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

Abstract

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.

Region-specific distribution of human immunodeficiency virus type 1 long terminal repeats containing specific configurations of CCAAT/enhancer-binding protein site II in brains derived from demented and nondemented patients

“…Previous studies have indicated that the C/EBP site II ConB variant was preferentially encountered in brain-and peripheral blood-derived LTRs (Hogan et al, 2003a(Hogan et al, , 2003bRoss et al, 2001a). In addition, the LTRs containing C/EBP site II 4C and 6G variants were found only in brain tissues derived from patients with HIVD (Hogan et al, 2003b).…”

“…We have previously demonstrated that the C/EBP binding site I 3T variant (C/EBP site I variant where position 3 contains a C to T nucleotide change from the consensus B sequence) recruited little, if any, C/EBP, whereas the C/EBP binding site I ConB variant recruited modestly higher levels of C/EBP factors (Hogan et al, 2003b;Ross et al, 2001a). In a similar manner, we report herein that the ConB and 6G variants of C/EBP binding site II recruited large quantities of C/EBP α and β, whereas the C/EBP binding site II 4C variant recruited little, if any, C/EBP α and β.…”

“…The high degree of C/EBP site II conservation in brain-derived LTRs was likely important in LTR regulation because the C/EBP binding site II consensus cladeB (ConB) variant exhibits a relatively high affinity for members of the C/EBP transcription factor family (Ross et al, 2001a). In addition, we have demonstrated that LTRs containing C/EBP site II 4C and 6G sequence variants were only found in brain tissues derived from patients with HIVD (Hogan et al, 2003b) and not in brain tissues derived from patients without HIVD. The studies reported herein have focused on sequence variation within C/EBP site II of HIV-1 LTRs derived from six regions of the brain obtained from patients with and without HIVD.…”

“…In addition, the LTRs containing C/EBP site II 4C and 6G variants were found only in brain tissues derived from patients with HIVD (Hogan et al, 2003b). The C/EBP site II variants were designated according to the position at which base pair change occurred relative to the clade B consensus sequence, followed by the actual base pair that was found at the variant position ( Figure 1A).…”

“…As a first approach to examine the properties of these binding sites, recombinant LTR luciferase reporter constructs containing the C/EBP site II ConB, 6G, or 4C variants were constructed with either a C/EBP site I 3T or ConB configuration. The C/EBP site I 3T variant was of particular interest because it has been previously shown to appear only in brains of patients with HIVD and not in those from non-HIVD individuals (Hogan et al, 2003b). Transient transfection analyses were performed utilizing the U-937 monocytic cell line, and for comparative purposes, all values are shown relative to the construct containing the ConB sequence at both C/EBP sites I and II.…”