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Genetics of Retroviral Replication and Retroviral Oncogenes
Summary: Stephen Goff is interested in the replication of retroviruses, and the cellular gene products that interact with these viruses.
My laboratory studies the replication of retroviruses, the interactions of these viruses with the host, and the signal transduction pathways that are activated in retrovirus-induced tumors. The retroviruses, a large family of RNA viruses found in all eukaryotes, include such important pathogens as the human immunodeficiency viruses (HIVs). These viruses have an extraordinary lifestyle: the RNA genome is converted into a double-stranded DNA, which is inserted into the host genome and then transcribed to make new viral RNAs.
As a direct result of the integration of the viral DNA into the host genome, retroviruses can cause an impressive variety of effects in an infected host. The viruses persist in the genome for the lifetime of an infected cell and an infected individual; they can even be transmitted to offspring in the germline as integrated DNA. Many cause leukemias and other malignancies by inappropriately activating genes near the site of viral DNA insertion. They can also acquire host genes and carry them into newly infected individuals, causing a range of distinctive and aggressive tumors. These properties make the retroviruses formidable pathogens. Blocking their replication will require a deep understanding of the functions of each of the viral proteins.
Early Events of Retrovirus Infection
We are using a combination of biochemical and genetic methods 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 and insertion into the host DNA. We have identified particular mutants of the viral gag gene that synthesize linear viral DNA but hold it in a form that is not properly guided to the nucleus and integrated. Furthermore, we found that the capsid protein of the mouse retroviruses must be modified by specific cellular machinery (the SUMO transferases) to allow the nuclear entry and integration of the viral DNA.
We have isolated mutant cell lines that are resistant to retroviral infection by both the murine leukemia viruses (MuLVs) and HIVs, and we are using DNA-mediated transfection and DNA microarrays to identify and characterize the host genes involved in these steps. Overexpression or underexpression of a number of host proteins—such as IQGAP, moesin, and FEZ1, regulators of the actin cytoskeleton—can profoundly block retroviral infection. Some of these cell lines exhibit abnormalities in cytoskeletal structures and intracellular trafficking of surface receptors that correlate with their virus resistance.
Late Stages of Infection
Our laboratory is also interested in the processes of viral gene expression and the assembly of virion particles, and many efforts have been directed toward identifying host genes involved in these stages of virus replication. We have isolated dominant-acting mutant versions of several nuclear host proteins that block HIV-1 RNA formation and processing, identified components of transcriptional silencing complexes that block MuLV transcription in embryonic stem cells, and characterized a highly structured RNA suppressor of translational termination (a so-called pseudoknot) that is required for synthesis of the retroviral Pol gene products.
We have screened large mammalian cDNA expression libraries for genes that can block virus replication and have identified ZAP, a novel zinc finger protein with potent antiviral activity. Analysis of infected cells shows that overexpression of ZAP causes a profound and specific reduction in the levels of viral mRNA in the cytoplasm, without affecting levels in the nucleus. ZAP exhibits potent activity against a number of other RNA viruses, including such alphaviruses as Sindbis virus, suggesting that it may represent part of an innate antiviral immune response. We have found that ZAP binds specifically to these viral RNAs and utilizes the exosome, a nuclease machine, to degrade them.
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 (see Figures 1 and 2). The major player in assembly is the viral Gag protein, often called "the particle-making machine." We have been particularly active in utilizing the two-hybrid system, a method for screening for protein-protein interactions between partners expressed in yeast, to identify 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 (RT), and the trimerization of the transmembrane subunits of the envelope protein.
We have also identified a number of novel host proteins that bind to the Moloney MuLV Gag precursor (including the endophilins, likely involved in inducing membrane curvature); to RT; to the viral integrase enzyme; and to the envelope protein. Some of these proteins are incorporated into virion particles, and dominant-negative forms of several can act to suppress virion assembly and release.
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 and lead to constitutive mitogenic signaling. We are especially interested in one such molecule, the activated Abl tyrosine kinase expressed by the Abelson murine leukemia virus in pre–B cell lymphomas. Activation of this kinase by chromosomal translocation in humans leads to chronic myelogenous leukemia, a disease whose treatment has been revolutionized by the advent of Gleevec, an Abl inhibitor. We are working to characterize the downstream signaling pathways activated by Abl, and we have identified several novel proteins that bind and may mediate Abl kinase activity. We are particularly interested in PST-PIP1, a molecule that directs a major phosphatase, the PEST protein-tyrosine phosphatase, to dephosphorylate Abl and so negatively regulate its kinase activity. Another interacting protein is UV-DDB (UV-damaged DNA-binding activity), a sensor of DNA damage and initiator of the DNA damage response, encoded by the XPE gene in humans. We have recently identified several new UV-DDB–interacting proteins that may contribute to Abl signaling.
To further probe Abl function, we have generated knockout mice deficient in the Abl kinase and characterized defects in several organ systems and tissues. These mice are deficient in pro–B and pre–B cells in the bone marrow and the B1 subclass in the peritoneum. They are osteoporotic, due to defects in development of osteoblasts, the cells responsible for bone deposition. Other conditional knockout mice depleted of the DDB1 protein have led us to the realization that this protein, regulated by Abl, is in turn a critical regulator of the cell cycle and of cell survival. Depletion of DDB1 in the brain, liver, or skin leads to profound failures of organogenesis and selective loss of dividing cells. Further analysis of knockout mice lacking these and other Abl targets is under way.
Grants from the National Institutes of Health and the Foundation Fighting Blindness supported some of our studies on the murine retroviruses and on the analysis of knockout mice.
Last updated: May 1, 2008
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