Preparation of Viral DNA from Nucleocapsids

Summary

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Abstract

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.^1,2,3

This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.^4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD_260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 μg or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections.

Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 10⁶ PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 10⁸ plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.⁶ For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 μl of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).

Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

Protocol

1. First Day: Viral Infection and Preparation of Buffers

1. Prepare 5-10 dishes (15 cm diameter) of tissue culture cells for infection, e.g. PK-15 cells for pseudorabies virus (PRV) or Vero cells for herpes simplex virus (HSV).
2. When cells are 95 - 100% confluent, infect them at a (MOI) of 5-10. To do this, inoculate each plate using virus stock in a total volume of 4 ml per plate, then incubate the plates for 1 hour at 37°C. Rock plates gently every 15 minutes to ensure that the cell monolayer remains fully coated by the virus inoculum. Meanwhile, warm the medium for the next step.
3. After one hour of infection, aspirate viral inoculum from ....

Discussion

Portions of this protocol were originally developed for viral DNA isolation in BSL4 conditions, but it adapts equally well to non-BSL conditions.⁴ We commonly use this protocol to isolate DNA from the alpha-herpesviruses PRV and HSV-1, which have DNA genomes enclosed in a proteinaceous capsid and surrounded by a lipid envelope.^7,8 However it is likely directly adaptable to other large DNA viruses, including beta- and gamma-herpesviruses and adenoviruses. Similar extractions are commonly used for RNA.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Freon (1,1,2-trichloro-1,2,2-trifluor–thane)	Fisher Scientific	T178-4	Check with your institution for guidelines on appropriate disposal of Freon-containing waste, or see Mendez et al. for potential Freon alternatives.9
Phase lock gel tubes, Heavy 15ml capacity	5 PRIME	2302850	Optional
Polyallomer ultracentrifuge tubes	Beckman Coulter Inc.	331372*	*Select tubes appropriate for your own ultracentrifuge; these are included as an example only
NP-40 / IGEPAL	Sigma-Aldrich	I-3021
PK-15 cells	ATCC	CCL-33
Vero cells	ATCC	CCL-81
PBS	Hyclone	SH30028.03