Scientific Report 2006

Molecular Biology

Molecular Biology of Retroviruses

J.H. Elder, A.P. de Parseval, Y.-C. Lin, S. de Rozieres, M. Sundstrom, K. Tam, M. Giffin,* H. Heaslet,* C.D. Stout, B.E. Torbett*

* Department of Molecular and Experimental Medicine, Scripps Research

Our research centers on the molecular characterization of retroviruses, with emphasis on feline immunodeficiency virus (FIV) and development of ways to interfere with the viral life cycle. FIV causes an AIDS-like syndrome in domestic cats and has structural and functional similarities to HIV, the causative agent of AIDS in humans. Discovery of ways to interfere with FIV infection may ultimately result in development of treatments for infections in both cats and humans. In recent studies, we continued to focus on the molecular characterization of receptor interactions and the molecular basis for the development of drug resistance in the aspartic protease encoded by FIV.

Receptor Studies

Like many strains of HIV, FIV uses the chemokine receptor CXCR4 to enter the primary target cell, the CD4⁺ T cell. However, unlike HIV, FIV does not use the cell-surface protein CD4 as a primary binding receptor. Rather, the feline lentivirus uses the activation antigen CD134 to initially bind to CD4⁺ T cells. CD134 is expressed on activated CD4⁺ T cells, a finding that explains why FIV can infect and kill CD4⁺ T cells, even though the virus does not bind CD4.

As reported last year, we showed that interaction of the FIV surface glycoprotein gp95 with a soluble version of CD134 allows productive infection of cells that bear the entry receptor CXCR4 but lack cell-surface CD134. This finding is consistent with the notion that binding of CD134 causes a conformational change in gp95, which in turn increases the affinity of interaction with CXCR4 to facilitate infection of the target cell. These effects are similar to the effects of binding of soluble CD4 by gp120, the surface glycoprotein of HIV and indicate that although different primary receptors are involved, the actual mechanism of infection of FIV and HIV is strikingly similar. We speculate that the benefit of this type of binding cascade is to limit exposure of critical regions of the surface glycoproteins to the immune system until the primary binding event has already occurred, thus reducing the likelihood of virus neutralization.

Using chimeric proteins consisting of feline and human CD134 (the human homolog does not bind FIV glycoprotein) and site-directed mutagenesis, we have mapped regions of feline CD134 involved in interaction with gp95. The results indicated that as few as 3 amino acids in the C-terminal part of outer domain 1 of feline CD134 are sufficient to impart FIV gp95 binding and receptor function to human CD134. Studies are in progress to map the regions of gp95 that bind CD134.

Importantly, we have now a panel of antibodies that bind and neutralize FIV only after CD134 is bound; we have used peptides to map the region in which these CD134-dependent neutralizing antibodies react. These studies effectively map regions of the viral glycoprotein critical for CD134 interaction. Cocrystallization studies are under way to determine the structure of the region surrounding the antibody-binding site. These experiments will contribute to our understanding of the nature of receptor binding and will define targets for vaccine development.

Protease Drug Resistance

The aspartic protease of lentiviruses is responsible for processing the viral Gag and Pol polyproteins into the final gene products required for viral replication and must function efficiently to generate infectious virus. Drugs against HIV protease are keys to the success of highly active antiretroviral therapy used to treat, but not cure, patients infected with HIV. The substrate and inhibitor specificities of FIV differ from those of HIV. We investigated the nature of these differences to better understand the structural basis of development of resistance to therapy, an ongoing problem with current drugs used to treat HIV disease.

In certain instances, similarities exist between amino acid positions that dictate differences in substrate specificity between FIV and HIV aspartic protease and those that mutate in response to drug treatment. Mutations in these sites increase the dissociation constant for a given drug, but at a cost in catalytic efficiency for the viral protease. Compensatory amino acid substitutions can then occur that increase the catalytic efficiency of the drug-resistant protease, thus increasing expression of virus despite drug treatment.

We prepared mutants of FIV protease in which amino acids found in drug-resistant HIV protease were placed in the equivalent positions in the FIV enzyme. Then, using cells transduced with gag/pol gene expression vectors encoding HIV-FIV hybrid proteases, we tested the mutants for relative drug sensitivity. We found that the Gag/Pol polyproteins are processed by the hybrid proteases and have drug sensitivity profiles similar to those of HIV protease. However, the order of site cleavage, which is critical to generation of infectious virus, is altered by these specific changes. Studies are under way to establish a structural basis for this phenomenon. The findings highlight yet another potential approach to interrupting the viral life cycle.

Publications

Brik, A., Alexandratos, J., Lin, Y.-C., Elder, J.H., Olson, A.J., Wlodawer, A., Goodsell, D.S., Wong, C.-H. 1,2,3-Triazole as a peptide surrogate in the rapid synthesis of HIV-1 protease inhibitors. Chembiochem 6:1167, 2005.

de Parseval, A., Bobardt, M.D., Chatterji, A., Chatterji, U., Elder, J.H., David, G., Zolla-Pazner, S., Farzan, M.R., Lee, T.-H., Gallay, P.A. A highly conserved arginine in gp120 governs HIV-1 binding to both syndecans and CCR5 via sulfated motifs. J. Biol. Chem. 280:39493, 2005.

de Parseval, A., Grant, C.K., Sastry, K.J., Elder, J.H. Sequential CD134-CXCR4 interactions in feline immunodeficiency virus (FIV): soluble CD134 activates FIV Env for CXCR4-dependent entry and reveals a cryptic neutralization epitope. J. Virol. 80:3088, 2006.

Gonzalez-Lira, B., Rueda-Orozco, P.E., Galicia, O., Montes-Rodriguez, C.J., Guzman, K., Guevara-Martinez, M., Elder, J.H., Prospero-Garcia, O. Nicotine prevents HIVgp120-caused electrophysiological and motor disturbances in rats. Neurosci. Lett. 394:136, 2006.

Heaslet, H., Kutilek, V., Morris, G.M., Lin, Y.-C., Elder, J.H., Torbett, B.E., Stout C.D. Structural insights into the mechanisms of drug resistance in HIV-1 protease NL4-3. J. Mol. Biol. 356:967, 2006.

Liang, F.-S., Brik, A., Lin, Y.-C., Elder, J.H., Wong, C.-H. Epoxide in water and screening in situ for rapid discovery of enzyme inhibitors in microtiter plates. Bioorg. Med. Chem. 14:1058, 2006.

Whiting, M., Muldoon, J., Lin, Y.-C., Silverman, S.M., Lindstrom, W., Olson, A., Kolb, H.C., Finn, M.G., Sharpless, K.B., Elder, J.H., Fokin, V.V. Inhibitors of HIV-1 protease by using in situ click chemistry. Angew. Chem. Int. Ed. 45:1435, 2006.