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The Elder Lab

Research

(TSRI Scientific Report)
Molecular Biology of Retroviruses
 
J.H. Elder, Y.C. Lin, M. Q-Y. Hu, Y. Hong, C.D. Stout$, B.E. Torbett,* C.K. Grant#, E. Fink, and M. Happer

  • Department of Molecular and Experimental Medicine, Scripps Research
  • Custom Monoclonals, Inc., W. Sacramento, CA
  • Department of Molecular Biology, Scripps Research
    Our laboratory studies the molecular biology of retroviruses, with the goal to development broad-based pharmacological and immunological intervention strategies. The majority of our experiments utilize feline immunodeficiency virus (FIV) for the study of lentivirus infections. FIV causes an AIDS-like syndrome in domestic cats and has many structural and functional similarities to HIV-1, the cause of AIDS in humans. Thus, FIV can serve as a vehicle to develop useful treatments for infections in both cats and humans. Primarily, we investigate the molecular mechanisms by which the major virus glycoprotein binds to its receptors and the how the host’s immune system targets this interaction as a way to limit virusspread.  In addition, we study a critical viral enzyme, the aspartic protease, as a target for development of antiviral drugs.

ENVELOPE STUDIES

FIV shares with certain strains of HIV-1 the use of the chemokine receptor CXCR4 to enter CD4+ T lymphocytes, the primary cell target for both lentiviruses. Most strains of both HIV and FIV also utilize a primary binding receptor to increase the effective local concentration of the incoming virus and to alter the conformation of the surface glycoprotein so as to increase the binding affinity for the CXCR4.  HIV uses the cell-surface protein CD4 as a primary binding receptor, whereas FIV accomplishes the same goal by using another T cell surface glycoprotein, CD134. Our previous studies using monoclonal antibodies against the viral glycoprotein have demonstrated that binding to CD134 causes a conformational change in gp95 necessary for high affinity binding to CXCR4.  Of importance, this conformational change in gp95 presents a window of opportunity to the host’s immune system in that cryptic neutralization sites become available for a brief time before the virus enters the cell.  Our goal is to utilize this information to develop vaccines that can take advantage of this Achilles Heel and limit virus load, if not totally prevent infection.
 
Our most recent studies have been a retrospective examination of a large bank of cat sera amassed from infected and uninfected pet cats as well as experimentally infected animals, with the mission to determine how infected cats react to the viral glycoprotein and how the immune response might relate to the ability of the cats to control virus infection.  We examined sera from approximately 600 cats, roughly half of which were positive for FIV infection.  Where data were available, we compared the level and quality of the immune response to the health and viral load in each animal.  As expected, only the FIV positive cats expressed antibodies reactive with the virus and all infected animals, if they did not die early from the infection, had antibodies to the viral glycoprotein.  Additionally, these cats had neutralizing antibodies as well, but surprisingly, these antibodies did not correlate significantly with reduced viral loads.  This is likely due to the generation of mutations in the viral glycoprotein, which allows the virus to stay one step ahead of the immune system.  Of great interest was the finding that a preponderance of long-lived infected cats also had antibodies to the virus receptor, CD134.  Furthermore, we noted a correlation between better health and lower viral loads in animals expressing high levels of anti-receptor antibodies.  Subsequent tests showed that these anti-CD134 antibodies could block virus infection by reacting with sites on the receptor only exposed when virus bound to the cell.  No adverse autoimmune responses have been reported for FIV infection, likely due to the normally cryptic nature of these CD134 epitopes.  Important to vaccine development, the receptor does not mutate and change like the virus and thus may offer a more stationary target for vaccine development. These findings offer an important new approach to vaccine development that we will attempt to exploit in the coming year.

PROTEASE SPECIFICITY

The viral protease is a critical enzyme of the virus that is responsible for processing the structural proteins and enzymes from long polyproteins. Processing must occur at the proper time and in the proper order to make infectious virus and thus the protease is an excellent drug target. As reported previously, we have prepared chimeric proteases in which substitutions of amino acids of HIV protease have been placed in equivalent positions of FIV protease, as a means to define residues critical to substrate and inhibitor specificity.  These chimeric proteases work well in the test tube and show increased HIV character, particularly as to drug sensitivity.  However, getting infectious virus has been problematic, due to subtle changes in the processing of the viral polyproteins by the mutant proteases.  In the past year, we have succeeded in producing infectious FIVs that express these chimeric proteases. Of importance to our goals, the chimeric FIVs have taken on the drug sensitivities of HIV.  These viruses can now be used to further investigate the molecular evolution of substrate/inhibitor specificity.  
 
PUBLICATIONS

de Rozìeres, S., Thompson, J., Sundstrom, M., Gruber, J., Stump, D.S., de Parseval, A.P., VandeWoude, S., Elder, J.H. Replication properties of clade A/C chimeric feline immunodeficiency viruses and evaluation of infection kinetics in the domestic cat. J. Virol. 2008 Aug;82(16):7953-63. PMID:PMC2519559.
 
Elder, J.H., Sundstrom, M., de Rozieres, S., de Parseval, A, Grant, C.K., Lin, Y.C. Molecular mechanisms of FIV infection. Vet Immunol Immunopathol. 2008 May 15;123(1-2):3-13 PMID:PMC2409060.
 
Heaslet H, Rosenfeld R, Giffin M, Lin YC, Tam K, Torbett BE, Elder JH, McRee DE, Stout CD. Conformational flexibility in the flap domains of ligand-free HIV protease.  Acta Crystallogr D Biol Crystallogr. 2007 Aug;63(Pt 8):866-7 PMID:17642513.
 
Khurana, S., Krementsov, D.N., de Parseval, A., Elder, J.H., Foti, M., Thali, M. Human immunodeficiency virus type 1 and influenza virus exit via different membrane microdomains. J Virol. 2007 Nov;81(22):12630-40 PMID:17855546.
 
Sundstrom, M., White, R.L., de Parseval, A., Sastry, K.J., Morris G., Grant, C.K., Elder J.H. Mapping of the CXCR4 binding site within variable region 3 of the feline immunodeficiency virus surface glycoprotein. J. Virol. 2008 Sep;82(18):9134-42 PMID:PMC2546885.
 
Sundstrom, M., Chatterji, U., Schaffer, L., de Rozières, S., Elder, J.H. Feline immunodeficiency virus OrfA alters gene expression of splicing factors and proteasome-ubiquitination proteins.  Virology. 2008 Feb 20;371(2):394-404 PMID:1796381.

Index terms
 
AIDS. See HIV infection.
Feline immunodeficiency virus
            as AIDS model
            proteases of
            receptors for
HIV infection
Receptors
            for FIV
Viruses
            feline immunodeficiency virus
            HIV
            lentiviruses
            retroviruses

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