Latency-associated degradation of the MRP1 drug transporter during latent human cytomegalovirus infection.

The reactivation of latent human cytomegalovirus (HCMV) infection after transplantation is associated with high morbidity and mortality. In vivo, myeloid cells and their progenitors are an important site of HCMV latency, whose establishment and/or maintenance require expression of the viral transcript UL138. Using stable isotope labeling by amino acids in cell culture-based mass spectrometry, we found a dramatic UL138-mediated loss of cell surface multidrug resistance-associated protein-1 (MRP1) and the reduction of substrate export by this transporter. Latency-associated loss of MRP1 and accumulation of the cytotoxic drug vincristine, an MRP1 substrate, depleted virus from naturally latent CD14(+) and CD34(+) progenitors, all of which are in vivo sites of latency. The UL138-mediated loss of MRP1 provides a marker for detecting latent HCMV infection and a therapeutic target for eliminating latently infected cells before transplantation.

BET bromodomain inhibition as a novel strategy for reactivation of HIV-1.

The persistence of latent HIV-1 remains a major challenge in therapeutic efforts to eradicate infection. We report the capacity for HIV reactivation by a selective small molecule inhibitor of BET family bromodomains, JQ1, a promising therapeutic agent with antioncogenic properties. JQ1 reactivated HIV transcription in models of latent T cell infection and latent monocyte infection. We also tested the effect of exposure to JQ1 to allow recovery of replication-competent HIV from pools of resting CD4(+) T cells isolated from HIV-infected, ART-treated patients. In one of three patients, JQ1 allowed recovery of virus at a frequency above unstimulated conditions. JQ1 potently suppressed T cell proliferation with minimal cytotoxic effect. Transcriptional profiling of T cells with JQ1 showed potent down-regulation of T cell activation genes, including CD3, CD28, and CXCR4, similar to HDAC inhibitors, but JQ1 also showed potent up-regulation of chromatin modification genes, including SIRT1, HDAC6, and multiple lysine demethylases (KDMs). Thus, JQ1 reactivates HIV-1 while suppressing T cell activation genes and up-regulating histone modification genes predicted to favor increased Tat activity. Thus, JQ1 may be useful in studies of potentially novel mechanisms for transcriptional control as well as in translational efforts to identify therapeutic molecules to achieve viral eradication.

Structural and functional studies of CCAAT/enhancer binding sites within the human immunodeficiency virus type 1 subtype C LTR.

Human immunodeficiency virus type 1 (HIV-1) subtype C, which is most predominant in sub-Saharan Africa as well as in Asia and India, is the most prevalent subtype worldwide. A large number of transcription factor families have been shown to be involved in regulating HIV-1 gene expression in T lymphocytes and cells of the monocyte-macrophage lineage. Among these, proteins of the CCAAT/enhancer binding protein (C/EBP) family are of particular importance in regulating HIV-1 gene expression within cells of the monocytic lineage during the course of hematologic development and cellular activation. Few studies have examined the role of C/EBPs in long terminal repeat (LTR)-directed viral gene expression of HIV-1 subtypes other than subtype B. Within subtype B viruses, two functional C/EBP sites located upstream of the TATA box are required for efficient viral replication in cells of the monocyte-macrophage lineage. We report the identification of three putative subtype C C/EBP sites, upstream site 1 and 2 (C-US1 and C-US2) and downstream site 1 (C-DS1). C-US1 and C-DS1 were shown to form specific DNA-protein complexes with members of the C/EBP family (C/EBPα, ß, and δ). Functionally, within the U-937 monocytic cell line, subtype B and C LTRs were shown to be equally responsive to C/EBPß-2, although the basal activity of subtype C LTRs appeared to be higher. Furthermore, the synergistic interaction between C/EBPß-2 and Tat with the subtype C LTR was also observed in U-937 cells as previously demonstrated with the subtype B LTR.

Japanese encephalitis virus down-regulates thioredoxin and induces ROS-mediated ASK1-ERK/p38 MAPK activation in human promonocyte cells.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes severe neurological disease with high mortality. Molecular mechanisms of JEV pathogenesis such as upstream apoptotic processes and pathways are not yet completely resolved or understood. In this study, JEV replication in human promonocyte cells induced time-dependent apoptosis and activated virus dose-dependent caspases 3, 8 and 9. Proteomic analysis demonstrated up- and down-regulated (more or less than 1.5-fold) proteins in JEV-infected promonocyte cells. Biological process categorization showed processes of antioxidation, free radical removal, and sulfur redox metabolism entailed many identified up- and down-regulated proteins. Down-regulation of thioredoxin, confirmed by using Western blotting, was involved in the apoptosis process of the oxidative stress response pathway. JEV infection caused increased intracellular ROS production and activation of ASK1-ERK/p38 MAPK signaling. ERK/p38 MAPK inhibitor PD98059 treatment definitely suppressed this apoptosis. Down-regulation of thioredoxin, increased intracellular ROS, and activation of ASK1-ERK/p38 MAPK signaling all were associated with JEV-induced apoptosis. These results are suggestive of an oxidative stress-pathway as a key element of JE pathogenesis.

Extracellular high mobility group box-1 inhibits R5 and X4 HIV-1 strains replication in mononuclear phagocytes without induction of chemokines and cytokines.

OBJECTIVE: High mobility group box-1 (HMGB1) is a nuclear chromatin protein. Furthermore, it induces chemotaxis and inflammation once released in the extracellular milieu, and it has been reported to upregulate, but also to inhibit HIV-1 replication in different cell types. We here investigated the potential role of extracellular HMGB1 in both R5 and X4 HIV-1 replication in primary human monocyte-derived macrophages (MDM) and U937 promonocytic cells, respectively. DESIGN: MDM or U937 cells were infected with R5 and X4 HIV-1 strains, respectively, in the presence or absence of endotoxin-free recombinant (r) HMGB1 or necrotic cell supernatants either containing or depleted of endogenous HMGB1. METHODS: HIV replication was measured by means of virion-associated reverse transcriptase activity in culture supernatants and cell-associated viral protein expression. Cytokine and chemokine production were measured by enzyme-linked immunosorbent assay; cell surface expression of CD4, CC chemokine receptor 5, receptor for advanced glycation end-products, Toll-like receptor-2 and Toll-like receptor-4 were analyzed by flow cytometry. RESULTS: Both rHMGB1 and necrotic cell supernatant-associated HMGB1 inhibited replication of R5 HIV-1 in MDM. Surprisingly enough, no upregulation of CC chemokine receptor 5-binding chemokines or of other chemokines and cytokines was observed in rHMGB1-stimulated MDM. HMGB1 also induced chemotaxis and strongly inhibited the replication of X4 HIV-1 in the 'Minus' subset of U937 cell clones expressing high levels of putative HMGB1 receptors (receptor for advanced glycation end-products, Toll-like receptors 2 and 4). CONCLUSION: Extracellular HMGB1 is a potent inhibitor of both R5 and X4 HIV-1 replication in mononuclear phagocytic cells without inducing the release of HIV-Modulatory chemokines or cytokines.