Mutations generated in human immunodeficiency virus type 1 long terminal repeat during vertical transmission correlate with viral gene expression.
ABSTRACT: We determined the effect of mutations generated in HIV-1 LTR on viral gene expression in six mother-infant pairs following vertical transmission. We show that the functional domains critical for LTR function, the promoter (TATAA), enhancers (three SpI and two NFkappaB sites), the modulatory region (two AP-I sites, two NFAT, one NF-IL6 site, one Ets-1, and one USF-1) and the TAR region were generally conserved among mother-infant pairs, although we observed several patient and pair specific mutations in these important domains. We then determined the promoter activity of our mother-infant LTR sequences by measuring CAT gene expression, which was driven by these LTRs and found that most of these HIV-1 LTRs derived from 6 mother-infant pairs were functional. However, mutations in the important transcription factor binding sites, including TATAA, SpI, NFkappaB, AP-I, NFAT, NF-IL6, Ets-1, USF-1 and TAR resulted in reduced LTR driven CAT gene expression. Taken together, conservation of functional domains in the LTR during vertical transmission supports the notion that a functional LTR is critical in viral replication and pathogenesis and mutations generated during the course of infection correlated with HIV-1 gene expression.
Project description:Functional mapping of the 5'LTR has shown that the U3 and the R regions (U3R) contain a cluster of regulatory elements involved in the control of HIV-1 transcription and expression. As the HIV-1 genome is characterized by extensive variability, here we aimed to describe mutations in the U3R from various HIV-1 clades and CRFs in order to highlight strain specific differences that may impact the biological properties of diverse HIV-1 strains. To achieve our purpose, the U3R sequence of plasma derived virus belonging to different clades (A1, B, C, D, F2) and recombinants (CRF02_AG, CRF01_AE and CRF22_01A1) was obtained using Illumina technology. Overall, the R region was very well conserved among and across different strains, while in the U3 region the average inter-strains nucleotide dissimilarity was up to 25%. The TAR hairpin displayed a strain-distinctive cluster of mutations affecting the bulge and the loop, but mostly the stem. Like in previous studies we found a TATAA motif in U3 promoter region from the majority of HIV-1 strains and a TAAAA motif in CRF01_AE; but also in LTRs from CRF22_01A1 isolates. Although LTRs from CRF22_01A1 specimens were assigned CRF01_AE, they contained two NF-kB sites instead of the single TFBS described in CRF01_AE. Also, as previously describe in clade C isolates, we found no C/EBP binding site directly upstream of the enhancer region in CRF22_01A1 specimens. In our study, one-third of CRF02_AG LTRs displayed three NF-kB sites which have been mainly described in clade C isolates. Overall, the number, location and binding patterns of potential regulatory elements found along the U3R might be specific to some HIV-1 strains such as clade F2, CRF02_AG, CRF01_AE and CRF22_01A1. These features may be worth consideration as they may be involved in distinctive regulation of HIV-1 transcription and replication by different and diverse infecting strains.
Project description:Mathematical models for the regulation of the Ca(2+)-dependent transcription factors NFAT and NFkappaB that are involved in the activation of the immune and inflammatory responses in T lymphocytes have been developed. These pathways are important targets for drugs, which act as powerful immunosuppressants by suppressing activation of NFAT and NFkappaB in T cells. The models simulate activation and deactivation over physiological concentrations of Ca(2+), diacyl glycerol (DAG), and PKCtheta using single and periodic step increases. The model suggests the following: (1) the activation NFAT does not occur at low frequencies as NFAT requires calcineurin activated by Ca(2+) to remain dephosphorylated and in the nucleus; (2) NFkappaB is activated at lower Ca(2+) oscillation frequencies than NFAT as IkappaB is degraded in response to elevations in Ca(2+) allowing free NFkappaB to translocate into the nucleus; and (3) the degradation of IkappaB is essential for efficient translocation of NFkappaB to the nucleus. Through sensitivity analysis, the model also suggests that the largest controlling factor for NFAT activation is the dissociation/reassociation rate of the NFAT:calcineurin complex and the translocation rate of the complex into the nucleus and for NFkappaB is the degradation/resynthesis rate of IkappaB and the import rate of IkappaB into the nucleus.
