viruses
Article
The Thiazole-5-Carboxamide GPS491 Inhibits HIV-1,
Adenovirus, and Coronavirus Replication by Altering RNA
Processing/Accumulation
Subha Dahal 1 , Ran Cheng 1 , Peter K. Cheung 2,3 , Terek Been 1 , Ramy Malty 4 , Melissa Geng 1 ,
Sarah Manianis 1 , Lulzim Shkreta 5 , Shahrazad Jahanshahi 1,4 , Johanne Toutant 5 , Rose Chan 1 , Sean Park 1 ,
Mark A. Brockman 2,3 , Mohan Babu 6 , Samira Mubareka 7 , Karen Mossman 8 , Arinjay Banerjee 8 ,
Scott Gray-Owen 1 , Martha Brown 1,7 , Walid A. Houry 4,9 , Benoit Chabot 5 , David Grierson 10
and Alan Cochrane 1, *
1
2
3
4
5
Citation: Dahal, S.; Cheng, R.;
Cheung, P.K.; Been, T.; Malty, R.;
6
Geng, M.; Manianis, S.; Shkreta, L.;
Jahanshahi, S.; Toutant, J.; et al. The
7
Thiazole-5-Carboxamide GPS491
Inhibits HIV-1, Adenovirus, and
8
Coronavirus Replication by Altering
RNA Processing/Accumulation.
Viruses 2022, 14, 60. https://doi.org/
9
10
10.3390/v14010060
*
Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada;
subha.dahal@mail.utoronto.ca (S.D.); ran.cheng@mail.utoronto.ca (R.C.); terek.been@mail.mcgill.ca (T.B.);
melissa.geng@mail.utoronto.ca (M.G.); sarah.manianis@mail.utoronto.ca (S.M.);
Shahrazad.Jahanshahi@mail.utoronto.ca (S.J.); rose.chan@mail.utoronto.ca (R.C.);
seonghungpark@mail.utoronto.ca (S.P.); scott.gray.owen@utoronto.ca (S.G.-O.);
martha.brown@utoronto.ca (M.B.)
British Columbia Centre for Excellence in HIV/AIDS, Vancouver, BC V6Z 1Y6, Canada;
pcheung@bccfe.ca (P.K.C.); mark.brockman@sfu.ca (M.A.B.)
Faculty of Health Sciences, Simon Fraser University, Burnaby, BC V5A 1S6, Canada
Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada;
ramy.malty@utoronto.ca (R.M.); walid.houry@utoronto.ca (W.A.H.)
Department of Microbiology and Infectious Diseases, Faculty of Medicine and Health Sciences, Université de
Sherbrooke, Sherbrooke, QC J1H 5N4, Canada; Lulzim.Shkreta@usherbrooke.ca (L.S.);
Johanne.toutant@usherbrooke.ca (J.T.); benoit.chabot@usherbrooke.ca (B.C.)
Research and Innovation Centre, Department of Biochemistry, University of Regina,
Regina, SK S4S 0A2, Canada; mohan.babu@uregina.ca
Department of Laboratory Medicine & Pathobiology, University of Toronto, Toronto, ON M5S 1A8, Canada;
samira.mubareka@sunnybrook.ca
Department of Medicine, McMaster University, Hamilton, ON L8N 3Z5, Canada; mossk@mcmaster.ca (K.M.);
arinjay.banerjee@usask.ca (A.B.)
Department of Chemistry, University of Toronto, Toronto, ON M5S 3H6, Canada
Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC V6T 1Z3, Canada;
david.grierson@ubc.ca
Correspondence: alan.cochrane@utoronto.ca; Tel.: +1-416-978-4550
Academic Editors: Cheng-Wen Lin
and Szu-Hao Kung
Received: 26 November 2021
Accepted: 20 December 2021
Published: 30 December 2021
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Abstract: Medicinal chemistry optimization of a previously described stilbene inhibitor of HIV-1,
5350150 (2-(2-(5-nitro-2-thienyl)vinyl)quinoline), led to the identification of the thiazole-5-carboxamide
derivative (GPS491), which retained potent anti-HIV-1 activity with reduced toxicity. In this report, we
demonstrate that the block of HIV-1 replication by GPS491 is accompanied by a drastic inhibition of
viral gene expression (IC50 ~ 0.25 µM), and alterations in the production of unspliced, singly spliced,
and multiply spliced HIV-1 RNAs. GPS491 also inhibited the replication of adenovirus and multiple
coronaviruses. Low µM doses of GPS491 reduced adenovirus infectious yield ~1000 fold, altered virus
early gene expression/viral E1A RNA processing, blocked viral DNA amplification, and inhibited
late (hexon) gene expression. Loss of replication of multiple coronaviruses (229E, OC43, SARS-CoV2)
upon GPS491 addition was associated with the inhibition of viral structural protein expression and
the formation of virus particles. Consistent with the observed changes in viral RNA processing,
GPS491 treatment induced selective alterations in the accumulation/phosphorylation/function of
splicing regulatory SR proteins. Our study establishes that a compound that impacts the activity
of cellular factors involved in RNA processing can prevent the replication of several viruses with
minimal effect on cell viability.
Keywords: HIV-1; adenovirus; coronavirus; RNA processing; inhibitor
4.0/).
Viruses 2022, 14, 60. https://doi.org/10.3390/v14010060
https://www.mdpi.com/journal/viruses
Viruses 2022, 14, 60
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1. Introduction
As of 2017, 75% of FDA-approved antivirals target discrete stages of the virus replication cycle (entry, genome replication, proteolytic cleavage of viral proteins) by inhibiting the
activity of virus-encoded proteins [1]. Of the remainder, most are derivatives of interferons
or target interactions with host cell surface receptors (i.e., maraviroc) [2]. Consequently,
currently approved antivirals display effectiveness against only a limited range of viruses
and can become ineffective by their selection for resistant viral strains. However, as obligate
parasites, viruses are dependent upon a common subset of host cell processes for their
replication [2,3]. Consequently, altering the ability of a virus to effectively use a host cell
process may provide an alternative strategy to impede virus replication, generating a more
robust barrier to virus resistance, and affect a broader spectrum of viruses with a single
agent. Examples of such host-targeted therapeutic (HTT) strategies include the modulation
of cell signaling, protein trafficking, lipid metabolism, epigenetic modifications, and RNA
processing [2–6].
Previously, our group demonstrated that the stilbene 2-(2-(5-nitro-2-thienyl)vinyl)quinoline (designated 5350150) inhibits HIV-1 replication by altering viral RNA accumulation [7].
