A mutant Tat protein inhibits infection of human cells by strains from diverse HIV-1 subtypes
© The Author(s). 2017
Received: 25 January 2017
Accepted: 10 February 2017
Published: 14 March 2017
Nullbasic is a mutant HIV-1 Tat protein that inhibits HIV-1 replication via three independent mechanisms that disrupts 1) reverse transcription of the viral RNA genome into a DNA copy, 2) HIV-1 Rev protein function required for viral mRNA transport from the nucleus to the cytoplasm and 3) HIV-1 mRNA transcription by RNA Polymerase II. The Nullbasic protein is derived from the subtype B strain HIV-1BH10 and has only been tested against other HIV-1 subtype B strains. However, subtype B strains only account for ~10% of HIV-1 infections globally and HIV-1 Tat sequences vary between subtypes especially for subtype C, which is responsible for ~50% HIV-1 infection worldwide. These differences could influence the ability of Tat to interact with RNA and cellular proteins and thus could affect the antiviral activity of Nullbasic. Therefore, Nullbasic was tested against representative HIV-1 strains from subtypes C, D and A/D recombinant to determine if it can inhibit their replication.
Nullbasic was delivered to human cells using a self-inactivating (SIN) γ-retroviral system. We evaluated Nullbasic-mCherry (NB-mCh) fusion protein activity against the HIV-1 strains in TZM-bl cell lines for inhibition of transactivation and virus replication. We also examined antiviral activity of Nullbasic-ZsGreen1 (NB-ZSG1) fusion protein against the same strains in primary CD4+ T cells. The Nullbasic expression was monitored by western blot and flow cytometry. The effects of Nullbasic on primary CD4+ T cells cytotoxicity, proliferation and apoptosis were also examined.
The results show that Nullbasic inhibits Tat-mediated transactivation and virus replication of all the HIV-1 strains tested in TZM-bl cells. Importantly, Nullbasic inhibits replication of the HIV-1 strains in primary CD4+ T cells without affecting cell proliferation, cytotoxicity or level of apoptotic cells.
A SIN-based γ-retroviral vector used to express Nullbasic fusion proteins improved protein expression particularly in primary CD4+ T cells. Nullbasic has antiviral activity against all strains from the subtypes tested although small differences in viral inhibition were observed. Further improvement of in γ-retroviral vector stable expression of Nullbasic expression may have utility in a future gene therapy approach applicable to genetically diverse HIV-1 strains.
The HIV-1/AIDS pandemic remains a huge social and economic burden. By 2014, 36.9 million people were living with HIV and 1.2 million AIDS related death cases were reported . One of the major obstacles in treating this disease is a high genetic diversity of HIV-1 that leads to different rates of disease progression and resistance to antiviral drugs [2, 3]. We have investigated an anti-HIV-1 agent that targets three different steps of virus replication by targeting viral and cellular proteins, and therefore may have efficacy against HIV-1 with diverse genetic backgrounds.
The agent is a Tat mutant protein derived from HIV-1 subtype B strain BH10 that strongly inhibits HIV-1 replication in human cells , and is referred to as Nullbasic. Wild type Tat is an essential HIV-1 protein required for transactivation of the HIV-1 long terminal repeat (LTR) promoter resulting in high levels of viral mRNA transcription by RNA polymerase II . It also plays a role in HIV-1 reverse transcription [6, 7] and in other cellular processes such as immune suppression, induction of inflammatory cytokines and apoptosis [8–10]. Nullbasic, which has been described previously [4, 11, 12], has a substitution mutation spanning the entire basic domain; amino acids 49 to 57, RKKRRQRRR, are replaced with GGGGGAGGG. Studies show that Nullbasic expressed in cells is located in the nucleus and cytoplasm , and inhibits HIV-1 replication by 1) inhibiting HIV-1 transcription by RNA polymerase II through interaction with the positive transcription elongation factor (p-TEFb) and causing epigenetic silencing of the HIV-1 LTR promoter [4, 12, 13], 2) inhibiting Rev-dependent viral mRNA transport from the nucleus by binding to DEAD/H-box helicase 1 (DDX1) [13, 14], and 3) inhibiting reverse transcription by directly interacting with reverse transcriptase (RT) leading to accelerated uncoating kinetics post-infection and defective viral DNA synthesis .
