasmic?dextran polymers. Thus, kinesin-1 uncoats viral DNA?and compromises NPC integrity, allowing viral genomes nuclear access to promote infection.     

The role of capsid maturation on adenovirus priming for sequential uncoating

Adenovirus assembly concludes with proteolytic processing of several capsid and core proteins. Immature virions containing precursor proteins lack infectivity because they cannot properly uncoat, becoming trapped in early endosomes. Structural studies have shown that precursors increase the network of interactions maintaining virion integrity. Using different biophysical techniques to analyze capsid disruption in vitro, we show that immature virions are more stable than the mature ones under a variety of stress conditions and that maturation primes adenovirus for highly cooperative DNA release. Cryoelectron tomography reveals that under mildly acidic conditions mimicking the early endosome, mature virions release pentons and peripheral core contents. At higher stress levels, both mature and immature capsids crack open. The virus core is completely released from cracked capsids in mature virions, but it remains connected to shell fragments in the immature particle. The extra stability of immature adenovirus does not equate with greater rigidity, because in nanoindentation assays immature virions exhibit greater elasticity than the mature particles. Our results have implications for the role of proteolytic maturation in adenovirus assembly and uncoating. Precursor proteins favor assembly by establishing stable interactions with the appropriate curvature and preventing premature ejection of contents by tightly sealing the capsid vertices. Upon maturation, core organization is looser, particularly at the periphery, and interactions preserving capsid curvature are weakened. The capsid becomes brittle, and pentons are more easily released. Based on these results, we hypothesize that changes in core compaction during maturation may increase capsid internal pressure to trigger proper uncoating of adenovirus.     

Insertion of Capsid Proteins from Nonenveloped Viruses into the Retroviral Budding Pathway

Retroviral Gag proteins direct the assembly and release of virus particles from the plasma membrane. The budding machinery consists of three small domains, the M (membrane-binding), I (interaction), and L (late or "pinching-off") domains. In addition, Gag proteins contain sequences that control particle size. For Rous sarcoma virus (RSV), the size determinant maps to the capsid (CA)-spacer peptide (SP) sequence, but it functions only when I domains are present to enable particles of normal density to be produced. Small deletions throughout the CA-SP sequence result in the release of particles that are very large and heterogeneous, even when I domains are present. In this report, we show that particles of relatively uniform size and normal density are released by budding when the size determinant and I domains in RSV Gag are replaced with capsid proteins from two unrelated, nonenveloped viruses: simian virus 40 and satellite tobacco mosaic virus. These results indicate that capsid proteins of nonenveloped viruses can interact among themselves within the context of Gag and be inserted into the retroviral budding pathway merely by attaching the M and L domains to their amino termini. Thus, the differences in the assembly pathways of enveloped and nonenveloped viruses may be far simpler than previously thought.     

A Direct and Versatile Assay Measuring Membrane Penetration of Adenovirus in Single Cells

Endocytosis is the most prevalent entry port for viruses into cells, but viruses must escape from the lumen of endosomes to ensure that viral genomes reach a site for replication and progeny formation. Endosomal escape also helps viruses bypass endolysosomal degradation and presentation to certain Toll-like intrinsic immunity receptors. The mechanisms for cytosolic delivery of nonenveloped viruses or nucleocapsids from enveloped viruses are poorly understood, in part because no quantitative assays are readily available which directly measure the penetration of viruses into the cytosol. Following uptake by clathrin-mediated endocytosis or macropinocytosis, the nonenveloped adenoviruses penetrate from endosomes to the cytosol, and they traffic with cellular motors on microtubules to the nucleus for replication. In this report, we present a novel single-cell imaging assay which quantitatively measures individual cytosolic viruses and distinguishes them from endosomal viruses or viruses at the plasma membrane. Using this assay, we showed that the penetration of human adenoviruses of the species C and B occurs rapidly after virus uptake. Efficient penetration does not require acidic pH in endosomes. This assay is versatile and can be adapted to other adenoviruses and members of other nonenveloped and enveloped virus families.     

Insight into the Mechanisms of Adenovirus Capsid Disassembly from Studies of Defensin Neutralization