However, the limited therapeutic index (CC50 /EC50 ratio) of 5350150 indicated that further
optimization was necessary for its application as an antiviral against HIV-1. Synthesis and
evaluation of a library of di(hetero)aryl amide analogs of 5350150 led to the selection of
the thiazole carboxamide compound GPS491 [8] (Figure 1a, Table 1) for further study as
an antiviral agent. In this report, we determine that, similar to 5350150, GPS491 blocks
HIV-1 protein expression in part due to changes in viral RNA accumulation and processing.
Additional tests determined that GPS491 also inhibits the replication of human adenovirus
type 5 and several coronaviruses, demonstrating pan-antiviral activity of the compound.
In the case of adenovirus, GPS491 addition altered E1A RNA processing and inhibited
expression of other viral early genes resulting in the loss of adenovirus DNA replication and
late viral gene expression. Consistent with the observed changes in viral RNA processing,
treatment with GPS491 induced selective changes in SR protein accumulation/modification
as well as function with minimal cytotoxic effects. Together, these findings support the
hypothesis that compounds affecting viral RNA processing/expression can be used to
suppress the growth of different viruses.
Table 1. Effect of GPS491 on replication of multiple mammalian viruses.
Virus
Strain
EC50
CC50
HIV-1
Assayed in CEM-GXR cell line
Clade A
Clade B
NL43 E0043 RTI
NL43 2918 PI
NL43 11845 INI
IIIB MVC res
176 nM
248 nM
235 nM
230 nM
203 nM
216 nM
12,052 nM
12,052 nM
12,052 nM
12,052 nM
12,052 nM
12,052 nM
HIV-1
Assayed in PBMCs
HIV-1 Bal (average of 3 donors)
HIV-1 IIIB (average of 3 donors)
248 nM
738 nM
2,509 nM
2,509 nM
Adenovirus
Assayed in A549 cell line
HAdV-C5
1000 nM
>40,000 nM
Coronavirus
Assayed in Huh7 cell line
229E
OC43
SARS-CoV2
250 nM
250 nM
100 nM
>10,000 nM
>10,000 nM
>10,000 nM
See Figure S1 for data of the effect of GPS491 on HIV-1 replication.
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Figure 1. GPS491 inhibits expression of multiple HIV-1 proteins. (a) Chemical structure of GPS491.
∆
HeLa rtTA HIV∆mls cells were treated with either 2.5 µM or 1.25 µM GPS491. Cells grown with or
without doxycycline (Dox) with 1% DMSO served as positive and negative controls, respectively.
Cells were harvested for HIV-1 protein and RNA analyses after 24 h of induction. (b) Shown are
representative Western blots showing the effects of compounds on HIV-1 Gag, Env, and Tat≤ levels.
Below
≤ is a summary≤of n = 3 independent experiments; data are indicated as mean ± SD; * p ≤ 0.05,
** p ≤ 0.01, and *** p ≤ 0.001. (c)∆Proteasome inhibition reverses the effect of GPS491
− on Tat but not
Gag expression. HeLa rtTA HIV∆mls cells were treated with DMSO or GPS491 +/− Dox for 24 h.
Cells were treated with or without MG132 8 h prior to harvest, lysates were fractionated on 10%
stain-free gels or 14% SDS PAGE gels, and the resulting blots were probed for HIV-1 Gag or Tat,
respectively. Band intensity was quantified relative to Dox-induced DMSO control and normalized
to total protein load (Stainfree) for Gag blot or normalized to GAPDH for Tat blot using Bio-Rad
ImageLab software.
2. Materials and Methods
2.1. Cell Lines and Virus Strains
Effects of compound treatment on HIV-1 gene expression were initially assessed in
the context of HeLa cells stably transduced with an inducible Tet-On HIV-1 system, as
previously described [9–11]. The provirus was modified by either deletion in the reverse
∆
transcriptase
and integrase region of the pol gene by an MlsI restriction digest (HeLa rtTA
HIV∆Mls cell line) or GFP fusion to Gag, deleting the PR- and RT-coding regions (HeLa rtTA
HIVGagGFP cell line). Tat and its TAR binding sites were inactivated so that HIV-1 gene
expression was only induced in the presence of doxycycline (Dox). HeLa cells containing
Viruses 2022, 14, 60
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HIV-1 provirus and J-Lat 10.6 [12] cells were maintained in Iscove’s Modified Dulbecco’s
Medium (IMDM), and Roswell Park Memorial Institute (RPMI) 1640 Medium (Wisent Corp.,
Saint-Jean Baptiste, QC, Canada), respectively, supplemented with 10% (v/v) fetal bovine
serum (FBS, Wisent Corp., Saint-Jean Baptiste, QC, Canada), 1% penicillin/streptomycin
(P/S, Wisent Corp., Saint-Jean Baptiste, QC, Canada), and 0.2% Amphotericin B (Wisent
Corp., Saint-Jean Baptiste, QC, Canada). The indicator cell line CEM-GXR was maintained
and used as described previously [13]. A549 cells (human lung carcinoma) were obtained
from the American Type Culture Collection (ATCC, Manassas, VA, USA) at passage level
76 and used between passages 89 and 110. HEK 293 (human embryonic kidney) cells were
obtained from F. Graham, McMaster University, Hamilton, Ontario, Canada, at passage
24 and were used between passages 58 and 90. A549 and HEK 293 cells were cultured in
Eagle’s minimum essential medium (MEM, Gibco, Waltham, MA, USA) supplemented with
10% FBS (Sigma-Aldrich, St. Louis, MO, USA) plus penicillin (100 U/mL) and streptomycin
(100 µg/mL). Huh7 cells for infection with coronaviruses were maintained in Dulbecco’s
Modified Eagle Medium (DMEM, Wisent Corp., Saint-Jean Baptiste, QC, Canada) with
high glucose (HyClone) supplemented with 10% heat-inactivated FBS and 1% P/S. All the
cell lines were maintained at 37 ◦ C in a humidified incubator with 5% CO2 . HIV-1 reverse
transcriptase-resistant virus (E00443) was obtained from Dr. Zabrina Brumme (Simon
Fraser University, Burnaby, BC, Canada). HIV-1 clade A (97USSN54 (N54)), HIV-1 clade B
(IIIB), integrase inhibitor-resistant virus (11845), and protease inhibitor-resistant virus (2948)
were obtained through the NIH AIDS Research and Reference Reagent Program, Division
of AIDS, NIAID, NIH. Adenovirus used in the studies was HAdV-C5. Coronaviruses 229E
and OC43 were obtained from ATCC. SARS-CoV2 used is the SB2 strain was obtained from
Dr. S. Mubareka.
