The focus of our research is to understand the molecular basis for the pathogenesis of mouse retroviruses. Our recent studies have concentrated on retroviruses that cause leukemia or neurological disease in rodents. The overall aim of our research is to obtain basic information on how these viruses induce molecular changes in normal cells that result in pathological consequences. To date, our studies have identified a number of molecular events associated with the induction of these retrovirus-induced diseases that we can potentially target for therapeutic intervention. We hope to use the information gained from these animal studies to develop rational strategies for treating similar diseases in man. Friend spleen focus-forming virus (SFFV) induces an acute erythroleukemia in mice and provides an important model for understanding how deregulation of hematopoietic pathways can lead to leukemia. Normal erythroid cells require the hormone erythropoietin (Epo) for their growth and differentiation, and when they are deprived of Epo, they undergo apoptosis. In contrast, erythroid cells infected with SFFV do not die when deprived of Epo, but continue to proliferate and differentiate, leading to erythroid hyperplasia and polycythemia. The SFFV envelope protein, gp55, interacts at the cell surface with the Epo receptor complex, and previous studies from our laboratory demonstrated that this results in the constitutive activation of signal transducing molecules normally activated by Epo, including Stat proteins, components of the Raf-1/MAP kinase pathway, PI 3-kinase, protein kinase C and Akt kinase. While PI 3-kinase and protein kinase C are required for Epo-independent proliferation, Stat proteins appear to be involved in the induction of Epo-independent differentiation by the virus. Our studies further suggest that SFFV-infected erythroid cells are protected from apoptosis during Epo withdrawal due to the upregulation in these cells of the anti-apoptotic protein Bcl-XL as well as by the constitutive phosphorylation of the pro-apoptotic protein BAD, which blocks its function. Our recent studies indicate that the Epo-independent proliferation and differentiation of SFFV-infected erythroid cells also requires the expression of a truncated form of the receptor tyrosine kinase Stk, termed sf-Stk, which was recently implicated in susceptibility to SFFV-induced erythroleukemia. We showed that SFFV gp55 covalently interacts with sf-Stk, which significantly extends the half life of the kinase and leads to its constitutive tyrosine phosphorylation and subsequent interaction with the Epo receptor and various signal transducing molecules. SFFV-infected erythroid cells expressing mutants of sf-Stk that lack kinase activity or specific tyrosine residues do not proliferate and differentiate in the absence of Epo. Interestingly, expression of a constitutively activated form of sf-Stk in erythroid cells in the absence of SFFV can induce their Epo-independent proliferation and diffentiation, suggesting that the induction of Epo-independence by SFFV is mediated by sf-Stk after it has been activated by its association with SFFV gp55. Studies to determine if constitutively activated sf-Stk can induce the same disease as SFFV in mice are in progress. Finally, we have isolated a novel Ste20/GCK-related protein kinase that is upregulated when SFFV-transformed cells are induced to polyploidy by growth in serum-free medium. Our studies indicate that this kinase, PAPK, functions upstream of MKK4 and MKK7 and modulates cytoskeletal organization and cell survival. PVC-211 murine leukemia virus, which is a variant of the leukemia-inducing Friend MuLV, induces rapid spongiform neurodegeneration when injected into newborn rats. It serves as a model not only for elucidating how retroviral-induced changes in the brain can lead to neurological disease but also for understanding how retroviruses can undergo genetic changes that alter their interaction with cells in the host to cause novel biological effects. The primary target of PVC-211 MuLV infection in the central nervous system (CNS) is the capillary endothelial cell (BCEC), which is resistant to F-MuLV infection. Our previous studies showed that changes in the envelope gene of PVC-211 MuLV confer BCEC tropism to the virus by creating a unique heparin binding site that may allow the virus to efficiently interact with the negatively charged surface of BCEC, facilitating receptor binding and entry. Within the CNS, BCEC are the only cells infected with virus, indicating that molecular events occurring in PVC-211 MuLV-infected BCEC play a crucial role in neurological disease induction. Since excessive production of nitric oxide (NO) can lead to neurotoxicity, we carried out studies to determine if infection of BCEC by PVC-211 MuLV results in the activation of NO, which is synthesized from L-arginine by NO-synthases (NOS). Our recent studies indicate that inducible NOS, iNOS, is specifically activated in BCEC from PVC-211 MuLV-infected rats. We also detect elevation of a 32 kDa cellular protein modified by tyrosine nitration, a hallmark of NO production. These changes were not detected in BCEC from rats infected with a BCEC-tropic, but nonpathogenic variant of the virus, suggesting that they are important for the development of neurological disease. The mechanism by which PVC-211 MuLV induces the expression of iNOS in BCEC remains to be elucidated. Expression of a viral protein in these cells does not appear to be sufficient to activate iNOS, since we failed to detect elevated iNOS protein in BCEC after in vitro infection with PVC-211 MuLV. Furthermore, PVC-211 MuLV, but not a BCEC-tropic, non-neuropathogenic variant of the virus, can be seen budding from the brain side of BCEC, suggesting that the virus needs to be present in the brain parenchyma to initiate the events leading to iNOS production and neurological disease. Activated microglia are found adjacent to PVC-211 MuLV-infected BCEC and may be involved in stimulating BCEC to make iNOS.
