Identification of binding sites for members of the CCAAT/enhancer binding protein transcription factor family in the simian immunodeficiency virus long terminal repeat.

Members of the CCAAT/enhancer binding protein (C/EBP) transcription factor family are necessary for human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) activity and viral replication in cells of monocyte/macrophage lineage. The integral roles that HIV-1-infected monocytes and macrophages play in the development and progression of HIV-1-associated disease in the immune and central nervous systems underscore the importance of the C/EBP transcription factor family within the context of regulating HIV-1 gene expression. Although there are considerable similarities between HIV-1 and simian immunodeficiency virus (SIV), including viral-induced immunopathogenesis and neurologic dysfunction, infection of CD4(+) T cells and cells of monocyte/macrophage origin, and LTR structure/function, the involvement of C/EBP factors in regulating SIV transcription has not been previously demonstrated. Analyses of the SIV(mac)239 LTR sequence indicated the presence of five putative C/EBP binding sites within the LTR. Electrophoretic mobility shift (EMS) analyses demonstrated that four of the five sites within the SIV LTR were able to bind C/EBP factors (alpha and beta) and compete for DNA-protein complexes formed by the HIV-1 C/EBP site located adjacent to the promoter-distal NF-kappaB site. DNase I protection assays indicated that purified C/EBPbeta specifically was able to occupy each of the four binding sites. These studies suggest that C/EBP factors may also have important roles in the regulation of SIV gene expression and replication, and that these factors and signal transduction pathways that regulate their activity may impact SIV-associated pathogenesis.

HIV-1 Vpr binding to HIV-1 LTR C/EBP cis-acting elements and adjacent regions is sequence-specific.

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is a 14 kDa virion-associated protein that transactivates the HIV-1 long terminal repeat (LTR) as well as other eukaryotic promoters. Vpr also functions in nuclear localization and import of the HIV-1 preintegration complex (PIC), cell cycle arrest at the G(2)/M interface, and virion packaging. Electrophoretic mobility shift analysis has been utilized to demonstrate a direct association between purified Vpr (strain pNL4-3) and HIV-1 LTR sequences that span the adjacent C/EBP site I, NF-kappaB site II, and ATF/CREB binding site (nt -95 to -130, relative to the start of transcription). A similar interaction has been observed between HIV-1 Vpr and LTR C/EBP site II (nt -167 to -175). A total of 94.7% of LTRs derived from peripheral blood contained C/EBP site I variants that displayed a high relative Vpr binding affinity phenotype, while only 5.3% exhibited a low relative Vpr binding affinity phenotype. All LTRs derived from peripheral blood exhibited a high relative Vpr binding phenotype at C/EBP site II. These results suggest a preference for the maintenance of two cis-acting elements with high affinity for Vpr within LTRs derived from peripheral blood. Additional studies have also demonstrated that naturally occurring sequence variation within C/EBP site I and II can dramatically alter the relative affinity of Vpr for these cis-acting elements. These studies suggest that Vpr may regulate the interaction of members of the C/EBP transcription factor family with the viral LTR.

C/EBP- and Tat-mediated activation of the HIV-1 LTR in CD34+ hematopoietic progenitor cells.

Human immunodeficiency virus type 1 (HIV-1) infection of cells of the monocyte/macrophage lineage within the bone marrow and peripheral blood plays an important role in the pathologic events leading to the development of the acquired immune deficiency syndrome (AIDS) as well as HIV-1 dementia (HIVD). The TF-1 erythro-myeloid cell line is being utilized as a model cellular phenotype to examine HIV-1 infection of a hematopoietic progenitor cell population. Expression of TF-1 cell surface marker RNAs and proteins was characterized by RT-PCR and FACS, respectively, and compared to those of the well characterized U-937 monocytic cell line. Transcription factors in TF-1 and U-937 cells that have been shown to be important for sustaining the expression of HIV-1 LTR activity were also examined. TF-1 cells were shown to contain the transcription factors C/EBP, Sp1, and NF-kappaB. C/EBP- and Tat-mediated induction of the YU-2 LTR was examined. Relative C/EBP induction of the HIV-1 strain YU-2 LTR was greater in TF-1 cells than in U-937 cells. When the C/EBP sites I and II were mutated to sequences with a low relative affinity for C/EBP factors, there was a reduction of Tat-mediated trans-activation in TF-1 cells, but not in U-937 cells. These studies form the foundation for investigations into the relationship between HIV-1 infection of bone marrow and peripheral blood precursor cells of the monocyte/macrophage lineage and pathogenesis associated with HIV-1 infection of the immune and central nervous system (CNS).

