Our laboratory is interested in the replication of retroviruses, the interactions of these viruses with the host, and in signal transduction pathways that are activated in retrovirus-induced tumors. A combination of biochemical and genetic methods are being used to analyze the steps occurring early in infection. Fractionation of cell extracts has helped define the components and properties of a large complex of proteins that mediate the reverse transcription of the viral RNA into DNA and its subsequent movement into the cell nucleus. The group has used panels of viral mutants to help identify key proteins; for example, Gag mutants have been identified that synthesize DNA but hold it in a form that is not properly guided to the nucleus and integrated. There are also indications that host gene products are important in these early steps of infection. We have isolated mutant cell lines that are resistant to retroviral infection by both MuLVs and HIVs, and is presently using DNA-mediated transfection to identify and characterize the host genes involved in these steps.
We are also interested in the steps involved in the assembly and maturation of virion particles. In a chronically infected cell, a number of viral proteins are brought together under the plasma membrane to form spherical particles that bud outward and are released from the cell surface. The major player in assembly is the viral Gag protein, often dubbed "the particle-making machine"; a number of Gag mutants are affected in virion assembly. Mutants lacking the p12 Gag domain do not efficiently form round particles but instead form long tubes. Other mutants with alterations in a specific sequence, the PPPY motif, form round particles but do not properly release them from the cell surface, producing chains of linked particles.
A number of protein-protein interactions are involved in the formation of virions. Our group has been particularly active in utilizing the two-hybrid system, a method for screening for protein-protein interactions between partners expressed in yeast, to monitor contacts that are important in virus assembly and to select for mutants with altered binding. These studies include analysis of Gag-Gag interactions, the dimerization of the subunits of reverse transcriptase, and the trimerization of the transmembrane subunits of the envelope. A number of novel host proteins that bind to the Moloney MuLV Gag precursor have also been found. Some of these proteins are incorporated into virion particles through their interactions with Gag, and dominant negative forms of others can act to suppress virion assembly and release.
A variation of the two-hybrid system, sometimes called the three-hybrid system, allows for the detection of RNA-protein interactions in yeast.This system has been used to demonstrate the specific binding of the HIV Gag protein to that portion of the genomic RNA required for its encapsidation into virion particles.
Signal Transduction by an Activated Retroviral Oncogene: the v-Abl Tyrosine Kinase
Retroviruses can recombine with host genes during their replication to form viral oncogenes with potent transforming activities. These gene products often stimulate signal transduction pathways inappropriately in infected cells, and lead to constitutive mitogenic signalling. We have been especially interested in one such molecule, the activated Abl tyrosine kinase expressed by the Abelson murine leukemia virus in pre-B cell lymphomas. We have identified several novel Abl-binding proteins that may mediate Abl signalling. One such protein, termed Abi-1, binds to Abl by a conserved SH3 (for src homology domain 3) motif, and is linked to a number of other interesting molecules. Abi-1 is bound to the receptor for the platelet-derived growth factor (PDGF), and may potentiate the mitogenic response to the ligand. In addition, Abi-1 binds tightly to SOS-2 (son of sevenless), a guanosine exchange protein involved in Ras activation. Immunostaining of cells expressing epitope-tagged versions of the protein show that Abi-1 is localized to the cytoskeleton and to punctate structures similar to those containing SOS. These studies suggest that Abi-1 may be a bridging molecule that links Abl to other proteins, both upstream and downstream of the kinase function. We are also interested in two other molecules that interact with Abl: Dokl, a member of the Dok family, which binds to Abl and can inhibit its signal transduction and transforming activities; and a novel protein, Aph2, which binds to Abl and may potentiate its ability to induce cell cycle arrest and cell death.
Although Abl is ubiquitously expressed in all cells, it may play essential roles in only a few specialized cell types. Knock-out mice deficient in the Abl kinase show defects in B cell development, with variable but often severe depletion of pro-B and pre-B compartments in the bone marrow. This deficiency may be mediated by the hypersensitivity of these cells to apoptotic stimuli. We have recently discovered that the mutant mice are also profoundly osteoporotic. The bone volume and density is reduced, and the rate of mineral apposition is much lower than that of control littermates. The major defect is attributable to osteoblasts, the fibroblastic cells responsible for bone deposition. Osteoblasts cultured from the mutant mice show delayed maturation, based on induction of three osteogenic markers in vitro. We are continuing to characterize these cell cultures to identify those signal transduction pathways that may be defective in the absence of the Abl kinase.