Inhibition of human endogenous retrovirus-K by antiretroviral drugs
© The Author(s) 2017
Received: 13 May 2016
Accepted: 9 March 2017
Published: 22 March 2017
Human endogenous retroviruses (HERVs) are genomic sequences of retroviral origin which were believed to be integrated into germline chromosomes millions of years ago and account for nearly 8% of the human genome. Although mostly defective and inactive, some of the HERVs may be activated under certain physiological and pathological conditions. While no drugs are designed specifically targeting HERVs, there are a panel of antiretroviral drugs designed against the human immunodeficiency virus and approved by the Federal Drug Administration (FDA).
We determined if these antiretroviral drugs may also be effective in inhibiting HERVs. We constructed a plasmid with consensus HERV-K sequence for testing the effect of antiretroviral drugs on HERV-K. We first determined the effects of nucleoside and non-nucleotide reverse transcriptase (RT) inhibitors on HERV-K by product enhanced reverse transcription assay. We found that all RT inhibitors could significantly inhibit HERV-K RT activity. To determine the effects of antiretroviral drugs on HERV-K infection and viral production, we pseudotyped HERV-K with VSV-G and used the pseudotyped HERV-K virus to infect HeLa cells. HERV-K production was measured by quantitative real time polymerase chain reaction. We found that RT inhibitors Abacavir and Zidovudine, and integrase inhibitor Raltegravir could effectively block HERV-K infection and production. However, protease inhibitors were not as effective as RT and integrase inhibitors.
In summary, we identified several FDA approved antiretroviral drugs that can effectively inhibit HERV-K. These antiretrovirals may open new prospects for studying HERV-K pathophysiology and potentially for exploring treatment of diseases in which HERV-K has been implicated.
Tremendous progress has been made in the development of antiretroviral drugs that target HIV replication. Initial drugs targeted reverse transcriptase (RT), however now drugs are available that target all major parts of the viral life cycle. This includes blockage at viral entry, integrase (IN) inhibitors that prevent integration of proviral DNA into the chromosomal DNA, protease inhibitors that prevent cleavage of the Gag–Pol polyprotein and maturation inhibitors. However, the effect of these drugs on endogenous retroviruses (ERV) remains unknown. ERV’s constitute nearly 8% of the human genome. While they play an important role in embryonic development , they remain silent in adults. Under pathological circumstances, these viral elements may get reactivated. For example, we previously showed that in patients with amyotrophic lateral sclerosis (ALS), human ERV-K (HERV-K) was expressed and this activation causes neurotoxicity [2, 3]. Hence it would be important to know if inhibition of HERV-K could alter the course of ALS. HERV-K activation has also been associated with schizophrenia and some cancers [4–7]. HERV-K is a beta-retrovirus. It has similarity to lentivirus HIV including 5′ and 3′ LTR region, an envelope (env), gag and pol genes. HERV-K is the most recently acquired ERV in the human genome and hence has several intact open reading frames . The pol gene encodes RT, protease and IN. Rec protein is encoded from an alternative spliced messenger RNA from env and is similar in function to Rev protein of HIV. In some HERV-K sequences there is a deletion of 292 base pairs at the pol–env junction as a result Rec is not formed . This resulted in an mRNA for a ~9 kDa fusion protein referred to as Np9, the function of which is not yet understood. HERV-K lacks the other regulatory genes such as tat, nef, vpr and vpu which are present in the HIV genome. Since HERV-K has its own RT, protease and IN, we screened a panel of FDA approved anti-HIV drugs that target these enzymes, for their ability to inhibit HERV-K.
