Capsid Protein VP4 of Human Rhinovirus Induces Membrane Permeability by the Formation of a Size-Selective Multimeric Pore

Abstract

Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.

Viroporins: structure, function and potential as antiviral targets

“…VP4 channels can be reconstituted in vitro using recombinant protein and their activity is amenable to liposome dye-release assays (Davis et al, 2008). Recent studies also support the formation of discrete multimeric complexes (pentameric and hexameric) of defined pore size, with activity enhanced by myristoylation and reduced pH, consistent with the scenario within the early endosome (Panjwani et al, 2014). The tantalising prospect of a smallmolecule inhibitor of enterovirus entry targeting VP4 is therefore a realistic possibility, which could have profound impact ranging from polio eradication to treating the common cold.…”

Antibody-induced uncoating of human rhinovirus B14

“…These observations suggest that the VP1 N-terminal residues might be externalized and that VP4 might be lost from the empty particle, because these residues reside in the interior of the full particle. It has been suggested that externalization of these internal residues facilitates interactions of the capsid with host cell membranes during cell entry of EVs (22,23).…”

Cryo-EM study of slow bee paralysis virus at low pH reveals iflavirus genome release mechanism

“…During cell entry, nonenveloped viruses have to breach a biological membrane to deliver their genomes to the cell cytoplasm. Previously, it has been shown that both myristoylated and unmodified VP4 minor capsid proteins (which are 60-80 residues long) of several picornaviruses and dicistroviruses can induce the lysis of liposomes (26,40,41). In contrast, the VP4 subunits of SBPV have been predicted to be only 20 residues long, and lack the myristoylation signal sequence (42).…”

“…The N-terminal regions of VP1 subunits of enteroviruses contain sequences, which were proposed to form amphipathic α-helices that disrupt endosome membranes, and together with VP4 subunits enable translocation of the virus genome to the cytoplasm (26,27,41). Twelve residues from the N terminus of SBPV VP1 become disordered upon genome release (Fig.…”

“…particle surface, and released VP4 subunits (19)(20)(21)(22)(23)(24). Amphipathic sequences from the N termini of VP1 and myristoylated VP4 subunits interact with endosome membranes and enable the delivery of enterovirus genomes into the cell cytoplasm (25)(26)(27). It has been previously proposed that the enterovirus genome leaves the capsid via a pore located at a twofold icosahedral axis (19)(20)(21).…”