CCMV VLP

CCMV CP sequence:

ATGTCTACAGTCGGAACAGGGAAGTTAACTCGTGCACAACGAAGGGCTGCGGCCCGTAAGAACAAGCGGAACACTCGTGTGGTCCAACCTGTTATTGTAGAACCCATCGCTTCAGGCCAAGGCAAGGCTATTAAAGCATGGACCGGTTACAGCGTATCGAAGTGGACCGCCTCTTGTGCGGCTGCCGAAGCTAAAGTAACCTCGGCTATAACTATCTCTCTCCCTAATGAGCTATCGTCCGAAAGGAACAAGCAGCTCAAGGTAGGTAGAGTTTTATTATGGCTTGGGTTGCTTCCCAGTGTTAGTGGCACAGTGAAATCCTGTGTTACAGAGACGCAGACTACTGCTGCTGCCTCCTTTCAGGTGGCATTAGCTGTGGCCGACAACTCGAAAGATGTTGTCGCTGCTATGTACCCCGAGGCGTTTAAGGGTATAACCCTTGAACAACTCGCCGCGGATTTAACGATCTACTTGTACAGCAGTGCGGCTCTCACTGAGGGCGACGTCATCGTGCATTTGGAGGTTGAGCATGTCAGACCTACGTTTGACGACTCTTTCACTCCGGTGTATTAG

MSTVGTGKLTRAQRRAAARKNKRNTRVVQPVIVEPIASGQGKAIKAWTGYSVSKWTASCAAAEAKVTSAITISLPNELSSERNKQLKVGRVLLWLGLLPSVSGTVKSCVTETQTTAAASFQVALAVADNSKDVVAAMYPEAFKGITLEQLAADLTIYLYSSAALTEGDVIVHLEVEHVRPTFDDSFTPVY

https://www.ncbi.nlm.nih.gov/nucleotide/AF325741.1

http://dx.doi.org/10.1016/j.jviromet.2015.08.023

Highly efficient strategy for the heterologous expression andpurification of soluble Cowpea chlorotic mottle virus capsid protein andin vitro pH-dependent assembly of virus-like particles http://dx.doi.org/10.1016/j.jviromet.2015.08.023

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. 

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

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

Method

The CP of CCMV is obtained from the virus by disrupting the qua-ternary structure of the capsid and then selectively precipitating the viral RNA. In the first step, CCMV is dialyzed against Disassembly Buffer where the neutral pH and high ionic strength result in disas-sembly of the capsid into noncovalent dimers of CP, and the high concentration of CaCl2 decreases the solubility of the viral RNA. In the second step, the disassembled CCMV is subjected to ultracentri-fugation which causes the viral RNA to precipitate from solution while the majority of the CP dimers remain in the supernatant. In the final step, the purified CP is dialyzed against Assembly Buffer I in preparation for reassembly with heterologous RNA.

1. Prepare a sealed dialysis bag containing 1 ml of 1 mg/ml puri-fied CCMV (seeNote 15).

2. Place the dialysis bag in a beaker containing 1 l of Disassembly Buffer at 4 °C. Add a magnetic stir bar to the beaker and stir gently on a stir plate at 4 °C overnight.

3. Remove the contents of the dialysis bag (i.e., disassembled CCMV in Disassembly Buffer) with a pipette and place in a sterile ultracentrifuge tube on ice (seeNote 16). Add addi-tional Disassembly Buffer to the sample until its total volume is 1.5 ml.

4. Prepare a balance tube by pipetting the appropriate weight (seeNote 17) of Disassembly Buffer into a separate, labeled ultra-centrifuge tube. Seal each tube with Parafilm to prevent evap-oration and spilling prior to centrifugation.

5. Prepare the centrifuge for a 100-min run at 100,000 × g at 4 °C.

6. Remove the Parafilm from each tube, load the tubes and rotor into the centrifuge, and immediately begin centrifugation.

7. Remove the sample tube immediately after centrifugation and quickly fractionate (seeNote 18) the sample solution from top to bottom into separate 300 μl volumes in labeled, sterile 1.5 ml tubes (seeNote 19)

8. Prepare five labeled, sealed, dialysis bags each containing one of the collected fractions (seeNote 15). Place each of the dial-ysis bags in a single beaker containing 1 l of Asssembly Buffer I at 4 °C. Add a magnetic stir bar to the beaker and stir gently on a stir plate at 4 °C overnight.

9. Collect 1 ml of the Assembly Buffer I dialysate for use in acquiring a blank UV-Vis spectrum in step 11.

10. Remove the contents of each dialysis bag (i.e., purified CP in Assembly Buffer I) with a pipette and place each sample in a separate labeled, sterile, 1.5 ml tube on ice.

11. Take UV-Vis spectra of each fraction using the aliquot of Assembly Buffer I dialysate from step 8 as a blank (seeNote 20).

