Garret C. Newbound

Endoplasmic reticulum and cis-Golgi localization of human T-lymphotropic virus type 1 p12I: Association with calreticulin and calnexin

ABSTRACT Human T-lymphotropic virus type 1 (HTLV-1) is a complex retrovirus encoding regulatory and accessory genes in four open reading frames (ORF I to IV) of the pX region. We have demonstrated an important role of pX ORF I expression, which encodes p12I, in establishment of HTLV-1 infection in a rabbit model and for optimal viral infectivity in quiescent primary lymphocytes. These data indicated that p12I may enhance lymphocyte activation and thereby promote virus infection. To further define the role of p12I in cell activation, we characterized the subcellular localization of p12I in transfected 293T cells and HeLa-Tat cells by multiple methods, including immunofluorescence confocal microscopy, electron microscopy, and subcellular fractionation. Herein, we demonstrate that p12I accumulates in the endoplasmic reticulum (ER) and cis-Golgi apparatus. The location of p12I was unchanged following treatments with both cycloheximide (blocking de novo protein synthesis) and brefeldin A (disrupting ER-to-Golgi protein transport), indicating that the protein is retained in the ER and cis-Golgi. Moreover, using coimmunoprecipitation assays, we identify the direct binding of p12I with both calreticulin and calnexin, resident ER proteins which regulate calcium storage. Our results indicate that p12I directly binds key regulatory proteins involved in calcium-mediated cell signaling and suggest a role of p12Iin the establishment of HTLV-1 infection by activation of host cells.

CCAAT/Enhancer-binding Protein δ Regulates Mammary Epithelial Cell G0 Growth Arrest and Apoptosis

CCAAT/enhancer-binding proteins (C/EBPs) are a highly conserved family of DNA-binding proteins that regulate cell-specific growth, differentiation, and apoptosis. Here, we show that induction of C/EBPδ gene expression during G0 growth arrest is a general property of mammary-derived cell lines. C/EBPδ is not induced during G0 growth arrest in 3T3 or IEC18 cells. C/EBPδ induction is G0-specific in mouse mammary epithelial cells; C/EBPδ gene expression is not induced by growth arrest in the G1, S, or G2 phase of the cell cycle. C/EBPδ antisense-expressing cells (AS1 cells) maintain elevated cyclin D1 and phosphorylated retinoblastoma protein levels and exhibit delayed G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. Conversely, C/EBPδ-overexpressing cells exhibited a rapid decline in cyclin D1 and phosphorylated retinoblastoma protein levels, a rapid increase in the cyclin-dependent kinase inhibitor p27, and accelerated G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. When C/EBPδ levels were rescued in AS1 cells by transfection with a C/EBPδ “sense” construct, normal G0 growth arrest and apoptosis were restored. These results demonstrate that C/EBPδ plays a key role in the regulation of G0 growth arrest and apoptosis in mammary epithelial cells.

Comparison of Gene Transfer Efficiencies and Gene Expression Levels Achieved with Equine Infectious Anemia Virus- and Human Immunodeficiency Virus Type 1-Derived Lentivirus Vectors

ABSTRACT This report compares gene transfer efficiencies as well as durations and levels of gene expression for human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) lentiviral vectors in a variety of human cell types in vitro. EIAV and HIV vectors transduced equivalent numbers of proliferating and G1/S- and G2/M-arrested cells, and both had very low efficiencies of transduction into G0-arrested cells. Analysis of the levels of both the enhanced green fluorescent protein (EGFP) and mRNA demonstrated that the HIV-transduced cells expressed greater levels of EGFP protein and RNA than the EIAV-transduced cells. Measurements of vector-derived EGFP RNA half-lives were fourfold higher with the HIV vector than with the EIAV vector. Long-term culture of EIAV-transduced human cells showed a significant decrease in the number of cells expressing the transgene; however, no corresponding loss was found in EIAV-transduced equine cells. In contrast, only a moderate decrease in the number of transgene-expressing cells was seen with the HIV vectors. Taken together, these results demonstrate that the EIAV vectors transduced human cells with efficiencies similar to those of the HIV vectors. However, our data indicate that transgene expression from EIAV vectors is limited by the instability of vector-derived RNA transcripts and silencing of the EIAV vectors over time.