HIV-1 sequence diversity is categorized by HIV-1 subtypes that are defined by comparisons of envelope genes. These subtype variations can also be observed as differences in viral proteins, such as Tat, Rev and RT. Amino acid sequence variation in the viral proteins of various HIV-1 subtypes can affect virus replication and virulence . For example, RT from subtype C isolates differs from subtype B by ~7–10%, which can affect drug susceptibility and cause drug resistance . Tat proteins from different subtypes can vary up to 40% without significantly affecting Tat transactivation ability , but the effects on many alternative functions of Tat  have not been studied in detail.
To date, Nullbasic antiviral activity has only been tested against HIV-1 subtype B strains such as HIV-1NL43 [4, 11]. However, subtype B strains only accounts for ~10% of HIV-1 infections globally and HIV-1 Tat sequences vary between subtypes especially for subtype C, which is responsible for ~50% HIV-1 infection worldwide [19, 20]. Subtype C is predominant in sub Saharan Africa, India and South America , while subtype D and recombinant A/D are increasing in sub-Saharan Africa [22, 23]. Whether sequence variations in different HIV-1 subtypes alter the susceptibility to the antiviral effect of Nullbasic has not been examined. Therefore, in this study, Nullbasic ability to inhibit replication of HIV-1 strains from different subtypes including C, D and A/D was evaluated. To enable protein expression detection in the targeted cells, Nullbasic was tested in the form of fusion proteins as NB-mCh  or NB-ZSG1 .
Cell lines and cultures
HEK 293T (ATCC), TZM-bl [24, 25] and Phoenix-Ampho  cell lines were grown in Dubelcco’s modified Eagle’s medium (DMEM; Life Technologies) supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 IU/ml) and streptomycin (100 μg/ml) (referred to as DF10 medium). TZM-bl expressing NB-mCh or mCh cell lines were established by transduction of NB-mCh or mCh virus-like particles (VLPs) and then selected by fluorescent activated cell sorter (FACS) for the top 10% of mCherry positive cells by mean fluorescent intensity (MFI).
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor’s buffy coat supplied by Australian Red Cross Blood service using Ficoll density gradient centrifugation. CD4+ cells were isolated from the PBMCs by using a magnetic-activated cell sorting human CD4+ cell isolation kit (Miltenyi Biotec) as per the manufacturer’s instruction. The selected cells were grown in 6 cm tissue culture dishes and stimulated using plates pre-coated with purified anti-human CD3 (clone HIT3a) and anti-human CD28 (clone CD28.2) antibodies (BioLegend) in RPMI medium supplemented with 20% (v/v) FBS and 5 ng/ml interleukin-2 (IL-2) (hereafter called RF20 IL-2) for 2 days. All cells were grown at 37 °C in humidified incubators with 5% CO2.
pSRS11-SF-γC-EGFP was a gift from Axel Schambach and Christopher Baum . pSRS11-SF-γC-NB-mCh or pSRS11-SF-γC-mCh or pSRS11-SF-γC-NB-ZSG1 or pSRS11- SF-γC-ZSG1 construct was made by replacing the enhanced green fluorescent protein gene in pSRS11-SF-γC-EGFP with NB-mCh or mCh or NB-ZSG1 or ZSG1. A proviral plasmid pGCH making HIV-1NL43 (GenBank accession number AF324493) was previously described . The proviral plasmid pZAC (GenBank accession number JN188292.1) was obtained from Jochen Bodem . The proviral plasmids pELI and pMAL (Los Alamos accession number A07108 and A07116 respectively) were provided by Damian Purcell . The exon tat genes with hemagglutinin epitope were synthesized by GenScript and ligated into pcDNA3.1+ plasmid (Thermofisher Scientific).
