Development of a Cellular Model Expressing Cloned HIV env Genes

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Jeffrey S. Johnson and Dr. Gregory F. Burton, Chemistry and Biochemistry

Highly Active Antiretroviral Therapy (HAART), a treatment that uses protease inhibitors and nucleoside analogs to block replication of HIV, has had a marked effect on decreasing the viral loads of HIV in infected individuals. However, studies have indicated that if a patient receiving HAART for long periods of time (2 to 3 years) is taken off treatment, HIV levels in the blood will increase drastically within several weeks (2). This problem indicates that reservoirs in the body harbor the virus and protect it from treatment until conditions allow the virus to replicate unchecked throughout the body. The Follicular Dendritic Cell (FDC) is one of three such reservoirs (1). FDCs reside in the germinal centers of secondary lymphoid tissue and function in the maintenance of specific immune responses. Studies have shown that mouse FDCs not only trap large quantities of HIV, but also maintain the trapped virus in an infectious state for at least 25 days in vitro and 9 months in vivo (3). Research by Smith-Franklin et al. has also shown that human FDCs maintain HIV in an infectious state in vitro and that HIV-specific antibodies (Abs) and antibody receptors on FDCs (FDC-FcgRs) are necessary in order to maintain this infectivity (4). However, much less is known about HIV trapped by human FDCs in vivo.

I hypothesize that FDC trapping of HIV in vivo is mediated by specific Abs and FDC-FcãRs and that HIV-specific Abs are required to maintain the infectivity of the trapped virus. If this is true, a patient with trapped HIV on his or her FDCs has to have Abs specific to those HIV quasispecies circulating in his or her serum. Specific Abs produced within a patient would react and bind to the proteins embedded in the viral envelope including those encoded by the env gene: glycoprotein 41 (gp 41) and glycoprotein 120 (gp 120). To test whether HIV-specific Abs in an infected person’s serum were reactive to the HIV quasi-species trapped on the individual’s FDCs, I worked to develop a cellular model in which specific proteins, gp41 and gp120, from HIV trapped on FDCs could be expressed. After development of this model is completed, the model will be used to analyze the reactivity of Abs in a patient’s serum with gp41 and gp120 from HIV trapped on the patient’s FDCs. This study will conclude whether HIV-specific Abs need to be circulating in the serum in order for HIV quasi-species to be trapped by FDCs.

Initially, I received several samples of a plasmid (a small circular piece of DNA) containing cloned env genes from patient 559 and a selectable marker for the antibiotic geneticin. Many of the samples did not contain the env gene in an orientation that would express the gene in a cellular model. I performed restriction digests on the plasmid samples using the restriction enzyme NSI and analyzed the results by gel electrophoresis to determine which samples were in the correct orientation. Since all the genes had been sequenced, I also analyzed the base sequences of the different samples to double check that the genes were in the right orientation.

The plasmid samples with env in the correct orientation were used to attempt transfections (insertion of foreign DNA into viable cells) of various cell lines in order to express gp41 and gp120 in the cell lines. I attempted to transfect four different cell lines: Hela, Cos-7, Jurkat, and Cho cells. Because the plasmid containing the env gene also contained a selectable marker for geneticin, I cultured in the transfected cells in geneticin, which would kill the cells not containing the plasmid. However, the geneticin did not appear to kill the non-transfected cells effectively, so I then attempted more experiments without it. The transfected cells were analyzed using Western Blot Assays and flow cytometry in which monoclonal Abs specific for gp41 and gp120 (Chessie 8-specific for gp41, Chessie 13-specific for gp120, and H902-specific for gp120) were used to probe for the expressed proteins. I was unable to find expression of gp41 or gp120 in any of the cell lines that I transfected.

I was unsuccessful in expressing env genes in a cellular model. I have several ideas as to where the problem is occurring in the development of a cellular model to express gp41 and gp120. First there might be problems with the transfection. If that is successful, there might be problems with either the transcription or translation of the gene message within the cell lines. To determine if a problem lies in the transcription or translation of the env gene, the transfected cells should be probed for mRNA (the message from DNA that encodes proteins of the env gene), which could be done by mRNA isolation and reverse transcriptase PCR. If no mRNAs of env are found, then the gene is obviously not being transcribed. If mRNAs of env are found, then the gene is being transcribed, and the problem may be in translation of the mRNA. Additionally, the Env proteins expressed in the cell lines might not be recognized by the monoclonal Abs (H902, Chessie 8, and Chessie 13), which were used as probes. Because these monoclonal Abs (H902, Chessie 8, and Chessie 13) were produced against lab-adapted strains of gp41 and gp120, these Abs might not bind effectively to Env proteins originating from primary strains of HIV, which are somewhat different from lab-adapted strains.

Although I was not able to develop this cellular model in which to express env genes, the development of such a model will be beneficial in studying the interactions between HIV and the human immune system. Specifically, it will help us understand if HIV-specific Abs are required to trap virus particles on FDCs.

References

  1. Burton, G. F., B. F. Keele, J. D. Estes, T. C. Thacker, S. Gartner. 2002. Follicular dendritic cell contributions to HIV pathogenesis. Semin. Immunol. 14(4):275.
  2. Davey, RT, N. Bhat, C. Yoder, T. W. Chun, J. A. Metcalf, R. Dewar, V. Natarajan, R. A. Lempicki, J. W. Adelsberger, K. D. Miller, J. A. Kovacs, M. A. Polis, R. E. Walker, J. Falloon, H. Masur, D. Gee, M. Baseler, D. S. Dimitrov, A. S. Fauci, H. C. and Lane. HIV-1 and T cell dynamics after interruption of highly active antiretroviral therapy (HAART) in patients with a history of sustained viral suppression. Proc Natl Acad Sci USA 1999, 96:15109-15114.
  3. Smith, B. A., S. Gartner, Y. Liu, A. S. Perelson, N. I. Stilianakis, B. F. Keele, T. M. Kerkering, A. Ferreira-Gonzalez, A. K. Szakal, J. G. Tew, and G. F. Burton. 2001. Persistence of infectious HIV on follicular dendritic cells. J. Immunol. 166: 690-696.
  4. Smith-Franklin, B. A., B. F. Keele, J. G. Tew, S. Gartner, A. K. Szakal, J. D. Estes, T. C. Thacker, and G. F. Burton. 2002. Follicular dendritic cells and the persistence of HIV infectivity: the role of antibodies and Fcg receptors. J. Immunol. 168: 2408-2414.