Molecular evolution of primate immunodeficiency viruses and hepatitis delta virus

Primate immunodeficiency viruses, or lentiviruses (HIV-1, HIV-2, and SIV), and hepatitis delta virus (HDV) are RNA viruses characterized by rapid evolution. Infection by primate immunodeficiency viruses usually results in the development of acquired immunodeficiency syndrome (AIDS) in humans and AIDS-like illnesses in Asian macaques. Similarly, hepatitis delta virus infection causes hepatitis and liver cancer in humans. These viruses are heterogeneous within an infected patient and among individuals. Substitution rates in the virus genomes are high and vary in different lineages and among sites. Methods of phylogenetic analysis were applied to study the evolution of primate lentiviruses and the hepatitis delta virus. The following results have been obtained: (1) The substitution rate varies among sites of primate lentivirus genes according to the two parameter gamma distribution, with the shape parameter $\alpha$ being close to 1. (2) Primate immunodeficiency viruses fall into species-specific lineages. Therefore, viral transmissions across primate species are not as frequent as suggested by previous authors. (3) Primate lentiviruses have acquired or lost their pathogenicity several times in the course of evolution. (4) Evidence was provided for multiple infections of a North American patient by distinct HIV-1 strains of the B subtype. (5) Computer simulations indicate that the probability of committing an error in testing HIV transmission depends on the number of virus sequences and their length, the divergence times among sequences, and the model of nucleotide substitution. (6) For future investigations of HIV-1 transmissions, using longer virus sequences and avoiding the use of distant outgroups is recommended. (7) Hepatitis delta virus strains are usually related according to the geographic region of isolation. (8) Evolution of HDV is characterized by the rate of synonymous substitution being lower than the nonsynonymous substitution rate and the rate of evolution of the noncoding region. (9) There is a strong preference for G and C nucleotides at the third codon positions of the HDV coding region. ^

Characterization of E2F1's oncogenic, apoptotic and tumor-suppressive activities utilizing the K5 mouse model

Though E2F1 is deregulated in most human cancers by mutations of the p16-cyclin D-Rb pathway, it also exhibits tumor suppressive activity. A transgenic mouse model overexpressing E2F1 under the control of the bovine keratin 5 (K5) promoter exhibits epidermal hyperplasia and spontaneously develops tumors in the skin and other epithelial tissues after one year of age. In a p53-deficient background, aberrant apoptosis in K5 E2F1 transgenic epidermis is reduced and tumorigenesis is accelerated. In sharp contrast, K5 E2F1 transgenic mice are resistant to papilloma formation in the DMBA/TPA two-stage carcinogenesis protocol. K5 E2F4 and K5 DP1 transgenic mice were also characterized and both display epidermal hyperplasia but do not develop spontaneous tumors even in cooperation with p53 deficiency. These transgenic mice do not have increased levels of apoptosis in their skin and are more susceptible to papilloma formation in the two-stage carcinogenesis model. These studies show that deregulated proliferation does not necessarily lead to tumor formation and that the ability to suppress skin carcinogenesis is unique to E2F1. E2F1 can also suppress skin carcinogenesis when okadaic acid is used as the tumor promoter and when a pre-initiated mouse model is used, demonstrating that E2F1's tumor suppressive activity is not specific for TPA and occurs at the promotion stage. E2F1 was thought to induce p53-dependent apoptosis through upregulation of p19ARF tumor suppressor, which inhibits mdm2-mediated p53 degradation. Consistent with in vitro studies, the overexpression of E2F1 in mouse skin results in the transcriptional activation of the p19ARF and the accumulation of p53. Inactivation of either p19ARF or p53 restores the sensitivity of K5 E2F1 transgenic mice to DMBA/TPA carcinogenesis, demonstrating that an intact p19ARF-p53 pathway is necessary for E2F1 to suppress carcinogenesis. Surprisingly, while p53 is required for E2F1 to induce apoptosis in mouse skin, p19ARF is not, and inactivation of p19ARF actually enhances E2F1-induced apoptosis and proliferation in transgenic epidermis. This indicates that ARF is important for E2F1-induced tumor suppression but not apoptosis. Senescence is another potential mechanism of tumor suppression that involves p53 and p19ARF. K5 E2F1 transgenic mice initiated with DMBA and treated with TPA show an increased number of senescence cells in their epidermis. These experiments demonstrate that E2F1's unique tumor suppressive activity in two-stage skin carcinogenesis can be genetically separated from E2F1-induced apoptosis and suggest that senescence utilizing the p19ARF-p53 pathway plays a role in tumor suppression by E2F1. ^

Oncogenic activation of c-Abl tyrosine kinase in non-small cell lung cancer: FUS1 tumor suppressor down-regulates c-Abl tyrosine kinase