Viral particle production with consensus HERV-K genome
Direct inhibition of HERV-K reverse transcriptase (RT) by HIV-1 RT inhibitors
Inhibition of HERV-K by HIV-1 RT inhibitors
Comparative modeling of HERV-K RT
Inhibition of HERV-K by HIV-1 protease inhibitors
Inhibition of HERV-K infection by integrase inhibitor
Currently there are three FDA-approved IN inhibitors: Dolutegravir, Elvitegravir, and Raltegravir. We tested the effect of Raltegravir on HERV-K replication. VSV-G pseudotyped HERV-K was used to infect HeLa cells. Raltegravir was added immediately after viral inoculation. After 6 days of infection, HERV-K gag gene was determined by qPCR. As shown in Fig. 3d, Raltegravir inhibited the replication of HERV-K in a dose-dependent manner, with an IC90 of 0.075 µM.
We used a consensus sequence of HERV-K for these studies since there are nearly 100 copies of the virus in the human chromosome  and multiple loci of HERV-K are expressed in patients with ALS [2, 11]. Similarly in patients with HIV infection there is wide variability in viral sequences among individuals but drug development has been successful using laboratory strains of the virus.
Currently there are 26 antiretroviral drugs approved by the FDA. Suppression of HIV infection requires the use of combination therapy with RT inhibitors, protease inhibitors, and IN inhibitors.
Comparison of HIV and HERV-K IC90 values for antiretroviral drugs
HERV-K IC90 (µM)
HERV-K IC50 (µM)
IC50 17 nM (no serum)
IC50 102 nM (50% serum)
HIV protease is crucial for the maturation of viral particles. It is a homodimer and belongs to the aspartate protease family . It cleaves Gag and Gag–Pol polyprotein precursor to produce capsid (CA) and active RT proteins. There are about 10 FDA-approved protease inhibitors. These inhibitors share similar chemical structures and similar binding property. They block the enzyme catalytic site by mimicking the transition state of the real substrate .
HERV-K protease is encoded in pol gene. It belongs to the aspartate protease family and includes a signature motif of aspartate–threonine–aspartate similar to HIV protease. Its core functional domain has about 106 amino acid residues and shares only 28% homology with HIV protease . However modeling of the HERV-K protease shows similarities between the active domains with the HIV-protease. The protease inhibitors could be docked to the active domain of HERV-K protease. Consistent with these observations, we found that Darunavir and Lopinavir were able to inhibit HERV-K replication in a dose-responsive manner. However the IC90 of protease inhibitors (Darunavir and Lopinavir) for HERV-K were 20–50 times higher than that for HIV (Table 1). This supports the notion that protease inhibitors are more virus specific than RT inhibitors. A report using HERV-K10 protease also showed that HIV protease inhibitors were not as effective against HERV-K protease . This suggests that HERV-K protease specific inhibitors may need to be discovered for effective antiretroviral therapy for HERV-K.
We found that IN inhibitor Raltegravir is highly effective against HERV-K, indicating that replication of HERV-K is integration dependent. IN is a key enzyme in the life cycle of the retrovirus and is coded in the pol gene. The catalytic core domain of HIV IN transfers the viral DNA into the chromosome with the help of both C- and N-terminal domains  HIV IN has similar structural similarity with other retroviral INs from avian sarcoma virus, rous sarcoma virus and simian immunodeficiency virus, indicating they may share a similar mechanism of action . Comparative modeling showed that there are almost no differences in the active site of HERV-K and simian PFV INs that can be inhibited by Raltegravir . The inhibitors target the DNA stand transfer process. Hence they are called strand transfer inhibitors. Raltegravir is one of the FDA approved stand transfer inhibitors and is highly potent in inhibiting both HIV and HERV-K replication (Table 1). Because IN inhibitors have less side effects compared to RT and protease inhibitors, it should be an important component in any antiretroviral regime.
Our data suggests that similar to HIV antiretroviral treatment, complete inhibition of HERV-K may require combination therapy that targets different parts of the life cycle of the virus particularly since most of these drugs are less potent in their activity against HERV-K compared to their effects on HIV. However, studies are needed to determine the efficacy of combined inhibitors on HERV-K replication. Currently, it is not clear if the activation of HERV-K expression in ALS patients leads to production of infectious viral particles. The anitretrovirals will be effective only if there was active infection and replication. For treatment of ALS patients with antiretroviral drugs, the drugs need to have good blood brain barrier penetration to target brain neurons with increased HERV-K expression.