Defensins are effectors of the innate immune response with potent antibacterial activity. Their role in antiviral immunity, particularly for non-enveloped viruses, is poorly understood. We recently found that human alpha-defensins inhibit human adenovirus (HAdV) by preventing virus uncoating and release of the endosomalytic protein VI during cell entry. Consequently, AdV remains trapped in the endosomal/lysosomal pathway rather than trafficking to the nucleus. To gain insight into the mechanism of defensin-mediated neutralization, we analyzed the specificity of the AdV-defensin interaction. Sensitivity to alpha-defensin neutralization is a common feature of HAdV species A, B1, B2, C, and E, whereas species D and F are resistant. Thousands of defensin molecules bind with low micromolar affinity to a sensitive serotype, but only a low level of binding is observed to resistant serotypes. Neutralization is dependent upon a correctly folded defensin molecule, suggesting that specific molecular interactions occur with the virion. CryoEM structural studies and protein sequence analysis led to a hypothesis that neutralization determinants are located in a region spanning the fiber and penton base proteins. This model was supported by infectivity studies using virus chimeras comprised of capsid proteins from sensitive and resistant serotypes. These findings suggest a mechanism in which defensin binding to critical sites on the AdV capsid prevents vertex removal and thereby blocks subsequent steps in uncoating that are required for release of protein VI and endosomalysis during infection. In addition to informing the mechanism of defensin-mediated neutralization of a non-enveloped virus, these studies provide insight into the mechanism of AdV uncoating and suggest new strategies to disrupt this process and inhibit infection.     

Viral Uncoating Is Directional: Exit of the Genomic RNA in a Common Cold Virus Starts with the Poly-(A) Tail at the 3′-End

Upon infection, many RNA viruses reorganize their capsid for release of the genome into the host cell cytosol for replication. Often, this process is triggered by receptor binding and/or by the acidic environment in endosomes. In the genus Enterovirus, which includes more than 150 human rhinovirus (HRV) serotypes causing the common cold, there is persuasive evidence that the viral RNA exits single-stranded through channels formed in the protein shell. We have determined the time-dependent emergence of the RNA ends from HRV2 on incubation of virions at 56°C using hybridization with specific oligonucleotides and detection by fluorescence correlation spectroscopy. We report that psoralen UV crosslinking prevents complete RNA release, allowing for identification of the sequences remaining inside the capsid. We also present the structure of uncoating intermediates in which parts of the RNA are condensed and take the form of a rod that is directed roughly towards a two-fold icosahedral axis, the presumed RNA exit point. Taken together, in contrast to schemes frequently depicted in textbooks and reviews, our findings demonstrate that exit of the RNA starts from the 3′-end. This suggests that packaging also occurs in an ordered manner resulting in the 3′-poly-(A) tail becoming located close to a position of pore formation during conversion of the virion into a subviral particle. This directional genome release may be common to many icosahedral non-enveloped single-stranded RNA viruses.     

Coxsackievirus A9 VP1 mutants with enhanced or hindered A particle formation and decreased infectivity

We have studied coxsackievirus A9 (CAV9) mutants that each have a single amino acid substitution in the conserved 29-PALTAVETGHT-39 motif of VP1 and a reduced capacity to produce infectious progeny virus. After uncoating, all steps in the infection cycle occurred according to the same kinetics as and similar efficiency to the wild-type virus. However, the particle/infectious unit ratio in the progeny was significantly increased. The differences were apparently due to altered stability of the capsid: there were mutant viruses with enhanced or hindered uncoating, and both of these characteristics were found to reduce fitness under standard passaging conditions. At 32 degrees C the instable mutants had an advantage, while the wild-type and the most stable mutant grew poorly. When comparing the newly published CAV9 structure and the other enterovirus structures, we found that the PALTAVETGHT motif is always in exactly the same position, in a cavity formed by the 3 other capsid proteins, with the C terminus of VP4 between this motif and the RNA. In the 7 enterovirus structures determined to date, the most conserved residues of the studied motif have identical contacts to neighboring residues of VP2, VP3, and VP4. We conclude that (i) the mutations affect the uncoating step necessary for infection, resulting in an untimely or hindered externalization of the VP1 N terminus together with the VP4, and (ii) the reason for the studied motif being evolutionarily conserved is its role in maintaining an optimal balance between the protective stability and the functional flexibility of the capsid.     

Wortmannin delays transfer of human rhinovirus serotype 2 to late endocytic compartments

Human rhinovirus 2 (HRV2) is internalized by members of the low-density lipoprotein receptor family into early endosomes (pH 6.2-6.0) where it dissociates from its receptors. After transfer into late endosomes, the virus undergoes a conformational change and RNA uncoating solely induced by pH < 5.6. Finally, virus capsids are degraded in lysosomes. To investigate the role of phosphatidylinositol 3-kinases (PI3K) in the HRV2 entry route, we used the inhibitor wortmannin. Although virus internalization was not altered by wortmannin, virus accumulated in enlarged early endosomes. Furthermore, the drug delayed HRV2 degradation and viral protein synthesis. Consequently, wortmannin-sensitive PI3K are involved in HRV2 transport from early to late compartments. However, wortmannin had no effect on the titer of infectious virus produced. Our data therefore suggest that virus retained in early endosomes for prolonged time periods can undergo the conformational change that otherwise occurs at pH < or = 5.6 in late endosomes.