Barriers to Infection of Human Cells by Feline Leukemia Virus: Insights into Resistance to Zoonosis.

The human genome displays a rich fossil record of past gammaretrovirus infections, yet no current epidemic is evident, despite environmental exposure to viruses that infect human cells in vitro Feline leukemia viruses (FeLVs) rank high on this list, but neither domestic nor workplace exposure has been associated with detectable serological responses. Nonspecific inactivation of gammaretroviruses by serum factors appears insufficient to explain these observations. To investigate further, we explored the susceptibilities of primary and established human cell lines to FeLV-B, the most likely zoonotic variant. Fully permissive infection was common in cancer-derived cell lines but was also a feature of nontransformed keratinocytes and lung fibroblasts. Cells of hematopoietic origin were generally less permissive and formed discrete groups on the basis of high or low intracellular protein expression and virion release. Potent repression was observed in primary human blood mononuclear cells and a subset of leukemia cell lines. However, the early steps of reverse transcription and integration appear to be unimpaired in nonpermissive cells. FeLV-B was subject to G→A hypermutation with a predominant APOBEC3G signature in partially permissive cells but was not mutated in permissive cells or in nonpermissive cells that block secondary viral spread. Distinct cellular barriers that protect primary human blood cells are likely to be important in protection against zoonotic infection with FeLV.IMPORTANCE Domestic exposure to gammaretroviruses such as feline leukemia viruses (FeLVs) occurs worldwide, but the basis of human resistance to infection remains incompletely understood. The potential threat is evident from the human genome sequence, which reveals many past epidemics of gammaretrovirus infection, and from recent cross-species jumps of gammaretroviruses from rodents to primates and marsupials. This study examined resistance to infection at the cellular level with the most prevalent human cell-tropic FeLV variant, FeLV-B. We found that blood cells are uniquely resistant to infection with FeLV-B due to the activity of cellular enzymes that mutate the viral genome. A second block, which appears to suppress viral gene expression after the viral genome has integrated into the host cell genome, was identified. Since cells derived from other normal human cell types are fully supportive of FeLV replication, innate resistance of blood cells could be critical in protecting against cross-species infection.

Disruption of pre-B-cell receptor signaling jams the WNT/ß-catenin pathway and induces cell death in B-cell acute lymphoblastic leukemia cell lines.

Targeting components of the B-cell receptor (BCR) pathway have dramatically improved clinical outcomes in a variety of B-cell malignancies. Despite the well-documented pathogenic role of BCR precursor (pre-BCR) pathway in B-cell acute lymphoblastic leukemia (B-ALL), there is limited available data of therapies that aim to disrupt this pathway. To investigate the role of protein kinase Cß (PKCß), a crucial mediator of BCR and pre-BCR signaling, in B-ALL survival, we studied the activity of the PKCß selective inhibitor enzastaurin (ENZ) in seven B-ALL cell lines. Treatment with ENZ resulted in a dose- and time-dependent growth inhibition in all cell lines with a relatively higher efficacy in pro-B ALL with translocation t(4;11)(q21;q23). The mechanism of growth inhibition was by apoptotic induction and cell cycle arrest. A rapid reduction in phosphorylation of AKT and its downstream target glycogen synthase kinase 3ß (GSK3ß) were observed at 30min after treatment and remaining for 48h. The reduction in GSK3ß phosphorylation was associated with a paradoxical accumulation of ß-catenin, which was due to a transient loss of ß-catenin phosphorylation at ser33-37. In addition, accumulation of ß-catenin was associated with downregulation of c-Myc, upregulatiuon of c-Jun, and a subsequent protective effect on the tumor suppressor p73. Data in this paper were presented in part at 2012 American Society of Hematology Annual Meeting, abstract 1350.

The surface glycoprotein of feline leukemia virus isolate FeLV-945 is a determinant of altered pathogenesis in the presence or absence of the unique viral long terminal repeat.

