Identification and characterization of a human herpesvirus 6 gene segment capable of transactivating the human immunodeficiency virus type 1 long terminal repeat in an Sp1 binding site-dependent manner.
ABSTRACT: The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) is transactivated by various extracellular signals and viral cofactors that include human herpesviruses. These transactivators are capable of transactivating the HIV-1 LTR through the transactivation response element, NF-kappa B, or other regulatory binding elements. Human herpesvirus 6 (HHV-6) is a potential cofactor of HIV-1. Here, we report that an HHV-6 gene segment, ZVH14, which can neoplastically transform NIH 3T3 and human keratinocytes, is capable of transactivating HIV-1 LTR chloramphenicol acetyltransferase constructs in an Sp1 binding site-dependent manner. Transactivation increased synergistically in the presence of multiple Sp1 sites and was dramatically reduced by cotransfection with oligomers designed to form triplex structures with HIV-1 LTR Sp1 binding sites. HIV-1 LTR NF-kappa B sites were not essential for ZVH14-mediated transactivation. A putative open reading frame in ZVH14, B115, which may encode a highly basic peptide consisting of 115 amino acid residues, showed transactivation capacity similar to that of ZVH14. This open reading frame also transactivated the HIV-1 LTR in an Sp1 site-dependent fashion in African green monkey kidney cells and human T cells. These data suggest that HHV-6 may stimulate HIV-1 replication via transactivation of Sp1 binding sites present in the HIV-1 promoter.
Project description:Sequencing studies have indicated that the unique component of the human herpesvirus 6 (HHV-6) genome and the unique long segment of the human cytomegalovirus genome are genetically colinear. Of particular interest is the identification of a region of local CpG dinucleotide suppression in the genome of HHV-6, a feature conserved in the genomes of human cytomegalovirus, murine cytomegalovirus, and simian cytomegalovirus, and a characteristic of the major immediate-early loci of these viruses. Adjacent to this region in HHV-6 are approximately 30 copies of a 103- to 108-bp sequence element, which contains consensus binding sites for the transcription factors AP2 and NF kappa B, in addition to a single KpnI recognition site. Together, these KpnI repeat units may compose an immediate-early enhancer, analogous to those found in the cytomegaloviruses. We present the sequence of this region of HHV-6 and demonstrate that a transactivating function is encoded by this region. We have used polymerase chain reaction to synthesize fragments containing open reading frames and 5' sequences with or without the upstream KpnI repeat units. Effector plasmids containing these HHV-6 coding and 5' sequences were able to effect activation of heterologous promoter-chloramphenicol acetyltransferase (CAT) constructs, including adenovirus E3-CAT and E4-CAT, human T-cell lymphotropic virus type I long terminal repeat (LTR)-CAT, and human immunodeficiency virus LTR-CAT, in cotransfection experiments in Vero cells and peripheral blood lymphocytes. Furthermore, we have identified the major open reading frame (RF4; 2.3 kb) as being essential for activation, and we have shown that the NF kappa B, SP1, and TATA box motifs in the human immunodeficiency virus LTR are all required for full induction of the promoter by the HHV-6-encoded transactivator.
