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

Subirats, Xavier; Weiss, Victor U.; Gösler, Irene; Puls, Christoph; Limbeck, Andreas; Allmaier, Günter; Kenndler, Ernst

doi:10.1002/elps.201200686

Cited by 7 publications

(9 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This stretch corresponds to roughly 10% of the total RNA, a number similar to an earlier estimate of ca. 7%, based on the UV absorbance ratios at 200 and 260 nm of the peaks of native virus and RNase-treated intermediate-particles in capillary electrophoresis (51).…”

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

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

show abstract

“…Addition of divalent cations (e.g. Mg 2+ ) known to stabilize the MB stem was omitted as these had been found to stabilize viral uncoating intermediates [ 9 ].…”

Section: Resultsmentioning

confidence: 99%

“…In vitro, the alteration of HRV-A2 can either be triggered by acidification (pH ≤5.8), which mimics the in vivo situation in endosomes, or by heating (≥10 min to ≥56 °C) [ 7 ]. Depending on the concentration of divalent cations, subviral A particles or empty viral capsids are formed [ 8 , 9 ]. Several orthogonal analytical methods like sucrose density centrifugation (e.g.…”

Section: Introductionmentioning

confidence: 99%

In vitro RNA release from a human rhinovirus monitored by means of a molecular beacon and chip electrophoresis

et al. 2016

Self Cite

View full text Add to dashboard Cite

Liquid-phase electrophoresis either in the classical capillary format or miniaturized (chip CE) is a valuable tool for quality control of virus preparations and for targeting questions related to conformational changes of viruses during infection. We present an in vitro assay to follow the release of the RNA genome from a human rhinovirus (common cold virus) by using a molecular beacon (MB) and chip CE. The MB, a probe that becomes fluorescent upon hybridization to a complementary sequence, was designed to bind close to the 3′ end of the viral genome. Addition of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a well-known additive for reduction of bleaching and blinking of fluorophores in fluorescence microscopy, to the background electrolyte increased the sensitivity of our chip CE set-up. Hence, a fast, sensitive and straightforward method for the detection of viral RNA is introduced. Additionally, challenges of our assay will be discussed. In particular, we found that (i) desalting of virus preparations prior to analysis increased the recorded signal and (ii) the MB–RNA complex signal decreased with the time of virus storage at −70 °C. This suggests that 3′-proximal sequences of the viral RNA, if not the whole genome, underwent degradation during storage and/or freezing and thawing. In summary, we demonstrate, for two independent virus batches, that chip electrophoresis can be used to monitor MB hybridization to RNA released upon incubation of the native virus at 56 °C.Graphical AbstractSchematic of the study strategy: RNA released from HRV-A2 is detected by chip electrophoresis through the increase in fluorescence after genom complexation to a cognate molecular beaconElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-016-9459-2) contains supplementary material, which is available to authorized users.

show abstract

“…The use of the surfactant SDS as BGE additive allows to overcome the common troubles of irreproducibility encountered in the CZE analyses of non‐enveloped icosahedral viruses . Although BGE additives may not be necessary in some cases , when the presence of SDS is required, it becomes problematic to understand the behavior of non‐enveloped icosahedral viruses since the way by which SDS interacts with the surface of such viruses is poorly known.…”

Section: Discussionmentioning

confidence: 99%

“…Consequently, non‐enveloped icosahedral viruses possess a global negative surface charge at pH higher than the isoelectric point contributing to proffer them an electrophoretic mobility . Exploiting this characteristic, it has been shown that capillary zone electrophoresis (CZE), a separative analytical technique based on the difference of the electrophoretic mobility of the analytes , can be applied in the separation, detection and quantification of viral, subviral and virus‐like particles in aqueous media . However, in practice, the CZE analysis of viruses is harder than of molecules, where uncontrolled viral particles aggregation and capillary wall adsorption responsible of irreproducible results having been often encountered .…”

Section: Introductionmentioning

confidence: 99%

MS2 and Qβ bacteriophages reveal the contribution of surface hydrophobicity on the mobility of non‐enveloped icosahedral viruses in SDS‐based capillary zone electrophoresis

et al. 2017

View full text Add to dashboard Cite

SDS is commonly employed as BGE additive in CZE analysis of non-enveloped icosahedral viruses. But the way by which SDS interacts with the surface of such viruses remains to date poorly known, making complicate to understand their behavior during a run. In this article, two related bacteriophages, MS2 and Qβ, are used as model to investigate the migration mechanism of non-enveloped icosahedral viruses in SDS-based CZE. Both phages are characterized by similar size and surface charge but significantly different surface hydrophobicity (Qβ > MS2, where '>' means 'more hydrophobic than'). By comparing their electrophoretic mobility in the presence or not of SDS on both sides of the CMC, we show that surface hydrophobicity of phages is a key factor influencing their mobility and that SDS-virus association is driven by hydrophobic interactions at the surface of virions. The CZE analyses of heated MS2 particles, which over-express hydrophobic domains at their surface, confirm this finding. The correlations between the present results and others from the literature suggest that the proposed mechanism might not be exclusive to the bacteriophages examined here.

show abstract

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

Cited by 7 publications

References 35 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

In vitro RNA release from a human rhinovirus monitored by means of a molecular beacon and chip electrophoresis

MS2 and Qβ bacteriophages reveal the contribution of surface hydrophobicity on the mobility of non‐enveloped icosahedral viruses in SDS‐based capillary zone electrophoresis

Contact Info

Product

Resources

About