Scientific Report 2005

Molecular Biology

Molecular Biology of Retroviruses

J.H. Elder, A.P. de Parseval, Y.-C. Lin, S. de Rozieres, U. Chatterji,* K. Tam, B.E. Torbett**

* Department of Immunology, Scripps Research
** Department of Molecular and Experimental Medicine, Scripps Research

Our research centers on the molecular characterization of retroviruses, with emphasis on feline immunodeficiency virus (FIV). FIV causes an AIDS-like syndrome in domestic cats, and although it does not infect humans, the feline retrovirus has many structural and functional similarities to HIV, the causative agent of AIDS in humans. Thus, study of FIV can yield insights into ways to interfere with the retrovirus life cycle that may ultimately result in the development of treatments for infections in both cats and humans.

During the past year, we focused on 2 major areas: the molecular characterization of cell-surface receptors for FIV 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 initially binds to another cell-surface molecule, CD134. In the past year, we characterized the expression of CD134 and showed that it is upregulated on CD4⁺ T cells. This observation explains why FIV can infect and kill this subset of T cells even though the virus’s surface glycoprotein does not interact with CD4.

In an extension of these studies, we found that interaction of the FIV surface glycoprotein gp95 with a soluble version of CD134 allows the productive infection of cells that bear the entry receptor, CXCR4, but lack surface expression of the binding receptor, CD134. The results are consistent with the notion that binding of CD134 causes a conformational change in gp95, which in turn increases the affinity of interaction with CXCR4 and facilitates infection of the target cell. These effects are similar to the effects of binding of soluble CD4 by gp120, the cell-surface glycoprotein of HIV, and indicate that although different molecules are involved, the actual mechanisms of infection of FIV and HIV are 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.

We also precisely mapped regions of CD134 involved in interaction with gp95. CD134 is a member of the TNF-α receptor superfamily and has a domain structure similar to that of the TNF-α receptor. Human CD134 does not bind FIV gp95, even though human CD134 shares considerable amino acid homology with feline CD134. Using chimeric proteins consisting of feline and human CD134 and site-directed mutagenesis, we showed 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. Structural studies of both receptor and ligand will establish a molecular basis for the putative conformational change induced by interaction with the binding receptor.

Development of Drug Resistance by FIV Aspartic Protease

The aspartic protease of lentiviruses is a particularly good target for drug therapy because its function in processing the viral Gag and Pol polyproteins is absolutely required for generation of infectious virus. Drugs active against the HIV protease have been keys to the success of highly active antiretroviral therapy used to treat patients infected with HIV. The substrate and inhibitor specificity of FIV differs from that of HIV, and we previously reported the identification of amino acids that define the different specificities. Comparing FIV with HIV offers a means to better understand the development of resistance to therapy, an ongoing problem with current drugs used to treat HIV disease.

Interestingly, parallels 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 complexes consisting of the protease and an inhibitor drug, but at a cost in catalytic efficiency for the protease. Over time, compensatory amino acid substitutions occur that result in an increase in catalytic efficiency, which results in increased 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. These “HIV-inized” FIV proteases had drug sensitivity profiles similar to those of HIV protease. In studies with cells transduced with gag/pol gene expression vectors encoding HIV-FIV hybrid proteases, the Gag/Pol polyproteins were processed with proper fidelity and had the expected drug sensitivities. When engineered into FIV, these hybrid proteases will offer a means to study drug resistance and to develop new inhibitors capable of blocking replication of drug-resistant viruses, without the biohazard associated with handling infectious HIV.

Publications

de Parseval, A., Chatterji, U., Morris, G., Sun, P., Olson, A.J., Elder, J.H. Structural mapping of CD134 residues critical for interaction with feline immunodeficiency virus. Nat. Struct. Mol. Biol. 12:60, 2005.

de Parseval, A., Chatterji, U., Sun, P., Elder, J.H. Feline immunodeficiency virus targets activated CD4⁺ T cells by using CD134 as a binding receptor. Proc. Natl. Acad. Sci. U. S. A. 101:13044, 2004.

de Rozieres, S., Swan, C.H., Sheeter, D.A., Clingerman, K.J., Lin, Y.-C., Huitrón-Reséndiz, S. Henriksen, S., Torbett, B.E., Elder, J.H. Assessment of FIV-C infection of cats as a function of treatment with the protease inhibitor, TL-3. Retrovirology 1:38, 2004.

Montes-Rodriguez, C.J., Alavez, S., Elder, J.H., Haro, R., Moran, J., Prospero-Garcia, O. Prolonged waking reduces human immunodeficiency virus glycoprotein 120- or tumor necrosis factor α-induced apoptosis in the cerebral cortex of rats. Neurosci. Lett. 360:133, 2004.