Project description:The sequence of a promoter within a genome does not uniquely determine gene expression levels and their variability; rather, promoter sequence can additionally interact with its location in the genome, or genomic context, to shape eukaryotic gene expression. Retroviruses, such as human immunodeficiency virus-1 (HIV), integrate their genomes into those of their host and thereby provide a biomedically-relevant model system to quantitatively explore the relationship between promoter sequence, genomic context, and noise-driven variability on viral gene expression. Using an in vitro model of the HIV Tat-mediated positive-feedback loop, we previously demonstrated that fluctuations in viral Tat-transactivating protein levels generate integration-site-dependent, stochastically-driven phenotypes, in which infected cells randomly 'switch' between high and low expressing states in a manner that may be related to viral latency. Here we extended this model and designed a forward genetic screen to systematically identify genetic elements in the HIV LTR promoter that modulate the fraction of genomic integrations that specify 'Switching' phenotypes. Our screen identified mutations in core promoter regions, including Sp1 and TATA transcription factor binding sites, which increased the Switching fraction several fold. By integrating single-cell experiments with computational modeling, we further investigated the mechanism of Switching-fraction enhancement for a selected Sp1 mutation. Our experimental observations demonstrated that the Sp1 mutation both impaired Tat-transactivated expression and also altered basal expression in the absence of Tat. Computational analysis demonstrated that the observed change in basal expression could contribute significantly to the observed increase in viral integrations that specify a Switching phenotype, provided that the selected mutation affected Tat-mediated noise amplification differentially across genomic contexts. Our study thus demonstrates a methodology to identify and characterize promoter elements that affect the distribution of stochastic phenotypes over genomic contexts, and advances our understanding of how promoter mutations may control the frequency of latent HIV infection.
Project description:Several gene fragments of human herpesvirus 6 (HHV-6) have been shown to activate the human immunodeficiency virus (HIV) type 1 long terminal repeat (LTR). An open reading frame (ORF) designated B701 (Y. Geng, B. Chandran, S. F. Josephs, and C. Wood, J. Virol. 66:1564-1570, 1992), found within a 22-kb HHV-6A strain GS [HHV-6A(GS)] genomic fragment and a 3.8-kb SalI subfragment, was shown to activate the HIV LTR. B701, also known as HHV-6 U16, is located in the immediate-early B (IE-B) region of the genome. The sequence of the 3.8-kb genomic fragment of HHV-6A(GS) is nearly identical to the published sequence of HHV-6A strain U1102, with minor differences. The HHV-6A(GS) B701 ORF (U16) was used to screen an HHV-6A(GS) cDNA library, and two different but overlapping cDNAs were identified. These cDNAs represent differently spliced transcripts ending at different polyadenylation signals. The ORFs included in the cDNAs are positionally homologous to the human cytomegalovirus (HCMV) UL36 ORF. The ORF in one cDNA was generated by splicing together in frame ORFs U17 and U16, and the second cDNA included ORFs U16 and U15. A third differentially spliced cDNA (U16+), was identified by 5' rapid amplification of cDNA ends. The predicted protein was identical to the U16 portion of the U17/U16 spliced gene product but did not include the U17 portion. 5'-extension analyses of the mRNAs demonstrated that at least two potential transcription initiation sites were used to express the transcripts encoding U17 and U16 gene products. Single-stranded U16 and U17 gene-specific RNA probes hybridized with at least five RNA species from infected cells and demonstrated that the expression of these transcripts was differentially regulated. The U17/U16 spliced gene products were expressed at IE times after infection, but a multiply spliced gene product encoded by U16 was expressed as a late gene. The U17/U16 and the U16+ gene products transactivated the HIV LTR. Thus, while there are similarities to the HCMV UL36-UL38 gene family, some of the IE-B U17/U16 transcripts are unique to HHV-6.
