The CD8 Antiviral Factor (CAF) can suppress HIV-1 transcription from the Long Terminal Repeat (LTR) promoter in the absence of elements upstream of the CATATAA box
© Shridhar et al.; licensee BioMed Central Ltd. 2014
Received: 26 February 2014
Accepted: 6 June 2014
Published: 21 July 2014
The CD8 Antiviral Factor (CAF) suppresses viral transcription from the HIV-1 Long Terminal Repeat (LTR) promoter in a non-cytolytic manner. However, the region on the LTR upon which CAF acts is unknown. Our objective was to determine the region on the LTR upon which CAF acts to suppress HIV-1 transcription.
Serial deletions of the LTR from the 5’ end and inactivating point mutations were made.
Serial deletions of the LTR from the 5’ end indicated the importance of a short ~120 bp segment, containing the 3 SpI sites, CATA box (used by HIV-1 instead of the TATA box) and TAR region, in the suppressive process. Introduction of deletions or inactivating point mutations in the SpI sites or deletion of the TAR region did not abolish CAF-mediated transcriptional suppression. Yet, CAF-mediated transcriptional suppression was still retained in the HIV-1 CATA-TAR segment.
CAF is able to suppress transcription from the LTR lacking all the elements upstream of the CATA box. Our results suggest that the HIV-1 CATA box may be responsible for CAF-mediated suppression of transcription from the HIV-1 LTR.
CD8+ T cells can control HIV-1 replication by non-cytolytic mechanisms [1, 2]. The first non-cytolytic antiviral CD8+ T cell response was described in Long Term Non-Progressers of HIV-1 infection  and the factor mediating it was termed “CD8 Antiviral Factor” (CAF) . CAF-mediated antiviral response has several characteristics: First, CAF suppresses HIV-1 mRNA production [4–8]. Second, CAF activity is not MHC-restricted or does not require direct contact between the CD8+ T cell and the target cell [9, 10]. Third, CAF has been found to be effective against a wide range of HIV-1 clades, as well as HIV-2 and SIV and its activity inversely correlates with the stage of disease [11–18], and finally, both HIV-1 R5 and X4 viruses can be equally well suppressed [19–21]. Although many non-cytolytic CD8+ T cell factors have since been described [2, 22–24], the identity and mechanism of action of CAF are as yet unknown.
The aim of this study was to elucidate the mechanism by which CAF mediates its HIV-1 transcription-suppressing effects. We hypothesized that CAF acts on and induces changes in the viral promoter to suppress transcription. Towards this, we focused on determining the region in viral promoter that was crucial for the suppressive effect of CAF. We performed serial progressive deletions on the LTR at 5’ end to identify the minimal region required for CAF-mediated transcriptional suppression. By a process of eliminating likely candidates, our data suggest that the HIV-1 CATA box (used by HIV-1 instead of the TATA box, motif: CATATAA, ref ) is the target for transcriptional suppression by CAF.
Results and discussion
CAF is HIV-1 LTR specific
LTR progressive deletion constructs narrow the region of the promoter needed for CAF action
We used constructs that had been deleted of these regions from the HIV-1 LTR to investigate the possibility of multiple transcription factor (TF)-binding sites acting in conjunction with each other to suppress transcription in response to CAF. These constructs have been described before  and include the full length Wt-LTR, the EcoRV construct (containing deletion of the NRE), the BstNI construct (containing deletions up to the SpI sites), and the HaeIII construct (deletions up to the 3rd Sp1 site and containing the 2 SpI sites most proximal to the CATA box, the TATA box and the TAR region). They were transfected into 293 T cells treated with either CAF or media control, and then stimulated with PMA. We found that CAF was able to suppress reporter gene expression from all of these constructs (Figure 1A). Sequential deletions of the LTR actually had higher transcriptional rates compared to the full-length promoter. This might be because of removal of negative regulatory regions from the LTR, leading to higher basal transcription in the absence of CAF.
These results indicate that CAF-mediated suppression of transcription is specific to HIV-1, is not affected by the type of reporter gene, and that the region encompassing the -69 to +83 of the LTR, containing 3 SpI sites, CATA box and the TAR region, is sufficient for the suppressive action of CAF.
Role of the TAR loop in conferring susceptibility to CAF
The TAR region is a very attractive candidate target for CAF action. It has a unique bulge-loop structure and is well-conserved across all clades of HIV-1 . Critical interactions between the TAR and the viral transactivator protein Tat take place on the bulge-loop structure on TAR [29, 30]. If TAR were the target for CAF, it would help explain the specificity of CAF for HIV. Previous investigations on the region of the LTR necessary for CAF action probed the role of TAR by introducing inactivating, point mutations to disrupt the Tat-TAR interaction axis . But if the structure of TAR, and not its sequence, were important for CAF action, point mutations might not indicate the importance of TAR in suppression.
Role of the SpI sites in CAF-mediated suppression
To confirm that the results we observed were a product of SpI site inactivation and not because of the deletion process itself, we next inactivated the SpI sites by point mutation. Inactivation of the SpI sites, either individually or in pairs, resulted when key G residues in the SpI binding sites were replaced with T. Mutation of the SpI sites did not transcriptionally inactivate the construct, in accordance with previous reports . While mutation of the various SpI sites was seen to affect transcription, all constructs were able to get suppressed in response to CAF (Figure 4C).
