Eryn Barber Hadley and Dr. Greg Burton, Microbiology
Human Immunodeficiency Virus (HIV) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS). There is currently no cure for HIV infection, and AIDS has been reported as the fourth highest killer in the world [1]. The initial success of the drug cocktail used in Highly Active Anti-Retroviral Therapy (HAART) has made the possibility of a cure for AIDS seem attainable, however the persistence of virus reservoirs makes this extremely difficult and leads to a rebound of viral load [2]. The known reservoirs of HIV include latently infected T cells, macrophages and follicular dendritic cells.
Any given population of HIV contains a variety of genetic sequences called quasi species. These quasi species are generated by the rapid mutation of the RNA genome as it is converted to DNA for use by the host cell polymerase. The resultant genetic variation provides a selective advantage to the virus because some of the variants thrive in spite of immune and therapeutic drug selection. It is our hypothesis that follicular dendritic cells (FDCs) trap multiple quasi species over the course of infection, acting as an archive of past virus. My initial proposal was to do a sequence analysis on murine quasi species trapped by FDCs, however we were fortunate enough to obtain actual patient tissue. Researchers from the Microbiology and Zoology departments are collaborating to extensively analyze the data from these HIV-positive patient samples.
Samples infected with HIV from three different deceased patients were obtained for analysis. It was our goal to distinguish quasi species from tissue samples of multiple conditions in each patient by PCR amplification of a specific gene, ligation into a bacterial plasmid, and transformation into bacterial E. coli cells. These cells multiply profusely providing us with an abundance of DNA, which could then be sequenced.
My focus was on patient number 2202. The DNA from infected cells was isolated from FDCs and T cells in the axillary lymph nodes, cervical lymph nodes and spleen. A Southern Blot was performed to confirm the presence of HIV in each of the six conditions. I began PCR amplification for the envelope gene of HIV, which is about 2,500 kilo bases long and carries the code for the outer shell of the virion. After multiple failed attempts with numerous dilutions of the patient DNA it was concluded that this gene was too large to amplify successfully from patient 2202’s DNA. New primers were ordered for the PCR reaction and I was able to successfully generate product on a shorter segment of the envelope gene about one half the size of the full envelope gene. This was then ligated and transformed into the bacterial clones and pure DNA was isolated. I have also followed this procedure for the polymerase gene and have been successful in isolating DNA for a few conditions.
The pure DNA isolated from multiple clones in the same condition is what gives us a picture of past mutations. This allows us to see how the virus has changed over time in a particular individual. In order to determine what mutations have occurred, the sequence of at least twenty different clones must be determined. Each condition is compared to the others and the T cells are compared to the FDCs.
A sequencing reaction was performed on the pure DNA isolated above. This added radiolabeled nucleotides to the amplified segments of the gene in question. The BYU DNA Sequencing Center took that DNA and was able to determine the sequence using the radiolabeled nucleotides. From the chromatograms that have been generated by the Sequencing Center our laboratory will be able to analyze the mutations in this portion of the envelope gene.
Because this is a lengthy procedure I have not yet begun analysis on the sequences that have been returned. I do expect that each of the three conditions, the axillary and cervical lymph nodes and the spleen will not be tremendously different. However, I do expect that the T cells and FDCs within the three conditions will be dramatically different. The FDCs should retain more ancient quasi species and display much more diversity between clones, while the virus from the T cells should be more closely related.
This has been an invaluable learning experience for me. I have learned skills related to my field and made contacts that will assist me as I enter the work force. The my mentor and especially the graduate students that I have worked with have been amazing and have opened my eyes to the tiny world within our cells.
References
- Balter, M. 1999. AIDS now world’s fourth biggest killer [news]. Science 284:1101.
- Chun, T.W. and A.S. Fauci. 1999. Latent reservoirs of HIV: obstacles to the redication of virus. PNAS 96:10958.