Project description:The HIV promoter within the viral long terminal repeat (LTR) orchestrates many aspects of the viral life cycle, from the dynamics of viral gene expression and replication to the establishment of a latent state. In particular, after viral integration into the host genome, stochastic fluctuations in viral gene expression amplified by the Tat positive feedback loop can contribute to the formation of either a productive, transactivated state or an inactive state. In a significant fraction of cells harboring an integrated copy of the HIV-1 model provirus (LTR-GFP-IRES-Tat), this bimodal gene expression profile is dynamic, as cells spontaneously and continuously flip between active (Bright) and inactive (Off) expression modes. Furthermore, these switching dynamics may contribute to the establishment and maintenance of proviral latency, because after viral integration long delays in gene expression can occur before viral transactivation. The HIV-1 promoter contains cis-acting Sp1 and NF-kappaB elements that regulate gene expression via the recruitment of both activating and repressing complexes. We hypothesized that interplay in the recruitment of such positive and negative factors could modulate the stability of the Bright and Off modes and thereby alter the sensitivity of viral gene expression to stochastic fluctuations in the Tat feedback loop. Using model lentivirus variants with mutations introduced in the Sp1 and NF-kappaB elements, we employed flow cytometry, mRNA quantification, pharmacological perturbations, and chromatin immunoprecipitation to reveal significant functional differences in contributions of each site to viral gene regulation. Specifically, the Sp1 sites apparently stabilize both the Bright and the Off states, such that their mutation promotes noisy gene expression and reduction in the regulation of histone acetylation and deacetylation. Furthermore, the NF-kappaB sites exhibit distinct properties, with kappaB site I serving a stronger activating role than kappaB site II. Moreover, Sp1 site III plays a particularly important role in the recruitment of both p300 and RelA to the promoter. Finally, analysis of 362 clonal cell populations infected with the viral variants revealed that mutations in any of the Sp1 sites yield a 6-fold higher frequency of clonal bifurcation compared to that of the wild-type promoter. Thus, each Sp1 and NF-kappaB site differentially contributes to the regulation of viral gene expression, and Sp1 sites functionally "dampen" transcriptional noise and thereby modulate the frequency and maintenance of this model of viral latency. These results may have biomedical implications for the treatment of HIV latency.
Project description:Jembrana disease virus (JDV) is a bovine lentivirus genetically similar to bovine immunodeficiency virus; it causes an acute and sometimes fatal disease in infected animals. This virus carries a very potent Tat that can strongly activate not only its own long terminal repeat (LTR) but also the human immunodeficiency virus (HIV) LTR. In contrast, HIV Tat cannot reciprocally activate the JDV LTR (H. Chen, G. E. Wilcox, G. Kertayadnya, and C. Wood, J. Virol. 73:658-666, 1999). This indicates that in transactivation JDV Tat may utilize a mechanism similar to but not the same as that of the HIV Tat. To further study the similarity of JDV and HIV tat in transactivation, we first tested the responses of a series of HIV LTR mutants to the JDV Tat. Cross-transactivation of HIV LTR by JDV Tat was impaired by mutations that disrupted the HIV type 1 transactivation response element (TAR) RNA stem-loop structure. Our results demonstrated that JDV Tat, like HIV Tat, transactivated the HIV LTR at least partially in a TAR-dependent manner. However, the sequence in the loop region of TAR was not as critical for the function of JDV Tat as it was for HIV Tat. The competitive inhibition of Tat-induced transactivation by the truncated JDV or HIV Tat, which consisted only of the activation domain, suggested that similar cellular factors were involved in both JDV and HIV Tat-induced transactivation. Based on the one-round transfection assay with HIV tat mutant proviruses, the cotransfected JDV tat plasmid can functionally complement the HIV tat defect. To further characterize the effect of JDV Tat on HIV, a stable chimeric HIV carrying the JDV tat gene was generated. This chimeric HIV replicated in a T-cell line, C8166, and in peripheral blood mononuclear cells, which suggested that JDV Tat can functionally substitute for HIV Tat. Further characterization of this chimeric virus will help to elucidate how JDV Tat functions and to explain the differences between HIV and JDV Tat transactivation.