HIV-1 and VLPs production
HIV-1 subtype B, C, D and A/D were produced from pGCH, pZAC, pELI and pMAL proviral plasmids respectively. HEK 293 T cells were grown on a 10 cm plate at ~80% confluency and transfected with 10 μg of each proviral plasmid then incubated for 24 h at 37 °C. On the next day, the transfected cells were washed with 1 x phosphate buffered saline (PBS) and the DF10 media was replaced. The supernatant containing HIV-1 VLPs was collected 48 and 72 h post transfection and the amount of HIV-1 capsid (CA) protein in each supernatant was measured by enzyme-linked immunosorbent assay (ELISA) (Zeptometrix) as recommended by the manufacturer.
NB-mCh or mCh or NB-ZSG1 or ZSG1 VLPs were produced in Phoenix-amphotropic retroviral packaging producer cell line by co-transfection of 7.5 μg of pSRS11-SF-γC vector expressing NB-mCh or mCh or NB-ZSG1 or ZSG1 and 1.5 μg of Gag-Pol expressing plasmid using X-tremeGENETM DNA transfection reagent (Roche) in a 10 cm plate. Six hours post transfection, the cells were washed with PBS and the media was replaced. The VLPs were collected 48 and 72 h post transfection and filtered through a 0.45 μm filter.
Western blot analysis
Cell lysates were made from 5 × 106 NB-mCh or mCh-TZM-bl cells, or from 3 × 106 CD4-NB-ZSG1, CD4-ZSG1 or non-transduced CD4 cells in cell lysis buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid and 1% (v/v) Triton X-100). The total protein concentration was measured by a Bradford assay using Bio Rad protein assay (Bio Rad) and equivalent amounts of protein were used for analysis. The blots were stained with a anti-mCherry rabbit antibody (BioVision), a rabbit anti-Tat antibody (Diatheva), a mouse anti-ZsGreen1 (Origene), a rabbit anti-β-tubulin antibody (Sigma Aldrich), or a goat anti-actin antibody (Santa Cruz) as indicated. Appropriate species-specific secondary antibodies conjugated to horse radish peroxidase (HRP) (Cell Signaling Technology) and followed detection by chemiluminescence (BioRad).
Tissue culture dishes (6 cm) were seeded with 5 × 105 TZM-bl cells expressing NB-mCh or mCh and then co-transfected with 1 μg of each subtype Tat plasmid or pCDNA3.1+ without an insert and 150 ng of Gaussia luciferase expression plasmid. After 48 h, the cells were washed with PBS and cell lysates were made using Glo Lysis buffer (Promega). Luciferase assays were performed in 96 well white polystyrene microplates as per the manufacturer’s instructions using 10 μl of the cell lysates and Dual-Glo® luciferase substrate (Promega). Luciferase activity in each sample was measured within 20 min by using a luminescence microplate reader and relative values were normalized to Gaussia luminescence in the sample.
Next, 3 × 105 TZM-bl cells expressing NB-mCh or mCh or non-transduced (NT) TZM-bl cells were seeded in 6 well plates. The next day, the cells were infected with HIV-1NL4–3 (subtype B), HIV-1ZAC (subtype C) , HIV-1ELI (subtype D) and HIV-1MAL (A/D recombinant subtype) virus supernatant containing 20 ng of CA, or a mock supernatant for 48 h. The cells were washed with PBS and then cell lysates were made using Glo Lysis buffer (Promega). Luciferase activity was measured as described above.
