Torbett Laboratory

Research Summary

Our group has three research areas: Regulation of normal and abnormal myeloid development and function by the ets transcription factor PU.1; Hematopoietic stem and T cell gene delivery strategies to block HIV-1 entry and limit infection; and Evolution of HIV-1 protease inhibitor resistance.



Defining PU.1 regulated genes and cellular pathways in hematopoietic cells

PU.1, an ets transcription factor family member, is expressed in hematopoietic (blood) cells. PU.1 gene-disrupted mice are devoid of B and dendritic cells, monocytes / macrophages, and mature neutrophils, but not T cells. Thus, PU.1 is necessary for controlling monocyte/macrophage and dendritic cell commitment and differentiation and neutrophil differentiation. PU.1 not only has a role in controlling development, but we and others have shown that it is also required for regulating genes necessary for monocyte / macrophage, dendritic, and neutrophil function. Finally, partial disruption of PU.1 or limited expression promotes a form of acute myelogenous leukemia in mice.

PU.1 is composed of three domains, an N-terminal transactivation domain, a PEST domain, and a C-terminal DNA binding (ets) domain. These domains interact with various cellular genes to promote differentiation and cellular function. To uncover pathways regulated by PU.1, we developed various PU.1 domain mutants and stably expressed these mutant PU.1 genes, as well as a wild type PU.1 gene, in a PU.1 negative cell line. The resulting cell lines were analyzed allowing the identification of novel genes, pathways, and functions regulated by PU.1 in a directly or indirectly. We are currently studying two PU.1 regulated genes identified in the screen, TREM1 and TREM2, and their pathways, in monocytes / macrophages.


Hematopoietic cell gene-delivery to control HIV-1 infection

Long-term antiretroviral treatment is effective at suppressing HIV-1 replication in patients and maintaining T cell function. However, long-term drug treatment can cause physiological changes, resulting in health problems. Moreover, drug resistance develops and salvage therapies are required. One possible remedy is to genetically modify an individual's hematopoietic and / or CD4 T cells such that they are refractory to HIV-1 infection or capable of controlling viral infection. Disruption of cellular pathways utilized by HIV-1 limits viral entry, viral production, cell death, and is difficult for the virus to evolve resistance around. Furthermore, not all HIV-1 susceptible cells would require protection, since CD4 T cells and other cells protected from HIV-1 infection should survive and expand during an infection.

Our group is evaluating the biological and virological effects of genomic modification of CXCR4 one of two chemokine receptors that are viral entry portals in hematopoietic and CD4 T cells. For genomic modification of chemokine receptors we have relied on gene delivery of zinc finger nucleases targeting CXCR4, as well as other genetic modification strategies. As mentioned, the rationale for this strategy is that cells without a viral entry portal would be protected from viral infection allowing preservation of the immune system in the presence of an ongoing infection.

A limitation to genomic modification of hematopoietic CD34+ stem cells is the partially refractory nature of these cells to gene delivery via lentiviral vectors, as well as by other methods. The inability to successfully target stem cells results in a low frequency of cells being genomically modified. To improve genomic modification of stem cells, we are determining what cellular pathways limit lentiviral gene delivery and how to overcome this limitation and increase the frequency of stem cell targeting.


Evolution of HIV-1 resistance to inhibitors

Combination antiretroviral treatment of HIV-1 infected individuals result in viral suppression. However, treatment interruption can result in treatment resistance to one or all of the inhibitors used. Protease inhibitors, commonly one of the three inhibitors used in combination therapy, are potent and suppress viral replication. However, resistance can arise to both the inhibitor used and other classes of protease inhibitors, termed cross-resistance.

Resistance is supported by mutations arising in protease and its substrate, Gag-Pol, which alters both biochemical and viral fitness. We and others have shown that initial drug resistance mutations are at the active site of the protease, decreasing the affinity for the inhibitor, as well as the enzymatic activity for viral substrates. Moreover, these initial drug resistant mutations destabilize the protease structure. The cumulative result of the resistant mutations produces a less fit virus. Continued drug pressure (treatment) results in the selection of distal mutations in the flap, elbow, and other protease regions. These distal, accessory mutations restore protease catalytic efficiency, as well as stabilize the protein.

Drug resistance mutations in Gag, which are components of the viral capsid necessary for viral maturation and a substrate of protease, occur at cleavage sites and in distal Gag regions. The drug resistance mutations in Gag cleavage sites facilitate protease cleavage by altering catalytic efficiency. We have shown that distal Gag mutations also enhance catalytic efficiency, presumably by altering Gag structure. Studies are underway to determine Gag structures to resolve the mechanisms whereby Gag mutations enhance catalytic efficiency.

To evolve viral resistance in protease and Gag, we have established high-throughput assays, and in collaboration with our colleagues in Chemistry Department at TSRI, have identified novel inhibitors of protease and Gag function. These novel inhibitors are used as chemical probes to drive resistance, allowing us to investigate and understand protein plasticity and dynamics of both protease and Gag as they co-evolve inhibitor resistance, while contributing to maintain viral function and fitness. Lastly, the biochemical and structural changes resulting from drug resistance are being investigated as to their resulting effect on viral fitness.

Insights gleaned from our studies will allow predication on mutational pathways resulting from protease inhibitor treatment. Moreover, our findings will provide information as to which locations in protease and Gag are difficult to mutate around when targeted by combinations of small molecules and if mutations occur, the resulting virus will be less fit to promote disease. We anticipate that by understanding the limits to resistance plasticity in protease and Gag we can corral and thus control the virus.



Department of Immunology and Microbiology

The Scripps Research Institute

Director, Molecular Basis of Viral Pathogenesis Training Program

Director, CFAR Protein Expression and Proteomics (PEP) Core

Co-Director, HIV Interaction and Viral Evolution (HIVE) Center

Co-Director, UCSD Center for AIDS Research (CFAR)


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