Project description:Jembrana disease virus (JDV) is a newly identified bovine lentivirus that is closely related to the bovine immunodeficiency virus (BIV). JDV contains a tat gene, encoded by two exons, which has potent transactivation activity. Cotransfection of the JDV tat expression plasmid with the JDV promoter chloramphenicol acetyltransferase (CAT) construct pJDV-U3R resulted in a substantial increase in the level of CAT mRNA transcribed from the JDV long terminal repeat (LTR) and a dramatic increase in the CAT protein level. Deletion analysis of the LTR sequences showed that sequences spanning nucleotides -68 to +53, including the TATA box and the predicted first stem-loop structure of the predicted Tat response element (TAR), were required for efficient transactivation. The results, derived from site-directed mutagenesis experiments, suggested that the base pairing in the stem of the first stem-loop structure in the TAR region was important for JDV Tat-mediated transactivation; in contrast, nucleotide substitutions in the loop region of JDV TAR had less effect. For the JDV LTR, upstream sequences, from nucleotide -196 and beyond, as well as the predicted secondary structures in the R region, may have a negative effect on basal JDV promoter activity. Deletion of these regions resulted in a four- to fivefold increase in basal expression. The JDV Tat is also a potent transactivator of other animal and primate lentivirus promoters. It transactivated BIV and human immunodeficiency virus type 1 (HIV-1) LTRs to levels similar to those with their homologous Tat proteins. In contrast, HIV-1 Tat has minimal effects on JDV LTR expression, whereas BIV Tat moderately transactivated the JDV LTR. Our study suggests that JDV may use a mechanism of transactivation similar but not identical to those of other animal and primate lentiviruses.
Project description:We have studied the effects of chromomycin and of a triple-helix-forming oligonucleotide (TFO) that recognizes Sp1 binding sites on protein-DNA interactions and HIV-1 transcription. Molecular interactions between chromomycin, the Sp1 TFO and target DNA sequences were studied by gel retardation, triplex affinity capture using streptavidin-coated magnetic beads and biosensor technology. We also determined whether chromomycin and a TFO recognizing the Sp1 binding sites of the HIV-1 long terminal repeat (LTR) inhibit the activity of restriction enzyme HaeIII, which recognizes a sequence (5'-GGCC-3') located within these Sp1 binding sites. The effects of chromomycin and the TFO on the interaction between nuclear proteins or purified Sp1 and a double-stranded oligonucleotide containing the Sp1 binding sites of the HIV-1 LTR were studied by gel retardation. The effects of both chromomycin and TFO on transcription were studied by using an HIV-1 LTR-directed in vitro transcription system. Our results indicate that low concentrations of chromomycin potentiate the effects of the Sp1 TFO in inhibiting protein-DNA interactions and HIV-1-LTR-directed transcription. In addition, low concentrations of chromomycin do not affect binding of the TFO to target DNA molecules. The results presented here support the hypothesis that both DNA binding drugs and TFOs can be considered as sequence-selective modifiers of DNA-protein interactions, possibly leading to specific alterations of biological functions. In particular, the combined use of chromomycin and TFOs recognizing Sp1 binding sites could be employed in order to abolish the biological functions of promoters (such as the HIV-1 LTR) whose activity is potentiated by interactions with the promoter-specific transcription factor Sp1.
Project description:Human immunodeficiency virus type 1 (HIV-1) gene expression and replication are regulated by the promoter/enhancer located in the U3 region of the proviral 5' long terminal repeat (LTR). The binding of cellular transcription factors to specific regulatory sites in the 5' LTR is a key event in the replication cycle of HIV-1. Since transcriptional activity is regulated by the posttranslational modification of transcription factors with the monosaccharide O-linked N-acetyl-D-glucosamine (O-GlcNAc), we evaluated whether increased O-GlcNAcylation affects HIV-1 transcription. In the present study we demonstrate that treatment of HIV-1-infected lymphocytes with the O-GlcNAcylation-enhancing agent glucosamine (GlcN) repressed viral transcription in a dose-dependent manner. Overexpression of O-GlcNAc transferase (OGT), the sole known enzyme catalyzing the addition of O-GlcNAc to proteins, specifically inhibited the activity of the HIV-1 LTR promoter in different T-cell lines and in primary CD4(+) T lymphocytes. Inhibition of HIV-1 LTR activity in infected T cells was most efficient (>95%) when OGT was recombinantly overexpressed prior to infection. O-GlcNAcylation of the transcription factor Sp1 and the presence of Sp1-binding sites in the LTR were found to be crucial for this inhibitory effect. From this study, we conclude that O-GlcNAcylation of Sp1 inhibits the activity of the HIV-1 LTR promoter. Modulation of Sp1 O-GlcNAcylation may play a role in the regulation of HIV-1 latency and activation and links viral replication to the glucose metabolism of the host cell. Hence, the establishment of a metabolic treatment might supplement the repertoire of antiretroviral therapies against AIDS.
