Characterization of rhinovirus subviral <scp>A</scp> particles via capillary electrophoresis, electron microscopy and gas‐phase electrophoretic mobility molecular analysis: Part I

Weiss, Victor U.; Subirats, Xavier; Pickl‐Herk, Angela; Bilek, Gerhard; Winkler, Wolfgang; Kumar, Mohit; Allmaier, Günter; Blaas, Dieter; Kenndler, Ernst

doi:10.1002/elps.201100647

Cited by 23 publications

(39 citation statements)

References 36 publications

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“…2, upper left panels). Rather, the particles continued to migrate identically to the previously characterized intermediate particles (27,40) and differently from native virus (N). We thus reasoned that the exposed RNA segments might not fold into double strands long enough to be recognized by MAb J2.…”

Section: Resultsmentioning

confidence: 75%

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

Harutyunyan

Kowalski

Blaas

2014

J Virol

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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.

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Section: Resultsmentioning

confidence: 75%

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

Harutyunyan

Kowalski

Blaas

2014

J Virol

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“…1B) contained similar proportions of empty particles. Negative stain EM and capillary electrophoresis (18) confirmed that no native virus remained in the acidified sample. The four types of particles (full and empty without and with acidification) were selected manually and a 3D reconstruction (3DR) was obtained for each set (Fig.…”

Section: Resultsmentioning

confidence: 87%

“…This suggests that acidification transformed native HRV2 into the A-particle stage and NEC into B-particles. Conversion of A-(and native HRV2) into B-particles at low pH is thus very inefficient in vitro, indicating that low pH does not suffice for productive uncoating (18). This process might be catalyzed in vivo by insertion of the amphipathic exposed N-terminal VP1 helices into membranes and/or by a specific ionic milieu (30,31) (Fig.…”

Section: Discussionmentioning

confidence: 99%

Uncoating of common cold virus is preceded by RNA switching as determined by X-ray and cryo-EM analyses of the subviral A-particle

Pickl‐Herk

Luque

Vives-Adrián

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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During infection, viruses undergo conformational changes that lead to delivery of their genome into host cytosol. In human rhinovirus A2, this conversion is triggered by exposure to acid pH in the endosome. The first subviral intermediate, the A-particle, is expanded and has lost the internal viral protein 4 (VP4), but retains its RNA genome. The nucleic acid is subsequently released, presumably through one of the large pores that open at the icosahedral twofold axes, and is transferred along a conduit in the endosomal membrane; the remaining empty capsids, termed B-particles, are shuttled to lysosomes for degradation. Previous structural analyses revealed important differences between the native protein shell and the empty capsid. Nonetheless, little is known of A-particle architecture or conformation of the RNA core. Using 3D cryo-electron microscopy and X-ray crystallography, we found notable changes in RNAprotein contacts during conversion of native virus into the A-particle uncoating intermediate. In the native virion, we confirmed interaction of nucleotide(s) with Trp 38 of VP2 and identified additional contacts with the VP1 N terminus. Study of A-particle structure showed that the VP2 contact is maintained, that VP1 interactions are lost after exit of the VP1 N-terminal extension, and that the RNA also interacts with residues of the VP3 N terminus at the fivefold axis. These associations lead to formation of a well-ordered RNA layer beneath the protein shell, suggesting that these interactions guide ordered RNA egress.genome uncoating | X-ray analysis | 3D cryo-EM | picornavirus H uman rhinoviruses (HRVs) cause the common cold. Although seldom severe, this disease is widespread and frequent in man; HRVs thus have considerable economic impact due to expenditure on medication and lost working days. More than 150 serotypes belong to the genus Enteroviruses (EVs) of the Picornaviridae family, which includes serious human and animal pathogens. In addition to phylogenetic classification into species A, -B, and -C, HRVs are divided into a minor receptor group (12 HRV-A) that bind low-density lipoprotein receptors (LDLRs), and a major receptor group (more than 89 HRV-A and -B serotypes) that use intercellular adhesion molecule 1 (ICAM-1) for cell entry (1). HRV-C binds an unknown receptor (2).The EV icosahedral shell is built from four viral proteins (VP1-4) that encase a single-stranded (+)-sense RNA genome. Sixty copies each of these four polypeptides assemble on a T = 1 (pseudo T = 3) lattice, ∼30 nm in diameter. VP1, VP2, and VP3 are surface-exposed; the small myristoylated VP4 is internal. In the mature virion, the N-terminal extensions of VP1, VP2, and VP3, together with the entire VP4, interact in an intricate network beneath the shell (Fig. S1) (3, 4).In the cytosol, the viral RNA is translated into a ∼250 kDa precursor polyprotein that is processed by viral proteinases. Assembly of the viral shell involves immature pentamers built from VP0, VP1, and VP3. VP2 and VP4 arise late in infection through VP0 cl...

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“…Native virus, A- and (empty) B-particles were identified according to their electrophoretic mobility as described previously [36]. As seen in Fig.…”

Section: Resultsmentioning

confidence: 99%

Viral Uncoating Is Directional: Exit of the Genomic RNA in a Common Cold Virus Starts with the Poly-(A) Tail at the 3′-End

et al. 2013

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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 viruses.

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Characterization of rhinovirus subviral A particles via capillary electrophoresis, electron microscopy and gas‐phase electrophoretic mobility molecular analysis: Part I

Cited by 23 publications

References 36 publications

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

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

Uncoating of common cold virus is preceded by RNA switching as determined by X-ray and cryo-EM analyses of the subviral A-particle

Viral Uncoating Is Directional: Exit of the Genomic RNA in a Common Cold Virus Starts with the Poly-(A) Tail at the 3′-End

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