Feline leukemia virus (FeLV) is a naturally transmitted gammaretrovirus that infects domestic cats. FeLV-945, the predominant isolate associated with non-T-cell disease in a natural cohort, is a member of FeLV subgroup A but differs in sequence from the FeLV-A prototype, FeLV-A/61E, in the surface glycoprotein (SU) and long terminal repeat (LTR). Substitution of the FeLV-945 LTR into FeLV-A/61E resulted in pathogenesis indistinguishable from that of FeLV-A/61E, namely, thymic lymphoma of T-cell origin. In contrast, substitution of both FeLV-945 LTR and SU into FeLV-A/61E resulted in multicentric lymphoma of non-T-cell origin. These results implicated the FeLV-945 SU as a determinant of pathogenic spectrum. The present study was undertaken to test the hypothesis that FeLV-945 SU can act in the absence of other unique sequence elements of FeLV-945 to determine the disease spectrum. Substitution of FeLV-A/61E SU with that of FeLV-945 altered the clinical presentation and resulted in tumors that demonstrated expression of CD45R in the presence or absence of CD3. Despite the evident expression of CD45R, a typical B-cell marker, T-cell receptor beta (TCRß) gene rearrangement indicated a T-cell origin. Tumor cells were detectable in bone marrow and blood at earlier times during the disease process, and the predominant SU genes from proviruses integrated in tumor DNA carried markers of genetic recombination. The findings demonstrate that FeLV-945 SU alters pathogenesis, although incompletely, in the absence of FeLV-945 LTR. Evidence demonstrates that FeLV-945 SU and LTR are required together to fully recapitulate the distinctive non-T-cell disease outcome seen in the natural cohort.

Protein kinase C-beta inhibition induces apoptosis and inhibits cell cycle progression in acquired immunodeficiency syndrome-related non-hodgkin lymphoma cells.

INTRODUCTION: Acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin lymphoma (NHL) constitutes an aggressive variety of lymphomas characterized by increased extranodal involvement, relapse rate, and resistance to chemotherapy. Protein kinase C-beta (PKCß) targeting showed promising results in preclinical and clinical studies involving a wide variety of cancers, but studies describing the role of PKCß in AIDS-NHL are primitive if not lacking. METHODS: In the present study, 3 AIDS-NHL cell lines were examined: 2F7 (AIDS-Burkitt lymphoma), BCBL-1 (AIDS-primary effusion lymphoma), and UMCL01-101 (AIDS-diffuse large B-cell lymphoma). RESULTS: Immunoblot analysis demonstrated expression of PKCß1 and PKCß2 in 2F7 and UMCL01-101 cells, and PKCß1 alone in BCBL-1 cells. The viability of 2F7 and BCBL-1 cells decreased significantly in the presence of PKCß-selective inhibitor at half-maximal inhibitory concentration of 14 and 15 µmol/L, respectively, as measured by tetrazolium dye reduction assay. In contrast, UMCL01-101 cells were relatively resistant. As determined using flow cytometric deoxynucleotidyl transferase dUTP nick-end labeling assay with propidium iodide staining, the responsiveness of sensitive cells was associated with apoptotic induction and cell cycle inhibition. Protein kinase C-beta-selective inhibition was observed not to affect AKT phosphorylation but to induce a rapid and sustained reduction in the phosphorylation of glycogen synthase kinase-3 beta, ribosomal protein S6, and mammalian target of rapamycin in sensitive cell lines. CONCLUSIONS: The results indicate that PKCß plays an important role in AIDS-related NHL survival and suggest that PKCß targeting should be considered in a broader spectrum of NHL. The observations in BCBL-1 were unexpected in the absence of PKCß2 expression and implicate PKCß1 as a regulator in those cells.

Viral determinants of FeLV infection and pathogenesis: lessons learned from analysis of a natural cohort.

Detailed analysis has been performed over many years of a geographic and temporal cohort of cats naturally infected with feline leukemia virus (FeLV). Molecular analysis of FeLV present in the diseased tissues and application of those viruses to experimental systems has revealed unique isolates with distinctive disease potential, previously uncharacterized virus-receptor interactions, information about the role of recombinant viruses in disease induction, and novel viral and cellular oncogenes implicated in pathogenesis, among other findings. The studies have contributed to an understanding of the selective forces that lead to predominance of distinctive FeLV isolates and disease outcomes in a natural population.

The surface glycoprotein of a natural feline leukemia virus subgroup A variant, FeLV-945, as a determinant of disease outcome.