Project description:The primary body of information on the structure of human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)/gag leader genotypes has been determined from the analysis of cocultivated isolates. Functional studies of this regulatory portion of the provirus have been derived from the study of in vitro-generated mutations of laboratory-adapted molecular clones of HIV-1. We have performed a longitudinal analysis of molecular clones from the LTR/gag leader region amplified directly from the peripheral blood of four patients over three years. We have found a remarkable number of point mutations and length polymorphisms in cis- and trans-acting regulatory elements within this cohort. Most of the length polymorphisms were associated with duplications of Sp1 and TCF-1 alpha sequences. These mutations were associated with a wide range of transcriptional activities for these genotypes in a reporter gene assay. Mutations in conserved Sp1 sequences correlated with a diminished capacity of such genotypes to bind purified Sp1 protein. Although no generalized trend in transcriptional activity was seen, a single patient accumulated mutations in NF-kappa B, Sp1, and TAR elements over this period. The analysis of naturally occurring mutations of LTR genotypes provides a means to study the molecular genetic consequences of virus-host interactions and to assess the functional impact of HIV therapeutics.
Project description:The HIV-2 long terminal repeat (LTR) region contains several transcription factor (TF) binding sites. Efficient LTR transactivation by cellular TF and viral proteins is crucial for HIV-2 reactivation and viral production. Proviral LTRs from 66 antiretroviral-naive HIV-2-infected patients included in the French ANRS HIV-2 CO5 Cohort were sequenced. High genetic variability within the HIV-2 LTR was observed, notably in the U3 subregion, the subregion encompassing most known TF binding sites. Genetic variability was significantly higher in HIV-2 group B than in group A viruses. Notably, all group B viruses lacked the peri-ETS binding site, and 4 group B sequences (11%) also presented a complete deletion of the first Sp1 binding site. The lack of a peri-ETS binding site was responsible for lower transcriptional activity in activated T lymphocytes, while deletion of the first Sp1 binding site lowered basal or Tat-mediated transcriptional activities, depending on the cell line. Interestingly, the HIV-2 cellular reservoir was less frequently quantifiable in patients infected by group B viruses and, when quantifiable, the reservoirs were significantly smaller than in patients infected by group A viruses. Our findings suggest that mutations observed in vivo in HIV-2 LTR sequences are associated with differences in transcriptional activity and may explain the small cellular reservoirs in patients infected by HIV-2 group B, providing new insight into the reduced pathogenicity of HIV-2 infection.IMPORTANCE Over 1 million patients are infected with HIV-2, which is often described as an attenuated retroviral infection. Patients frequently have undetectable viremia and evolve at more slowly toward AIDS than HIV-1-infected patients. Several studies have reported a smaller viral reservoir in peripheral blood mononuclear cells in HIV-2-infected patients than in HIV-1-infected patients, while others have found similar sizes of reservoirs but a reduced amount of cell-associated RNA, suggesting a block in HIV-2 transcription. Recent studies have found associations between mutations within the HIV-1 LTR and reduced transcriptional activities. Until now, mutations within the HIV-2 LTR region have scarcely been studied. We conducted this research to discover if such mutations exist in the HIV-2 LTR and their potential association with the viral reservoir and transcriptional activity. Our study indicates that transcription of HIV-2 group B proviruses may be impaired, which might explain the small viral reservoir observed in patients.