We identified that FDA approved RT and IN inhibitors can effectively inhibit HERV-K virus, while protease inhibitors were not as effective in inhibiting HERV-K virus as HIV. Development of new protease inhibitors for HERV-K may be required. These antiretrovirals may open new prospects for studying HERV-K pathophysiology and potentially for exploring treatment of diseases in which HERV-K has been implicated.
DNA constructs and HIV inhibitors
HERV-K whole genome consensus sequence  was synthesized and cloned into pcDNA3.1 vector (Invitrogen). HIV-1 Rev plasmid was reported previously . To increase the production of HERV-K viral particles, the Rev expression cassette was inserted to the pcDNA3.1-HERV-K construct. The resulting plasmid was called pCD-HK/Rev. All HIV inhibitors and VSV-G plasmid were obtained from NIH AIDS reagent program (http://www.aidsreagent.org). A stock of 10 mM was made by diluting the inhibitors in dimethyl sulfoxide (DMSO). For further use serial dilutions for each inhibitor was made in complete media: Dulbecco’s modified Eagle’s medium; DMEM + 10% fetal bovine serum (FBS) and penicillin–streptomycin.
Cell culture and transfection
The human cell lines 293T and Hela were maintained in DMEM supplemented with 10% FBS and penicillin–streptomycin. For testing activity of HIV-RT inhibitors in a cell free system, Hela cells were transiently transfected with pCD-HK/Rev in 24-well plates at 0.2 × 106 cells/well using lipofectamine 2000 (Invitrogen) according to the manufacture’s protocol. Virus particle-containing supernatants were collected after 24 and 48 h. Control experiments included mock transfection with empty vector pcDNA3.1. Cell culture supernatants were assayed for RT activity using a PERT assay as described below. At the time of reverse transcription, HIV nucleoside or non-nucleotide RT Inhibitors were added to the supernatant at six different doses ranging from 0.001 to 0.25 µM. Any change in RT activity was expressed as percent inhibition relative to no treatment control.
For testing the activity of HIV protease inhibitors against HERV-K, HeLa cells were transiently transfected with pCD-HK/Rev as described above. Six hours post-transfection, culture medium was completely replaced with fresh medium containing HIV protease inhibitors in a twofold serial dilution ranging from 31.25 nM to 1 µM. After 48 h, cell culture supernatants were collected and RT activity in the culture supernatant was determined by PERT assay. Darunavir and Lopinavir were identified as the two most potent drugs and were further screened in a tenfold-serial dilution treatment ranging from 0.001 to 100 µM.
Recombinant virus production and infection
293T cells were cultured in DMEM with 10% FBS and penicillin–streptomycin. Cells were transiently transfected in 10 cm plates at 5 × 106 cells/plate using lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Briefly, cells were co-transfected with pCD-HK/Rev with or without pCD-VSV-G. After 24 h, the transfection medium was completely removed and cells were washed with phosphate-buffered saline (PBS) to eliminate any residual plasmid and then fresh medium was added to the cells. Virus particle-containing supernatants were harvested after an additional 24–48 h and cleared of any cellular debris with two centrifugations at 1000×g at 4 °C. The clarified samples were then subjected to DNase treatment using the RNase-free DNase kit (Qiagen). Cleared supernatant was concentrated using Retro-X™ Concentrator (Clontech) as per manufacturer’s instructions. Briefly, viral supernatant was mixed with the Retro-X Concentrator and incubated overnight at 4 °C. The mixture was then centrifuged at 1500×g for 45 min at 4 °C to obtain a virus-containing pellet. The viral pellet was gently resuspended using complete DMEM and titrated using the PERT assay. An absolute amount of RT was determined using HIV RT as standard and 80 pg of HERV-K virus was used for each infection. At the time of transduction of target cells, the concentrated virus was again treated with RNase free DNase to ensure there was no plasmid DNA contamination. Infection was performed by exposing the resuspended DNase treated viral samples with fresh 293T or HeLa cells that had been plated in 24-well plates 24 h earlier in 5 μg/ml of polybrene in the presence or absence of Abacavir, Zidovudine or Raltegravir. Total RNA was extracted 6 days post infection and HERV-K Gag gene expression was quantified using QPCR. Any change in RT inhibitor treated wells compared to untreated was expressed as percent inhibition. To determine the effect of protease inhibitors on HERV-K infection, 293T cells were transfected with pCD-HK/Rev and pCD-VSV-G in the presence of 1 µM of Darunavir or Lopinavir. After 24 h, the transfection medium was completely removed and cells were washed with PBS. Fresh medium with 1 µM of Darunavir or Lopinavir was then added back. After another 24 h, viral particles were harvested and used to infect 293T cells using the same method mentioned above, but without any further PI treatment during infection.
