Determining the Role of Kaposi’s Sarcoma-associated Herpesvirus LANA Protein in the Development of Persistent Infection

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Jamie Gardiner and Dr. Brad Berges, Department of Microbiology and Molecular Biology

Kaposi’s Sarcoma-associated Herpesvirus (KSHV) is a human cancer virus that causes Kaposis’ sarcoma, the most common cancer found in AIDS patients. Our lab is working to develop humanized mice as a novel animal model to study KSHV infection and related diseases, since there has previously been no animal model established. The purpose of my project was to determine the role of the viral protein LANA (latencyassociated nuclear antigen) in establishing a latent KSHV infection in humanized mice. My project included two major aims:

  1. Develop three different strains of virus (KSHV wild-type, KSHV LANA-deleted, and KSHV LANA-rescued) and use them to infect humanized mice. Using these three strains will help us determine the role of the LANA protein in establishing a latent infection. (Achieving this aim was the major focus of another member of the lab.)
  2. Use RNA in situ hybridization (ISH) to monitor the expression five KSHV genes: LANA, RTA, ORF 65, vIL6, and vCyclin. Knowing the expression patterns of these genes will help us determine whether the strain of virus is in a lytic or latent viral replication state, as some of these genes are expressed lytically while others are expressed latently. We will observe gene expression at two different time points (4 and 32 days post infection). We expect to see lytic and latent gene expression in all strains at 4 days post infection. However, at 32 days post infection we do not expect to see any latent gene expression in the LANA-deleted strain.

We encountered a few setbacks in the development of one of our three virus strains. The KSHV wild-type and KSHV LANA-rescued viruses were successfully propagated and collected; they are ready to be used to infect our humanized mice. The KSHV LANA-deleted virus, however, did not propagate as expected. We are currently employing new strategies to induce the generation of this virus strain.

More progress was made with the use of RNA ISH. Both antisense and sense (control) probes were synthesized for each of the five viral genes. In order to establish the optimal conditions for each of these probes, we performed RNA ISH on uninfected humanized mouse tissue (negative control), BCBL-1 tumor tissue (positive control), and KSHV wild-type-infected tissue (experimental). Conditions were properly established for expression of LANA, a latent gene. This gene was detected in humanized mouse spleen tissue. When we moved on to establish the conditions for the lytic gene, RTA, we encountered more obstacles. After initially obtaining results that were the opposite of what we expected, we determined that our sense and antisense probes were reversed. Upon correcting this mistake, we obtained promising findings but still struggled to obtain consistent, repeatable results.

The remaining probes have yet to be tested, but the protocol has been optimized so that hopefully the only adjustment that needs to be made for each probe is the hybridization temperature. Although I personally did not complete this project, I have done all in my power to make it easy for someone else to finish where I left off.