Editing CCMV VLP (section)

== E coli Expression ==

Chemically competent cells of E. coli strain Rosetta 2(DE3)pLysS®(Novagen, Germany) were transformed with the T7::9×HisTag-PPSsite-CPC CMV construct for protein induction and expression. This strain is resistant to chloramphenicol and is designed to enhance protein expression using rare codons not normally used in a regular E. coli strain. The transformed cells were grown in LB solid medium supplemented with carbenicillin(100 mg ml−1) and chloramphenicol (34 mg ml−1). One colony was selected and a pre-inoculum was prepared in 100 ml of LB medium supplemented with carbenicillin (100 mg ml−1) and chloramphenicol (34 mg ml−1). The culture was grown at 37◦C for 16 h with constant stirring. This pre-inoculum was seeded into 1 l of liquidLB medium, supplemented with carbenicillin and chlorampheni-col, at the same concentration as before, and grown at 37◦C with constant agitation and up to an optical density of 0.6 absorbance units. Recombinant expression of the CCMV CP was induced after the addition of IPTG, ata final concentration of 0.5 mM and incubation for 16 h at 20 ◦C with constant stirring. Induced cells were collected by centrifugation at 15,000 × g for 20 min at 4◦C. The bacteria pellet was storedat −80◦C.

Chromatography

The pellet was re-suspended in 60 ml of lysis buffer (100 mMTris–HCl, 500 mM NaCl, pH 6.0). Lysozyme was added to a finalconcentration of 1 mg ml−1and incubated for 1 h at 4◦C with con-stant stirring. The solution was frozen and thawed quickly in fourconsecutive cycles by immersion in dry ice with ethanol and thenin water at 37◦C. Phenylmethylsulfonyl fluoride (PMSF) was thenadded to a final concentration of 1 mM. The solution was soni-cated for 10 min, using 20-s pulses, at an intensity of 70% (SonicsVibra-Cell model VCX130). Centrifugation at 13,000 × g was donefor 30 min at 4◦C. The pellet was discarded and the soluble fraction was filtered through a 45 um membrane and further loadedinto a Pierce®cobalt affinity column (HisPurTMCobalt Chromatog-raphy Cartridge) previously equilibrated with 10 volumes of lysis buffer. Consecutive washes were made using three different buffers varying the imidazole concentration (100 mM Tris–HCl, 500 mMNaCl and L1 = 15 mM imidazol, pH 7.0; L2 = 20 mM imidazol pH 7.2; L3 = 35 mM imidazol, pH 7.3). Each wash was done with 20 volumes of the cobalt affinity column. Recombinant CP was eluted usingelution buffer (100 mM Tris–HCl, 500 Mm NaCl, 500 mM imidazol,pH 8.0) in 1.5 ml fractions. The eluates containing the recombinant protein were dialyzed for 24 h at 4◦C in 1 l of dialysis buffer(100 mM Tris–HCl, 500 mM NaCl, 0.1 mM PMSF, pH 7.5). In order to effectively retain the CP, a dialysis membrane of 6–8 kDa MWCO (Spectrum Labs, CA, USA) was used. The dialyzed protein was stored at 4◦C before use.