RNA extraction and quantitative PCR
Primer sequence (5′–3′)
Product enhanced reverse transcriptase (PERT) assay
PERT assay was used as described  with minor modifications. Briefly, cell culture supernatant was collected and centrifuged to pellet any cell debris. The cleared supernatant was then supplemented with 0.25% Triton X-100, 5 mM dithiothreitol and 0.25 mM ethylene-diamine-tetra-acetate as the source of HERV-K RT. Bacteriophage MS2 genomic RNA was used as template for the reverse transcription reaction. Quantitative PCR was performed with TaqMan primers (MS2-Forward and MS2-Reverse) and probe (MS2-Probe) using Applied Biosystems Vii 7. RT activity was expressed as fold change compared to control or as pg/ml RT determined by standard curve generated from PERT using HIV-1 RT.
Western blot analysis, immunoflourescence and antibody production
For Western blot analysis of HERV-K viral protein expression and cleavage, 293T cells were transiently transfected with either the HERV-K expression vectors or empty vector using lipofectamine 2000 (Invitrogen). After 48 h transfection, cells were washed with PBS and lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (Roche). The insoluble pellet was removed by a 10 min centrifugation at 12,000×g. The harvested lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis using Novex 4–12% Bis–Tris gels (Invitrogen), followed by transfer onto polyvinylidene fluoride membranes. The blots were incubated overnight at 4 °C with either anti-HERV K Env antibody (Austral biologicals) or anti HERV K Gag antibody (Austral biologicals) followed by 1 h incubation with a secondary antibody linked to horseradish peroxidase. After 30 min washing, the blot was developed with SuperSignal™ West Femto ECL reagent (ThermoFisher), and imaged with FluoroM imaging machine (ProteinSimple). Immunofluorescence analysis for the co-localization of Gag and Pol was performed on 293T cells transiently transfected with the HERV-K expression vector or empty vector (negative control). Twenty-four hours post-transfection cells were fixed with 4% paraformaldehyde, permeabilized, and stained with a rabbit polyclonal anti-Pol serum and a mouse monoclonal anti-Gag antibody. Alexa 488-conjugated goat anti-rabbit IgG and Alexa 594-conjugated goat anti-mouse IgG were used as secondary antibodies (molecular probes); nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; molecular probes).
A polyclonal anti-HERV-K Pol antibody using amino acids 57–245 as an immunogen was developed by SDIX with its proprietary Advanced GAT technologies. The monoclonal antibodies against the full length HERV-K Env and Gag were obtained from Austral Biologicals. Rabbit antisera against HERV-K envelope protein were developed by Genscript, using peptides QRKAPPRRRRHRNRC, CSDLTESLDKHKHKK, and CSKRKGGNVGKSKRD as immunogens.
