Non-human papilloma viruses selected for this study.

<p>Animal papilloma viruses that were produced as VLPs and PsVs.</p

Effect of ι-carrageenan on transduction with PcPV1 and MfPV11.

<p>(A) Different doses of ι-carrageenan were added to the cell culture medium immediately before transduction of HEK293TT cells with PcPV1 PsVs carrying a G.Luc reporter plasmid. Luciferase assay was performed 72h after transduction. Shown are mean values and standard deviation of three independent PcPV1 PsV preparations. Statistical analysis was performed by 2way ANOVA and Tukey’s multiple comparison test. (B) Additionally, HEK293TT cells were transduced with PcPV1 and MfPV11 PsVs carrying a pEGFP reporter plasmid with (+) and without (-) addition of 10μg/ml ι-carrageenan. GFP-positive cells were counted 72h after transduction and transducing units were calculated. Each data point represents one experiment with one PsV preparation. Dashed line indicates limit of detection. Statistical analysis was performed using t-test.</p

Transmission electron microscopy of HPV16, PcPV1 and MfPV11 VLPs.

<p>Purified VLPs were fixed for 24h at room temperature with formaldehyde, contrasted with phosphotungstic acid and analyzed by transmission electron microscopy.</p

Titration of PsVs by transduction of HEK293TT cells.

<p>(A) HEK293TT cells were transduced with the indicated PsVs carrying pEGFP as reporter plasmid. 72h after transduction, GFP-positive cells were counted and titer was calculated as transducing units per ml PsVs. Each data point represents one PsV preparation. (B) To compare the amount of transducing units with the amount of particles carrying the pEGFP reporter plasmid, plasmid DNA was isolated from PsVs and quantified by qPCR to obtain the pEGFP copy number in the sample.</p

Bioluminescence imaging after intramuscular application.

<p>50μl of the indicated PsV preparation were injected into the left thigh muscle. Bioluminescent imaging was performed approx. 3h after injection (day 0) to test for any free F.Luc and subsequently in a weekly manner.</p

Western blot analysis of purified HAs treated/untreated with PNGase F.

<p>Purified HAs from leaves were deglycosylated using the commercial PNGase F enzyme described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099347#s2" target="_blank">Materials and Methods</a> section. PNGase F-treated and untreated proteins were then separated in 10% SDS-PAGE. Recombinant proteins were detected using an anti-c-myc monoclonal antibody. “−” and “+” indicate PNGase F-untreated and treated samples, respectively.</p

Influence of hydrophobin and elastin-like polypeptide on hemagglutinin accumulation in transgenic plants under the control of either the CaMV 35S promoter (A) or the seed-specific USP promoter (B).

<p>Each column shows the mean value of expression level of the transgenic plants, and the error bars indicate the standard deviation. TSP: total soluble protein.</p

Immunofluorescence analysis of recombinant HAs in plant leaves.

<p>Leaves were fixed, embedded in PEG and sectioned. Recombinant HAs were immunodecorated with an anti-c-myc monoclonal antibody followed by incubation with secondary antibody (anti-mouse-IgG conjugated with AlexaFluor488) and counterstaining with DAPI. A. H5; B. H5-HFBI; C. H5-ELP; D. wild type. Bars represent 50 µm.</p

Expression cassettes for hemagglutinin (HA) in plants.

<p>HAs were stably expressed in both leaves and seeds under the control of the CaMV 35S and the seed-specific promoters as the naked form (H5), hydrophobin I fusion protein (H5-HFBI) and ELPylated H5 (H5-ELP). All recombinant HAs contained His and c-myc tags for affinity chromatography purification and Western blotting, respectively. The LeB4 signal peptide and KDEL motif were used to ensure ER retention.</p

Membrane Condensation and Curvature Induced by SARS-CoV‑2 Envelope Protein

The envelope (E) protein of SARS-CoV-2 participates in virion encapsulation and budding at the membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC). The positively curved membrane topology required to fit an 80 nm viral particle is energetically unfavorable; therefore, viral proteins must facilitate ERGIC membrane curvature alteration. To study the possible role of the E protein in this mechanism, we examined the structural modification of the host lipid membrane by the SARS-CoV-2 E protein using synchrotron-based X-ray methods. Our reflectometry results on solid-supported planar bilayers show that E protein markedly condenses the surrounding lipid bilayer. For vesicles, this condensation effect differs between the two leaflets such that the membrane becomes asymmetric and increases its curvature. The formation of such a curved and condensed membrane is consistent with the requirements to stably encapsulate a viral core and supports a role for E protein in budding during SARS-CoV-2 virion assembly