2.2. GPS491 Inhibition of Virus Replication
The compound was prepared as outlined in Zamiri, M., et al. [8] and dissolved to
10 mM or 1 mM stock concentration in dimethyl sulfoxide (DMSO) and stored at −20 ◦ C
for subsequent experiments. Compound purity was determined to be >95% as assessed by
high-resolution mass spectrometry. For assays involving cell lines containing the integrated
HIV-1 provirus, cells were seeded at a confluence of 70% on 6-well plates in IMDM or
RPMI 1640 complete medium and treated with GPS491 at the desired concentration for 24 h.
Control wells contained DMSO at 1% final concentration. Necessary cells were induced
with Dox or prostratin at a final concentration of 2 µg/mL and 1 µg/mL, respectively, at
the same time the compound was added. Twenty-four hours post compound treatment,
cells were harvested in 2 mM EDTA and 1×PBS for Western blotting and RNA analyses.
To assess the activity of compounds against HIV-1 (WT or strains resistant to various antiretroviral drugs), assays were performed in the context of CEM-GXR cells, an
immortalized CD4+ T-lymphocyte line that expresses GFP due to the activation of an
integrated Tat-dependent promoter upon HIV-1 infection [13]. HIV-1 spread in culture
was monitored using flow cytometry. CEM-GXR cells naturally express the HIV-1 receptor
CD4 and coreceptor CXCR4 and have been engineered to express HIV-1′ s other coreceptor,
CCR5. Cultures were infected with different HIV-1 strains of interest, differing in their
subtypes (A and B), and resistance to four major drug targets (reverse transcriptase, protease, integrase, and coreceptor antagonist). The HIV-1 inhibition assay was performed
in a 96-well plate, where CEM-GXR cells were inoculated via co-culture with CEM-GXR
cells infected with different viral strains of interest (1%, initial proportion of cells). GPS491
was then immediately added to the inoculated cultures such that the final compound
concentrations were between 100 nM and 10 µM. The percentage of infected cells in the
culture was determined by flow cytometry on day 3 p.i. The inhibition curves were fitted
by nonlinear regression using GraphPad Prism software (San Diego, CA, USA), allowing
for EC50 calculation. All data are reported as the mean ± s.e.m. from three independent
determinations. The cytotoxicity of GPS491 was investigated by exposing uninfected CEMGXR cells to GPS491 and assessing viability using the Guava ViaCount flow cytometry
Viruses 2022, 14, 60
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assay according to the manufacturer’s instructions (Luminex Corp., Toronto, ON, Canada).
In brief, CEM-GXR cells were seeded into 96-well plates at 8 × 104 cells/well in the absence
or presence of GPS491 at the indicated concentrations. Three days later, 25 µL of cell
suspension was mixed with 225 µL of Guava ViaCount reagent, the mixture was incubated for 5 min at room temperature, and then analyzed on a Guava easyCyte 8HT flow
cytometer (Luminex Corp., Toronto, ON, Canada). Data analysis was performed using
the EasyFit analysis feature in the ViaCount software module in GuavaSoft v2.7 (Luminex
Corp., Toronto, ON, Canada).
Studies involving peripheral blood mononuclear cells (PBMCs) were performed as
described by Mwimanzi et al. [14]. Briefly, PBMCs from three HIV-1-negative donors were
activated for 3 days using 5 µg/mL phytohemagglutinin in RPMI media supplemented
with 10% heat-inactivated FBS (R10+), infected with HIV-1IIIB or HIV-1BaL for 6 h, washed
and resuspended in R10+ media supplemented with 100 U/mL IL-2 (Sigma-Aldrich,
St. Louis, MO, USA), seeded into 96-well U-bottom plates at 150,000 cells/well, and treated
with GPS491 or 0.1% DMSO. On day 4 and day 7 p.i., the culture media, compound,
and IL-2 were replenished. Supernatant p24Gag levels were quantified on day 11 p.i. by
ELISA (Xpress Bio, Frederick, MD, USA) as per manufacturer’s instructions. To quantify
cytotoxicity of the compound on PBMCs, the above procedure was repeated with uninfected
PBMCs from the same donors. On day 11 post treatment, culture viability was assessed
using Guava ViaCount reagent (Luminex Corp., Toronto, ON, Canada) and flow cytometry,
as per the manufacturer’s instructions.
To examine the effect of GPS491 on adenovirus replication, A549 cells were seeded in
a 6-well plate at a density of 5 × 105 cells/well. Cells were infected one day post seeding
at an input multiplicity of infection (MOI) of 100 IU/cell of human adenovirus serotype 5
(HAdV-C5). After 1 h of adsorption at 37 ◦ C, inoculum was removed and replaced with a
fresh culture medium containing 1% DMSO (solvent control) or compound dissolved in
DMSO (duplicate wells per condition). Progeny virus was harvested 24 h p.i. by scraping
the cells into the culture fluid, followed by freeze-thaw of suspension five times with
vortexing. The lysate was clarified by centrifugation at 500× g for 5 min and titrated by
endpoint dilution in HEK 293 cells [15].
To examine the dose-dependent effects of GPS491 on coronavirus replication, Huh7
cells were seeded in a 96-well plate and infected at an input MOI of 0.1 and 1 for 229E and
OC43, respectively. To study the effects of the compound on viral protein levels and viral
RNA in media (sups), cells were seeded in a 6-well plate and infected with 229E, OC43,
or SARS-CoV2 [16] at an input MOI of 0.03, 0.3, and 1, respectively. Infection with virus
inoculum was done in serum-free DMEM for an hour with gentle rocking of the plate back
and forth every 20 min during the course of infection. Virus inoculum was subsequently
removed after an hour, cells washed with 1× PBS, and treated with DMSO or a compound
diluted in DMEM supplemented with 2% FBS and 1% P/S. Cell media were harvested
at 2 d p.i. for 229E or SARS-CoV2 infection and at 4 d p.i. for OC43 infection, followed
by heat inactivation at 95 ◦ C for 5 min. For time of addition studies, cells were infected
at an input MOI of 2, DMSO or GPS491 added at various times post virus removal, and
media harvested at 24 h post infection to assay viral RNA levels. Media were directly
used to detect viral RNA using Luna® Universal One-Step RT-qPCR Kit (New England
Biolabs, Ipswich, MA, USA) as per the manufacturer’s instructions. Each reaction was
set up as follows in qPCR 384-well plate: 5 µL Luna Universal One-Step Reaction Mix
(2×), 0.5 µL Luna WarmStart® RT Enzyme (20×), 0.2 µL of each 5′ and 3′ primers (10 µM),
and 1 µL of media (template RNA) in a total reaction volume of 10 µL. Sequences used
to detect coronavirus RNA in media are provided in Supplementary Table S1. The qPCR
cycling conditions were as follows: reverse transcription at 55 ◦ C for 10 min and initial
denaturation at 95 ◦ C for 1 min, followed by 40 cycles of denaturation at 95 ◦ C for 10 s and
extension at 60 ◦ C for 30 s. The melting curve protocol followed with 15 s at 95 ◦ C and then
15 s each at 0.2 ◦ C increments between 60 ◦ C and 95 ◦ C. Melting and standard curves were
generated by the CFX Maestro Software (version 1.1, Bio-Rad, Mississauga, ON, Canada).