HeLa cells were cultured in microplates (tissue culture grade, 96 wells, flat bottom) in a final volume of 100 µl/well culture medium in a humidified atmosphere (e.g., 37 °C, 5% CO2). 24 h later, the cells were treated with HIV inhibitors at dosage ranging from 0.01 to 10 µM. Six days post-treatment Cell Proliferation Reagent WST-1(Roche) was used per manufacturer’s instructions to determine drug toxicity. Briefly, 10 µl of Cell Proliferation Reagent WST-1 was added to each well and the plate was shaken thoroughly for 1 min on a shaker. The cells were incubated for 0.5–4 h in a humidified atmosphere (37 °C, 5% CO2). The absorbance of the samples against a background control as blank was measured using a microplate reader at 420–480 nm using a FlexStation microplate reader (molecular devices).
All the comparative modeling was performed using the homology modeling protocols implemented in the program molecular operating environment (MOE) . The sequences of target and templates were initially aligned with clustalW  and manually adjusted after inspection to place insertions and deletions in favorable regions. An AMBER10HT force field was used for energy calculations and minimization. Ramachandran’ plot showed 95% of the residues of the final model are in allowed regions, and no rotamer outliers are present. When complexes between template and target inhibitors are available, the poses of the inhibitors in HERV-K targets were based on that of the template’s complexes. When required to improve the pose, rotamers of selected residues 4.5 Å apart from the drug were explored to relieve the few clashes observed and to improve contacts, as well as drug and nearby residues relaxed by minimization. The drug minimization in the active site environment of the HERV-K targets was performed, tethering the protein atoms to their initial position with a weak harmonic potential (0.5 kCal/mol) during minimization.
In the case of the protease, the structures of a dimer of HIV-1 (PDBId: 2HS1, 0.85 Å) and Rous sarcoma virus (RSV; 1BAI, 2.4 Å) proteases were used as templates. The RSV protease structure was used to model the insertion in the loop between β4–5 because it displays similar characteristics to the one in HERV-K protease and, the HIV-1 protease was used for the rest of the model (Fig. 4b). Three features common to retroviral proteases were carefully maintained in the alignment and model: (1) the active site triad (26-DTG-28), (2) the highly conserved triad GRN/D unique to retroviral proteases , and (3) the intra- and inter-subunit salt bridge between R89, D30, and R9′. To model complexes of HERV-K protease with inhibitors the structures of the highest resolution complexes of the HIV-1 protease with Lopinavir (2OS4), and Darunavir (2HS1) were used. Models of the complexes with Darunavir and Lopinavir were prepared by overlaying the respective complexes structures with the HERV-K model.
In the case of the HERV-K RT, HIV-1 RT crystal structure (4W1E) was used as template, and the crystal structures of the complexes with Efavirenz (1JKH), Nevirapine (3QIP), and Etravirine (3MEC) were used to model the inhibitors bound to HERV-K RT.
In the case of the HERV-K IN the simian prototypical foamy virus in complex with magnesium, DNA, and Elvitegravir (3L2U) with an 18% of identity with the target was used as template. This elvitegravir complex and the complexes with ratelgravir (3L2) and dolutegravir (3S3M) were used to model these inhibitors complexes with HERV-K IN (Fig. 8c–e).
Data analysis and statistics
All the experiments were repeated at least three times. Representative results were shown and plotted as mean ± SEM. Student’s t test was used for pair-wise comparison.
RT: conducted most of the experiments and analyzed the data. WL: designed and supervised the experiments and wrote the manuscript. DP: conducted the experiment on protease inhibitors. MB: conducted the comparative modeling. AN: conceived and supervised the project, helped in writing the manuscript, and provided laboratory space and financial support. All authors read and approved the final manuscript.
The authors would like to thank the NIH AIDS Reagent Program for providing all the HIV antiretroviral drugs and the reverse transcriptase recombinant protein.
The authors declare that they have no competing interests.
This work was supported by intramural funds from the National Institute of Neurological Disorders and Stroke at the National Institutes of Health.
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