Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Feb 6;13(2):e1006197.
doi: 10.1371/journal.ppat.1006197. eCollection 2017 Feb.

Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes

Elisabetta Groppelli  1 , Hazel C Levy  2 , Eileen Sun  3 , Mike Strauss  2 , Clare Nicol  1 , Sarah Gold  4 , Xiaowei Zhuang  5 , Tobias J Tuthill  4 , James M Hogle  2 , David J Rowlands  1
Affiliations

Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes

Elisabetta Groppelli et al. PLoS Pathog. .

Abstract

Picornaviruses are non-enveloped RNA viruses that enter cells via receptor-mediated endocytosis. Because they lack an envelope, picornaviruses face the challenge of delivering their RNA genomes across the membrane of the endocytic vesicle into the cytoplasm to initiate infection. Currently, the mechanism of genome release and translocation across membranes remains poorly understood. Within the enterovirus genus, poliovirus, rhinovirus 2, and rhinovirus 16 have been proposed to release their genomes across intact endosomal membranes through virally induced pores, whereas one study has proposed that rhinovirus 14 releases its RNA following disruption of endosomal membranes. For the more distantly related aphthovirus genus (e.g. foot-and-mouth disease viruses and equine rhinitis A virus) acidification of endosomes results in the disassembly of the virion into pentamers and in the release of the viral RNA into the lumen of the endosome, but no details have been elucidated as how the RNA crosses the vesicle membrane. However, more recent studies suggest aphthovirus RNA is released from intact particles and the dissociation to pentamers may be a late event. In this study we have investigated the RNase A sensitivity of genome translocation of poliovirus using a receptor-decorated-liposome model and the sensitivity of infection of poliovirus and equine-rhinitis A virus to co-internalized RNase A. We show that poliovirus genome translocation is insensitive to RNase A and results in little or no release into the medium in the liposome model. We also show that infectivity is not reduced by co-internalized RNase A for poliovirus and equine rhinitis A virus. Additionally, we show that all poliovirus genomes that are internalized into cells, not just those resulting in infection, are protected from RNase A. These results support a finely coordinated, directional model of viral RNA delivery that involves viral proteins and cellular membranes.

PubMed Disclaimer

Conflict of interest statement

I have read the journal's policy and the authors of this manuscript have the following competing interests: Eileen Sun now works for Aspyrian Therapeutics.