Viruses 2022, 14, 60
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2.3. Effect on Viral and Host Protein Expression
To assess the effects on viral proteins by Western blot, cell extracts were prepared in
RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate,
0.1% SDS) and fractionated on 10% TGX acrylamide stain-free (Bio-Rad, Mississauga, ON,
Canada) or 14% SDS-PAGE gels. Stain-free gels were directly imaged on ChemiDoc MP Imager (Bio-Rad) to measure total protein levels, which served as a loading control. Following
imaging for total protein levels, protein was transferred to PVDF using the Trans-Blot Turbo
Transfer System (Bio-Rad, Mississauga, ON, Canada). Blots were blocked in either 5% MilkTBS-T (5% Milk, 0.05% Tween-20, 1× TBS) or 3% BSA-TBS-T (3% BSA, 0.05% Tween-20,
1× TBS) prior to incubating with primary antibodies (all diluted in either 5% BSA-TBS-T
or 5% Milk-TBS-T). Primary antibodies used to evaluate the effect of GPS491 on HIV-1
protein levels were purified mouse anti-p24 supernatant from anti-HIV-1 Gag hybridoma
183, mouse anti-gp120 hybridoma 902, mouse α-Tat hybridoma (1D9) (all obtained from
NIH AIDS Reagent Program), and rabbit α-GAPDH (Sigma-Aldrich, St. Louis, MO, USA).
To evaluate the effects of GPS491 on SR protein expression levels, blots were probed with
one of the following antibodies; mouse α-SRSF1 (Life Technologies, Waltham, MA, USA
Cat: 32-4500), rabbit α-SRSF2 (BD Pharmingen, San Diego, CA, USA, Cat: 556363), mouse
α-SRSF3 (Life Technologies, Waltham, MA, USA, Cat: 33-4200), rabbit α-SRSF4 (Novus Biologicals, Toronto, ON, Canada, Cat: NBP2-04144), rabbit α-SRSF5 (MBL, Woburn, MA, USA
RN082PW), rabbit α-SRSF6 (Novus Biologicals, Toronto, ON, Canada, Cat: NBP2-04142),
rabbit α-SRSF7 (Abcam, Cambridge, UK, Cat: ab137247), rabbit α-SRSF9 (MBL, Woburn,
MA, USA, Cat: RN081PW), mouse α-SRSF10 (Novus Biologicals, Toronto, ON, Canada, Cat:
H00010772-M07), rabbit α-Tra2β (Abcam, Cambridge, UK, Cat: ab31353), mouse α-CLK1
(Santa Cruz, Biotechnology Inc., Dallas, TX, USA Cat: 515897), rabbit α-CLK2 (Abcam, Cambridge, UK, Cat: ab65082), mouse α-CLK3 (Abnova, Walnut, CA, USA Cat: H0001198-M05),
and rabbit α-SRPK1 (Cedarlane, Burlington, ON, Canada Cat: OAAN01583). Adenovirus
E1A and hexon proteins were detected using rabbit α-E1A (Santa Cruz Biotechnology
Inc., Dallas, TX, USA Cat: sc-430) and undiluted hybridoma (2Hx-2) media, respectively,
as previously detailed [17]. To measure the effect of the compound on 229E, OC43, or
SARS CoV-2 nucleocapsid (N) protein expression, either mouse α-coronavirus antibody
clone FIPV3-70 (Novus Biologicals, Toronto, ON, Canada Cat: NB10064754), mouse αcoronavirus group antigen antibody clone 542-7D (Millipore Sigma, Burlington, MA, USA,
Cat: MAB9013), or rabbit anti-SARS CoV2 nucleocapsid antibody (SinoBiological, Beijing,
China, Cat: 40143-R019) was used, respectively. Changes in OC43 spike protein expression
were assessed using mouse monoclonal antibody 541-8F (Sigma-Aldrich, St. Louis, MO,
USA, Cat: MAB9012). Following overnight incubation with primary antibodies at 4◦ C,
blots were washed and incubated in appropriate secondary antibody (either α-mouse or
α-rabbit horseradish peroxidase (HRP)-conjugated IgG (Jackson ImmunoResearch, West
Grove PA, USA) for an hour at room temperature. After subsequent washes, signals were
detected by ECL/ECL Plus (Perkin-Elmer, Waltham, MA, USA), or Clarity Western ECL
reagent (BioRad, Mississauga, ON, Canada) and imaged using a ChemiDoc MP Imager
(Bio-Rad, Mississauga, ON, Canada). To test whether changes in protein migration were
attributable to altered phosphorylation, extracts were treated with lambda phosphatase
(New England Biolabs, Ipswich, MA, USA Cat: P0753S) prior to running on gels. Relative
intensity of detected bands was normalized to corresponding bands of the loading control
(GAPDH or total protein using Bio-Rad ImageLab software, Mississauga, ON, Canada).
To examine the effects of GPS491 on adenoviral protein (hexon) expression by immunofluorescence, cells were fixed at 24 h p.i. with 3.7% PFA for 15 min, washed with
1× PBS, and permeabilized for 15 min with 0.1% Triton X-100 in PBS (PBT), followed by
blocking for 45 min with 5% BSA in PBT (BSA-PBT). Cells were then incubated in primary
antibody (undiluted culture fluid from 2HX-2 hybridoma cells to probe for hexon) for
45 min followed by three washes with 1× PBS and incubation in secondary antibody (goat
anti-mouse labeled with AlexaFluor488 (Jackson ImmunoResearch, West Grove, PA, USA)
at 1:200 dilution) for 45 min. After subsequent washes in PBT and PBS, coverslips were
Viruses 2022, 14, 60
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mounted on a glass slide with PBS containing 0.25 µg/mL DAPI. Cells were viewed with
10× objective using a Leica DMR microscope and images captured with OpenLab imaging
software version 2.0.7 (Santa Clara, CA, USA).
2.4. Toxicity Assays
Cytotoxicity of GPS491 was assessed by using a CellTiter-Glo luminescent cell viability
assay (Promega, Madison, WI, USA), alamarBlue or Trypan blue exclusion (Life Technologies, Waltham, MA, USA) and expressed relative to cells treated with DMSO (1%) alone.
alamarBlue was added to culture medium prior to harvest/fixation, cells were incubated at
37 ◦ C in a 5% CO2 humidified incubator for 2–6 h, and fluorescence reflecting cell metabolic
rate was measured using Bio Tek Cytation 5 (Winooski, VT, USA or TECAN infinite 200Pro
fluorescence plate reader (Meilen, Zurich, Switzerland) (fluorescence detection, 560 nm
(ex)/590 nm (em)).