The FUS1 tumor suppressor gene (TSG) has been found to be deficient in many human non-small cell lung cancer (NSCLC) tissue samples and cell lines (1,2,3). Studies have shown potent anti-tumor activity of FUS1 in animal models where FUS1 was delivered through a liposomal vector (4) and the use of FUS1 as a therapeutic agent is currently being studied in clinical human trials (5). Currently, the mechanisms of FUS1 activity are being investigated and my studies have shown that c-Abl tyrosine kinase is inhibited by the FUS1 TSG.^ Considering that many NSCLC cell lines are FUS1 deficient, my studies further identified that FUS1 deficient NSCLC cells have an activated c-Abl tyrosine kinase. C-Abl is a known proto-oncogene and while c-Abl kinase is tightly regulated in normal cells, constitutively active Abl kinase is known to contribute to the oncogenic phenotype in some types of hematopoietic cancers. My studies show that the active c-Abl kinase contributes to the oncogenicity of NSCLC cells, particularly in tumors that are deficient in FUS1, and that c-Abl may prove to be a viable target in NSCLC therapy.^ Current studies have shown that growth factor receptors play a role in NSCLC. Over-expression of the epidermal growth factor receptor (EGFR) plays a significant role in aggressiveness of NSCLC. Current late stage treatments include EFGR tyrosine kinase inhibitors or EGFR antibodies. Platelet-derived growth factor receptor (PDGFR) also has been shown to play a role in NSCLC. Of note, both growth factor receptors are known upstream activators of c-Abl kinase. My studies indicate that growth factor receptor simulation along deficiency in FUS1 expression contributes to the activation of c-Abl kinase in NSCLC cells. ^

Acceleration of the PanIN Development in Mice Expressing Oncogenic K-Ras Due to a High Fat Diet

Obesity is postulated to be one of the major risk factors for pancreatic cancer, and recently it was indicated that an elevated body mass index (BMI correlates strongly with a decrease in patient survival. Despite the evident relationship, the molecular mechanisms involved are unclear. Oncogenic mutation of K-Ras is found early and is universal in pancreatic cancer. Extensive evidence indicates oncogenic K-Ras is not entirely active and it requires a triggering event to surpass the activity of Ras beyond the threshold necessary for a Ras-inflammation feed-forward loop. We hypothesize that high fat intake induces a persistent low level inflammatory response triggering increased K-Ras activity and that Cox-2 is essential for this inflammatory reaction. To determine this, LSL-K-Ras mice were crossed with Ela-CreER (Acinar-specific) or Pdx-1-Cre (Pancreas-specific) to “knock-in” oncogenic K-Ras. Additionally, these animals were crossed with Cox-2 conditional knockout mice to access the importance of Cox-2 in the inflammatory loop present. The mice were fed isocaloric diets containing 60% energy or 10% energy from fat. We found that a high fat diet increased K-Ras activity, PanIN formation, and fibrotic stroma significantly compared to a control diet. Genetic deletion of Cox-2 prevented high fat diet induced fibrosis and PanIN formation in oncogenic K-Ras expressing mice. Additionally, long term consumption of high fat diet, increased the progression of PanIN lesions leading to invasive cancer and decreased overall survival rate. These findings indicate that a high fat diet can stimulate the activation of oncogenic K-Ras and initiate an inflammatory feed forward loop requiring Cox-2 leading to inflammation, fibrosis, and PanINs. This mechanism could explain the relationship between a high fat diet and elevated risk for pancreatic cancer.

Isolation and characterization of revertant cell lines expressing oncogenic rat {\it neu\/}

The neu gene encodes the transmembrane tyrosine kinase growth factor receptor, p185. To study neu induced cellular transformation, we developed revertant cells from the neu transformed NIH 3T3 cell line, B104-1-1, by treating the cells with the chemical mutagen ethylmethane sulfonate. The morphologically normal revertant cells were first selected by their ability to either attach to culture plates or survive in the presence of the cytotoxic reagents colchicine or 5-fluoro-2deoxyuridine. Two of the 21 candidate revertant cell lines isolated were further characterized and were found to lose their anchorage independence and ability to grow in 1% calf serum, indicating that they were nontransformed even though they still expressed p185 oncoprotein. The tyrosine residues of p185 in these two revertants were underphosphorylated, which may have contributed to their nontransformed status. Also, the p185 oncoprotein lacked significant tyrosine kinase activity. In addition, these revertants also resisted transformation by neu and several additional oncogenes (H-ras, N-ras, v-mos, v-abl, and v-fos) as determined by focus forming assays. These results indicated that we had successfully developed, from neu transformed cells, revertants which exhibited defective tyrosine phosphorylation and kinase activity of the neu oncoprotein. The results also suggested that neu and several other oncogenes may share common elements in their pathways for the induction of cellular transformation. ^

Structural conservations and diversities of dsRNA viruses: Structural studies of the cytoplasmic polyhedrosis virus by electron cryomicroscopy and 3D reconstruction

