Biophysical Studies on Rhinovirus and Poliovirus

McGregor, Sandy; Mayor, Heather D.

doi:10.1128/jvi.2.2.149-154.1968

Cited by 35 publications

(1 citation statement)

References 30 publications

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“…POAS nucleoprotein fibers and virus particles were visualized by electron microscopy after adsorbing the diluted sample onto glow-discharged grids and stained with 1% uranyl acetate. The RNase-resistant nature of POAS fibers was determined after incubating the heated PV sample for 1 h at 37°C in the presence of RNase A, as previously described (39,40). The grids were imaged at 80 kV in a JEOL 1200EX electron microscope equipped with an AMT 2k charge-coupled device (CCD) camera.…”

Section: Methodsmentioning

confidence: 99%

Real-Time Imaging of Polioviral RNA Translocation across a Membrane

Karunatilaka

Filman

Strauß

et al. 2021

mBio

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Genome transfer from a virus into a cell is a critical early step in viral replication. Enveloped viruses achieve the delivery of their genomes into the cytoplasm by merging the viral membrane with the cellular membrane via a conceptually simple mechanism called membrane fusion. In contrast, genome translocation mechanisms in nonenveloped viruses, which lack viral membranes, remain poorly understood. Although cellular assays provide useful information about cell entry and genome release, it is difficult to obtain detailed mechanistic insights due both to the inherent technical difficulties associated with direct visualization of these processes and to the prevalence of nonproductive events in cellular assays performed at a very high multiplicity of infection. To overcome these issues, we developed an in vitro single-particle fluorescence assay to characterize genome release from a nonenveloped virus (poliovirus) in real time using a tethered receptor-decorated liposome system. Our results suggest that poliovirus genome release is a complex process that consists of multiple rate-limiting steps. Interestingly, we found that the addition of exogenous wild-type capsid protein VP4, but not mutant VP4, enhanced the efficiency of genome translocation. These results, together with prior structural analysis, suggest that VP4 interacts with RNA directly and forms a protective, membrane-spanning channel during genome translocation. Furthermore, our data indicate that VP4 dynamically interacts with RNA, rather than forming a static tube for RNA translocation. This study provides new insights into poliovirus genome translocation and offers a cell-free assay that can be utilized broadly to investigate genome release processes in other nonenveloped viruses. IMPORTANCE The initial transfer of genomic material from a virus into a host cell is a key step in any viral infection. Consequently, understanding how viruses deliver their genomes into cells could reveal attractive therapeutic targets. Although conventional biochemical and cellular assays have provided useful information about cell entry, the mechanism used to deliver the viral genomes across the cellular membrane into the cytoplasm is not well characterized for nonenveloped viruses such as poliovirus. In this study, we developed a fluorescence imaging assay to visualize poliovirus genome release using a synthetic vesicle system. Our results not only provide new mechanistic insights into poliovirus genome translocation but also offer a cell-free assay to bridge gaps in understanding of this process in other nonenveloped viruses.

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Section: Methodsmentioning

confidence: 99%

Real-Time Imaging of Polioviral RNA Translocation across a Membrane

Karunatilaka

Filman

Strauß

et al. 2021

mBio

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Capillary Electrophoresis, Gas-Phase Electrophoretic Mobility Molecular Analysis, and Electron Microscopy: Effective Tools for Quality Assessment and Basic Rhinovirus Research

Weiss

Subirats

Kumar

et al. 2014

Methods in Molecular Biology

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We describe standard methods for propagation, purification, quality control, and physicochemical characterization of human rhinoviruses, using HRV-A2 as an example. Virus is propagated in HeLa-OHIO cells grown in suspension culture and purified via sucrose density gradient centrifugation. Purity and homogeneity of the preparations are assessed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis (CE), gas-phase electrophoretic mobility molecular analysis (GEMMA), and electron microscopy (EM). We also briefly describe usage of these methods for the characterization of subviral particles as well as for the analysis of their complexes with antibodies and soluble recombinant receptor mimics.

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The Structure and Replication of Rhinoviruses

Macnaughton¹

1982

Current Topics in Microbiology and Immunology

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Biophysical Studies on Rhinovirus and Poliovirus

Cited by 35 publications

References 30 publications

Real-Time Imaging of Polioviral RNA Translocation across a Membrane

Real-Time Imaging of Polioviral RNA Translocation across a Membrane

Capillary Electrophoresis, Gas-Phase Electrophoretic Mobility Molecular Analysis, and Electron Microscopy: Effective Tools for Quality Assessment and Basic Rhinovirus Research

The Structure and Replication of Rhinoviruses

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