Transduction of NB-ZSG1 or ZSG1 VLP in CD4+ T cells
NB-ZSG1 or ZSG1 VLPs were concentrated using the precipitation method through the addition of 20% (v/v) of 34% polyethylene glycol 8000 (Sigma Aldrich) and 10% (v/v) of 0.3 M NaCl solution. The solution mixture was incubated at 4 °C for 1.5 h, mixed every 30 min and then centrifuged at 1500 × g for 1 h at 10 °C. The supernatant was discarded and the precipitate was resuspended in 600 μl RF20 IL-2 medium. The concentrated VLP (150 μl) was added to Retronectin (Takara) coated 24 well plate and incubated at 37 °C for 30 min. 5 × 105 stimulated CD4+ cells were added to each well and incubated for 3 days. Transduced cells were processed by FACS to collect ZSG1 positive cells which were grown for 3 days further. The RF20-IL2 media was replaced every day.
Infection of HIV-1NL4–3 (subtype B), HIV-1ZAC (subtype C), HIV-1ELI (subtype D) and HIV-1MAL (A/D recombinant subtype) in TZM-bl cell lines and primary CD4+ T cells
TZM-bl cells expressing NB-mCh or mCh or NT (3 × 105 cells/well) cultured in a 6 well plate were infected with a virus stock containing 20 ng CA of HIV-1NL4–3, HIV-1ELI and HIV-1MAL or 40 ng CA of HIV-1ZAC, or a mock supernatant for 2 h at 37 °C. A larger amount of HIV-1ZAC was required to yield measurable infections. The virus was then removed by washing the cells 3 times with PBS and the infected cells were cultured at 37 °C with 5% of CO2. The culture supernatants were sampled on day 3 and 5 post infection. The amount of HIV CA present was measured using a CA ELISA kit (Zeptometrix) according to the manufacturer’s instruction.
Primary CD4+ T cells (5 × 105 NB-ZSG1 or ZSG1 or NT) were infected with virus stocks containing 2 ng CA of each HIV-1 subtype for 2 h at 37 °C. After infection, the cells were washed with PBS and cultured in RF20 IL-2 medium. Cell and supernatant samples were collected on days 0, 3, 7, 10 and 14 by centrifugation at 500 × g for 5 min. The amount of viral CA in the supernatant was measured by ELISA. The cells were fixed with 1% paraformaldehyde in PBS solution and NB-ZSG1 or ZSG1 expression was measured by flow cytometry.
Cell metabolic activity was measured by MTS assay using a CellTiter 96® aqueous one solution cell proliferation reagent (Promega) according to the manufacturer’s instructions. Cell proliferation was quantified using a Violet Proliferation Dye 450 (BD HorizonTM) assay in accordance with the manufacturer’s instructions and violet fluorescence was measured using a violet laser-equipped BD LSRFortessaTM IV flow cytometer. Apoptosis events were quantified using a PE Annexin V apoptosis detection kits (BD PharmingenTM) as per the manufacturer’s instructions. Camptothecin, which induces apoptosis in CD4+ T cells, was used as a positive control.
Mean values of percentage of transactivation inhibition between strains were compared using Kruskal-Wallis one-way analysis of variance. A 95% confidence interval was used, therefore a p value less than 0.05 was considered to be significant.
Inhibition of transactivation and replication of HIV-1 strains from diverse subtypes in TZM-bl cells by NB-mCh fusion protein
Tat mediates HIV-1 transactivation by binding to trans-activation response (TAR) RNA in the R region of HIV-1 LTR and recruiting P-TEFb  that then binds a super elongation complex (SEC) [32, 33]. P-TEFb consists of cyclin T1 and cyclin dependent kinase 9 (CDK9) . In Tat, K41 is important for intramolecular hydrogen bonding and structural integrity of the Tat core  and is present in all Tat proteins shown except HIV-1ZAC which has T41. A crystal structure of the Tat-P-TEFb complex showed that the surface of 37% amino acids 1–49 are complementary to the kinase complex, and this model indicates that the interactions between Tat and P-TEFb can accommodate substitutions commonly present in different Tat genes . However, Tat interacts with other cellular proteins many of which are important for HIV transcription . Therefore, it is possible that these subtle differences could affect the ability of Nullbasic to inhibit the transactivation by Tat from the HIV-1 strains shown.