12. Compare the ratio of the absorbance values at 280 nm (A280) and 260 nm (A260) to determine the purity of the CP in each fraction with respect to viral RNA contamination (see Fig. 2a). A280/A260 ratios above 1.5 correspond to less than 5% RNA contamination and are suitable for use in reassembly reactions.

13. Calculate the concentration of CP in suitably pure fractions using the formula (CP)mg/ml = (Abs280 nm*1197.7–24.032)/1000

VLPs are synthesized by dialyzing an aqueous mixture of CP and RNA against a series of buffer solutions that collectively pro-mote the coassembly of RNA-filled capsids (see Fig. 3). In the first step, a specific stoichiometry—6:1 by mass—of CP and RNA is mixed in Assembly buffer I where the neutral pH and high ionic strength disfavor intermolecular interaction. It has been shown [20, 26] that this particular stoichiometry leads to essentially complete encapsidation of any length of RNA by pro-viding electrostatic charge-matching between the cationic N-termini of the CP and the negatively charged phosphate back-bone of the RNA. In the second step, the mixture is dialyzed against Assembly Buffer II where the ionic strength is an order of magnitude lower and CP–RNA attraction is strong. These conditions lead to the formation of amorphous structures con-sisting of a single molecule of RNA decorated by a disordered arrangement of bound CP [27]. This intermediate stage is cru-cial for avoiding kinetically trapped aggregate structures. In the final step, the mixture is dialyzed against Assembly Buffer III where the acidic (4.8) pH strengthens the CP–CP attraction. Under these conditions well-formed capsids form with sizes that depend on the length of the RNA [20].3.2VLP SynthesisFig. 3 The two-step assembly process. We start with noninteracting CP and RNA in Assembly Buffer I (second from the left). We then turn on CP–RNA attractions by dialyzing against Assembly Buffer II, yielding amorphous CP–RNA complexes (second from the right). Finally, we turn on CP–CP attractions by dialyzing against Assembly Buffer III, yielding well-formed capsids (rightmost). However, if we instead dialyze the initial mixture of nonin-teracting CP and RNA directly against Assembly Buffer III, disordered aggregate structures form (leftmost)Cell-Free Synthesis of CCMV-VLPs Containing Heterologous RNAs

1. Mix 54 μg of purified CP with 9 μg of RNA in a total volume of 300 μl of Assembly Buffer I and place in a sealed dialysis bag (seeNote 15). The total volume and the absolute concentra-tions specified above can be scaled up by a factor of 2 or 3 (higher values have not been systematically tested), or scaled down by more than an order of magnitude, as long as the rela-tive concentration of CP and RNA remains constant at 6 to 1.

2. Place the dialysis bag in a beaker containing 1 l of Assembly Buffer II at 4 °C. Add a magnetic stir bar to the beaker and stir gently on a stir plate at 4 °C for at least 6 h.

3. Remove the dialysis bag from Assembly Buffer II and place it in a beaker containing 1 l of Assembly Buffer III at 4 °C. Add a magnetic stir bar to the beaker and stir gently on a stir plate at 4 °C overnight

CCMV CP is particularly prone to cleavage when it is not assembled as cap-sids: solutions of CCMV or CCMV VLPs can be stored for years at −80 °C (or −20 °C) prior to use, but purified CP should not be frozen and should be stored at 4 °C and used within 2–3 weeks. All portions of the protocol involving purified CP should be carried out on ice or at 4 °C. In certain steps of the protocol we add PMSF to inhibit proteases.

RNA to be encapsidated can be from any source as long as it is between 100 and 4000 nt in length and sufficiently pure. RNAs much shorter than 100 nt do not result in stable capsids (Comas-Garcia, personal communication), and RNAs much longer than 4000 nt are encapsidated by multiple cap-sids that are “strung” together by exposed RNA [20]. Common impurities associated with in vitro transcribed RNAs are left-over template DNA and free nucleotides—both must be completely removed. Free nucleotides are especially prob-lematic because they are difficult to detect and they interfere with the UV-absorbance measurements used to quantify the concentration of RNA. To remove free nucleotides, dilute the RNA sample fivefold and reconcentrate with a 100 kDa. Centricon filtration unit (EMD Millipore, Billerica, MA, USA)—repeat the dilution-concentration cycle five times to ensure complete removal of free nucleotides.

About 20% of the mass of CCMV is RNA. Thus 2.5 μg of CCMV corresponds to about 0.5 μg of RNA.Rees F. Garmann et al.

Absorbance at 260 nm of 1 mg/ml of CCMV5.88 OD (A260/A280 = 1.6) Absorbance at 260 nm of 1 mg/ml of ssRNA25 OD (A260/A280 > 2 in TE buffer) Absorbance at 280 nm of 1 mg/ml of CP0.81 OD (A260/A280≤ 0.67)

CCMV as DNA cargo

https://www.sciencedirect.com/science/article/pii/S0168365919303293

CCMV for self-amplifying mRNA delivery

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0215031