Recombinant VP4 of Human Rhinovirus Induces Permeability in Model Membranes

Davis, Matthew P; Bottley, Graham; Beales, Lucy; Killington, R. A.; Rowlands, David J.; Tuthill, Tobias J.

doi:10.1128/jvi.01070-07

Cited by 48 publications

(62 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, VP4 activity is not membrane-disruptive and induces discrete channel events in artificial bilayers (Danthi et al, 2003). 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).…”

Section: Other Rna Virus Viroporinsmentioning

confidence: 76%

Viroporins: structure, function and potential as antiviral targets

Scott

Griffin

2015

Journal of General Virology

124

146

View full text Add to dashboard Cite

The channel-forming activity of a family of small, hydrophobic integral membrane proteins termed 'viroporins' is essential to the life cycles of an increasingly diverse range of RNA and DNA viruses, generating significant interest in targeting these proteins for antiviral development. Viroporins vary greatly in terms of their atomic structure and can perform multiple functions during the virus life cycle, including those distinct from their role as oligomeric membrane channels. Recent progress has seen an explosion in both the identification and understanding of many such proteins encoded by highly significant pathogens, yet the prototypic M2 proton channel of influenza A virus remains the only example of a viroporin with provenance as an antiviral drug target. This review attempts to summarize our current understanding of the channel-forming functions for key members of this growing family, including recent progress in structural studies and drug discovery research, as well as novel insights into the life cycles of many viruses revealed by a requirement for viroporin activity. Ultimately, given the successes of drugs targeting ion channels in other areas of medicine, unlocking the therapeutic potential of viroporins represents a valuable goal for many of the most significant viral challenges to human and animal health.

show abstract

Section: Other Rna Virus Viroporinsmentioning

confidence: 76%

Viroporins: structure, function and potential as antiviral targets

Scott

Griffin

2015

Journal of General Virology

124

146

View full text Add to dashboard Cite

show abstract

“…The natural precursor protein is myristoylated at the N terminus. The same modification of recombinant proteins in E. coli is achieved by the coexpression of N-myristoyl transferase (21), a system used routinely in our laboratory for the production of myristoylated proteins (6,18,19). The myristoylation of capsid precursor was also tested by the incorporation of radiolabeled myristic acid into proteins expressed in cell-free translation reactions (Fig.…”

Section: Resultsmentioning

confidence: 99%

Foot-and-Mouth Disease Virus Assembly: Processing of Recombinant Capsid Precursor by Exogenous Protease Induces Self-Assembly of Pentamers In Vitro in a Myristoylation-Dependent Manner

Goodwin

Tuthill

Arias

et al. 2009

J Virol

Self Cite

View full text Add to dashboard Cite

The assembly of foot-and-mouth disease virus (FMDV) particles is poorly understood. In addition, there are important differences in the antigenic and receptor binding properties of virus assembly and dissociation intermediates, and these also remain unexplained. We have established an experimental model in which the antigenicity, receptor binding characteristics, and in vitro assembly of capsid precursor can be studied entirely from purified components. Recombinant capsid precursor protein (P1 region) was expressed in Escherichia coli as myristoylated or unmyristoylated protein. The protein sedimented in sucrose gradients at 5S and reacted with monoclonal antibodies which recognize conformational or linear antigen determinants on the virion surface. In addition, it bound the integrin ␣ v ␤ 6 , a cellular receptor for FMDV, indicating that unprocessed recombinant capsid precursor is both structurally and antigenically similar to native virus capsid. These characteristics were not dependent on the presence of 2A at the C terminus but were altered by N-terminal myristoylation and in mutant precursors which lacked VP4. Proteolytic processing of myristoylated precursor by recombinant FMDV 3C pro in vitro induced a shift in sedimentation from 5S to 12S, indicating assembly into pentameric capsid subunits. Nonmyristoylated precursor still assembled into higher-order structures after processing with 3Cpro , but these particles sedimented in sucrose gradients at approximately 17S. In contrast, mutant precursors lacking VP4 were antigenically distinct, were unable to form pentamers, and had reduced capacity for binding integrin receptor. These studies demonstrate the utility of recombinant capsid precursor protein for investigating the initial stages of assembly of FMDV and other picornaviruses.Foot-and-mouth disease virus (FMDV) is the etiological agent of a highly infectious disease of cattle and other clovenhoofed animals. It is of economic importance, because the presence of the disease results in severe restrictions of international trade. In many parts of the developing world, the disease is endemic, and it continues to pose a serious threat to livestock industries globally, as exemplified by recent major outbreaks in the United Kingdom, Argentina, and Uruguay.FMDV is a small, nonenveloped, positive-strand RNA virus belonging to the genus Aphthovirus within the family Picornaviridae. Other members of the family include poliovirus (PV; genus Enterovirus) and hepatitis A virus (genus Hepatovirus). The mature picornavirus comprises 60 copies each of four structural proteins, termed VP1, VP2, VP3, and VP4, which encapsidate a single, positive-sense RNA genome. The proteins form a pseudo Tϭ3 icosahedral capsid with VP1 located close to the fivefold axes of symmetry and VP2 and VP3 alternating around the threefold axes (24). VP4, which is myristoylated at the N terminus, is an internal component of the capsid (13,48).Although the morphogenesis of picornaviral particles is not completely understood, it is known that capsid...

show abstract

“…Egress of the RNA is believed to occur after the A-particle dissociates from the receptor (52); concomitantly, the particle might be handed over to the endosomal membrane, where the amphipathic N-terminal segments of VP1, and presumably VP4 set free during the process, insert into the membrane for its destabilization and pore formation (37,(53)(54)(55). Our observation that the poly(A) stretch already exits upon acidification adds a new dimension to the uncoating process; the 3= end of the RNA might be actively participating in the correct positioning of the viral particle onto the inner endosomal membrane, thereby initiating and directing its transfer.…”

Section: Discussionmentioning

confidence: 99%

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

Harutyunyan

Kowalski

Blaas

2014

J Virol

View full text Add to dashboard Cite

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.

show abstract

Recombinant VP4 of Human Rhinovirus Induces Permeability in Model Membranes

Cited by 48 publications

References 34 publications

Viroporins: structure, function and potential as antiviral targets

Viroporins: structure, function and potential as antiviral targets

Foot-and-Mouth Disease Virus Assembly: Processing of Recombinant Capsid Precursor by Exogenous Protease Induces Self-Assembly of Pentamers In Vitro in a Myristoylation-Dependent Manner

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

Contact Info

Product

Resources

About