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:BACKGROUND: Retroviral LTRs, paired or single, influence the transcription of both retroviral and non-retroviral genomic sequences. Vertebrate genomes contain many thousand endogenous retroviruses (ERVs) and their LTRs. Single LTRs are difficult to detect from genomic sequences without recourse to repetitiveness or presence in a proviral structure. Understanding of LTR structure increases understanding of LTR function, and of functional genomics. Here we develop models of orthoretroviral LTRs useful for detection in genomes and for structural analysis. PRINCIPAL FINDINGS: Although mutated, ERV LTRs are more numerous and diverse than exogenous retroviral (XRV) LTRs. Hidden Markov models (HMMs), and alignments based on them, were created for HML- (human MMTV-like), general-beta-, gamma- and lentiretroviruslike LTRs, plus a general-vertebrate LTR model. Training sets were XRV LTRs and RepBase LTR consensuses. The HML HMM was most sensitive and detected 87% of the HML LTRs in human chromosome 19 at 96% specificity. By combining all HMMs with a low cutoff, for screening, 71% of all LTRs found by RepeatMasker in chromosome 19 were found. HMM consensus sequences had a conserved modular LTR structure. Target site duplications (TG-CA), TATA (occasionally absent), an AATAAA box and a T-rich region were prominent features. Most of the conservation was located in, or adjacent to, R and U5, with evidence for stem loops. Several of the long HML LTRs contained long ORFs inserted after the second A rich module. HMM consensus alignment allowed comparison of functional features like transcriptional start sites (sense and antisense) between XRVs and ERVs. CONCLUSION: The modular conserved and redundant orthoretroviral LTR structure with three A-rich regions is reminiscent of structurally relaxed Giardia promoters. The five HMMs provided a novel broad range, repeat-independent, ab initio LTR detection, with prospects for greater generalisation, and insight into LTR structure, which may aid development of LTR-targeted pharmaceuticals.
Project description:LTR retrotransposons constitute a significant part of plant genomes and their evolutionary dynamics play an important role in genome size changes. Current methods of LTR retrotransposon age estimation are based only on LTR (long terminal repeat) divergence. This has prompted us to analyze sequence similarity of LTRs in 25,144 LTR retrotransposons from fifteen plant species as well as formation of solo LTRs. We found that approximately one fourth of nested retrotransposons showed a higher LTR divergence than the pre-existing retrotransposons into which they had been inserted. Moreover, LTR similarity was correlated with LTR length. We propose that gene conversion can contribute to this phenomenon. Gene conversion prediction in LTRs showed potential converted regions in 25% of LTR pairs. Gene conversion was higher in species with smaller genomes while the proportion of solo LTRs did not change with genome size in analyzed species. The negative correlation between the extent of gene conversion and the abundance of solo LTRs suggests interference between gene conversion and ectopic recombination. Since such phenomena limit the traditional methods of LTR retrotransposon age estimation, we recommend an improved approach based on the exclusion of regions affected by gene conversion.
Project description:The CRISPR/Cas9 system has been proposed as a cure strategy for HIV. However, few published guide RNAs (gRNAs) are predicted to cleave the majority of HIV-1 viral quasispecies (vQS) observed within and among patients. We report the design of a novel pipeline to identify gRNAs that target HIV across a large number of infected individuals. Next generation sequencing (NGS) of LTRs from 269 HIV-1-infected samples in the Drexel CARES Cohort was used to select gRNAs with predicted broad-spectrum activity. In silico, D-LTR-P4-227913 (package of the top 4 gRNAs) accounted for all detectable genetic variation within the vQS of the 269 samples and the Los Alamos National Laboratory HIV database. In silico secondary structure analyses from NGS indicated extensive TAR stem-loop malformations predicted to inactivate proviral transcription, which was confirmed by reduced viral gene expression in TZM-bl or P4R5 cells. Similarly, a high sensitivity in vitro CRISPR/Cas9 cleavage assay showed that the top-ranked gRNA was the most effective at cleaving patient-derived HIV-1 LTRs from five patients. Furthermore, the D-LTR-P4-227913 was predicted to cleave a median of 96.1% of patient-derived sequences from other HIV subtypes. These results demonstrate that the gRNAs possess broad-spectrum cutting activity and could contribute to an HIV cure.
Project description:FoxP3 determines the development of CD4+CD25+ regulatory T (Treg) cells and represses interleukin-2 (IL-2) expression in Treg cells. However, human immunodeficiency virus type 1 (HIV-1) infects and replicates efficiently in FoxP3+ Treg cells. We report that, while inhibiting IL-2 gene expression, FoxP3 enhances gene expression from HIV-1 long terminal repeat (LTR). This FoxP3 activity requires both the N- and C-terminal domains and is inactivated by human IPEX (immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome) mutations. FoxP3 enhances HIV-1 LTR via its specific NFkappaB binding sequences in an NFkappaB-dependent fashion in T cells but not in HEK293 cells. FoxP3 decreases level of histone acetylation at the interleukin-2 locus but not at the HIV-1 LTR. Although NFkappaB nuclear translocation is not altered, FoxP3 enhances NFkappaB-p65 binding to HIV-1 LTR. These data suggest that FoxP3 modulates gene expression in a promoter sequence-dependent fashion by modulating chromatin structure and NFkappaB activity. HIV-1 LTR has evolved to both highjack the T-cell activation pathway for expression and to resist FoxP3-mediated suppression of T-cell activation.
