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. 2019 Dec 1;33 Suppl 2(Suppl 2):S145-S157.
doi: 10.1097/QAD.0000000000002268.

Presence of Tat and transactivation response element in spinal fluid despite antiretroviral therapy

Lisa J Henderson  1 , Tory P Johnson  2 , Bryan R Smith  1 , Lauren Bowen Reoma  1 , Ulisses A Santamaria  1 , Muzna Bachani  3 , Catherine Demarino  4 , Robert A Barclay  4 , Joseph Snow  5 , Ned Sacktor  2 , Justin Mcarthur  2 , Scott Letendre  6 , Joseph Steiner  3 , Fatah Kashanchi  4 , Avindra Nath  1
Affiliations

Presence of Tat and transactivation response element in spinal fluid despite antiretroviral therapy

Lisa J Henderson et al. AIDS. .

Abstract

Objective: The aim of this study was to measure the protein concentration and biological activity of HIV-1 Tat in cerebrospinal fluid (CSF) of individuals on suppressive antiretroviral therapy (ART).

Design: CSF was collected from 68 HIV-positive individuals on ART with plasma viral load less than 40 copies/ml, and from 25 HIV-negative healthy controls. Duration of HIV infection ranged from 4 to more than 30 years.

Methods: Tat levels in CSF were evaluated by an ELISA. Tat protein and viral RNA were quantified from exosomes isolated from CSF, followed by western blot or quantitative reverse transcription PCR, respectively. Functional activity of Tat was assessed using an LTR transactivation assay.

Results: Tat protein was detected in 36.8% of CSF samples from HIV-positive patients. CSF Tat concentration increased in four out of five individuals after initiation of therapy, indicating that Tat was not inhibited by ART. Similarly, exosomes from 34.4% of CSF samples were strongly positive for Tat protein and/or TAR RNA. Exosomal Tat retained transactivation activity in a CEM-LTR reporter assay in 66.7% of samples assayed, which indicates that over half of the Tat present in CSF is functional. Presence of Tat in CSF was highly associated with previous abuse of psychostimulants (cocaine or amphetamines; P = 0.01) and worse performance in the psychomotor speed (P = 0.04) and information processing (P = 0.02) cognitive domains.

Conclusion: Tat and TAR are produced in the central nervous system despite adequate ART and are packaged into CSF exosomes. Tat remains biologically active within this compartment. These studies suggest that Tat may be a quantifiable marker of the viral reservoir and highlight a need for new therapies that directly inhibit Tat.

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Conflict of interest statement

Conflicts of Interest:

For the remaining authors none were declared.