Feline leukemia virus (FeLV) is a natural retrovirus of domestic cats associated with degenerative, proliferative and malignant diseases. Studies of FeLV infection in a cohort of naturally infected cats were undertaken to examine FeLV variation, the selective pressures operative in FeLV infection that lead to predominance of natural variants, and the consequences for infection and disease progression. A unique variant, designated FeLV-945, was identified as the predominant isolate in the cohort and was associated with non-T-cell diseases including multicentric lymphoma. FeLV-945 was assigned to the FeLV-A subgroup based on sequence analysis and receptor utilization, but was shown to differ in sequence from a prototype member of FeLV-A, designated FeLV-A/61E, in the long terminal repeat (LTR) and the surface glycoprotein gene (SU). A unique sequence motif in the FeLV-945 LTR was shown to function as a transcriptional enhancer and to confer a replicative advantage. The FeLV-945 SU protein was observed to differ in sequence as compared to FeLV-A/61E within functional domains known to determine receptor selection and binding. Experimental infection of newborn cats was performed using wild type FeLV-A/61E or recombinant FeLV-A/61E in which the LTR (61E/945L) or LTR and SU (61E/945SL) were exchanged for that of FeLV-945. Infection with either FeLV-A/61E or 61E/945L resulted in T-cell lymphoma of the thymus, although 61E/945L caused disease significantly more rapidly. In contrast, infection with 61E/945SL resulted in the rapid induction of a multicentric lymphoma of B-cell origin, thus recapitulating the outcome of natural infection and implicating FeLV-945 SU as a determinant of disease outcome. Recombinant FeLV-B was detected infrequently and at low levels in multicentric lymphomas, and was thereby not implicated in disease induction. Preliminary studies of receptor interaction indicated that virus particles bearing FeLV-945 SU bind to the FeLV-A receptor more efficiently than do particles bearing FeLV-A/61E SU, and that soluble SU proteins expressed from the viruses demonstrate the same differential binding phenotype. Preliminary mutational analysis of FeLV-945 was performed by exchanging regions containing either the primary receptor binding determinant, VRA, the secondary determinant, VRB, or a proline-rich region, PRR, with that of FeLV-A/61E. Results implicated a region containing VRA as a minor contributor, while a region containing VRB largely conferred increased binding efficiency.

Distinctive receptor binding properties of the surface glycoprotein of a natural feline leukemia virus isolate with unusual disease spectrum.

BACKGROUND: Feline leukemia virus (FeLV)-945, a member of the FeLV-A subgroup, was previously isolated from a cohort of naturally infected cats. An unusual multicentric lymphoma of non-T-cell origin was observed in natural and experimental infection with FeLV-945. Previous studies implicated the FeLV-945 surface glycoprotein (SU) as a determinant of disease outcome by an as yet unknown mechanism. The present studies demonstrate that FeLV-945 SU confers distinctive properties of binding to the cell surface receptor. RESULTS: Virions bearing the FeLV-945 Env protein were observed to bind the cell surface receptor with significantly increased efficiency, as was soluble FeLV-945 SU protein, as compared to the corresponding virions or soluble protein from a prototype FeLV-A isolate. SU proteins cloned from other cohort isolates exhibited increased binding efficiency comparable to or greater than FeLV-945 SU. Mutational analysis implicated a domain containing variable region B (VRB) to be the major determinant of increased receptor binding, and identified a single residue, valine 186, to be responsible for the effect. CONCLUSIONS: The FeLV-945 SU protein binds its cell surface receptor, feTHTR1, with significantly greater efficiency than does that of prototype FeLV-A (FeLV-A/61E) when present on the surface of virus particles or in soluble form, demonstrating a 2-fold difference in the relative dissociation constant. The results implicate a single residue, valine 186, as the major determinant of increased binding affinity. Computational modeling suggests a molecular mechanism by which residue 186 interacts with the receptor-binding domain through residue glutamine 110 to effect increased binding affinity. Through its increased receptor binding affinity, FeLV-945 SU might function in pathogenesis by increasing the rate of virus entry and spread in vivo, or by facilitating entry into a novel target cell with a low receptor density.

Apoptotic induction in B-cell acute lymphoblastic leukemia cell lines treated with a protein kinase Cß inhibitor.