Figures

Fig 1
Fig 1. Section through a subtomograms from a cryoelectron tomographic reconstruction of a warmed virus-receptor- liposome complex showing RNA being translocated across the liposome membrane.
The samples were produced by heating virus-receptor-liposome complexes at 37°C for 4 min, mixed with colloidal gold, placed on carbon-coated Quantifoil holey grids and flash frozen, and cryo tomographic data were acquired and processed as in [22] The central section through a representative subtomogram containing a single complex from this data set is presented to summarise the path of the viral RNA from the interior of the virus, across the liposome membrane, and into the lumen of the liposomes during uncoating. The left panel shows a section through raw averaged subtomogram showing a virus particle (center) attached to a liposome (bottom right), with density for the RNA clearly extending from the middle of the particle across the membrane and into the lumen of the liposome. The bright feature to the left of the virus is a colloidal gold particle. The right panel shows the same section of the tomogram segmented to highlight the virus capsid (light-blue), the membrane bilayer (pink), and the RNA (gold). The scale bar in both panels is 25 nm.
Fig 2
Fig 2. Receptor-decorated liposomes containing fluorescent dye detect PV RNA release.
A) Representative images of YoPro-1 encapsulating receptor-decorated liposomes (YRDLs) complexed with PV in the presence or absence of RNase A (50 μg/ml). Note that RNase A was added to the extra-liposomal space after PV-YRDL complexes were formed, but prior to heating the samples for 20 min at 37°C. Images were collected at room temperature using a 20X objective as described in Materials and Methods. Scale bars are 200 μm. B) Normalized histograms showing the number of pixels (y-axis) with a given level of fluorescence (in arbitrary units) (x-axis) of PV-YRDL complexes shown in A in the absence (green curve) and presence (black curve) of RNase A. (C and D) Representative images of PV RNA (in the absence of liposomes) in the presence of YoPro-1 dye following induction of uncoating by sPVR at 37°C (C) or by heating at 52°C (D) for 20 min in the presence or absence of RNase A (50 μg/ml). Images were collected using a 100X objective as described in Materials and Methods. Scale bars are 40 μm. E) Representative still frames from a time lapse of PV-YRDLs gradually heated from room temperature to 42°C. Average time lapse for averaged image is indicated. After 15 min of imaging a single field of view, a second region of interest was imaged in order to evaluate the influence of photobleaching on the fluorescence intensity (second ROI at 20 min shown on the right-side panel). Images were captured at 100x magnification using a custom built Total Internal Reflectance Fluorescence Microscopy (TIR-FM) setup, attached to an Olympus IX-71 microscope, as described in Material and Methods. Scale bars are 5 μm. F) PV-YRDLs integrated fluorescence intensity obtained as indicated in Materials and Methods (expressed as fold change of T = 1 min, left y-axis) during a 20 min time course (time in min along the x-axis) when the sample was heated from room temperature to 42°C. The temperature of the lens (right y-axis) is shown as a function of time (grey dashed line). Because the objective lens and the sample are 1.18 mm apart (with oil connecting the lens to the sample slide), the temperature of the lens is used to estimate the temperature of the sample. 42°C is the upper limit of the imaging apparatus. The black triangle shows the integrated fluorescence intensity of a region of interest that was imaged at a single time point of 20 min in order to assess photobleaching. G) Representative images of YoPro-1 encapsulating RDL using the same microscope setup described for E. YRDLs were incubated at 37°C for 10 min, alone with no PV (left), or were pre-incubated with PV at room temperature for 10 min to allow complex formation, and then incubated 37°C for 10 min (right). Scale bars are 5 μm.
Fig 3
Fig 3. PV infectivity and RNA integrity are not affected by the presence of high levels of RNase A during the infection process.
A) Representative image of HeLa Ohio cells infected with PV-Cy2 (green) in the presence of Dextrans-10 kDa conjugated to Alexa-594 (red) fixed 15 min post-infection. The degree of co-internalization (right-hand side panel) was measured on 10 random cells, R = 0.89 +/- 0.09 (SD). Scale bar is 5 μm. B) Plaque assay of PV in the presence of 0–1 mg/ml RNase A. Plaque forming units were expressed as percentage of no RNase A control. C) Scintillation counting of internalized vs unattached 3H-U-PV in HeLa Ohio cells in the presence of RNase A (1 mg/ml) or PBS carrier control. D) Scintillation counting of recovered and flow-through samples after a column-based RNA purification procedure of 3H-U-PV RNA internalized into HeLa Ohio cells in the presence of RNase A (1 mg/ml) or PBS carrier control. E) Scintillation counting of sucrose density gradient (15–30% sucrose, 0.1% SDS, 0.1 M Na acetate. Fraction 1 = top, 15% sucrose) of 3H-U-PV RNA recovered from HeLa Ohio cells 30 min post-infection in the presence or absence of 1 mg/ml RNase A (PV+HeLa+A, red line, and PV+HeLa, blue line, respectively). Data is expressed as percentage of the total counts per minutes (cpm) loaded onto the gradient. All data are from three independent experiments and error bars show standard error.
Fig 4
Fig 4. PV infectivity is not affected by covalent linkage of RNase A to the virus.
A) RNase A is covalently linked to PV VP1. Conjugation reactions containing 35S-Met/Cys radiolabelled PV and/or RNase A (as indicated at the top of the image) were subjected to SDS-PAGE and western blot with antisera against RNase A. The major over-exposed bands correspond to RNase A monomer and dimer (indicated by arrows). Bands in the middle lane are the expected size for radiolabelled virus proteins VP1, VP2 and VP3. The upper band in the left hand lane is the expected size for RNase A covalently attached to VP1 (as indicated by arrow). Molecular weight standards (kDa) are shown on the left. B) Plaque assay of PV conjugated to RNase A (0, 90, 300, 600 molar ratio). Plaque forming units (pfu) were expressed as percentage of no RNase A control and data pooled from three independent experiments. C) Ribonuclease activity was measured by quantifying tRNA fluorescence (Relative Fluorescence Units, RFU) with Ribogreen in the presence of RNase A, purified PV, PV conjugated to RNase A with EDC (PV-A + EDC) and mock conjugation reaction (PV-A—EDC). D) Sucrose gradient profile of 3H-U-PV RNA (as in Fig 3E) uncoated in vitro at 50°C for 10 min in the presence of RNase A (1mg/ml) or PBS carrier control (representative of two independent experiments). E) Sucrose gradient profile (as in Fig 3E) of 3H-U-PV RNA from viral particles directly conjugated to RNase A with the cross-linker EDC (PV-A + EDC) or mock conjugated (no EDC, PV-A—EDC) uncoated in vitro at 50°C for 10 min (representative of two independent experiments). F) Representative image of HeLa Ohio cells infected with PV conjugated to Cy2 (green, left panel) and RNase A-DyLight594 (red, middle panel) fixed 15 min post infection. The degree of co-internalization (Merge, right panel) was measured for 10 random cells (R = 0.92 +/- 0.06 (SD). Nuclei were stained with Hoechst (blue). Scale bar is 5 μm. G) RNase activity (as in C) of individual and mixed components of the RNase S system. H) RNase activity (as in C and G) and I) virus titre (as in B) of PV conjugated to individual or mixed components of the RNase S system. (All data from three independent experiments with error bars showing standard error, unless stated).
Fig 5
Fig 5. ERAV is co-internalized with RNase A but infectivity is not compromised.
A) Representative images of HeLa Ohio cells infected with ERAV conjugated to Cy2 (green, left panel) and RNase A-DyLight594 (red, middle panel) fixed 15 min post infection. The degree of co-internalization (Merge, right panel) was measured for 10 random cells (R = 0.86 +/- 0.09 (SD). Nuclei were stained with Hoechst (blue). Scale bar is 5 μm. B) Plaque assay of ERAV in the presence of 0–1 mg/ml RNase A. Plaque forming units (pfu) were expressed as percentage of no RNase A control. Data are from three independent experiments with error bars showing standard error.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Tuthill TJ, Groppelli E, Hogle JM, Rowlands DJ. Picornaviruses. Curr Top Microbiol Immunol. 2010;343: 43–89. 10.1007/82_2010_37 - DOI - PMC - PubMed
    1. Chow M, Newman JF, Filman D, Hogle JM, Rowlands DJ, Brown F. Myristylation of picornavirus capsid protein VP4 and its structural significance. Nature. Nature Publishing Group; 1987;327: 482–486. - PubMed
    1. Hogle JM, Chow M, Filman DJ. Three-dimensional structure of poliovirus at 2.9 A resolution. Science. 1985;229: 1358–1365. - PubMed
    1. Hogle JM. Poliovirus cell entry: common structural themes in viral cell entry pathways. Annu Rev Microbiol. Annual Reviews 4139 El Camino Way, P.O. Box 10139, Palo Alto, CA 94303–0139, USA; 2002;56: 677–702. 10.1146/annurev.micro.56.012302.160757 - DOI - PMC - PubMed
    1. Mendelsohn CL, Wimmer E, Racaniello VR. Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily. Cell. 1989;56: 855–865. - PubMed

Publication types