Chemical labelling of CCMV

Sulfo-Cy5 NHS ester(Lumiprobe) was conjugated to CCMV through NHS chemistry to the exterior surface lysines. The reaction was performed using a 100 molar excess of dye with CCMV in 0.1 M HEPES pH 7.0, 5 mM MgCl2 buffer containing 10% (v/v) DMSO. The reaction was allowed to proceed overnight at room temperature with gentle agitation. The reaction was purified with ultracentrifugation at 150,000 x g for 1 hourover a 30% (w/v) sucrose cushion.The peptide m-lycotoxin was conjugated to CCMV via an SM(PEG)4 crosslinker (Thermo Scientific) through NHS chemistry to the exterior surfacelysines. The reaction was performed using a 600 molar excess of SM(PEG)4 with CCMV in 0.1 M HEPES pH 7.0, 5 mM MgCl2 buffer for 2 hours at room temperature with gentle agitation. Excess SM(PEG)4was removed via centrifugation through a PD MidiTrap G-25 Sample Preparation Column (GE Healthcare). M-lycotoxin was reacted to CCMV-SM(PEG)4at 600 molar excess with gentle agitation over night at room temperature. The reaction was purified with ultracentrifugation at 150,000 x g for 1 hourover a 30% (w/v) sucrose cushion.Gel Electrophoresis For denaturing gel electrophoresis, samples were denatured by heating at 100ºC for 10 minutes in NuPage 4x LDS Sample loading buffer (Thermo Scientific). CCMV (10 μg) were loaded on 12% NuPage Bis-Tris protein gels (Thermo Scientific) and run in 1x MOPs buffer at 200V for 35 minutes. Gels were stained with Coomassie Blue. For native gel electrophoresis, 10 μg of sample was loaded into 0.8% (w/v) TAE agarose gels with 1X GelRed (Biotium) in 1x TAE buffer and run at 90V for 40 minutes. All gels were imaged on anAlphaImager HP(Protein Simple) and analyzed with Fiji.Cell culture HeLa and MCF-7 cells were obtained from the ATCC (Manassas,VA).HeLa/GFP cells were obtained from Cell Biolabs, Inc(San Diego, CA). Cells were grown and maintained in Dulbecco’s Modified Eagle’s medium (DMEM, Cellgro) supplemented with 10% (v/v) fetal bovine serum (Atlanta Biologicals) and 1% (v/v) penicillin/streptomycin (Gibco). Cells were grown at 37ºCin a 5% CO2 humidified incubator.Flow cytometryHeLacells were collected using enzyme-free Hank’s cell dissociation buffer (Gibco) and resuspended to 2.5 x 106cells mL-1. Cells (5 x 105cells in 0.2 mL) were added to 96-well V-bottom plates (Corning 3897). CCMV particles (1 x 105particles per cell) were added in triplicate and incubated for 6 hours at 37ºCin a 5% CO2humidified incubator. Following incubation,half of the cells were centrifuged at 500 x g andresuspended in DPBS with 1 mg mL-1pronase (Sigma-Aldrich) and treated at room temperature for 15 minutes. All cells were thencentrifuged at 500 x g and washed with FACS buffer (1mM EDTA, 1% (v/v)



Recombinant production and purification of CCMV coat proteins in E. coliThe CCMV coat protein (573bp) was cloned into the vector pET28a(+) (Novagen) via NdeI and BamHI. The pET281/CCMV-CP construct was transformed into the E. colistrain ClearColi BL21(DE3) (Lucigen). 2 mL of an overnight culture was transferred to 400 mL of LB-Miller broth with 50 mg L-1kanamycin and grown at 37ºC until OD600 reached 0.6-0.8. Protein expression was induced using 0.5 mM IPTG and the culture was allowed to grow at 22ºC for 16 hours. The cultures were then placed on ice for 10 minutes and cells were harvested by centrifugation at 15,000 x g for 20 min at 4ºC. The supernatantwas discarded and the pellet was resuspended in 20 mL bacteria lysis buffer (GoldBio) and incubated on ice for 5 minutes. Lysozyme was then added at a final concentration of 1 mg mL-1and the cell suspension was incubated at 37ºC for 1 hour. PMSF was thenadded at a final concentration at 1 mM and the solution was sonicated on a Q500 Sonicator (QSonic) for 15 minutes using 5 second pulses at an intensity of 40%. The solution was centrifuged at 15,000 x g for 30 min at 4ºC and the supernatant was passed through a 0.45 μm filter. The cell suspension was loaded through a HisPur Cobalt Chromatography Cartridge(Thermo Scientific) and His-tagged CCMV coat proteins were collected through affinity purification as per manufacturer’s protocol. 1.5 mL elutions were collected from the column using elution buffer (50mM sodium phosphate, 300mM sodium chloride, 250mM imidazole; pH 7.4). Elution fractions were measured using UV-visible spectroscopy to verify that proteins were present in the fractions, and fractions werepooled accordingly. Pooled fractions of CCMV coat proteins were then dialyzed in protein buffer (1 M NaCl, 20 mM Tris pH 7.2, 1 mM EDTA, 1 mM DTT, 1 mM PMSF) for 24 hoursat4ºC using a 3.5K MWCOSlide-a-Lyzer dialysis cassette (Thermo Scientific). Dialyzed coat proteins were stored at 4ºC.