Role of the CATA-box in CAF mediated HIV-1 suppression
In conclusion, our report shows that CAF can suppress transcription from all regions of the HIV-1 LTR upstream of the CATA box. We did not study epigenetic changes or changes in the CATA-box binding proteins on the HIV-1 LTR in response to CAF. Future work to elucidate the mechanism of CAF in HIV-1 transcription suppression may include these avenues.
Materials and methods
No patients were specifically recruited for the purposes of this study. The CD8+ T-cell line, TG, was previously established by herpesvirus saimiri (HVS)-transformation of CD8+ T cells from a chronically infected HIV-1-infected subject from the Multicenter AIDS Cohort Study (MACS) .
SpI deletion construct oligos and primers
Name of construct
DNA oligos used
Del 1, del 2
SpI inactivating point mutation construct oligos and primers
Oligos used (Oligo 2 is the same for all constructs)
Oligo 1: TCGGAGGACAGTACTCCGACCCGGTCGAAGGGATTCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAA
Oligo 2: GAGCCCTCAGATCCTGCATATAA GCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATC CGAGC
Oligo 1: TCGGAGGACAGTACTCCGACCCGGTCGAAGGGATTCGTGGCCTGGGCGGGACTGGTTAGTGGCGAGCCCTCAGATCCTGCATATAA
Oligo 1: TCGGAGGACAGTACTCCGACCCGGTCGAAGGGATTCGTGGCCTGTTCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAA
Oligo 1: TCGGAGGACAGTACTCCGACCCGGTCGAAGGGAGGCGTGGCCTGTTCGGGACTGGGGAGTGGCGAGCCCTCAGATCCTGCATATAA
Oligo 1: TCGGAGGACAGTACTCCGACCCGGTCGAAGGGAGGCGTGGCCTGTTCGGGACTGGTTAGTGGCGAGCCCTCAGATCCTGCATATAA
Oligo 1: TCGGAGGACAGTACTCCGACCCGGTCGAAGGGAGGCGTGGCCTGGGCGGGACTGGTTAGTGGCGAGCCCTCAGATCCTGCATATAA
Cells, cell culture and CAF preparations
293 T cells were obtained from the ATCC and cultured in DMEM, supplemented with 10% FBS, 2 mM Glutamine and 1 mM sodium pyruvate. TZM-bl cells were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: TZM-bl from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. The CD8+ T cell line, TG, was previously established by Herpes virus saimiri (HVS)-transformation of CD8+ T cells from a chronically HIV-1 infected subjected from the MACS. TG cells were grown in RPMI with 20% FBS (100 nm filtered, Invitrogen Life Sciences, Carlsbad, CA), supplemented with 25 mM HEPES, Penicillin (100 U/mL) and Streptomycin (100 ug/mL) and rIL2 (50 U/mL, Roche Diagnostics). CAF from these transformed CD8+ T cells was prepared as described before . Briefly, TG cells were cultivated for 14 days, after which, the cells were centrifuged at 300 g and the resulting supernatant was then further centrifuged at 4°C at the following speeds: 2000 g for 30 minutes, 6000 g for 20 minutes and 15000 g for 1 hour to remove other debris. This conditioned media was used for further investigations on CAF.
293 T cells were plated at a concentration of either 200,000 cells/mL in a 6- well plate (if the reporter gene assay was CAT), or 20,000 cells/100 uL in a 96-well plate (if the reporter gene assay was luciferase), in triplicate. 24 hours after plating, 10% vol/vol of CAF from TG cells was added to the culture. 24 hours later, either 1ug or 10 ng (depending on whether the assay was for CAT or luciferase respectively) of the relevant plasmid/s was transfected into all cells, using Lipofectamine Plus (Invitrogen, Carlsbad, CA), according to manufacturer’s instructions, along with the transfection control, CMV-Renilla plasmid. 24 hours following transfection, the cells were stimulated with 100 ng/mL phorbolmyristoylacetate (PMA) for 18 hours or 6 hours (depending of whether the assay was for CAT or luciferase respectively), after which they were lysed and the reporter gene product was measured. Measurement of the reporter gene was done after normalizing for total protein content and Renilla expression. For the experiment detailed in Figure 5, with ICP0 to boost transcription of the Mnl construct, 293 T cells were plated at a density of 200,000/mL in a 6 well plate, in triplicate. 24 hours after plating, 10% vol/vol of CAF from TG cells was added to the culture. 1ug of Mnl-CAT with or without 0.5 ug of ICP0 expression plasmid was transfected, into designated wells. Expression from the Mnl-LTR construct was induced either with co-transfection with CMV-tat expression plasmid (0.5 ug per well) or with PMA (100 ng/mL for 18 hours). CMV-renilla was transfected as a control. 36 hours after transfection, cells were lysed and CAT protein content quantified after normalizing for total protein (as measured by Bradford Assay) and Renilla luciferase levels. CAT and luciferase protein expressions were quantified by CAT ELISA (Roche) and Bright Glo systems (Promega), respectively, according to manufacturer’s instructions. Renilla content was measured using Stop-and-Glo kit (Promega).
The authors wish to acknowledge the contributions of Ashwin Tumne, Saleem Khan, Ronald Montelaro, Paul Robbins and Velpandi Ayyavoo.
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