B-cell acute lymphoblastic leukemia (B-ALL) in adults exhibits a 5-year disease-free survival rate of only 25-40% after currently available treatment. Protein kinase Cß (PKCß) is under active consideration as a rational therapeutic target in several B-cell malignancies, but studies of its possible utility in B-ALL are lacking. Expression of PKCß1 and PKCß2 isoforms was demonstrated in five B-ALL cell lines characterized by distinctive chromosomal translocations, and sensitivity to PKCß-selective inhibition was examined. Inhibitor treatment resulted in a dose-dependent reduction in viability in all cell lines, although pro-B ALL with t(4;11)(q21;q23) was most sensitive. Apoptotic induction was evident after 24-48 h of treatment, and an inhibition of cell cycle progression was detected in one cell line. Treatment resulted in a rapid induction of poly(ADP-ribose) polymerase (PARP) cleavage, indicating caspase-3-mediated apoptosis, and a rapid reduction in phosphorylation of AKT and its downstream target glycogen synthase kinase 3ß (GSK3ß). These results indicate that PKCß targeting should be considered as a potential treatment option in B-ALL.

The frequency of occurrence and nature of recombinant feline leukemia viruses in the induction of multicentric lymphoma by infection of the domestic cat with FeLV-945.

During feline leukemia virus (FeLV) infection in the domestic cat, viruses with a novel envelope gene arise by recombination between endogenous FeLV-related elements and the exogenous infecting species. These recombinant viruses (FeLV-B) are of uncertain disease association, but have been linked to the induction of thymic lymphoma. To assess the role of FeLV-B in the induction of multicentric lymphoma and other non-T-cell disease, the frequency of occurrence and nature of FeLV-B were examined in diseased tissues from a large collection of FeLV-infected animals. Diseased tissues were examined by Southern blot and PCR amplification to detect the presence of FeLV-B. Further analysis was performed to establish the recombination junctions and infectivity of FeLV-B in diseased tissues. The results confirmed the frequent association of FeLV-B with thymic lymphoma but showed infrequent generation, low levels and lack of infectivity of FeLV-B in non-T-cell diseases including multicentric lymphoma.

Advances in understanding molecular determinants in FeLV pathology.

Feline leukemia virus (FeLV) occurs in nature not as a single genomic species but as a family of closely related viruses. The disease outcome of natural FeLV infection is variable and likely reflects genetic variation both in the virus and the naturally outbreeding host population. A series of studies have been undertaken with the objectives of examining natural FeLV genetic variation, the selective pressures operative in FeLV infection that lead to predominance of natural variants, and the consequences for infection and disease progression. Genetic variation among FeLV isolates was examined in a cohort of naturally infected cats with thymic lymphoma of T-cell origin, non-T-cell multicentric lymphoma, myeloproliferative disorder or anemia. The predominant isolate in the cohort, designated FeLV-945, was identified exclusively in disorders of non-T-cell origin. The FeLV-945 LTR was shown to contain a unique 21-bp repeat element, triplicated in tandem downstream of enhancer. The 21-bp triplication was shown to act as a transcriptional enhancer and to confer a replicative advantage through the assembly of a distinctive transcription factor complex. Oncogene utilization during tumor induction by FeLV-945 was studied using a recombinant Moloney murine leukemia virus containing the FeLV-945 LTR. This approach identified novel loci of common proviral integration in tumors, including the regulatory subunit of PI-3Kgamma. Mutational changes identified in FeLV-945 SU were shown not to alter receptor usage as measured by host range and superinfection interference, but to significantly increase the efficiency of receptor binding. To determine whether the unique sequence elements of FeLV-945 influence the course of infection and disease in vivo, recombinant viruses were constructed in which the FeLV-945 LTR alone, or the FeLV-945 SU gene and LTR were substituted into the prototype isolate FeLV-A/61E. Longitudinal studies of infected animals showed that substitution of the FeLV-945 LTR into FeLV-A/61E resulted in a significantly more rapid disease onset, but did not alter the tumorigenic spectrum. In contrast, substitution of both the FeLV-945 LTR and SU gene changed the disease outcome entirely. Together, these observations indicate that the distinctive LTR and SU gene of FeLV-945 mediate a rapid pathogenesis with distinctive clinical features and oncogenic mechanisms.

Diminished potential for B-lymphoid differentiation after murine leukemia virus infection in vivo and in EML hematopoietic progenitor cells.

