Scientific Report 2007

Molecular and Experimental Medicine

Division of Blood Cell and Vascular Biology

Control of HIV Type 1, Gene Delivery, and Regulation of Hematopoietic Development

B.E. Torbett, G. Cauvi, L. Crisa, K.M. Fischer, G.E. Foos, M.J. Giffin, D. Vanitha John, P.A. McClintock, S. Miller, R.C. Prinsen, J.H. Savage, R. Schrier, C.H. Swan, J.A. Witkowski, A. Brik,* J.H. Elder,** H. Heaslet,** N.L. Letvin,*** Y.-C. Lin,** C.D. Stout,** M.P. Tschan,**** C.-H. Wong*

* Department of Chemistry, Scripps Research
** Department of Molecular Biology, Scripps Research
*** Beth Israel Deaconess Medical Center,, Boston, Massachusetts
**** University of Bern, Bern, Switzerland

Our research interests include the structural and biochemical evolution of the resistance of HIV type 1 (HIV-1) proteases, gene delivery strategies to disrupt cellular entry of HIV-1, and normal and abnormal regulation of myeloid development by the transcription factors PU.1 and cyclin D–interacting Myb-like protein (DMP1).

HIV-1 Protease Resistance

In patients infected with HIV-1, treatment with inhibitors of HIV reverse transcriptase, integrase, and protease suppresses replication of the virus. However, in some patients, HIV-1 variants evolve that escape the approved drug treatments by developing a broad-based resistance to the protease inhibitors. A molecular understanding of the resistance to protease inhibitors is needed so that new inhibitors can be developed to that target drug-resistant viruses and, importantly, are less likely to induce inhibitor-resistant viruses.

In collaboration with J.H. Elder, C.D. Stout, and H. Heaslet, Department of Molecular Biology, we showed that evolution of HIV-1 protease from a form susceptible to inhibitors to a form that is broadly resistant resulted in profound changes in the protease structure. Structural changes in the resistant proteases included alterations in the flap and basal regions and alteration from a symmetric to an asymmetric protease. To better understand how selected inhibitors disrupt function of resistant proteases, we used an inhibitor that targets and inhibits a resistant protease to probe the structure of the protease. We determined the necessity for interactions between the inhibitor and the protease backbone in the resistant protease. The structural changes that occur during the development of resistance and the use of new protease inhibitors as chemical probes provide insight into the biochemical basis for the loss of activity of protease inhibitors. To better understand how structure contributes to the biochemical basis of resistance, we are continuing investigations on the relationship between structure and function in our wild-type proteases and in mutant proteases that are broadly resistant to inhibitors.

HIV-1 Vector Delivery of CCR5-Intrabody Genes to Human Hematopoietic Cells

CXCR4 and CCR5 are the main chemokine receptors for HIV-1 entry into cells, and blocking these receptors limits entry of the virus. Naturally occurring polymorphisms of the gene for CCR5 indicate that disruption of the gene provides protection from viruses that use CCR5 to gain entry. Because polymorphisms are present in healthy persons, the use of genetic intervention strategies that prevent or limit expression of CCR5 may provide protection from initial infection and limit the spread of the virus.

With C.F. Barbas, Department of Molecular Biology, we showed that intracellular expression of a CCR5-specific single-chain antibody (intrabody) efficiently disrupted expression of CCR5 on the T-cell surface and protected cells from HIV-1 infection. Moreover, we found that human stem cells expressing the CCR5-intrabody develop into T cells and that the decreased expression of CCR5 protected cells against HIV-1 challenge and imparted a survival advantage in the presence of HIV-1 infection. Thus, it seems that gene delivery can provide gene programs that will protect and allow expansion of protected cells during HIV-1-infection.

Currently, we are disrupting the function of viruses that use either the CXCR4 or the CCR5 receptor for entry, the so-called R5X4 viruses. To accomplish our goals, we are using combination vectors that genetically target chemokine receptors and viral and cellular pathways critical for viral entry and replication.

