Bart's Cookbook--

Infection with retroviruses

It is well established with avian retroviruses that cells are most efficiently infected just after they are trypsinized. Trypsinization does two things. First, it apparently exposes the receptor to which the virus binds. Additionally, it stimulates DNA synthesis and cell division, two processes that are essential for the establishment of retroviral infection.

Additionally, it is important to do the infection in as small a volume of medium as possible.

It is likely that these principles are also relevant to the infection of mammalian cells with murine retroviruses.

Infection with retroviruses is facilitated greatly by polybrene. This is a small, positively charged molecule that binds to cell surfaces and neutralizes surface charge. This apparently allows the viral glycoproteins to bind more efficiently to their receptors, because it reduces the repulsion between sialic acid-containing molecules. To use polybrene optimally, the cells to be infected should be pre-treated with polybrene and the virus adsorption should be done in the presence of polybrene.

Cells vary in the amount of polybrene that they will tolerate. From 1 to 10 µg per ml is usual. Some of these values can be found in the Cell Line HyperCard stack.

There are two ways to infect adherent cells.

What I like is to seed the cells to be infected in medium containing polybrene and wait until the cells attach to the dish. This can take from 1 to 4 hrs. Then remove the medium, add the virus, let it adsorb for 30 to 60 min and then add back medium containing polybrene.

Alternatively, you can suspend trypsinized cells in a small volume of medium containing the virus and let adsorption occur while the cell suspension is incubated at 37 °C. The infected cells can then be seeded on dishes.

Retroviral infection requires cellular DNA synthesis--because viral DNA synthesis requires many of the same components and enzymes as cellular DNA synthesis, and mitosis--because the viral DNA must gain access to chromosomal DNA and cannot enter interphase nuclei. What is usually done is to infect cells at a density such that they will grow for about 3 days before reaching confluence. The plating efficiency of mammalian cell lines is usually high, close to 100%. The doubling time is approximately 18 to 24 hr. Therefore, if you seed 1 x 105 cells on a 50 mm dish, you will probably have 2 x 105 24 hours later. Confluence is at approximately 106 3T3 cells per 50 mm dish.

Infecting lymphoid cells.

Non-adherent cells can be pelleted and then resuspended in medium containing a retrovirus and an appropriate concentration of polybrene and incubated with the virus for 30 to 60 min. The cells can then be diluted with medium and allowed to grow.

We have had good success recently infecting lymphoid cells by co-cultivation of them with virus-producing cells for 18 hours in medium containing polybrene. In the case of COS cells, the transfected COS cells don't survive long enough to contaminate the lymphoid cells.

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