Ericf Burgon and Dr. Greg Burton, Biochemistry
Recombination is a major mechanism that generates variation in populations of human immunodeficiency type 1 (HIV-1). Mutations often confer replication advantages, such as drug resistance, which can lead to increased pathogenicity. This project is the first phase of a larger projected intended to study the effects of follicular dentritic cells (FDCs) on recombination rates.
Recombination is a major cause of genetic shuffling in HIV-1. It redistributes mutations made by reverse transcriptase leading to greater variation in genome. Recently, great attention has been placed on elucidating the mechanisms for recombination in HIV-1. In order for recombination to occur, specific requirements must be met. A cell must be co-infected with two different quasi species simultaneously. These two quasi species must then replicate and their progeny genomes must be package in the same virion creating a heterodipliod virion.
FDCs have been shown to be reservoirs for HIV-1 (1). They maintain the ability to present antigens to immune response cells which may provided a mechanism to facilitate the creation of heterodipliod virions and thus facilitate recombination.
FDCs harbor HIV-1 on their surface for extended periods of time. (1) It has also been shown that different quasi species exist on the surface of FDCs throughout infection. (1) HIV-1 has been shown to affectively infect through cell-to-cell contact. Because FDCs harbor many different quasi species on their surface there is an increased chance that FDC will co-infect a cell with two different species of HIV-1 leading to the creation of a heterodipliod virion capable of recombination.
Materials and Methods
In order to identify and quantify recombination rates in HIV-1, I intend to look at recombination rates of two different subtypes that are known to recombine in the env region. I will then use PCR primers to pull out recombination products in the env gene.
The first phase of my project is intended to produce an artificial positive of two known subtypes to test if these primers would select for the recombinants.
The subtypes used were HXB2 nef+, a B type virus with a correction in the nef. The second was 90cf402.1.8 an A/E recombinant with the env region being completely E.
First I transformed the STBL II cells with the HXB2 nef+ containing plasmid. The 90cf402.1.8 was in glycerol stock. Once prepared, I grew up the cells in a shaker at 37C for 20 hours.
I then streaked out the two plates of each sample. I plated out a 10 micro liter sample and a 25 micro liter sample for each virus and incubated them at 37C for 18 hours. From those plates I picked individual colonies and prepared a starter culture according to Qiagen Plasmid Maxi Kit. I then performed the Maxi Prep according to the Qiagen Protocol for high copy plasmid using a Qiagen-tip 500.
After the maxi prep I preformed a spectral analysis with a 1/50 dilution of the product. My OD readings for TBE buffer were 0.00128 and for the dilutions I got 0.00148 for HXB2 nef and 0.001345. I essentially did not have any DNA. When I performed the agarose gel analysis I got these results:
See Figure 1
This analysis indicates that I DNA in the first step for the HXB2 nef+ but not for 90cf402.1.8. All the DNA was lost in all subsequent steps.
Because it was the first time I performed a maxi prep I was unsure if I had made a mistake in the procedure or if I did not in fact have plasmid.
I performed a mini prep to check for plasmid. It appeared that I had plasmid DNA. When I performed an EcoR I digest it did not appear to cut out the insert. I ran the digest again and I also ran undigested DNA to get a better understanding of what my results were. It got these results:
See figure 2.
From this it appears that I do have plasmid DNA, but EcoR I is not cutting out the insert. I have analyzed the 90cf402.1.8 vector and identified two other restriction enzymes that can cut out my insert, Sac I and Nco I.
See Figure 3.
I did a digest with these and successfully cut out my insert.
Project Continuation
The next steps in my project are to successfully identify the HXB2 plasmid and find alternative restriction enzymes to cut out the plasmid. I also designed primers and have submitted them to be synthesized. If I can PCR amplify my product I may be able to bypass identifying it through a restriction digest.
Once I have PCR amplified both of my products, I will use the primers I already designed that have an Eco R I restriction site on the 5’ side. Once amplified, I will be able to do a restriction digest and ligate the two halves of the HXB2 nef+ and 90cf402.1.8 together.
Then I will be able to test my primers to see if they can identify recombinants.