Rhinovirus RNA uncoationg and membrane translocation

Human rhinoviruses (HRVs) are non-enveloped viruses with a RNA genome packaged into a capsid composed of viral proteins VP1-VP4. HRVs are the main cause of the usually harmless common cold - infections that are not life threatening, but may pose a threat for patients with chronic respiratory diseases. Today, there are still no effective and specific treatments against HRV infections available. Thus, exact knowledge of the molecular mechanism of the infection cycle is required to develop specific inhibitors of HRV infections. Therefore, this project focuses on the most important step in viral infections: uncoating and penetration of the viral genome, which is the prerequisite for viral replication in the cytoplasm. For these studies we have chosen the minor group human rhinovirus serotype 2 (HRV2) since it requires endocytosis and low endosomal pH to uncoat its RNA and to infect cells. HRV2 binds to and is internalized by members of the LDL receptor family into early endosomes where mildly acidic pH induces virus-receptor dissociation. Solely due to pH < 5.6 in late endosomes native viruses undergo a conformational change to C-antigenic particles: VP4 is expelled, the N-terminus of VP1 is externalized and the viral RNA is released. Our previous data are in accordance with RNA penetration into the cytoplasm through a pore in the endosomal membrane that involves hydrophobic domains of VP1. Presumably, also VP4 is involved in RNA translocation. So far, the cellular requirements for transfer of a viral genome across intact endosomes in vitro have not been analyzed for any non-enveloped virus. Thus, HRV2 is used as a representative for low pH induced endosomal uncoating and RNA penetration into the cytoplasm by pore formation. To gain insight into the molecular mechanism and driving forces of this process the aims of this proposal are as follows: 1. Requirements and driving forces for RNA translocation in vivo: The role of the endosomal pH gradient and membrane potential for productive uncoating will be investigated by determining cleavage of eIFG4, the earliest event in productive infection. 2. In vivo RNA transfer into the cytoplasm and intracellular fate of the viral RNA: We will apply fluorescent in situ hybridization (FISH) to detect uncoated RNA; its transport from the site of uncoating to the site of replication will be characterized by immuno co-localization with compartment specific markers. 3. In vitro RNA translocation: requirements and interaction of viral components with endosomal membranes: Endosomes containing native HRV2 will be isolated and acidified in vitro to induce the conformational change and RNA translocation. The amount of C-antigen will be correlated with RNA transfer from the endosome interior into the medium as determined by quantitative real-time PCR. The role of a pH gradient, membrane potential, endosomal and cytosolic proteins and ribosomes will be investigated. Viral proteins interacting with the viral RNA during translocation through the endosomal membrane will be cross-linked and analyzed. The results of the proposed experiments should reveal the mechanism and cellular requirements for RNA translocation into the cytosol, the identities of viral and cellular proteins that interact during this process and the route of the viral RNA after translocation to the site of replication. This knowledge then enables the design of short peptides or drugs that block RNA translocation and thus infection.

Human rhinoviruses (HRVs) are non-enveloped viruses with a RNA genome packaged into a capsid composed of viral proteins VP1-VP4. HRVs are the main cause of the usually harmless common cold - infections that are not life threatening, but may pose a threat for patients with chronic respiratory diseases. Today, there are still no effective and specific treatments against HRV infections available. Thus, exact knowledge of the molecular mechanism of the infection cycle is required to develop specific inhibitors of HRV infections. Therefore, this project focuses on the most important step in viral infections: uncoating and penetration of the viral genome, which is the prerequisite for viral replication in the cytoplasm. For these studies we have chosen the minor group human rhinovirus serotype 2 (HRV2) since it requires endocytosis and low endosomal pH to uncoat its RNA and to infect cells. HRV2 binds to and is internalized by members of the LDL receptor family into early endosomes where mildly acidic pH induces virus-receptor dissociation. Solely due to pH < 5.6 in late endosomes native viruses undergo a conformational change to C-antigenic particles: VP4 is expelled, the N-terminus of VP1 is externalized and the viral RNA is released. Our previous data are in accordance with RNA penetration into the cytoplasm through a pore in the endosomal membrane that involves hydrophobic domains of VP1. Presumably, also VP4 is involved in RNA translocation. So far, the cellular requirements for transfer of a viral genome across intact endosomes in vitro have not been analyzed for any non-enveloped virus. Thus, HRV2 is used as a representative for low pH induced endosomal uncoating and RNA penetration into the cytoplasm by pore formation. To gain insight into the molecular mechanism and driving forces of this process the aims of this proposal are as follows: 1. Requirements and driving forces for RNA translocation in vivo: The role of the endosomal pH gradient and membrane potential for productive uncoating will be investigated by determining cleavage of eIFG4, the earliest event in productive infection. 2. In vivo RNA transfer into the cytoplasm and intracellular fate of the viral RNA: We will apply fluorescent in situ hybridization (FISH) to detect uncoated RNA; its transport from the site of uncoating to the site of replication will be characterized by immuno co-localization with compartment specific markers. 3. In vitro RNA translocation: requirements and interaction of viral components with endosomal membranes: Endosomes containing native HRV2 will be isolated and acidified in vitro to induce the conformational change and RNA translocation. The amount of C-antigen will be correlated with RNA transfer from the endosome interior into the medium as determined by quantitative real-time PCR. The role of a pH gradient, membrane potential, endosomal and cytosolic proteins and ribosomes will be investigated. Viral proteins interacting with the viral RNA during translocation through the endosomal membrane will be cross-linked and analyzed. The results of the proposed experiments should reveal the mechanism and cellular requirements for RNA translocation into the cytosol, the identities of viral and cellular proteins that interact during this process and the route of the viral RNA after translocation to the site of replication. This knowledge then enables the design of short peptides or drugs that block RNA translocation and thus infection.