Purification and Analysis of Pseudovirions

At 40-48 h post infection with the recombinant VVs, cells become rounded up and can easily be harvested by simple pipeting. Although some cells have already released pseudovirions into the culture supernatant at that time, the bulk of the DNA-containing pseudovirions is still captured in cell nuclei. DNA-containing pseudovirions and empty virus-like particles may be prepared from nuclear lysates.

3.4.1. Preparation of Nuclear Lysates

1. Harvest VV-infected cells by centrifugation (10 min, 300g).

2. Wash cells with PBS and resuspend in 10 mL hypotonic dounce buffer (see Note 10).

3. Disrupt cells in a tight-fitting dounce homogenizer (40-50 strokes, 4°C).

4. Sediment cell nuclei by centrifugation (e.g., 10 min, 1000 g in a Sorvall SS34 Rotor).

5. Resuspend pellet in dounce buffer (10 mL).

6. Disrupt cell nuclei by sonification (three times, 45 s; 40% output).

7. Remove nuclear debris by centrifugation at 4°C (10 min, 8000 g, Sorvall SS34 Rotor).

8. Save supernatant for further purification steps.

3.4.2. CsCl Density Gradient Purification

Pseudovirions are further purified from nuclear lysates by density gradient centrifugation. During this procedure, pseudovirions are separated from nuclear debris not only but also from the high content of DNA-free VLPs still present in these preparations (Fig. 2).

1. Transfer supernatants (see Subheading 3.2.5.) to 15-mL conical tubes, and add 0.4 g CsCl per mL solution to obtain a density of 1.29 g/cm3.

2. Incubate for 60-90 min at RT. (This step is required for complete destruction of contaminating VVs.)

3. Transfer solution to 5-mL polyallomer centrifugation tubes (e.g., OptiSeal®, Beckman).

4. Ultracentrifugation: Vti65, 350,000 g, 12°C overnight.

5. Collect 0.25-mL fractions from the bottom of the centrifuge tubes.

3.4.3. Analysis of Pseudovirus-Containing Fractions

CsCl density gradient fractions are analyzed for the presence of pseudoviri-ons by refractive index determination, Western blot analysis, and pseudovirus infection assays (Fig. 2). Determination of the fraction density by measuring the refraction index in a density refractometer allows separation of DNA-con-taining pseudovirus fractions (1.33 g/cm3) and fractions containing VLPs (1.29 g/cm3). This separation may be confirmed by Western blot analysis (5 |L of each fraction) using an HPV capsid protein L1-specific antibody. A detailed description of pseudovirus infection assays is presented in Subheading 3.5. The encapsidated marker plasmid can be quantified by the following procedure.

1. Dialyse 100 |L each of fractions with densities from 1.35 to 1.30 g/cm3 for at least 6 h.

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