The Rhinovirus Subviral A-Particle Exposes 3′-Terminal Sequences of Its Genomic RNA

Abstract

Enteroviruses, which represent a large genus within the family Picornaviridae, undergo important conformational modifications during infection of the host cell. Once internalized by receptor-mediated endocytosis, receptor binding and/or the acidic endosomal environment triggers the native virion to expand and convert into the subviral (altered) A-particle. The A-particle is lacking the internal capsid protein VP4 and exposes N-terminal amphipathic sequences of VP1, allowing for its direct interaction with a lipid bilayer. The genomic single-stranded (؉)RNA then exits through a hole close to a 2-fold axis of icosahedral symmetry and passes through a pore in the endosomal membrane into the cytosol, leaving behind the empty shell. We demonstrate that in vitro acidification of a prototype of the minor receptor group of common cold viruses, human rhinovirus A2 (HRV-A2), also results in egress of the poly(A) tail of the RNA from the A-particle, along with adjacent nucleotides totaling ϳ700 bases. However, even after hours of incubation at pH 5.2, 5=-proximal sequences remain inside the capsid. In contrast, the entire RNA genome is released within minutes of exposure to the acidic endosomal environment in vivo. This finding suggests that the exposed 3=-poly(A) tail facilitates the positioning of the RNA exit site onto the putative channel in the lipid bilayer, thereby preventing the egress of viral RNA into the endosomal lumen, where it may be degraded. IMPORTANCEFor host cell infection, a virus transfers its genome from within the protective capsid into the cytosol; this requires modifications of the viral shell. In common cold viruses, exit of the RNA genome is prepared by the acidic environment in endosomes converting the native virion into the subviral A-particle. We demonstrate that acidification in vitro results in RNA exit starting from the 3=-terminal poly(A). However, the process halts as soon as about 700 bases have left the viral shell. Conversely, inside the cell, RNA egress completes in about 2 min. This suggests the existence of cellular uncoating facilitators.

Impairing the function of MLCK, myosin Va or myosin Vb disrupts Rhinovirus B14 replication

“…The immunoprecipitation assay included a crosslink to stabilize protein-RNA complexes, and to prevent myosin Va from dissociating from the BrU-RV-B14 genome during the procedure 71 . Our observations of uncoated BrU-RNA (Figs 3 and 4 ) did not exclude the possibility that we actually detected A-particles with partially released RNA 72 . Nevertheless, our data strongly argue that myosin Va was playing an essential role in the release of the RV-B14 genome.…”

Divergent Requirement for a DNA Repair Enzyme during Enterovirus Infections

“…In particular, transfection of viral RNA bypasses normal cellular receptor-mediated entry pathways. It is likely that the uncoating of the vRNA following a normal infection determines the cytoplasmic delivery site of the vRNA and which end of the vRNA is initially exposed to the cytoplasm ( 37 ). To circumvent this potential limitation, we generated MEF-TDP2 +/+ and MEF-TDP2 −/− cell lines stably expressing the human poliovirus receptor (PVR) ( 38 , 39 ) under blasticidin selection.…”

Viral entry pathways: the example of common cold viruses

“…Recently, it was demonstrated that RNA exit starts from the 3’-end on heating to 56 °C in vitro [ 155 ], as well as on physiologic endosomal acidification in vivo [ 156 ]. Interestingly, upon acidification in vitro , exit halted after about 700 bases had egressed.…”