Quantification of human T-cell lymphotropic virus type 1 proviral load by quantitative competitive polymerase chain reaction

The polymerase chain reaction (PCR) has been established as a highly sensitive technique for detection of viral DNA or RNA. However, due to inherent limitations of PCR the amount of amplified product often does not correlate with the initial amount of template DNA. This is particularly true for PCR detection of viral infections that are characterized by low in vivo viral copy numbers in certain stages of the infection, such as human T-cell lymphotropic virus type 1 (HTLV-1) and simian T-cell lymphotropic virus type 1 (STLV-1). Therefore, we developed a quantitative competitive polymerase chain reaction (qcPCR) for detection of HTLV-1 and STLV-1 proviral DNA. The assay was optimized using an infectious HTLV-1 clone, ACH, HTLV-1 infected cell lines, MT-2.6 and HUT-102 and STLV-1 infected lines Kia and Matsu. Applicability of this system was demonstrated by determining HTLV-1 proviral load in peripheral blood mononuclear cells (PBMC) of human subjects with HTLV-1 associated diseases and an asymptomatic carrier as well as rabbits infected experimentally. This qcPCR method, the first designed specifically for HTLV-1 and STLV-1, will provide an important tool for pathogenesis studies of HTLV-1 and for evaluating the efficacy of antiviral drugs and vaccines against the viral infection using animal models.

Repression of tax-mediated human t-lymphotropic virus type 1 transcription by inducible cAMP early repressor (ICER) protein in peripheral blood mononuclear Cells

Human T‐lymphotropic virus type 1 (HTLV‐1) infection causes adult T‐cell leukemia and is characterized by long periods of clinical latency with low levels of viral production. Transcription of HTLV‐1 is controlled through sequences in the promoter and enhancer regions of the long terminal repeat of the integrated provirus. Important among these sequences are three 21 bp imperfect repeats responsive to the viral oncogenic protein Tax (TRE). Members of the CREB/ATF‐1/CREM family of transcription factors bind to TRE‐1 and are critical for HTLV‐1 transcription. Other less studied family members include the inducible cAMP early repressor (ICER) proteins. ICER proteins lack phosphorylation and activation domains and are potent inhibitors of transcription. The ability of ICER to bind TRE‐1 and its effects on HTLV‐1 Tax mediated transcription have not been studied in the natural cell targets of the virus, peripheral blood mononuclear cells (PBMC). We show that ICER mRNA levels are low in quiescent PBMC, but rise and remain elevated for up to 18 hr after mitogenic stimulation of these cells. Electrophoretic mobility shift assays using recombinant Tax and ICER demonstrate that ICER binds TRE‐1 and that binding is increased in the presence of Tax. Furthermore, over expression of ICER IIγ suppressed Tax‐mediated transcription whereas an anti‐sense ICER II plasmid designed to block endogenous ICER enhanced Tax‐mediated transcription in activated PBMC. Together our data indicate that ICER inhibits Tax‐mediated transcription in activated PBMC and suggest a role for ICER in maintenance of HTLV‐1 persistence. J. Med. Virol. 62:286–292, 2000.

Analysis of gene transfer efficiency of retrovirus producer cell transplantation for in situ gene transfer to hematopoietic cells.