Infection with a recombinant murine-feline gammaretrovirus, MoFe2, or with the parent virus, Moloney murine leukemia virus, caused significant reduction in B-lymphoid differentiation of bone marrow at 2 to 8 weeks postinfection. The suppression was selective, in that myeloid potential was significantly increased by infection. Analysis of cell surface markers and immunoglobulin H gene rearrangements in an in vitro model demonstrated normal B-lymphoid differentiation after infection but significantly reduced viability of differentiating cells. This reduction in viability may confer a selective advantage on undifferentiated lymphoid progenitors in the bone marrow of gammaretrovirus-infected animals and thereby contribute to the establishment of a premalignant state.

Growth regulation of simian and human AIDS-related non-Hodgkin's lymphoma cell lines by TGF-beta1 and IL-6.

BACKGROUND: AIDS-related non-Hodgkin's lymphoma (AIDS-NHL) is the second most frequent cancer associated with AIDS, and is a frequent cause of death in HIV-infected individuals. Experimental analysis of AIDS-NHL has been facilitated by the availability of an excellent animal model, i.e., simian Acquired Immunodeficiency Syndrome (SAIDS) in the rhesus macaque consequent to infection with simian immunodeficiency virus. A recent study of SAIDS-NHL demonstrated a lymphoma-derived cell line to be sensitive to the growth inhibitory effects of the ubiquitous cytokine, transforming growth factor-beta (TGF-beta). The authors concluded that TGF-beta acts as a negative growth regulator of the lymphoma-derived cell line and, potentially, as an inhibitory factor in the regulatory network of AIDS-related lymphomagenesis. The present study was conducted to assess whether other SAIDS-NHL and AIDS-NHL cell lines are similarly sensitive to the growth inhibitory effects of TGF-beta, and to test the hypothesis that interleukin-6 (IL-6) may represent a counteracting positive influence in their growth regulation. METHODS: Growth stimulation or inhibition in response to cytokine treatment was quantified using trypan blue exclusion or colorimetric MTT assay. Intracellular flow cytometry was used to analyze the activation of signaling pathways and to examine the expression of anti-apoptotic proteins and distinguishing hallmarks of AIDS-NHL subclass. Apoptosis was quantified by flow cytometric analysis of cell populations with sub-G1 DNA content and by measuring activated caspase-3. RESULTS: Results confirmed the sensitivity of LCL8664, an immunoblastic SAIDS-NHL cell line, to TGF-beta1-mediated growth inhibition, and further demonstrated the partial rescue by simultaneous treatment with IL-6. IL-6 was shown to activate STAT3, even in the presence of TGF-beta1, and thereby to activate proliferative and anti-apoptotic pathways. By comparison, human AIDS-NHL cell lines differed in their responsiveness to TGF-beta1 and IL-6. Analysis of a recently derived AIDS-NHL cell line, UMCL01-101, indicated that it represents immunoblastic AIDS-DLCBL. Like LCL-8664, UMCL01-101 was sensitive to TGF-beta1-mediated inhibition, rescued partially by IL-6, and demonstrated rapid STAT3 activation following IL-6 treatment even in the presence of TGF-beta1. CONCLUSION: These studies indicate that the sensitivity of immunoblastic AIDS- or SAIDS-DLBCL to TGF-beta1-mediated growth inhibition may be overcome through the stimulation of proliferative and anti-apoptotic signals by IL-6, particularly through the rapid activation of STAT3.

Matrix attachment regions as targets for retroviral integration.

BACKGROUND: The randomness of retroviral integration has been debated for many years. Recent evidence indicates that integration site selection is not random, and that it is influenced by both viral and cellular factors. To study the role of DNA structure in site selection, retroviral integration near matrix attachment regions (MARs) was analyzed for three different groups of retroviruses. The objective was to assess whether integration near MARs may be a factor for integration site selection. RESULTS: Results indicated that MLV, SL3-3 MuLV, HIV-1 and HTLV-1 integrate preferentially near MARs, specifically within 2-kilobases (kb). In addition, a preferential position and orientation relative to the adjacent MAR was observed for each virus. Further analysis of SL3-3 MuLV insertions in common integration sites (CISs) demonstrated a higher frequency of integration near MARs and an orientation preference that was not observed for integrations outside CISs. CONCLUSION: These findings contribute to a growing body of evidence indicating that retroviral integration is not random, that MARs influence integration site selection for some retroviruses, and that integration near MARs may have a role in the insertional activation of oncogenes by gammaretroviruses.