Antiviral activity of Nullbasic-ZsGreen1 (NB-ZSG1) in primary CD4+ T cells against 4 HIV-1 subtypes
We previously used an MLV-based γ-retroviral vector, pGCsamEN , containing NB-ZSG1 or ZSG1 to transduce primary CD4+ cells. In those experiments, cells that expressed NB-ZSG1 significantly delayed HIV-1NL4–3 replication compared to cells expressing ZSG1. However, pGCsamEN is not a SIN vector and expression of a transgene is via the MLV-LTR promoter. Non-SIN γ-retroviral vector can be transcriptionally repressed in cells , which can be lessened by SIN-based γ-retroviral vectors that used strong constitutively expressed internal promoters. Therefore, SRS11-SF-γC-NB-ZSG1 VLPs were used to transduce CD4+ T cells in this study. The transduced CD4+ T lymphocytes were selected by FACS using parameters previously described and a western blot was performed to confirm NB-ZSG1 and ZSG1 expressions (hereafter referred to as CD4-NB-ZSG1 and CD4-ZSG1, respectively) in the sorted cells (see Additional file 1).
In summary, here we show that Nullbasic can inhibit replication of HIV-1 strains from different subtypes. The outcome indicates that the replication pathways affected by Nullbasic are most likely shared by these HIV-1 strains.
In this study, we investigated if Nullbasic could inhibit viral gene expression and virus replication of four different strains representing four HIV-1 subtypes in human cells. We previously showed that Nullbasic has three independent antiviral properties at different stage of HIV-1 replication cycle ; 1) inhibition of transactivation of virus gene expression by HIV-1 Tat [4, 11], 2) inhibition of HIV-1 Rev activity by sequestration of DDX1 [12, 13], and 3) binding to HIV-1 reverse transcriptase in the virion leading to premature uncoating and defective reverse transcription in newly infected cells . Given that Nullbasic inhibits HIV-1 by binding to both cellular (P-TEFb and DDX1) and viral (RT) targets, we found that, as expected, NB fusion proteins had antiviral activity against all strains tested although some small differences were observed.
Using TZM-bl cells, the effect of Nullbasic on transactivation and virus replication was examined in three ways. Briefly, wild type Tat mediates HIV-1 transactivation by binding and recruiting the SEC and P-TEFb (composed of cyclin T1 and CDK9) to nascent viral mRNA where CDK9 can phosphorylate RNA polymerase II leading to highly processive RNA transcription . A crystal structure of the Tat-P-TEFb complex showed that Tat tightly binds to P-TEFb as 37% of its folded N-terminal domain (amino acids 1–49) surface is complementary to the kinase. In Nullbasic, amino acids 1–48 are wild type but amino acids 49–57 are mutated. Hence, Nullbasic is able to bind P-TEFb , but not recruit the protein complex to nascent viral mRNA, which requires the RNA binding function of the Tat basic domain (amino acids 49–57) .
Firstly, in TZM-bl transfection experiments where equivalent amounts of each Tat expression plasmid were used, the overall inhibition of transactivation had a similar range (70–90% inhibition), but TatZAC was consistently inhibited the least by NB-mCh. Interestingly, a consensus subtype C Tat was reported to have superior transactivation capacity compared to a consensus subtype B Tat , whereas TatZAC was a weaker transcriptional activator here compared to the other Tat proteins tested. This may be due to a TatZAC K41T substitution that may affect TatZAC structure and interaction between TatZAC and P-TEFb . Secondly, TZM-bl cells were infected by each HIV-1 strain and transactivation of the TZM-bl LTR-luciferase reporter was inhibited at similar levels (~90% inhibition of TatZAC, TatELI and TatMAL). Thirdly, replication of all four viral strains was strongly inhibited by NB-mCh, which ranged from 97 to 99%. It is interesting that NB-mCh inhibited virus replication of each strain better than it inhibited transactivation LTR-Luciferase reporter, but the reason for this is unclear. It could be due to Nullbasic effects on Rev activity or reverse transcription, or perhaps differences in the TAR RNAs of the various viral strains. Further work will be required to elucidate the reasons. The data clearly shows that viral transcription and replication mediated by the four different Tat variant proteins, representing different HIV-1 subtypes, was inhibited by NB-mCh under the conditions tested.