OBJECTIVE The aim of this study was to assess the gene transfer efficiency of an in situ administration protocol for hematopoietic stem/progenitor cells in the rhesus macaque (Macaca mulatta) animal model. MATERIALS AND METHODS Moloney murine leukemia virus amphotropic vector producer cells (1--2 x 10(8) cells/animal) were transplanted into the femoral bone marrow cavities of six macaques. To determine if the levels of gene transfer could be increased, a second injection at the same dose of producer cells was performed into the iliac crest in three of the six macaques. RESULTS We demonstrated that 0.02-0.1% of peripheral blood mononuclear cells contained the vector transgene for up to 12 months following the initial administration of producer cells. Hematopoietic progenitor cell assays indicated that the neomycin phosphotransferase gene was detected in 10--30% of progenitor cell colonies. A humoral immune response directed toward viral particles was demonstrated in all animals. Additionally, we demonstrated that an increase in the levels of transduced cells, up to 1% of circulating peripheral blood mononuclear cells and granulocytes, contain the transgene following producer cell readministration. CONCLUSIONS These data demonstrate the successful in situ gene transfer to hematopoietic stem/progenitor cells and circulating peripheral blood mononuclear cells that persists as long as 12 months postinjection, in the absence of any preconditioning.

Comparison of HTLV-I basal transcription and expression of CREB/ATF-1/CREM family members in peripheral blood mononuclear cells and Jurkat T cells.

HTLV-I is the etiologic agent of adult T-cell leukemia/lymphoma and is associated with tropical spastic paraparesis/HTLV-I-associated myelopathy. Following integration into the host cell genome, HTLV-I replication is regulated by both host and viral mechanisms that control transcription. Low levels of viral transcription (basal transcription) occur before expression of the virally encoded Tax protein (Tax-mediated transcription). Members of the cyclic adenosine monophosphate (cAMP) response element binding (CREB)/activating transcription factor 1 (ATF-1) family of transcription factors bind three 21-bp repeats (Tax-responsive element-1, or TRE-1) within the viral promoter and are important for basal and Tax-mediated transcription. Using mitogen stimulated and quiescent peripheral blood mononuclear cells (PBMC) and Jurkat cells, we compared differences in basal transcription and amounts and binding of transcription factors with TRE-1. We demonstrate that amounts of transcriptionally active phosphorylated CREB protein (P-CREB) differ between activated PBMC and Jurkat cells. Following stimulation, P-CREB levels remain elevated in PBMC for up to 24 hours whereas CREB is dephosphorylated in Jurkat cells within 4 hours following stimulation. The differences in P-CREB levels between PBMC and Jurkat cells were directly correlated with basal transcription of HTLV-I in the two cell types. Using electrophoretic mobility shift assays, we determined that the pattern of band migration differed between the two cell types. These data demonstrate that PBMC differentially regulate basal HTLV-I transcription compared with Jurkat T cells, and this differential regulation is due, in part to differential phosphorylation and binding of CREB/ATF-1 to TRE-1 in the HTLV-I promoter. We demonstrate the utility of using primary lymphocyte models to study HTLV-I transcription in the context of cell signaling and suggest that activated PBMC maintain elevated levels of P-CREB, which promote basal HTLV-I transcription and enhance viral persistence in vivo.