Unique long terminal repeat and surface glycoprotein gene sequences of feline leukemia virus as determinants of disease outcome.

The outcome of feline leukemia virus (FeLV) infection in nature is variable, including malignant, proliferative, and degenerative disorders. The determinants of disease outcome are not well understood but are thought to include viral, host, and environmental factors. In particular, genetic variations in the FeLV long terminal repeat (LTR) and SU gene have been linked to disease outcome. FeLV-945 was previously identified as a natural isolate predominant in non-T-cell neoplastic and nonneoplastic diseases in a geographic cohort. The FeLV-945 LTR was shown to contain unique repeat elements, including a 21-bp triplication downstream of the enhancer. The FeLV-945 SU gene was shown to encode mutational changes in functional domains of the protein. The present study details the outcomes of infection with recombinant FeLVs in which the LTR and envelope (env) gene of FeLV-945, or the LTR only, was substituted for homologous sequences in a horizontally transmissible prototype isolate, FeLV-A/61E. The results showed that the FeLV-945 LTR determined the kinetics of disease. Substitution of the FeLV-945 LTR into FeLV-A/61E resulted in a significantly more rapid disease onset but did not alter the tumorigenic spectrum. In contrast, substitution of both the FeLV-945 LTR and env gene changed the disease outcome entirely. Further, the impact of FeLV-945 env on the disease outcome was dependent on the route of inoculation. Since the TM genes of FeLV-945 and FeLV-A/61E are nearly identical but the SU genes differ significantly, FeLV-945 SU is implicated in the outcome. These findings identify the FeLV-945 LTR and SU gene as determinants of disease.

Subtle mutational changes in the SU protein of a natural feline leukemia virus subgroup A isolate alter disease spectrum.

FeLV-945 is a representative isolate of the natural feline leukemia virus (FeLV) variant predominant in non-T-cell malignant, proliferative, and degenerative diseases in a geographic cohort. The FeLV-945 surface glycoprotein (SU) is closely related to natural horizontally transmissible FeLV subgroup A (FeLV-A) but was found to differ from a prototype to a larger extent than the members of FeLV-A differ among themselves. The sequence differences included point mutations restricted largely to the functional domains of SU, i.e., VRA, VRB, and PRR. Despite the sequence differences in these critical domains, measurements of receptor utilization, including host range and superinfection interference, confirmed the assignment of FeLV-945 to subgroup A. Other proviruses isolated from the cohort contained similar sequence hallmarks and were assigned to FeLV subgroup A. A provirus from cat 1046 contained a histidine-to-proline change at SU residue 6 within an SPHQ motif that was previously identified as a critical mediator of fusion events during virus entry. The 1046 pseudotype virus entered cells only in the presence of the soluble cofactor FeLIX provided in trans, but it retained an ecotropic host range even in the presence of FeLIX. The mutational changes in FeLV-945 were shown to confer significant functional differences compared to prototype FeLV-A viruses. The substitution of FeLV-945 envelope gene sequences for FeLV-A/61E sequences conferred a small but statistically significant replicative advantage in some feline cells. Moreover, substitution of the unique FeLV-945 long terminal repeat and envelope gene for those of FeLV-A/61E altered the disease spectrum entirely, from a thymic lymphoma of a T-cell origin to an as yet uncharacterized multicentric lymphoma that did not contain T cells.

Substitution of feline leukemia virus long terminal repeat sequences into murine leukemia virus alters the pattern of insertional activation and identifies new common insertion sites.