2.5. RNA/DNA Analysis
Total RNA was extracted using the BioRad Aurum Total RNA Lysis Kit (BioRad,
Mississauga, ON, Canada) as per the manufacturer’s instructions. For HIV-1 samples,
purified RNA (0.5–2 µg) was reverse transcribed using M-MLV (Invitrogen) to generate
complementary DNA (cDNA). HIV-1 and β-actin mRNA levels in DMSO- or compoundtreated samples were quantified by qPCR as described [9]. Target quantification was
evaluated using the absolute quantification method, normalized to β-actin expression, and
expressed relative to DMSO treatment. Adenovirus cDNA was produced from extracted
RNA by reverse transcription (RT) with the Bio-Rad iScript cDNA Synthesis Kit, according
to the manufacturer’s protocol, using 1 µg of RNA in a reaction volume of 20 µL. The
conditions were as follows: 5 min at 25 ◦ C, 20 min at 46 ◦ C, and 1 min at 95 ◦ C. Subsequent
qPCR analysis of the cDNA, diluted 1/100, was done in technical triplicates using Bio-Rad
SsoAdvanced Universal SYBR Green Supermix with primers (Supplementary Table S1) at
500 nM final concentration, according to the manufacturer’s protocol. Cycling parameters
were 98 ◦ C for 3 min, then 40 cycles of 95 ◦ C for 15 s, and 60 ◦ C for 30 s. The abundance
of adenovirus RNA in treated samples, relative to TBP RNA (control), was normalized to
RNA abundance at 24 h p.i. in 0.1% DMSO.
The effect of compound treatment on splice site selection within the HIV-1 MS RNA
class was analyzed by RT-PCR as described previously [18]. Amplicons were resolved on
7% polyacrylamide gels (1× TBE) and signals detected using ethidium bromide staining in a
BioRad ChemiDoc imager (Mississauga, ON, Canada). Analysis of images was performed
using ImageLab to calculate levels of HIV-1 MS mRNA isoforms, measured as signal
intensity of an individual isoform divided by the total signal of all visible viral RNA species
in a sample. Analysis of E1A RNA splicing was performed as previously described [17].
For adenovirus DNA analysis, cells were seeded on 6-well plates at a density of
5 ×105 cells per well. The next day, cells were infected with HAdV-C5 (input MOI 100 IU/cell)
for 1 h, and inoculum was removed and replaced with a culture medium containing DMSO
or GPS491. At the indicated times p.i., the culture fluid was removed, and cells were lysed
in situ with lysis buffer supplied with Norgen Biotek DNA extraction kit (Winooski, VT,
USA, Cat: 53100). Adenovirus DNA levels were determined using the E2B primer pair (see
Supplementary Table S1). Experiments were done using biological duplicates, each measured
in technical triplicates. Reactions contained 6 µL of Bio-Rad SsoAdvanced Universal SYBR
Green Supermix, 0.5 µL reverse primer (16.5 µM), 0.5 µL forward primer (16.5 µM), and
5 µL of the DNA sample diluted 1/10 in sterile distilled water. Cycling parameters were
as follows: 95 ◦ C for 30 s, and 40 cycles of 95 ◦ C for 15 s and 60 ◦ C for 30 s (as detailed in
Bio-Rad SsoAdvanced Universal SYBR Green Supermix user manual). Data were analyzed
using Bio-Rad Maestro software (Mississauga, ON, Canada).
Viruses 2022, 14, 60
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2.6. Role of Proteasome in Reducing HIV-1 Tat Expression
To determine whether the effect of GPS491 on HIV-1 Tat expression could be reversed
by inhibition of the proteasome, a compound treatment assay was performed as previously
described with the addition of 10 µM MG132 (Sigma-Aldrich, St. Louis, MO, USA) to
DMSO/compound-treated cells 8 h prior to harvesting [19]. Lysates were fractionated
on 10% TGX stain-free (Bio-Rad, Mississauga, ON, Canada) or 14% SDS-PAGE gels, and
blotted and probed with antibodies for Gag, Tat, or GAPDH in at least three independent
experiments.
2.7. Statistical Analysis
Data are representative of at least three independent experiments, each performed
in duplicate, and values are represented as mean ± standard deviation unless otherwise
stated. Statistical significance comparisons between two samples were calculated using the
paired two-tailed student’s t test (Microsoft Excel).
3. Results
3.1. GPS491 Inhibits HIV-1 Replication by Disrupting Viral RNA Accumulation
To assess the HIV-1 inhibitory activity of GPS491, we initially examined its ability to
suppress replication of multiple strains of the virus in the CEM-GXR cell line that expresses
GFP upon HIV-1 infection [13]. As summarized in Table 1 (see Supplementary Figure S1
for titration data), treatment of HIV-1-infected CEM-GXR cultures with nanomolar doses of
GPS491 suppressed replication of all HIV-1 strains tested including clade A and B wild-type
HIV-1 strains as well as variants resistant to different antiretroviral drugs (reverse transcriptase, protease, integrase, or entry inhibitors) (EC50 ~250 nM, CC50 = 12,052 nM). Inhibition
of HIV-1 replication was also observed in the context of human PBMCs (Table 1). Taken together, these data suggest that GPS491 may be used in conjunction with other combination
antiretroviral therapy (cART) regimens to enhance the control of HIV-1 infection.
To gain insight into its mechanism of action, we assessed the effect of GPS491 addition
on HIV-1 gene expression using a HeLa cell line (HeLa rtTA HIV∆mls) transduced with a
doxycycline-inducible HIV-1 provirus [9–11]. Western blots revealed >90% reduction in the
accumulation of all HIV-1 proteins examined (Gag, Env, Tat) (Figure 1b) at low µM doses
of GPS491. Parallel studies revealed a similar pattern of altered HIV-1 protein expression in
the context of the J-Lat 10.6 cell line (a Jurkat CD4+ T cell line infected with a replication
defective HIV-1 provirus expressing GFP from the position of Nef [12]), with a reduction of
both Gag and GFP synthesis at low µM doses of GPS491 (see Supplementary Figure S2).
