Lipopolysaccharide(LPS)-Induction of the HTLV-1 LTR in Monocytes

Abstract 2167

The human leukemia virus type 1 (HTLV-1) gene expression is regulated by the viral proteins and various cellular transcription factors. HTLV-1 genome encodes not only structural proteins, but also non-structural proteins such as Tax, a transcriptional activator for STAT5 p12^I, and HTLV-1 bZIP factor (HBZ) encoded by the minus strand of the viral genome. The functional analysis of the viral proteins such as Tax has shed light on the pathogenesis of adult T cell leukemia/lymphoma (ATL). Expression of Tax is enhanced by T-cell activation stimuli such as phorbol ester (PMA), phytohemagglutinin (PHA) or sodium butyrate in chronically HTLV-1-infected CD4+T-cells. Transgenic mouse studies with Tax expression under the control of the granzyme B promoter or the proximal Lck promoter showed that disease progression is associated with infiltration of activated T- and inflammatory cells, and dysregulated inflammatory cytokine production. More recently, a transgenic mouse model with Tax expression regulated by the LTR (LTR-Tax) showed that LTR-Tax CD4 positive T-cells are hyper-proliferative and hyper-responsive to immune stimulation and strongly produce Th1-, Th2- and Th17-associated cytokines. In addition, HTLV-1 infection causes inflammatory disease of the central nerve system, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) as well as ATL. Aberrant cytokine gene expression is the hallmark of HTLV-1-associated diseases.

HTLV-1 infection is widely distributed among mammalian cells. We previously demonstrated that Tax transactivates the promoter of human proIL-1β gene (IL1B) gene through association with two transcription factors, NF-IL6 (C/EBPβ) and Spi-1 (PU.1) in monocytes. Tax synergized with lipopolysaccharide (LPS) to induce IL1B promoter activity. Spi-1 is an Ets family protein restricted in expression to monocytes/myelocytes, B cells, mast cells and erythrocyte stem cells, while NF-IL6 is widely expressed. LPS, a component of the gram-negative bacterial cell wall involved in the activation of monocytes, binds to TLR4, leading to activation of TRAF6, IRAK and MyD88. We now extend these studies to elucidate roles of LPS and Tax on HTLV-1 LTR promoter regulation in monocytes. When HTLV-1 LTR reporter was transfected into THP-1 monocytic cells, LPS dose-dependently induced HTLV-1 LTR. The mid-LTR of the HTLV-1 gene possesses three potential Ets binding elements centered on a GGAA motif (PuB1, pets and PuB2). Elf-1 has been shown to be the predominant protein binding to the HTLV-1 Ets sites in Jurkat and peripheral blood T-cells. In the present study, mutation of the Ets sites, especially pets and PuB2 caused significant inhibition of LPS-induced LTR activity in THP-1 cells. EMSA studies using THP-1 nuclear extracts showed binding of Spi-1 to the HTLV-1 Ets sites in THP-1 cells. Anti-Spi-1 Ab, but not anti-Elf-1 Ab or anti-ets-1 Ab supershifted the complex generated by THP-1 nuclear extract and HTLV-1 LTR Ets site. However, when migration pattern of the complex was compared with recombinant Spi-1 in vitro translated in a reticulocyte lysate system, the THP-1 complex migrated slower than recombinant Spi-1 protein. In this regard, anti-IRF-8 Ab further recognized the slow complex. Several reports recently showed that IRF-8 functions as a heterodimeric complex with spi-1 for expression of relevant genes.

On the other hand, when Tax expression vector was cotransfected into THP-1 cells along with HTLV-1 LTR reporter, Tax synergized with LPS to activate LTR. Spi-1 protein has three independent transcriptional activation domains (TAD); a TBP binding region, a Q domain, and a PEST region. GST pull-down studies using GST-Tax and ³⁵S-labeled recombinant Spi-1 revealed that mutant Spi-1 lacking the TADs still retains the ability to interact with Tax. In contrast to THP-1 cells, Jurkat T-cells showed only a marginal increase in IL1B promoter activity following Tax expression. Mutations of the Spi-1 binding site in the IL1B promoter did not affect Tax-induced activity in Jurkat cells. Any factors did not bind to the IL1B Spi-1 site in Jurkat cells. Thus, our data suggest that LPS cooperates with Tax to activate the viral and various cellular genes in HTLV-1-infected monocytes. Spi-1 is a key player in the monocyte-specific gene regulation in HTLV-1 infection.