Project description:We have previously reported that the long terminal repeat (LTR) region of feline leukemia viruses (FeLVs) can enhance expression of certain cellular genes such as the collagenase IV gene and MCP-1 in trans (S. K. Ghosh and D. V. Faller, J. Virol. 73:4931-4940, 1999). Genomic DNA of all healthy feline species also contains LTR-like sequences that are related to exogenous FeLV LTRs. In this study, we evaluated the cellular gene transactivational potential of these endogenous FeLV LTR sequences. Unlike their exogenous FeLV counterparts, neither nearly full-length endogenous FeLV molecular clones (CFE-6 and CFE-16) nor their isolated LTRs were able to activate collagenase IV gene or MCP-1 expression in transient transfection assays. We had also demonstrated previously that production of an RNA transcript from exogenous FeLV LTRs correlates with their transactivational activity. In the present study, we demonstrate that the endogenous FeLV LTRs do not generate LTR-specific RNA transcripts in the feline embryo fibroblast cell line AH927. Furthermore, infection of AH927 cells by an exogenous FeLV subgroup A virus did not induce production of such LTR-specific transcripts from the endogenous proviral genomes, although the LTR-specific transcripts from the exogenous virus were readily detected. Finally, LTR-specific transcripts were not generated in BALB/3T3 cells transiently transfected with isolated CFE-6 LTR, in contrast to transfections with LTRs from exogenous viruses. Our data thus suggest that the inability of endogenous FeLV LTRs in gene transactivation is not due to cell line specificity or presence of any upstream inhibitory cis-acting element. Endogenous, nonleukemogenic FeLV LTRs, therefore, do not transactivate cellular gene expression, and this property appears to be specific to exogenous, leukemogenic FeLVs.
Project description:Human endogenous retroviruses (HERVs) and other long terminal repeat (LTR)-type retrotransposons (HERV/LTRs) have regulatory elements that possibly influence the transcription of host genes. We systematically identified and characterized these regulatory elements based on publicly available datasets of ChIP-Seq of 97 transcription factors (TFs) provided by ENCODE and Roadmap Epigenomics projects. We determined transcription factor-binding sites (TFBSs) using the ChIP-Seq datasets and identified TFBSs observed on HERV/LTR sequences (HERV-TFBSs). Overall, 794,972 HERV-TFBSs were identified. Subsequently, we identified "HERV/LTR-shared regulatory element (HSRE)," defined as a TF-binding motif in HERV-TFBSs, shared within a substantial fraction of a HERV/LTR type. HSREs could be an indication that the regulatory elements of HERV/LTRs are present before their insertions. We identified 2,201 HSREs, comprising specific associations of 354 HERV/LTRs and 84 TFs. Clustering analysis showed that HERV/LTRs can be grouped according to the TF binding patterns; HERV/LTR groups bounded to pluripotent TFs (e.g., SOX2, POU5F1, and NANOG), embryonic endoderm/mesendoderm TFs (e.g., GATA4/6, SOX17, and FOXA1/2), hematopoietic TFs (e.g., SPI1 (PU1), GATA1/2, and TAL1), and CTCF were identified. Regulatory elements of HERV/LTRs tended to locate nearby and/or interact three-dimensionally with the genes involved in immune responses, indicating that the regulatory elements play an important role in controlling the immune regulatory network. Further, we demonstrated subgroup-specific TF binding within LTR7, LTR5B, and LTR5_Hs, indicating that gains or losses of the regulatory elements occurred during genomic invasions of the HERV/LTRs. Finally, we constructed dbHERV-REs, an interactive database of HERV/LTR regulatory elements (http://herv-tfbs.com/). This study provides fundamental information in understanding the impact of HERV/LTRs on host transcription, and offers insights into the transcriptional modulation systems of HERV/LTRs and ancestral HERVs.
Project description:The dynamic modification of nuclear and cytoplasmic proteins with O-linked beta-N-acetylglucosamine (O-GlcNAc) by the O-linked N-acetylglucosaminyltransferase (OGT) is a regulatory post-translational modification that is responsive to various stimuli. Here, we demonstrate that OGT is a central factor for T- and B-lymphocytes activation. SiRNA-mediated knockdown of OGT in T cells leads to an impaired activation of the transcription factors NFAT and NFkappaB. This results in a reduction of IL-2 production consistent with prevention of T-cell activation. OGT is also required for the early activation of B cells mediated by stimulation of the B-cell receptor. Mechanistically, we demonstrate that NFkappaB as well as NFAT are glycosylated with O-GlcNAc after direct binding to OGT. Moreover, kinetic experiments show that O-GlcNAc modification prominently increased shortly after activation of lymphoid cells and it might be required for nuclear translocation of the transcription factors NFkappaB and NFAT.