To understand the basis for the observed alterations in viral protein expression, we
examined the effect of GPS491 on HIV-1 RNA accumulation. In the case of HIV-1, the 9 kb
primary viral transcript is alternatively spliced into ~69 mRNAs to express nine encoded
viral proteins [20–22]. The viral mRNAs are classified into three groups: unspliced (US,
encoding Gag and Gagpol), singly spliced (SS, used to make Vif, Vpr, Vpu, Tat p14, or
Env), or multiply spliced (MS, encoding Tat p16, Rev, or Nef) RNAs. RT-qPCR analysis
revealed that treatment with GPS491 reduced HIV-1 US and SS viral RNA accumulation
while slightly increasing MS RNA levels (Figure 2a). The reduced accumulation of HIV-1
US and SS RNAs accounts for the loss of Gag, Env, and Tat p14, but the loss of Tat p16
was unexpected given the significant levels of MS RNA present. One possibility was that
GPS491 induced an alteration in splice site usage in the MS class of viral RNAs, similar to
the related compound 5350150 [7]. RT-PCR analysis [18] of the HIV-1 MS RNAs (Figure 2b)
determined that a dose of GPS491 (1.25 µM), which reduced HIV-1 protein expression,
shifted the relative abundance of MS RNAs, increasing the abundance of Tat1 mRNA while
decreasing levels of Rev1/2 mRNAs. MS RNA relative abundance was similar to the
DMSO control at a lower dose of GPS491 (0.5 µM). With GPS491 addition increasing the
levels of Tat mRNA, the basis for the loss of Tat p16 protein (encoded by Tat1 mRNA) was
not immediately clear. An alternative explanation for the loss of Tat protein expression
is that GPS491 induced changes in the rate of Tat synthesis and/or degradation as seen
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previously with other modulators of HIV-1 replication [19,23]. To test this hypothesis,
we examined whether treatment with the proteasome inhibitor MG132 could restore Tat
protein expression. As shown in Figure 1c, addition of MG132 to cultures 8 h prior to
harvest resulted in a significant increase in Tat (both p16 and p14, expressed from MS and SS
viral RNAs, respectively) in the presence of DMSO as well as expression of predominantly
p16 Tat in the presence of GPS491. Although expression of Tat p16 was increased with
MG132 and GPS491 addition, there was no detectable change in Gag levels consistent with
the reduced level of its mRNA.
∆
Figure 2. Effects of GPS491 on HIV-1 RNA accumulation and processing. HeLa rtTA HIV∆mls
cells were treated with DMSO or varying concentrations of GPS491 −+/− doxycycline (Dox) for
24 h. Cells were then harvested, total RNA was extracted, and cDNA was generated. Samples were
subsequently analyzed (a) by RT-qPCR to measure levels of HIV-1 US, SS, and MS RNA levels or
(b) by RT-PCR to measure levels of different MS RNA isoforms. Shown is a representative gel and
summary quantitation of results from n > 3 independent assays. Data are indicated as mean ± SD,
* p ≤ 0.05, ** p ≤ 0.01, ns—not significant.
3.2. GPS491 Inhibits Adenovirus Replication
The ability of GPS491 to induce changes in HIV-1 RNA accumulation that correlate
with inhibition of virus replication suggested that we examine whether GPS491 affected
replication of adenovirus, another virus with a similar dependence on the host RNA
processing machinery. As shown in Figure 3a, addition of GPS491 to cultures 1 h after
HAdV-C5 virus inoculation reduced adenovirus yield ~1000-fold with an IC50 of ~1 µM.
Analysis of cells following GPS491 treatment in A549 cells over 24 h revealed some reduc-
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tion in metabolic activity at high concentrations (alamarBlue, Figure 3b). However, at the
concentration (2.5 µM) required to achieve maximal inhibition of adenovirus replication,
cell metabolism was 90% of the control (Figure 3b). No change in cell viability as assessed
by trypan blue was observed over the full range of doses tested (0–40 µM). To gain greater
insight into the basis for the reduction in virus replication by GPS491, we examined adenovirus early (E1A) and late (hexon) protein expression. Immunofluorescence analysis of
hexon expression 24 h post infection (p.i.) revealed a reduction in the proportion of hexonpositive cells and extent of staining upon the addition of GPS491 (Figure 4a). Subsequent
Western blot analysis of cell lysates at 8, 16, or 24 h p.i. determined that E1A expression
was delayed upon GPS491 addition, being readily detected at 16 h p.i. rather than 8 h as in
the control (Figure 4b). In contrast, although hexon expression was detectable as early as
16 h p.i. in the DMSO-treated samples, hexon expression was undetectable by Western blot
up to 24 h p.i. in the presence of GPS491 (Figure 4b).
Figure 3. GPS491 inhibits adenovirus (HAdV-C5) replication in A549 cells. (a) A549 cells were
uninfected or infected with human adenovirus serotype 5 (HAdV-C5) at an input MOI of 100 IU/cell
for 1 h after which the virus inoculum was removed and replaced with media containing DMSO or
the indicated concentrations of GPS491. After 24 h, the virus was harvested by scraping cells into
the culture fluid and the cell suspension was collected for titration of infectious virus by endpoint
dilution (blue line). T0 (dotted line, orange dots) is a sample harvested immediately after the 1 h
adsorption period to measure the residual virus from the inoculum. Error bars represent the standard
deviation of three independent experiments. (b) A549 cells were treated with the indicated doses of
GPS491 for 24 h and then assessed for changes in metabolic activity by alamarBlue (grey line) or for
viability by staining with trypan blue (blue line).
To assess whether the delayed expression of E1A upon GPS491 addition could be
attributed to changes in viral RNA accumulation or splicing, both RT-qPCR and RT-PCR
analyses were performed. Analysis of the kinetics of adenovirus early gene RNA accumulation revealed that, with the exception of E1A, the induction of multiple adenovirus
early genes (E1B, E2A, E2B, E4) was significantly reduced in the presence of GPS491, with
E2A and E2B RNAs being the most affected (Figure 5a). In contrast, E1A RNA abundance
increased in the presence of GPS491 over 24 h. Given the disparity between the effect of
GPS491 on adenovirus E1A protein and RNA accumulation, we asked whether some of
the effects are due to changes in E1A RNA processing. The E1A transcript is the first viral
RNA generated upon adenovirus infection and can be processed into multiple mRNAs
by alternative splicing: the major variants are designated as 13S, 12S, and 9S (Figure 5b).
However, the abundance of the different E1A RNA isoforms changes over the course of
infection; 13S and 12S predominate at early times p.i., while 9S is the major E1A RNA at
later times [24–26] (Figure 5b). As shown in Figure 5b, analysis of E1A RNAs over 24 h
revealed a marked alteration in E1A RNA processing in the presence of GPS491. At 8 h p.i.,
13S and 12S RNAs were the dominant isoforms in DMSO control and GPS491-treated samples, but GPS491 addition increased the relative abundance of 13S RNA. At later times p.i.,
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while DMSO-treated cells shifted E1A RNA accumulation to 9S RNA, GPS491 treatment
increased the predominance of 13S RNA, with this isoform representing >90% of all E1A
RNAs by 16 h p.i. (Figure 5b).
