Editing CCMV VLP (section)

== 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)