The overall goal of this procedure is to perform in vitro ENC coating of HIV one cause This is accomplished by first producing HIV one particles. The variants are then concentrated by Ultracentrifugation ation. The next step is to isolate cause by sucrose gradient ultracentrifugation.
Finally, a kinetic assay of HIV V one uncoating is performed. Ultimately results show time dependent uncoating of HIV one cause through quantification of ca content in the palate and supernatant by Eliza. This matter can help answer key questions in the HIV one fill, such as the role of cellular factors in encoding and the effect of mutations on virus capture stability before proline.
You must obtain permission and follow guidelines from the biosafety office of your institution to work in a biosafety facility for infectious H HIV V one. Proper handling and care should be taken while working in a biosafety laboratory. In this demonstration, HIV one particles are produced by transient transfection of 2 9 3 T cells with HIV one proviral DNA.
Using a calcium phosphate transfection method the day before transfection obtained previously cultured 2 9 3 T cells and detached them from the plates using 0.5%trips in EDTA seed six 100 millimeter culture dishes for production of variants The following day, the cell monolayer should be about 25%cofluent and ready for transfection for six dishes of cells. To be transfected, use 120 micrograms of proviral DNA to prepare a mixture containing calcium chloride and best buffered saline as described in the written protocol, add one milliliter of the resulting mixture, dropwise to the center of each dish while swirling gently to mix. Place the dishes overnight in an incubator set at 35 degrees Celsius and 3%carbon dioxide.
After overnight incubation, aspirate the culture medium and gently add five milliliters of PBS, then aspirate the PBS and replace with five milliliters of fresh medium. At this point, culture the cells in an incubator at 37 degrees Celsius and 5%carbon dioxide for an additional 24 to 48 hours. Next, collect the culture supinate containing viral particles and transfer to a 50 milliliter conical centrifuge tube.
After combining the S supernatants from all dishes, centrifuge the sample to pellet cells and debris, then filter the SUP natant using 0.45 micron pore size syringe filters. Begin concentration of HIV one furion by placing the cultures supernatant. In a 38.5 milliliter poly alam centrifuge tube.
Gently underlay the virus containing SUP natant with five milliliters of 20%sucrose in PBS using a pipette centrifuge for three hours at 32, 000 RPM and four degrees Celsius to pellet virus particles. Carefully remove the SUP natant without disturbing the pellet at the bottom of the tube. Add 0.5 milliliters of one times STE buffer to the tube.
Place the samples at four degrees Celsius for one hour to loosen the pellet After an hour, use a wide ball one milliliter pipette tip to detach the pellet from the bottom of the tube by gently pipetting up and down a few times. Then transfer the loosened pellet to a 1.5 milliliter micro centrifuge tube. Taking care to avoid foaming incubate at four degrees Celsius for another one to three hours to allow the small clumps of virus to disperse following incubation.
Gently pipette the virus suspension up and down several times. Finally, centrifuge the suspension at 8, 000 RPM in a refrigerated micro fuge for one minute to remove residual clumps to prepare a 12 milliliter linear 30 to 70%sucrose gradient. Use a 20 milliliter gradient former and place six milliliters of 70%sucrose solution on the near side closest to the outlook port.
Then place six milliliters of 30%sucrose solution on the far side. Ensure that air bubbles are not trapped in the channel. Connecting the two chambers with an auto density flow gradient.
Former pump the gradient from the bottom to the top of a 14.5 milliliter poly aamer centrifuge tube containing one milliliter of 85%Sucrose solution at the bottom. Gently place the gradient at four degrees Celsius until cooled. Once the gradient has cooled, use a wide bore one milliliter pipette tip to gently overlay it with 0.25 milliliters of 15%sucrose solution in STE containing 1%Triton X 100.
Then gently overlay with 0.25 milliliters of 7.5%sucrose solution in STE. Be careful not to mix the layers. Now gently overlay the gradient with 0.5 milliliters of virus suspension.
This step should be performed carefully to avoid any disturbance in the gradient and mixing of the underlaid sucrose layers. Next, place the tubes a preco centrifuge bucket. Once the vacuum in the centrifuge reaches 250 microns, centrifuge the samples at 32, 000 RPM overnight at four degrees Celsius.
Following centrifugation, collect one milliliter fractions from top to bottom of the gradient using the auto density flow gradient fractionator. Place the tubes on ice immediately upon collection. Mix the contents of the tubes by inverting several times.
Then withdraw 50 microliters of each fraction for quantification by e LSA or reverse transcriptase activity assay. Before performing on coating assays, determine which fractions contain intact HIV V one cause and combine the aliquots as described in the written protocol. Pre dilute the aliquot of HIV one cause with an equal volume of cold one times STE buffer to reduce the viscosity of the suspension and minimize pipetting errors.
Add 0.15 milliliters of cold one times STE buffer to enough micro centrifuge tubes for the desired amount of time points to be tested and a zero minute control. The STE buffer can be supplemented with BSA to prevent sticking of the cause to the walls of the tube. Then further dilute 100 microliters of the cause into each prepared tube mixed by inverting the tube several times.
Place one tube on ice for the zero minute control, which is used to determine the increase in uncoating. With time in the samples. Place the remaining tubes in a water bath at 37 degrees Celsius.
Make sure that the tubes are immersed in the water bath at least up to the level of the internal liquid. In order to ensure even warming, incubate the samples for timed intervals. Gently mix the contents of the tube periodically by flicking the tube at the end of the incubation period.
Stop the uncoating process by placing the tubes in ice for 10 minutes. In a preco centrifuge rotor centrifuge the sample tubes and control of 45, 000 rotations per minute for 20 minutes of four degrees Celsius. This will pellet the intact cause, whereas the free ca released as a result of uncoating of cause will remain in the s supernatant following centrifugation.
Transfer the snat to separate tubes and re suspend the pellets. In 250 microliters of Eli, a sample diluent quantify the CA content in both pellet and supernatant using P 24 Eliza as described in the written protocol. Finally, the extent of uncoating can be obtained by calculating the fraction of ca present in the supernatant.
An example of a result from the ELIZA for determining fractions containing cause is shown here. The total ca also called P 24 obtained by adding the values for 12 fractions was 14.5 micrograms with fractions, eight to 10 containing the cause. Fraction one or two will always contain the highest amount of P 24, which represents the free ca and is not associated with the cause.
This is followed by a gradual reduction in P 24 values with each subsequent fraction, then a sharp increase and finally a sharp drop in the P 24 values. If proper care is taken while loading the gradient, then the peak should always be close to fraction 10. Shown here is a typical result obtained by performing kinetics of uncoating of HIV one cause.
This graph shows a time dependent increase in uncoating of wild type HIV one cause the percent on coating value was obtained by calculating the fraction of ca present in the supinate, the basal level of uncoating, as well as the rate of uncoating could vary slightly with batches of cause. Nevertheless, the assay is highly reproducible and is useful for determining stability of viral cause in vitro. While attending this procedure, it is important to remember that the reproducibility of this assay is highly dependent on proper handling of the samples.
Since HIV V one course, our heat lab, it is important to keep the samples at four degrees throughout the assay except during the incubation period.