Figures

Figure 1.
Figure 1.
(A-B) Antiretroviral therapy (darunavir) inhibits viral release but does not diminish Tat protein expression in macrophages. Monocyte-derived macrophages (MDM) were infected with HIVSF162 followed by treatment with 1 uM darunavir or an equal volume of DMSO (vehicle). Medium was spiked with DMSO or darunavir every 48h hours. At 7 days post-infection supernatants were analyzed (A) for reverse transcriptase activity by a PERT assay and (B) cells were lysed for Tat by ELISA. Results indicate mean ± standard deviation from three independent experiments. **p<0.01 as determined by unpaired, two-tailed Student’s t-test; N.S. = not significant. (C-F) Presence of Tat protein in patients on antiretroviral therapy. Cerebrospinal fluid (C-F) was analyzed by ELISA for detection of Tat protein. Plots show (C) CSF Tat concentrations from five patients pre-ART initiation (Pre-ART) and two to five months after ART (Post-ART). (D) CSF Tat concentrations from 68 patients virologically well controlled on ART. (E) CSF Tat concentrations (pg/ mL) from 15 patients followed for at least three years. Samples were collected from patients at sequential yearly visits (Patient Visit). Data shown in (D) represent the highest CSF Tat level measured for that patient. For patients with longitudinal samples available, these data are repeated as a single time point in (E). (F) Correlation between Tat protein levels and HIV viral RNA (vRNA) in patients experiencing CNS viral escape without detectable virus in blood at the indicated time points. LOD = limit of detection for the ELISA (200 pg/ mL), as determined by background O.D. readings from 25 HIV-negative CSF samples + 2 standard deviations. All samples were run in triplicate with at least two negative controls per plate and quantitated relative to standard curve generated using recombinant Tat protein (rTat). Anti-Tat antibody specificity was validated by western blot using rTat and lysates from Tat-transfected or untransfected HEK293T/17.
Figure 2.
Figure 2.. Presence of Tat protein and TAR RNA in exosomes isolated from CSF.
A) Immunoblot of isolated exosomes for the Tat protein, the exosome marker CD63, actin (loading control), and IgG (negative control) for background subtraction. The top of the image shows patient identification number and the bottom of the image indicates lane number.C1 and C11 are control samples from HIV seronegative patients. B) Levels of TAR RNA were measured in CSF exosomes by quantitative RT-PCR. Results indicate copies of TAR per 500 μl of CSF used for exosome isolation. Error bars represent ± S.D. of three technical replicates. C) Summary of exosome data showing number of patient samples positive for Tat protein and/or TAR RNA. Results shown in Tat column represent western blot densitometry relative to negative IgG control: - = <1%; +/− = 1%−14.99%; + = 15%−32.99%; ++ = 33%−65.99%; +++ = >66%. “TAR+” column indicates the number of patient samples positive for TAR RNA in each Tat category.
Figure 3.
Figure 3.. Tat protein isolated from CSF exosomes is capable of transactivating the HIV promoter.
Scatter plots showing collected flow cytometry data. Side scatter is on the X-axis and GFP is on the y-axis. Exosomes from HIV-negative controls (control CSF #1, control CSF #2) or HIV-positive patients with detectable CSF Tat as measured by western blot (#27, #24, #17, #19, #25, #3) were directly added to CEM-GFP reporter cells, which contain a GFP gene under the control of an HIV promoter. 72 hours later, flow cytometry was used to detect GFP expression. As a positive control, cells were infected with HIV. Cells with no treatment or treatment with beads alone were used as negative controls. Sample ID is indicated in the top right corner and the gated number represents the percent of GFP-positive cells for a given sample. Bolded numbers indicated samples with gated events that exceed the negative control.
Figure 4.
Figure 4.. Presence of Tat in CSF is associated with a history of drug abuse.
(A) Charts comparing percent of patients in the NIH cohort with Tat-positive (top) or Tat-negative (bottom) CSF who have a known history of drug abuse. Data on Illicit drug use was captured by a patient questionnaire asking each participant if he or she has ever used a specific substance. “Abuse of illicit drugs” includes patients who responded “yes” to questions regarding past drug use that affected their work or life but were negative for illegal substances by urine screening at their first visit. B) Summary of patient demographics for the NIH cohort (n=68), including sub-categories for patients with (n=25) or without (n=43) detectable CSF Tat. Significant differences between Tat-positive and Tat-negative individuals were determined by Chi-square analysis using Yate’s continuity correction, as appropriate for the sample size.
Figure 5.
Figure 5.. Model depicting possible mechanisms of elevated Tat levels in the presence of antiretroviral therapy.
A) in a cell that contains an integrated, actively transcribed provirus, protease inhibitors prevent cleavage of HIV polyproteins (Gag-Pol), abrogating assembly and release of new virions. However Tat, which does not require cleavage by the viral protease, may continue to be transcribed, translated and released from the cell. Tat can be secreted directly into the extracellular space, or it may be packaged into exosomes. TAR RNA, which is also transcribed under treated conditions, is also packaged into exosomes alone or in addition to Tat. B) Tat- and TAR-containing exosomes can be endocytosed by other uninfected or HIV-infected cells to cause altered signaling. In HIV-infected cells which contain an inactive (latent) provirus, exogenous Tat can reactivate the latent provirus via induction of host signaling pathways such as NF-κB and transactivation of the HIV LTR. Productive replication is blocked in the newly transcribing cell due to the presence of protease inhibitors, but Tat protein and TAR RNA are produced and can be packaged into exosomes for delivery to other target cells.
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