The replication of all HIV-1 strains in stimulated primary CD4+ T cells was also inhibited by Nullbasic, but differences were noted here too. The replication of HIV-1ZAC in the presence of NB-ZSG1 was below the limit of detection (~4 pg/ml), whereas HIV-1NL4–3, HIV-1ELI and HIV-1MAL were strongly inhibited. We also noted that although the FACS isolated CD4-NB-ZSG1 and CD4-ZSG1 cells were >97% ZSG1 positive, the percentage of ZSG1 positive cells was maintained by CD4-ZSG1 population over 14 days whereas the percentage of ZSG1 positive cells in the CD4-NB-ZSG1 population decreased by about 10–20%. Our data indicates that cell proliferation of CD4-NB-ZSG1 and CD4-ZSG1 are similar, and levels of cytotoxic effects and apoptotic cells were unchanged as well. NB-ZSG1 may affect cellular pathways other than those assayed. For example, we recently reported that NB-ZSG1 strongly suppressed HIV-1 transcription in Jurkat cells , so one possible cause of this NB-ZSG1 decreased expression level is that NB-ZSG1 also negatively affects transcription by the constitutively active spleen focus-forming virus (SFFV) promoter. Given that NB-ZSG1 is able to target P-TEFb, it may impede an SEC required for HIV-1 transcription , and perhaps SEC complexes that stimulate transcription by the SFFV promoter as well. Testing these possibilities will require determining if the NB-ZSG disrupts the P-TEFb-SEC complexes, and further understanding of transcriptional activation of the SFFV promoter.
Our data shows that HIV-1 replication increased in CD4-NB-ZSG1 cells as the percentage of NB-ZSG1 positive CD4 T cells decreased, as we observed previously . It is possible that alternative promoters used to express Nullbasic in the retroviral vector may provide sustained expression of NB-ZSG1, and lead to better viral control. In addition, it would be also interesting to introduce a Nullbasic-type mutation into other Tat variants and test if a strain-specific custom Nullbasic gene is a better inhibitor of specific strains.
SIN-based γ-retroviral vectors improved expression of Nullbasic and inhibited HIV-1 replication. The study shows that Nullbasic can inhibit replication of the HIV-1 strains from different HIV-1 subtypes tested in TZM-bl cells line as well as in primary CD4+ T cells. Stable expression of Nullbasic may have utility in a future gene therapy approach applicable to genetically diverse HIV-1 strains.
DEAD/H-box helicase 1
Enzyme-linked immunosorbent assay
Fluorescent activated cell sorter
Fetal bovine serum
Horse radish peroxidase
Long terminal repeat
Mean fluorescent intensity
Peripheral blood mononuclear cells
Phosphate buffered saline
Positive transcription elongation factor
Relative luminescence unit
Super elongation complex
Spleen focus-forming virus
Woodchuck hepatitis virus post transcription regulatory element
We thank Ting Wei for providing information to advance this project. TZM-bl cell was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH, from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc.
This research was supported by the National Health and Medical Research Council Project grant (1085359). LR was supported by Prime Minister’s Australia Asia Endeavour Postgraduate (PhD) Award funded by the Australian Government, Department of Education and Training, UQ international scholarship (UQI) and UQ Centenial scholarship (UQCent).
Availability of data and materials
LR and DH designed the experiments in the study. LR performed the experiments. LR and DH analyzed the data and drafted the manuscript. HJ, MHL, ML and DR contributed reagents, materials, and analytic tools. All the authors have read and approved the final manuscript.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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