CD2 SIGNALLING INDUCES PHOSPHORYLATION OF CREB IN PRIMARY LYMPHOCYTES

Promoter sequences responsive to cyclic AMP (cAMP) are found in a number of cellular genes, and bind transcription factors of the cAMP response element binding protein (CREB)/activating transcription factor‐1 (ATF‐1) family. We have used a human T‐lymphotropic virus type 1 (HTLV‐1) model of cAMP response element (CRE) transcription to investigate the influence of lymphocyte activation on transcription from homologous regions in the viral promoter. We previously demonstrated increased HTLV‐1 transcription following CD2 but not CD3 receptor cross‐linking. We hypothesized that this increased viral transcription was mediated, in part, through the phosphorylation of CREB. Therefore, we investigated CD2 and CD3 receptor‐mediated signalling in primary human peripheral blood mononuclear cells (PBMC). CD2, but not CD3, cross‐linking increased cAMP detected by competitive enzyme‐linked immunosorbent assay (ELISA) approximately fourfold. CD2 cross‐linking concurrently increased phosphorylation of CREB detected by immunoblot assay eightfold. Consistent with post‐translational regulation, no change in total level of CREB protein was observed. Phosphorylation of CREB occurred through a herbimycin A and Rp‐cAMP‐sensitive pathway, suggesting phosphorylation required antecedent activation of both protein tyrosine kinases (PTK) and protein kinase A (PKA). Both CD2 and CD3 cross‐linking increased binding of nuclear proteins to a radiolabelled CRE oligonucleotide probe in electrophoretic mobility shift assays suggesting that lymphocyte activation enhances binding independently of phosphorylation of CREB at serine 133. These data indicate specific modulation of the CREB/ATF‐1 family of transcription factors by the CD2 signalling pathway and suggest CD2 receptor modulation of CRE‐mediated transcription following ligand engagement (e.g. cell‐to‐cell contact).

Stimulation of the CD2 receptor pathway induces apoptosis in human T lymphotropic virus type I-infected cell lines.

We demonstrate that CD2 receptor engagement, but not CD3 crosslinking, induces apoptosis in lymphocytes transformed by human T-cell lymphotrophic virus type I (HTLV-I). Mitogenic pairs of anti-CD2 monoclonal antibodies inhibited [3H]thymidine incorporation from 25 to 62% in CD2+ HTLV-I-infected lymphocytes. This inhibition was associated with a 20-40% reduction in cell number and viability over a 3-day period, morphologic evidence of apoptosis, and irreversible DNA fragmentation. While cyclosporin A abrogated CD2-mediated proliferation in peripheral blood mononuclear cells, it had no effect on CD2-induced apoptosis in the HTLV-I-infected cell lines. Since HTLV-I is mitogenic to resting lymphocytes through CD2 activation pathways, these results suggest that HTLV-I-infected lymphocytes are primed for apoptosis following additional CD2 stimulation. This CD2-mediated apoptosis might be a factor in immune regulation of HTLV-I-associated diseases or might offer a novel adjunctive approach to treatment.

Co-stimulation of human peripheral blood mononuclear cells with IL-2 and anti-CD3 monoclonal antibodies induces phosphorylation of CREB.

Phosphorylation of the cAMP-response element binding protein CREB within 1 h of CD2 but not CD3 cross-linking of human PBMC was recently demonstrated. The absence of P-CREB following CD3 cross-linking was unexpected, as other laboratories reported increased phosphorylation of CREB following CD3 cross-linking of the Jurkat lymphocyte cell line. Due to Jurkat T-cells being IL-2-independent, it was postulated that IL-2 might provide a necessary co-stimulus for phosphorylation of CREB in primary lymphocytes. Therefore, P-CREB was evaluated following co-stimulation of human PBMC through the IL-2 and CD2 or CD3 receptors. IL-2 did not further augment phosphorylation of CREB following CD2 cross-linking. However, while neither IL-2 nor CD3 cross-linking alone induced P-CREB, a 4.5-fold increase in phosphorylation of CREB within 1 h of IL-2/CD3 co-stimulation was observed. Phosphorylation was not associated with the induction of cAMP, and inhibition of PKA signaling had no effect on P-CREB. Consistent with signal transduction through p56lck or p59fyn, inhibition of PTK signaling reduced phosphorylation 50%. Interestingly, inhibiting PKC signaling with calphostin C further increased P-CREB levels 3-fold over that observed in IL-2/CD3 co-stimulated cells not pretreated with a PKC inhibitor. In contrast to previous studies performed in the absence of exogenous IL-2, no increase in binding of CREB to a 32P-labeled oligonucleotide probe was observed by electrophoretic mobility shift assay. These data suggest that the IL-2 and CD3 signaling pathways provide a necessary and co-operative stimulus promoting phosphorylation of CREB following receptor cross-linking.