The recombinant retrovirus, MoFe2-MuLV (MoFe2), was constructed by replacing the U3 region of Moloney murine leukemia virus (M-MuLV) with homologous sequences from the FeLV-945 LTR. NIH/Swiss mice neonatally inoculated with MoFe2 developed T-cell lymphomas of immature thymocyte surface phenotype. MoFe2 integrated infrequently (0 to 9%) near common insertion sites (CISs) previously identified for either parent virus. Using three different strategies, CISs in MoFe2-induced tumors were identified at six loci, none of which had been previously reported as CISs in tumors induced by either parent virus in wild-type animals. Two of the newly identified CISs had not previously been implicated in lymphoma in any retrovirus model. One of these, designated 3-19, encodes the p101 regulatory subunit of phosphoinositide-3-kinase-gamma. The other, designated Rw1, is predicted to encode a protein that functions in the immune response to virus infection. Thus, substitution of FeLV-945 U3 sequences into the M-MuLV long terminal repeat (LTR) did not alter the target tissue for M-MuLV transformation but significantly altered the pattern of CIS utilization in the induction of T-cell lymphoma. These observations support a growing body of evidence that the distinctive sequence and/or structure of the retroviral LTR determines its pattern of insertional activation. The findings also demonstrate the oligoclonal nature of retrovirus-induced lymphomas by demonstrating proviral insertions at CISs in subdominant populations in the tumor mass. Finally, the findings demonstrate the utility of novel recombinant retroviruses such as MoFe2 to contribute new genes potentially relevant to the induction of lymphoid malignancy.

Regulation of FeLV-945 by c-Myb binding and CBP recruitment to the LTR.

BACKGROUND: Feline leukemia virus (FeLV) induces degenerative, proliferative and malignant hematologic disorders in its natural host, the domestic cat. FeLV-945 is a viral variant identified as predominant in a cohort of naturally infected animals. FeLV-945 contains a unique sequence motif in the long terminal repeat (LTR) comprised of a single copy of transcriptional enhancer followed by a 21-bp sequence triplicated in tandem. The LTR is precisely conserved among independent cases of multicentric lymphoma, myeloproliferative disease and anemia in animals from the cohort. The 21-bp triplication was previously shown to act as a transcriptional enhancer preferentially in hematopoietic cells and to confer a replicative advantage. The objective of the present study was to examine the molecular mechanism by which the 21-bp triplication exerts its influence and the selective advantage responsible for its precise conservation. RESULTS: Potential binding sites for the transcription factor, c-Myb, were identified across the repeat junctions of the 21-bp triplication. Such sites would not occur in the absence of the repeat; thus, a requirement for c-Myb binding to the repeat junctions of the triplication would exert a selective pressure to conserve its sequence precisely. Electrophoretic mobility shift assays demonstrated specific binding of c-Myb to the 21-bp triplication. Reporter gene assays showed that the triplication-containing LTR is responsive to c-Myb, and that responsiveness requires the presence of both c-Myb binding sites. Results further indicated that c-Myb in complex with the 21-bp triplication recruits the transcriptional co-activator, CBP, a regulator of normal hematopoiesis. FeLV-945 replication was shown to be positively regulated by CBP in a manner dependent on the presence of the 21-bp triplication. CONCLUSION: Binding sites for c-Myb across the repeat junctions of the 21-bp triplication may account for its precise conservation in the FeLV-945 LTR. c-Myb binding and CBP recruitment to the LTR positively regulated virus production, and thus may be responsible for the replicative advantage conferred by the 21-bp triplication. Considering that CBP is present in hematopoietic cells in limiting amounts, we hypothesize that FeLV-945 replication in bone marrow may influence CBP availability and thereby alter the regulation of CBP-responsive genes, thus contributing to altered hematopoiesis and consequent hematologic disease.

Feline leukaemia virus LTR variation and disease association in a geographical and temporal cluster.

Feline leukaemia virus (FeLV)-945 was previously identified in natural multicentric lymphomas and contains a 21 bp tandem triplication in the LTR. In the present study, FeLV LTR variation was examined in the cohort from which FeLV-945 was identified. The objectives of the study were to evaluate FeLV LTR variation within the cohort, to determine whether the FeLV-945 LTR was associated uniquely with multicentric lymphoma and to evaluate functional attributes that may have contributed selective advantage to the predominant LTR variants observed. T-cell tumours uniformly contained LTRs with duplicated enhancer sequences, although enhancer duplications conferred little transcriptional advantage. Non-T-cell malignant, proliferative and degenerative diseases contained LTRs with two, three or four tandemly repeated copies of the 21 bp sequence originally identified in FeLV-945. While the length and termini of enhancer duplications were variable, the 21 bp repeat unit was invariant. Triplication of the 21 bp repeat conferred the optimal replicative advantage in feline cells.