Marshall Sheide and Dr. Bradford Berges, Department of Microbiology and Molecular Biology

Kaposi’s sarcoma herpes virus (KSHV) is one of seven known cancer causing viruses which have the capacity to persist in the host for many years. This particular virus can undergo a long latent infection which ensures that the virus can hide by not expressing its genes. Last year, we proposed an experiment that would show the importance of a particular gene in the viral genome, LANA, in establishing a latent infection. The experiment required us to create stocks of a mutant virus that did not have the LANA gene inside (bac36deltaLANA) and then infect humanized mice with it. After infection, we planned on examining the mice to see if the capacity to establish a latent infection would be inhibited. However, as we proceeded with the experiment, we found that the bac36deltaLANA virus was very difficult to propagate. Other researchers had been able to transfect the viral DNA into cells but had been unable to keep it there. In other words, we could get the DNA into the cells but the cells would go into their lytic cycle and die instantly (because LANA was gone) or they would lose the DNA over subsequent divisions. We were suddenly faced with an unanticipated challenge that we have had to overcome in this last year. The journey to uncover a means to create the virus has been interesting research all by itself. The following are a list of experiments that we attempted and what we have learned from them:

Attempt 1

I initially transfected 293 T cells with bac36deltaLANA DNA. I used a generic protocol and 24 hours later, we saw evidence of a low yield retention of the viral DNA. I selected with Hygromycin, an agent that is lethal to all the cells that did not have bac36deltaLANA. We observed that the infected cells that divided, were unable to pass the genome along to the two daughter cells. We also observed that several of the infected cells were destroyed by the virus.

Attempt 2

From the previous experiment, I concluded that the best way to get any stock of virus was to attempt to stimulate cell lysis right after discovering bac36deltaLANA DNA in the cells. I believed that I could remove a small amount of virus and then reinfect a larger quantity of 293 cells with the actual virus and obtain a larger yield that way. We re-transfected and, 24 hours later, extracted by introducing BaculaK50 to the media. After removing the supernatant, I placed the small virus stalk into a beaker with a large concentration of 293 cells and used Polybrene to aid the virus in tethering to the cells. None of the cells were infected and we were forced to admit that the experiment had not worked. I later found out that, although standard protocol suggests bacK50 to coax out the virions from the cell, adenoK50 would have been a better and more efficient choice.

Attempt 3

I concluded that something must have gone wrong with the initial low yield of bac36deltaLANA in the original culture and I decided to tinker with using a higher ratio of DNA concentration and increase transfection time. I got a much higher initial yield after this personal change in protocol but again the cells lost their ability to retain the foreign genome. I was forced to admit that we would have to use a different method to obtain the virus.

Attempt 4

A fellow researcher in Arizona had discovered that he could insert a tethering LANA gene in 293 cells to help them to cling to foreign DNA. This fit in perfectly with what we were trying to find and we obtained some of his stock (called HEK293YfP). We believed that the tethering protein would discourage release of our bac36deltaLANA genome and that we could finally establish a system in order to get a high yield of virus. We transfected the HEK293 cells and observed the presence of bac36deltaLANA in several of the cells. Furthermore, we found that the gene persisted in the cells and daughter cells could retain the genome. This was a huge improvement and we finally believed that we had found our solution. I selected with Puromycin and Hygromycin, two agents meant to single out HEK293 cells with bac36deltaLANA, but the cells looked unhealthy and began to die out again until we lost the whole stock. It was disappointing but we had finally seen that our work was paying off and we were getting closer to our goal. We have concluded that the following protocol should be successful:

Proposal for Attempt 5

From the beginning, we will select for pure HEK293 stock by introducing Puromycin for one week. This should accustom the cells to a more intense living condition from the beginning and they won’t suffer so much when we select with the heavier drug, Hygromycin, later. After one week and with my higher yield protocol that I have developed, I will transfect the cells. 36 hours later, I will begin selection with Hygromycin. It is here that our protocol can go two ways: I can either extract at that moment to get virus stock and then reinfect other HEK293 cells or I can wait for the cells to divide and then encourage lysis to get viral stocks. We plan on doing both simultaneously to ensure the best results.

In summary

This last year, we have unexpectedly been forced to go into uncharted territory in order to someday proceed with our original experiment. We have learned so much and come so far from where we once were. We believe that what we have already learned, along with what we will learn in these next few months, will have been well worth the wait. Forward!