Reassembly of CCMV

For reassembly, coat proteins subunits and the desired dicer substrate siRNA (IDT) were mixed in a 6:1 (w/w) ratio in protein buffer. The mixture was dialyzed in a 7K MWCO Slide-a-Lyzerdialysis cassette(Thermo Scientific)against RNA assembly buffer (50 mM TrispH 7.2, 50 mM NaCl, 10 mM KCl, 5 mM MgCl2, 1 mM DTT) for at least 6 hours at 4ºC, thenimmediatelydialyzed against virus suspension buffer (50 mM sodium acetate buffer pH 4.5, 8 mM magnesium acetate) for at least 6 hours at 4ºC. The assembly was purifiedbycentrifugation through a 100k Amicon Ultra-0.5 mLcentrifugal filter (EMD Millipore) at 3000 x g for 5 minutes, followed by 3 washes with virus suspension buffer



Disassembly CCMV particles to obtain coat proteins

To disassemble CCMV to get coat proteins, virions were dialyzed using a 3.5K MWCO Slide-a-Lyzer dialysis cassette (Thermo Scientific) in disassembly buffer (0.5 M CaCl2, 50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF) at 4ºCfor 24 hours.Following dialysis, the solution was centrifuged at 12,000 x g for 30 minutes at 4ºC to pellet the viral RNA. The supernatant was then centrifuged at 220,000 x g for 2 hours at 4ºCto pellet any non-disassociated virus particles. The supernatant containing coat proteins was then dialyzed using a 3.5K MWCO Slide-a-Lyzer dialysis cassette in protein buffer (1 M NaCl, 20 mM Tris pH 7.2, 1 mM EDTA, 1 mM DTT, 1 mM PMSF) for 24 hours and stored at 4ºC.The concentration of the CCMV coat proteinswas determined at A280 and ε = 1.27 μl μg−1cm−1.&nbsp;



CCMV VLPs self-assembly dependence on pHIn order to investigate the pH dependence of the CCMV CP self-assembly process, two protein samples incubated in buffer withdifferent pH values were compared. In each case, 3 ml of CP at aconcentration of 0.5 mg ml−1were incubated in assembly buffer(pH 4.8) or dialysis buffer (pH 7.5) for 24 h at 4◦C. Each of thesamples was passed through a syringe filter with a 25 nm poremembrane (Millipore). The sample fractions that passed throughthe filter were collected in order to detect the presence of proteinby SDS gel electrophoresis at 12%.

1, N-His tag does not affect VLP formation 2. Induction at 20 oC o/n with 0.5 M IPTG 3, Wash column with 15, 20 and 35 mM I before eluting with 500 mM I 4, 25 mg/L yield.

VLP Disassembly Buffer

50 mM Tris–HCl (pH 7.5), 500 mM CaCl2, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), 0.5 mM PMSF (seeNote 5). Add DTT and PMSF directly before use

=== Assembly Buffer I ===

50 mM Tris–HCl (pH 7.2), 1 M NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF (seeNote 5). Add DTT and PMSF directly before use

=== <br/>Assembly Buffer II ===

50 mM Tris–HCl pH 7.2, 50 mM NaCl, 10 mM KCl, 5 mM MgCl2, 1 mM DTT. Add DTT directly before use

=== Assembly Buffer III ===

50 mM sodium acetate, 8 mM magne-sium acetate (pH 4.8)

Sterile 6–8 kDa MWCO dialysis tubing