Variation in cell-associated unspliced HIV RNA on antiretroviral therapy is associated with the circadian regulator brain-and-muscle-ARNT-like-1

Abstract

Objective(s): To determine whether variation in cell-associated unspliced (CA-US) HIV RNA in HIV-infected individuals on antiretroviral therapy (ART) has a circadian basis.

Methods: Prospective observational study of HIV-infected individuals on ART. Blood was collected on three occasions and CA-US HIV RNA and mRNA of the circadian-locomotor-output-cycles-kaput (CLOCK)-associated genes quantified by real time PCR. CLOCK-associated proteins were over-expressed in a cell line stably transfected with an HIV long-terminal repeat (LTR) luciferase reporter.

Results: Using a mixed effects model, there was a significant increase in log-CA-US RNA at the third visit compared with the first visit (effect size of 0.619 with standard error (SE) of 0.098, P < 0.001) and an independent effect of time of blood draw (effect size 0.051 (SE 0.025), P = 0.040). The CLOCK-associated gene, brain-and-muscle-ARNT-like-1 (BMAL-1) had a significant relationship with log CA-US HIV RNA (effect size 8.508 (SE 3.777), P = 0.028) and also with time (P = 0.045). Over expression of BMAL-1 and CLOCK in a cell line stably transfected with an HIV-LTR luciferase reporter resulted in an increase in luciferase expression and this was reduced following mutation of the second E-box in the HIV-LTR.

Conclusion: The basal level of HIV transcription on ART can vary significantly and is modulated by the circadian regulator BMAL-1, amongst other factors.

Figure 1

Relationship of CA-US HIV RNA in CD4+ T-cells, visit and time of blood collection (n=30). Results from three time points collected from each participant are connected by a line. The color of the symbol denotes the pre-intervention time-point. These were prior to receiving the study treatment (screening [B1; black]), immediately before the first dose of an intervention, here being disulfiram [B3; red], and a time-point between [B2; grey].

Figure 2

Relationship of CLOCK-associated genes and visit. Expression of CLOCK-associated genes in HIV-infected individuals on suppressive ART at three time-points (n=30). Data were normalized to GAPDH expression level by the ΔΔCT method. The median and inter-quartile range are shown. Nominal p-values from Wilcoxon matched pairs signed rank tests are shown for significant associations. The color of the symbol denotes the pre-intervention time-point. These were prior to receiving the study treatment (screening [B1; black]), immediately before the first dose of an intervention, here being disulfiram [B3; red], and a time-point between [B2; grey].

Figure 3

CD4+ T-cell composition and plasma cortisol and thyroid stimulating hormone (TSH) in HIV-infected individuals on suppressive ART. A: CD4+ T-cell subset proportions in participants are shown for n=28, 27 and 29 at B1, B2 and B3 respectively. The median and interquartile range are shown and comparisons are made with Wilcoxon matched pairs signed rank tests. B: Plasma cortisol and C: TSH in participants are shown for n=27 at B1 and 30 at B2 and B3. The mean and standard deviation are shown and comparisons are made with paired t-tests. The color of the symbol denotes the pre-intervention time-point. These were prior to receiving the study treatment (screening [B1; black]), immediately before the first dose of an intervention, here being disulfiram [B3; red], and a time-point between [B2; grey].

Figure 4

The circadian transcription factors CLOCK and BMAL1 upregulate HIV LTR activity. A: Raw luciferase activity in 293T cells stably transfected with an HIV-LTR (HXB2)-luciferase reporter and then co-transfected with CLOCK, BMAL1, CLOCK and BMAL1 or Tat. B: Fold change in luciferase activity. Comparisons are made to the HIV-LTR alone. C: Schematic of the HIV LTR highlighting the position of E-box elements. D: The effect of E-box mutations on upregulation of HIV LTR luciferase activity following co-transfection of CLOCK/BMAL-1. Fold change in luciferase activity of the mutated LTRs in the presence of CLOCK and BMAL1 compared to baseline expression. Box plots are shown which demonstrate the median, 25^th and 75^th percentiles (box) and minimum and maximum values (whiskers), n=13 (A,B) and n=5 (D) experiments are shown. Comparisons were made using a linear regression analysis. * p<0.05, **p<0.01, ***p<0.001. WT, wild type

Figure 5

Proposed model for interaction of visit, time and circadian genes in HIV transcription on ART. Effects of visit alone on CA-US RNA remains unexplained but could potentially be secondary to stress. The CLOCK-BMAL-1 heterodimer binds to a six-nucleotide motif, the E-box ^{[14, 15]}, to mediate transcription. HIV-1 is known to encode multiple E-Boxes in the LTR ^[16] and therefore changes in HIV transcription on ART, may plausibly be mediated directly through CLOCK-BMAL-1.