Structural and functional evolution of human immunodeficiency virus type 1 long terminal repeat CCAAT/enhancer binding protein sites and their use as molecular markers for central nervous system disease progression.

The appearance and progression of human immunodeficiency virus type 1 (HIV-1)-associated pathogenesis in the immune and central nervous systems is dependent on the ability of the virus to replicate in these compartments, which is, in turn, controlled by numerous factors, including viral binding and entry, receptor and coreceptor usage, and regulation of viral expression by the long terminal repeat (LTR). The LTR promotes viral expression in conjunction with viral and cellular regulatory proteins, including members of the CCAAT/enhancer binding protein (C/EBP) family, which modulate LTR activity through at least two cis-acting binding sites. Previous studies have shown that these sites are necessary for HIV-1 replication in cells of the monocyte/macrophage lineage, but dispensable in T lymphocytes. To establish potential links between this important family of transcription factors and HIV-1-associated pathogenesis, C/EBP site I and II sequence variation in peripheral blood mononuclear cell (PBMC)-derived LTRs from HIV-1-infected patients with varying degrees of disease severity was examined. A high prevalence of C/EBP site variants 3T (site I) and consensus B (site II) within PBMC-derived HIV-1 LTRs was shown to correlate with late stage disease in HIV-1-infected patients. These results suggest that the increased prevalence in the PBMCs of HIV-1 LTRs containing the 3T C/EBP site I variant and the consensus B site II variant may serve as a molecular marker for disease progression within the immune system. The relative low or high binding affinity of C/EBP beta to sites I and II in electrophoretic mobility shift (EMS) analyses correlated with low or high LTR activity, respectively, in transient expression analyses during both early and late disease stages. The 3T C/EBP site I was the only variant examined that was not found in LTRs derived from PBMCs of patients at early stages of HIV-1 disease, but was found at increasing frequencies in patients with late stage disease. Furthermore, the 3T C/EBP site I was not found in brain-derived LTRs of patients without HIV-1-associated dementia (HIVD), but was found in increasing numbers in brain-derived LTRs from patients diagnosed with HIVD. The C/EBP site I 3T variant appears to be exclusive to patients progressing to increasingly severe HIV-1-associated immunologic and neurologic disease.

Regulation of human immunodeficiency virus type 1 gene expression and pathogenesis by CCAAT/enhancer binding proteins in cells of the monocyte/macrophage lineage.

CCAAT/enhancer binding proteins (C/EBPs) are transcription factors that regulate a variety of cellular genes involved in broad range of physiological processes, including immune cell functions that involve cytokine expression and release as well as immune cell differentiation and inflammation. In addition, C/EBP factors regulate many viral promoters, including the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). The dependence of HIV-1 gene expression and replication on C/EBP factors in cells of myeloid origin positions these factors as important regulators of HIV-1 during the course of disease, because cells of monocyte/macrophage origin are critical components of viral pathogenesis in the peripheral blood and in other tissue compartments, including the central nervous system (CNS). Recent studies also indicate potentially important correlates between HIV-1 LTR C/EBP site sequence variation (which alters factor recruitment and activity), sequence compartmentalization, and the severity of HIV-1-induced immunologic dysfunction and neuropathogenesis. Cumulatively, studies of C/EBP factors and their roles in the regulation of host and viral gene expression indicate the importance of these factors in the progression of HIV-1-associated disease and, specifically, the genesis of CNS disease and HIV-1-associated dementia.