Featured Research Projects

Identification of Direct Transcriptional Targets of the Kaposi’s sarcoma-associated herpesvirus (KSHV) Rta Lytic Switch Protein by conditional nuclear localization

Lytic reactivation from latency is critical for the pathogenesis of Kaposi’s sarcoma-associated herpesvirus (KSHV). We previously demonstrated that the 691-amino-acid (aa) KSHV Rta transcriptional transactivator is necessary and sufficient to reactivate the virus from latency. Viral lytic cycle genes, including those expressing additional transactivators and putative oncogenes, are induced in a cascade fashion following Rta expression.  In this study, we sought to define Rta’s direct targets during reactivation by generating a conditionally nuclear variant of Rta. Wild-type Rta protein is constitutively localized to cell nuclei and contains two putative nuclear localization signals (NLSs). Only one NLS (NLS2; aa 516 to 530) was required for the nuclear localization of Rta, and it relocalized enhanced green fluorescent protein exclusively to cell nuclei. The results of analyses of Rta NLS mutants demonstrated that proper nuclear localization of Rta was required for transactivation and the stimulation of viral reactivation. RTA with NLS1 and NLS2 deleted was fused to the hormone-binding domain of the murine estrogen receptor to generate an Rta variant whose nuclear localization and ability to transactivate and induce reactivation were tightly controlled posttranslationally by the synthetic hormone tamoxifen. We used this strategy in KSHV-infected cells treated with protein synthesis inhibitors to identify direct transcriptional targets of Rta. Rta activated only eight KSHV genes in the absence of de novo protein synthesis. These direct transcriptional targets of Rta were transactivated to different levels and included the genes nut-1/PAN, ORF57/Mta, ORF56/Primase, K2/viral interleukin-6 (vIL-6), ORF37/SOX, K14/vOX, K9/vIRF1, and ORF52. Our data suggest that the induction of most of the KSHV lytic cycle genes requires additional protein expression after the expression of Rta.

Bu, W. Palmeri, D., Krishnan, R., Marin, R., Aris, V.M., Soteropoulos, P. and Lukac, D.M. (2008) Identification of Direct Transcriptional Targets of the Kaposi’s sarcoma-associated herpesvirus (KSHV) Rta Lytic Switch Protein by conditional nuclear localization.  Journal of Virology 82:10709-23

 

Transcriptional Profiling of Mycobacterium tuberculosis and host inflammatory response

During lung infection Mycobacterium tuberculosis resides inside macrophages and subverts the bactericidal mechanisms of these professional phagocytes. Comprehension of this host-pathogen relationship is fundamental for the development of new therapies to cure and prevent tuberculosis.  In this project we compared the gene expression profiling of M. tuberculosis strain H37Rv and a mutant strain with a deletion of the Sigma factor E (SigE) after infection of human macrophage-like cells (THP-1).  We conversely analyzed the gene expression profiles of the THP1-1 cells and resting and IFN-gamma treated mouse bone marrow macrophages after infection with the sigE mutant or the wild type strain H37Rv.  We were able to conclude that the gene expression profile of M. tuberculosis inside THP-1 cells suggests the perturbation of the cell envelope is one of the major intracellular stresses inside THP-1 macrophages, and that SigE controls the expression of Mtb factors related to maintenance of cell envelope function.  We also determined that inflammatory response of both, the THP-1 cells and the mouse bone marrow macrophages was enhanced after infection with the sigE mutant strain as compared to the wild type strain.  Our results demonstrate that the Mtb sigma factor E regulates the expression of bacterial components important for the maintenance of the cell envelope that helps the microorganism to cope with environmental stress and to suppress the host immune system and the antibacterial response.

Fontán, P., Aris, V., Ghanny, S., Soteropoulos, P. and Smith, I. (2008) The global transcriptional profile of Mycobacterium tuberculosis during THP-1 human macrophage infection. Infect. Immun. 76:717-25.

Fontán, P., Aris, V., Alvarez, M.E. Ghanny, S., Cheng, J., Soteropoulos, P. Trevani, A., Pine, R. and Smith, I. (2008) Mycobacterium tuberculosis SigE Regulon Modulates the Host Inflammatory Response. Journal of Infectious Diseases 198:877-85

 

Previously Featured Projects