The Reoviridae virus family is a group of economically and pathologically important viruses that have either single-, double-, or triple-shelled protein layers enclosing a segmented double stranded RNA genome. Each virus particle in this family has its own viral RNA dependent RNA polymerase and the enzymatic activities necessary for the mature RNA synthesis. Based on the structure of the inner most cores of the viruses, the Reoviridae viruses can be divided into two major groups. One group of viruses has a smooth surfaced inner core, surrounded by complete outer shells of one or two protein layers. The other group has an inner core decorated with turrets on the five-fold vertices, and could either completely lack or have incomplete outer protein layers. The structural difference is one of the determinant factors for their biological differences during the infection. ^ Cytoplasmic polyhedrosis virus (CPV) is a single-shelled, turreted virus and the structurally simplest member in Reoviridae. It causes specific chronic infections in the insect gut epithelial cells. Due to its wide range of insect hosts, CPV has been engineered as a potential insecticide for use in fruit and vegetable farming. Its unique structural simplicity, unparalleled capsid stability and ease of purification make CPV an ideal model system for studying the structural basis of dsRNA virus assembly at the highest possible resolution by electron cryomicroscopy (cryoEM) and three-dimensional (3D) reconstruction. ^ In this thesis work, I determined the first 3D structure of CPV capsids using 100 kV cryoEM. At an effective resolution of 17 Å, the full capsid reveals a 600-Å diameter, T = 1 icosahedral shell decorated with A and B spikes at the 5-fold vertices. The internal space of the empty CPV is unoccupied except for 12 mushroom-shaped densities that are attributed to the transcriptional enzyme complexes. The inside of the full capsid is packed with icosahedrally-ordered viral genomic RNA. The interactions of viral RNA with the transcriptional enzyme complexes and other capsid proteins suggest a mechanism for RNA transcription and subsequent release. ^ Second, the interactions between the turret proteins (TPs) and the major capsid shell protein (CSPs) have been identified through 3D structural comparisons of the intact CPV capsids with the spikeless CPV capsids, which were generated by chemical treatments. The differential effects of these chemical treatment experiments also indicated that CPV has a significantly stronger structural integrity than other dsRNA viruses, such as the orthoreovirus subcores, which are normally enclosed within outer protein shells. ^ Finally, we have reconstructed the intact CPV to an unprecendented 8 Å resolution from several thousand of 400kV cryoEM images. The 8 Å structure reveals interactions among the 120 molecules of each of the capsid shell protein (CSP), the large protrusion protein (LPP), and 60 molecules of the turret protein (TP). A total of 1980 α-helices and 720 β-sheets have been identified in these capsid proteins. The CSP structure is largely conserved, with the majority of the secondary structures homologous to those observed in the x-ray structures of corresponding proteins of other reoviruses, such as orthoreovirus and bluetongue virus. The three domains of TP are well positioned to play multifunctional roles during viral transcription. The completely non-equivalent interactions between LPP and CSP and those between the anchoring domain of TP and CSP account for the unparalleled stability of this structurally simplest member of the Reoviridae. ^

Requirement of the Jak2 tyrosine kinase in Bcr -Abl oncogenic transformation

Philadelphia chromosome (Ph)-positive chronic myeloid leukemia is caused by a clonal myeloproliferative expansion of malignant primitive hematopoietic progenitor cells. The Ph results from the reciprocal translocation of the ends of chromosome 9 and 22, which generate Bcr-Abl fusion proteins. The Bcr-Abl proteins possess a constitutively activated Abl tyrosine kinase, which is the driving force responsible for causing leukemia. The activated Bcr-Abl tyrosine kinase stimulates multiple signal transduction pathway affecting growth, differentiation and survival of cells. It is known that the Bcr-Abl tyrosine kinase activates several signaling proteins including Stat5, which is a member of the Jak/Stat pathway that is activated by cytokines that control the growth and differentiation of normal hematopoietic cells. Our laboratory was the first one to report that Jak2 tyrosine kinase is activated in a human Bcr-Abl positive hematopoietic cell line. In this thesis, we further investigated the activation of Jak2 by Bcr-Abl. We found that Jak2 is activated not only in cultured Bcr-abl positive cell lines but also in blood cells from CML blast crisis patients. We also demonstrated that SH2 domain of Bcr-Abl is required for efficient activation Jak2. We further showed that Jak2 binds to the C-terminal domain of Bcr-Abl; tyrosine residue 1007, which is critical for Jak2 activation, is phosphorylated by Bcr-Abl. We searched downstream targets of Jak2 in Bcr-Abl positive cells. We treated Bcr-Abl positive cells with a Jak2 kinase inhibitor AG490 and found that c-Myc protein expression is inhibited by AG490. We further demonstrated that Jak2 inhibitor AG490 not only inhibit C-MYC transcription but also protect c-Myc protein from proteasome-dependent degradation. We also showed that AG490 did not affect Bcr-Abl kinase activity and Stat5 activation and its downstream target Bcl-xL expression. AG490 also induced apoptosis of Bcr-Abl positive cells, similar to Bcr-Abl kinase inhibitor STI571 (also termed Gliveec, a very effective drug for CML), but unlike STI571 the apoptosis effects induced by AG490 can not be rescued by IL-3 containing WEHI conditioned medium. We further established several Bcr-Abl positive clones that express a kinase-inactive Jak2 and found that these clones had reduced tumor formation in nude mice assays. Taken together, these results establish that Jak2 is activated in Bcr-Abl positive CML cells and it is required for c-Myc induction and the oncogenic effects of Bcr-Abl. Furthermore, Jak2 and Stat5 are two independent targets of Bcr-Abl. ^