Productive Entry Pathways of Human Rhinoviruses

Currently, complete or partial genome sequences of more than 150 human rhinovirus (HRV) isolates are known. Twelve species A use members of the low-density lipoprotein receptor family for cell entry, whereas the remaining HRV-A and all HRV-B bind ICAM-1. HRV-Cs exploit an unknown receptor. At least all A and B type viruses depend on receptor-mediated endocytosis for infection. In HeLa cells, they are internalized mainly by a clathrin- and dynamin-dependent mechanism. Upon uptake into acidic compartments, the icosahedral HRV capsid expands by ~4% and holes open at the 2-fold axes, close to the pseudo-3-fold axes and at the base of the star-shaped dome protruding at the vertices. RNA-protein interactions are broken and new ones are established, the small internal myristoylated capsid protein VP4 is expelled, and amphipathic N-terminal sequences of VP1 become exposed. The now hydrophobic subviral particle attaches to the inner surface of endosomes and transfers its genomic (+) ssRNA into the cytosol. The RNA leaves the virus starting with the poly(A) tail at its 3′-end and passes through a membrane pore contiguous with one of the holes in the capsid wall. Alternatively, the endosome is disrupted and the RNA freely diffuses into the cytoplasm

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 viruse

Dynamic force microscopy for imaging of viruses under physiological conditions

Dynamic force microscopy (DFM) allows imaging of the structure and the assessment of the function of biological specimens in their physiological environment. In DFM, the cantilever is oscillated at a given frequency and touches the sample only at the end of its downward movement. Accordingly, the problem of lateral forces displacing or even destroying bio-molecules is virtually inexistent as the contact time and friction forces are reduced. Here, we describe the use of DFM in studies of human rhinovirus serotype 2 (HRV2) weakly adhering to mica surfaces. The capsid of HRV2 was reproducibly imaged without any displacement of the virus. Release of the genomic RNA from the virions was initiated by exposure to low pH buffer and snapshots of the extrusion process were obtained. In the following, the technical details of previous DFM investigations of HRV2 are summarized

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

Wiener Medizinische Wochenschrift / Viral entry pathways : the example of common cold viruses

For infection, viruses deliver their genomes into the host cell. These nucleic acids are usually tightly packed within the viral capsid, which, in turn, is often further enveloped within a lipid membrane. Both protect them against the hostile environment. Proteins and/or lipids on the viral particle promote attachment to the cell surface and internalization. They are likewise often involved in release of the genome inside the cell for its use as a blueprint for production of new viruses. In the following, I shall cursorily discuss the early more general steps of viral infection that include receptor recognition, uptake into the cell, and uncoating of the viral genome. The later sections will concentrate on human rhinoviruses, the main cause of the common cold, with respect to the above processes. Much of what is known on the underlying mechanisms has been worked out by Renate Fuchs at the Medical University of Vienna.(VLID)346748

Nonneutralizing Human Rhinovirus Serotype 2-Specific Monoclonal Antibody 2G2 Attaches to the Region That Undergoes the Most Dramatic Changes upon Release of the Viral RNA

The monoclonal antibody 2G2 has been used extensively for detection and quantification of structural changes of human rhinovirus serotype 2 during infection. It recognizes exclusively A and B subviral particles, not native virus. We have elucidated the basis of this selectivity by determining the footprint of 2G2. Since viral escape mutants obviously cannot be obtained, the structures of complexes between Fab fragments of 2G2 and 80S subviral B particles were determined by cryoelectron microscopy. The footprint of the antibody corresponds to the capsid region that we predicted would undergo the most dramatic changes upon RNA release