Figure 4. GPS491 delays E1A production and inhibits adenovirus hexon expression. A549 cells
were infected with HAdV-C5 at an input MOI of 100 IU/cell for 1 h after which the virus inoculum
was removed and replaced with media containing DMSO or GPS491 (2.5 µM). (a) Cells were fixed
24 h after infection and hexon expression assessed by immunofluorescence. Scale is the same for all
images. (b) Cells were harvested 8 h, 16 h, or 24 h p.i. and lysates were fractionated on SDS-PAGE
gels. Shown are the representative Western blots showing the expression levels of E1A and hexon
proteins in the control versus treated lysates.
While the changes in E1A RNA processing could explain the altered kinetics of E1A
protein expression, the basis for the loss of hexon remained unclear. During adenovirus
infection, transcription of the late genes is dependent upon viral DNA replication [27,28].
To determine whether the loss of hexon synthesis could be explained by an inhibition
of viral DNA synthesis, qPCR was used to measure the relative abundance of the adenovirus genome in the presence of DMSO or GPS491. Consistent with the loss of late gene
expression, GPS491 addition maintained viral DNA levels comparable to that observed immediately after virus addition (Figure 5c). Together, these observations indicate that GPS491
affects early events in adenoviral gene expression essential for viral genome replication.
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Figure 5. GPS491 alters adenovirus RNA expression/processing and inhibits adenovirus DNA amplification. A549 cells were infected with HAdV-C5 at an input MOI of 100 IU/cell for 1 h after which
virus inoculum was removed and replaced with media containing DMSO or GPS491 (2.5 or 5 µM).
(a) Total RNA was extracted at 8 h, 16 h, or 24 h after virus infection. After cDNA generation, RTqPCR was performed using primers for E1A, E1B, E2A, E2B, and E4 RNAs (Supplementary Table S1).
Values are expressed relative to RNA abundance observed 24 h p.i. in the presence of 1% DMSO.
Shown are data from samples treated with 2.5 µM GPS491. (b) The effect of GPS491 on E1A RNA
processing. Shown on the top is the schematic diagram of E1A RNA processing indicating the major
E1A mRNA isoforms generated by alternative splicing. In the middle is a representative gel of the
E1A RNA amplicons generated from cDNA. The graph on the bottom represents quantitation of
amplicons generated across n > 3 independent assays. (c) At 16 h, 20 h, and 24 h p.i., total DNA
≤
was ≤isolated from cells
≤ treated with DMSO or 2.5 µM GPS491 and levels of adenoviral DNA were
determined by qPCR. Data are indicated as mean ± SD, * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.
3.3. GPS491 Alters Host SR Protein Abundance, Modification, and Function
Given the shift in HIV-1 and adenovirus RNA processing/accumulation upon GPS491
addition, we examined whether some of the effects could be attributed to changes in
abundance or modification of SR proteins, cellular factors with known roles in regulating
RNA splicing as well as other facets of RNA processing [29–31]. Lysates from HeLa rtTA
HIV∆mls cells treated with either DMSO or GPS491 for 24 h were fractionated on SDSPAGE gels and individual SR proteins detected by Western blotting. Shown in Figure 6a
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are representative blots as well as a summary of the data from the three independent
assays. Treatment with GPS491 selectively altered the abundance of several members
of the SR protein family, increasing levels of both SRSF5 and SRSF9 by 1.5-fold, while
reducing the abundance of SRSF6 by 50%, respectively. Limited changes in the levels
of other SR/SR-related factors were observed. However, in addition to changes in SR
protein abundance, we also noted a shift in SRSF4 migration consistent with an increase
in its extent of phosphorylation (Figure 6b). To address whether this was indeed the case,
cell extracts were treated with or without alkaline phosphatase prior to fractionation on
SDS-PAGE and Western blotting. As indicated in Figure 6b, treatment with phosphatase
increased SRSF4 mobility and eliminated the mobility difference observed between DMSOand GPS491-treated samples, consistent with the altered mobility being due to increased
phosphorylation. Given that SR hyperphosphorylation is also associated with changes
in its subnuclear distribution (moving from nuclear speckles to more uniform nuclear
staining [32–35]), we examined the effect of GPS491 treatment on both SRSF2 and SRSF4
subcellular distribution. Despite the change in SRSF4 phosphorylation upon GPS491
addition, SRSF4 and SRSF2 localization to nuclear speckles remained unaffected (Figure 6c).
Figure 6. GPS491 alters expression and phosphorylation of selected SR proteins. HeLa rtTA HIV∆mls
∆
cells were treated with DMSO or GPS491 (2.5 µM) and induced with Dox. Twenty hours post induction,
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cells were harvested in RIPA buffer containing a phosphatase inhibitor cocktail and fractionated on
10% stain-free SDS-PAGE gels. (a) Representative Western blots for each SR protein are shown. At
the right, a summary of Western blots from three independent assays measuring the effect of GPS491
on SR proteins levels relative to DMSO-treated cells. Asterisks (*) represent changes in expression
level at p < 0.05. (b) Cell lysates were mock-treated or treated with alkaline phosphatase (PPase) prior
to loading onto 10% stain-free gels. After transfer onto PVDF, blots were probed for SRSF4. Blots
are representative of n > 3 independent assays. (c) Cells treated with DMSO or GPS491 were fixed
and permeabilized followed by staining with antibodies to either SRSF2 or SRSF4. DAPI was used to
stain for cell nuclei. Images were captured on Leica DMR Microscope at 63× oil immersion.
To evaluate the effect of GPS491 on SR function, we also examined whether the compound altered SR protein activity as measured by their modulation of Bcl-x RNA splicing.
In this instance, cells were transfected with a Bcl-x reporter vector (Bcl-x 2.13, Figure 7a)
along with control vector plasmids (CTRL, CMVmyc) or ones expressing individual SR
proteins. As indicated in Figure 7c and Supplementary Figure S3a, plasmid-derived expression of most SR proteins in the absence of the compound decreased the use of the
Bcl-xS 5′ ss, only transfection of SRSF10 yielding the opposite effect. The addition of 10 µM
GPS491 had a limited or no effect on the response to most SR proteins tested (Figure 7c),
except for SRSF10, where it decreased the ability of SRSF10 to promote Bcl-xS 5′ ss usage. As a confirmation of this effect, we examined the impact of GPS491 on endogenous
Bcl-x splicing (Figure 7b) and noted that GPS491 also prevented the SRSF10-mediated
switch towards more endogenous Bcl-xS RNA (Figure 7d and Supplementary Figure S3b).
