Analysis of the HIV-1 LTR NF-κB-Proximal Sp Site III: Evidence for Cell Type-Specific Gene Regulation and Viral Replication

Abstract

It has been widely demonstrated that the human immunodeficiency virus type 1 (HIV-1) envelope, specifically the V3 loop of the gp120 spike, evolves to facilitate adaptation to different cellular populations within an infected host. Less energy has been directed at determining whether the viral promoter, designated the long terminal repeat (LTR), also exhibits this adaptive quality. Because of the unique nature of the cell populations infected during the course of HIV-1 infection, one might expect the opportunity for such adaptation to exist. This would permit select viral species to take advantage of the different array of conditions and factors influencing transcription within a given cell type. To investigate this hypothesis, the function of natural variants of the NF-kappaB-proximal Sp element (Sp site III) was examined in human cell line models of the two major cell types infected during the natural course of HIV-1 infection, T cells and monocytes. Utilizing the HIV-1 LAI molecular clone, which naturally contains a high-affinity Sp site III, substitution of low-affinity Sp sites in place of the natural site III element markedly decreased viral replication in Jurkat T cells. However, these substitutions had relatively small effects on viral replication in U-937 monocytic cells. Transient transfections of HIV-1 LAI-based LTR-luciferase constructs into these cell lines suggest that the large reduction in viral replication in Jurkat T cells, caused by low-affinity Sp site III variants, may result from reduced basal as well as Vpr- and Tat-activated LTR activities in Jurkat T cells compared to those in U-937 monocytic cells. When the function of Sp site III was examined in the context of HIV-1 YU-2-based LTR-luciferase constructs, substitution of a high-affinity element in place of the natural low-affinity element resulted in increased basal YU-2 LTR activity in Jurkat T cells and reduced activity in U-937 monocytic cells. These observations suggest that recruitment of Sp family members to Sp site III is of greater importance to the function of the viral promoter in the Jurkat T cell line as compared to the U-937 monocytic cell line. These observations also suggest that other regions of the LTR may compensate for Sp recruitment defects in specific cell populations.

Functional Incompatibility between the Generic NF-κB Motif and a Subtype-Specific Sp1III Element Drives the Formation of the HIV-1 Subtype C Viral Promoter

“…1B) (31). Importantly, experimental evidence is suggestive that the Sp1III site is functionally more important for the viral promoter than the other two (18,19,32). Given the biological significance of the centrally located Sp1III motif and the subtype-associated variations within this motif among HIV-1 subtypes, we asked if the heterologous Sp1III sequences derived from the other HIV-1 subtypes can substitute for the original Sp1III sequence in C-LTR.…”

“…Of the three motifs, the Sp1III site plays a more deterministic role in gene expression from the LTR, as Sp1 protein binding at this site must physically interact with the p65 protein of NF-B recruited to the H-B site located immediately upstream (17). Thus, the Sp1III site critical for the regulation of gene expression for the viral promoter demonstrates subtype-specific sequence variations, and the influence of such variations has not been adequately examined (18,19). Third, in contrast to the large genetic variation of the Sp1 sites, the sequence context of the NF-B binding sites is invariable in the viral strains regardless of the copy number difference.…”

“…In previous studies, an active functional interaction in the B-LTR was demonstrated between the centrally positioned NF-B and Sp1III sites in such a way that not only the spacing between the sites but also the relative orientation of each site with respect to the other cannot be altered, suggesting that the host factors must bind the specific elements in a specific orientation (17,21). Furthermore, the centrally positioned NF-B and Sp1III motifs play a role, more critical than that of the flanking sites (one upstream H-B site and two downstream Sp1 motifs), in controlling viral gene expression (18,19). Given the special significance attached to the central NF-B and the Sp1III motifs in the HIV-1 LTR, it is enigmatic that a genetically different NF-B site (the C-B motif) is inserted in the subtype C-LTR, disrupting the original association between the H-B and Sp1III motifs.…”

Human Immunodeficiency Virus Type 1 Subtypes Have a Distinct Long Terminal Repeat That Determines the Replication Rate in a Host-Cell-Specific Manner

“…On the one hand, there is redundancy and overlap in the TFBS function that ensures replication in several cellular environments. On the other hand, the LTR also contains sites that enhance replication in specific cellular environments (1,28,34,42,47,51). It seems likely that the subtype-specific variation in HIV-1 represents different adaptational solutions as a result of a changing and/or fluctuating environment.…”

Impact of Naturally Occurring Genetic Variation in the HIV-1 LTR TAR Region and Sp Binding Sites on Tat-Mediated Transcription

“…In this regard, deletion of the LTR Sp binding sites or GC box array has been shown to dramatically reduce Tat transactivation [16]. In clinical samples derived from HIV-1-infected patients in years prior to or after the implementation of effective combination antiretroviral therapy(cART), specific single-nucleotide polymorphisms have been observed within the NF-κB-proximal Sp transcription factor binding sites that exhibit a very low affinity for their cognate factor and alter LTR-driven viral gene activation [17,18]. Specifically, these studies reported that LTR sequence variants within the Sp site III (5T; a C-to-T change at position 5 of the binding site) were prevalent during the course of HIV-1 infection and exhibit very low binding affinities for members of the Sp transcription factor family [19,20].…”