Myeloid Differentiation

PU.1, a member of the Ets family of transcription factors, is expressed solely in hematopoietic cells and is necessary for directing myeloid development and for regulating genes required for monocyte/macrophage and neutrophil function. PU.1 has 3 major domains: the transactivation, PEST, and Ets/DNA-binding domains. PU.1 interacts with other transcription factors, and domains of PU.1 have been implicated in its function.

Myeloid development is controlled by temporal gene expression of PU.1 and interactions among specific transcription factors. We are addressing which PU.1 domains regulate myeloid lineage–specific commitment, differentiation, and function. To determine which transcription factors interact with PU.1 and direct myeloid development, we use a strategy in which the gene for PU.1 is expressed only under certain conditions and a gene discovery and proteomics approach. These studies are enabling us to identify gene programs regulated by PU.1.

Cancer often originates from inactivation and/or deregulation of the control of gene expression. The transcription factor DMP1 positively regulates expression of human p14^ARF and CD13/aminopeptidase N, thus playing a role in cell-cycle control, differentiation, and function of hematopoietic and nonhematopoietic cells. The tumor suppressor ARF is critical for positive regulation of p53, which in turn controls cellular proliferation and modulates apoptosis. We have identified 2 novel and developmentally expressed human DMP1 splice variants: β and γ. We found that the β variant functions as a dominant-negative regulator of the originally reported DMP1 protein. Currently, we are investigating the molecular and biological roles of the various isoforms in the development of normal and leukemic cells.

Publications

Heaslet, H., Lin, Y.-C., Tam, K., Torbett, B.E., Elder, J.H., Stout, C.D. Crystal structure of an FIV/HIV chimeric protease complexed with the broad-based inhibitor, TL-3. Retrovirology 4:1, 2007.

Heaslet, H., Rosenfeld, R., Giffin, M., Lin, Y.C., Tam, K., Torbett, B.E., Elder, J.H., McRee, D.E., Stout, C.D. Conformational flexibility in the flap domains of ligand-free HIV protease. Acta Crystallogr. D Biol. Crystallogr. 63(Pt. 8):866, 2007.

Lin, Y.-C., Brik, A., Parseval, A., Tam, K., Torbett, B.E., Wong, C.-H., Elder, J.H. Altered gag polyprotein cleavage specificity of feline immunodeficiency virus/human immunodeficiency virus mutant proteases as demonstrated in a cell-based expression system. J. Virol. 80:7832, 2006.

Manuel, E.R., Charini W.A., Sen, P., Peyerl, F.W., Kuroda, M.J., Schmitz, J.E., Autissier, P., Sheeter, D.A., Torbett, B.E., Letvin, N.L. Contribution of T-cell receptor repertoire breadth to the dominance of epitope-specific CD8⁺ T-lymphocyte responses. J. Virol. 80:12032, 2006.

Rizzi, M., Tschan, M.P., Britschgi, C., Britschgi, A., Hügli, B., Grob, T.J., Leupin, N., Mueller, B.U., Simon, H.U., Ziemiecki, A., Torbett, B.E., Fey, M.F., Tobler, A. The death-associated protein kinase 2 is up-regulated during normal myeloid differentiation and enhances neutrophil maturation in myeloid leukemic cells. J. Leukoc. Biol. 81:1599, 2007.

Swan, C.H., Bühler, B., Steinberger, P., Tschan, M.P., Barbas, C.F. III, Torbett, B.E. T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery [published correction appears in Gene Ther. 14:626, 2007]. Gene Ther. 13:1480, 2006.

Swan, C.H., Torbett, B.E. Can gene delivery close the door to HIV-1 entry after escape? J. Med. Primatol. 35:23, 2006.

Tschan, M.P., Britschgi, C., Moser, B.A., Reddy, V.A., Sheeter, D.A., Fischer, K.M., Sun, P., Torbett, B.E. Human DMP1β is a negative regulator of the p14^ARF tumor suppressor and promotes cellular proliferation. J. Biol. Chem., in press.

Yeamans, C., Wang, D., Paz-Priel, I., Torbett, B.E., Tenen, D.G., Friedman, A.D. C/EBPα binds and activates the PU.1 distal enhancer to induce monocyte lineage commitment. Blood, in press.