Given the changes in SR protein abundance, phosphorylation, and activity observed, we
examined whether any of the changes were due to alteration of SR kinase expression.
As shown in Supplementary Figure S4, tests in the JLat 10.6 cell line did not detect any
significant alteration in CLK1-3 expression, but SRPK1 expression increased 2.5-fold upon
GPS491 addition.
3.4. GPS491 Inhibits Coronavirus Replication
To explore whether GPS491 could impact the replication of other viruses, we tested
its effect against coronaviruses. As positive strand RNA viruses, the replication cycle
of coronaviruses is restricted to the cell cytoplasm and has no direct dependence on
cellular RNA splicing [36–39]. However, GPS491 addition 1 h p.i. resulted in a dosedependent reduction (EC50 ~250 nM/CC50 >10 µM in Huh7 cells, Figure 8a) of coronavirus
replication as evidenced by loss of 229E and OC43 viral RNA accumulation in the media
(indicative of viral particle formation). Subsequent analyses (Figure 8b,c) determined
that GPS491 addition drastically reduced 229E and OC43 nucleocapsid (N) and spike (S)
protein expression in cell lysates. Success against the seasonal coronaviruses led us to
test whether GPS491 had a similar capacity to suppress replication of SARS-CoV2. As
shown in Figure 8d,e, GPS491 reduced SARS-CoV2 genome replication/expression in a
dose-dependent manner (EC50 ~100 nM) in Huh7 cells as evidenced by loss of viral genomic
RNA accumulation in the media and nucleocapsid expression in the cell.
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Figure 7. GPS491 alters the function of only a subset of SR proteins. (a) Map depicting the Bcl-x
pre-mRNA and the Bcl-x minigene 2.13 as well as the position of the primers used for RT-PCR.
(b) Map of the endogenous Bcl-x gene and spliced products Bcl-xS and Bcl-xL. Positions of primers
for RT-PCR are indicated. (c) HEK 293 cells were transfected with Bcl-x reporter along with plasmids
expressing the indicated SR proteins, followed by treatment with DMSO or 10 µM GPS491. After
48 h, total RNA was isolated and RT-PCR was performed. The relative abundance of the xL and
xS products was determined following fractionation on PAGE gels. (d) Assays were repeated with
overexpression of SRSF10 in the presence of DMSO and 10 or 20 µM GPS491, evaluating the effect
on alternative splicing of the endogenous Bcl-x gene (Bcl-x ENDO). Representative gel images are
provided in Supplementary Figure S3.
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Figure 8. GPS491 inhibits replication of 229E, OC43, and SARS-CoV2 coronaviruses. (a) Huh7
cells were infected with either 229E or OC43 at an input MOI of 0.1 and 1, respectively, for 1 h.
≤
≤
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Virus inoculum was removed, cells were washed, and fresh media containing 1% DMSO or varying
concentrations of GPS491 (from 0 µM to 10 µM) were added. Then, 2 days p.i (229E) or 4 days p.i.
(OC43), media were harvested to quantitate viral genomic RNA levels by RT-qPCR assay. All the
values are expressed relative to the values detected in virus-infected DMSO-treated samples. Effect
of GPS491 on cell viability was assessed 2 days post compound addition using alamarBlue at the
indicated doses of GPS491. Data are indicated as mean ± SD, ** p ≤ 0.01, and *** p ≤ 0.001. (b,c) Huh7
cells were infected with (b) 229E at an MOI of 0.03 or (c) OC43 at an MOI of 0.3 for 1 h after which
the virus inoculum was removed, cells were washed with 1× PBS, and media containing 1% DMSO
or 1.25 µM GPS491 were added. Cells and media were harvested 2 days (229E) or 4 days (OC43)
p.i., and the levels of viral proteins (N, S) were determined by Western blot and virus production
was assessed by RT-qPCR of the media (RNA). Shown are representative Western blots and their
respective quantitation indicating the effects of GPS491 following was removed, cells were washed
with 1× PBS, and media containing 1% DMSO or 1.25 µM GPS491 were added. Cells and media
were harvested 2 days (229E) or 4 days (OC43) p.i., and the levels of viral proteins (N, S) were
determined by Western blot and virus production was assessed by RT-qPCR of the media (RNA).
Shown are representative Western blots and their respective quantitation indicating the effects of
GPS491 following infection with 229E and OC43, respectively. Band intensity was quantified relative
to virus-infected control and normalized to total protein load (stain-free gels). Data are indicated as
mean ± SD and generated from n = 3 independent assays, each performed in duplicate. (d,e) Huh7
cells were infected with SARS-CoV2 at an MOI of 1 for 1 h. Virus inoculum was removed, and
fresh media containing DMSO, chloroquine (CQ, 10 µM), or indicated doses of GPS491 were added.
Two days post infection, (d) media were harvested, and levels of virion production determined by
RT-qPCR or (e) lysates from cells treated with 1% DMSO or 0.3 µM GPS491 were blotted to measure
expression of viral N protein. Results shown were generated from n = 3 independent assays.
To gain a greater understanding of the stage of coronavirus replication affected by
GPS491, we first studied 229E viral replication kinetics by measuring nucleocapsid expression in cell lysates and virion RNA release in media at different times p.i. (at an input
MOI of 2). Analysis of 229E kinetics of nucleocapsid expression determined that viral
nucleocapsid was first detectable at 12 h p.i, gradually increasing to a maximum at 24 h p.i.
(Figure 9a). Accumulation of viral genomic RNA in the media, an indicator of virion assembly and release, was first evident at 16 h p.i., reaching a maximum at 48 h p.i. (Figure 9a).
We next performed time of addition assays, in which the compound was added at different
time points (0, 6, 9, 12, or 16 h p.i.) to determine whether the compound was effective at
all stages of the virus replication cycle. These analyses (Figure 9b) revealed that delaying
GPS491 addition to cells had minimal effect on its ability to inhibit virus replication, with
addition at 12 or 16 h p.i. being almost as effective as 0 h p.i. in reducing the accumulation
of viral genomic RNA in the media 24 h p.i.
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Figure 9. GPS491 inhibits 229E virus replication post entry. (a) Kinetics of 229E coronavirus N protein
expression and virus release. Huh7 cells were infected at an input MOI of 2. After incubation of cells
for 1 h, cells were washed to remove residual virus and fresh media were added. At indicated times
post infection (p.i.), cells and media were harvested. Cell extracts were analyzed by Western blot for
N protein expression, while RTqPCR was used to monitor levels of viral RNA in the media. Shown
is a representative Western blot as well as a graphical summary of the quantitative results obtained
from n = 3 independent assays. (b) Addition of GPS491 at late stages of 229E replication suppresses
virus growth. Huh7 cells were infected at an input MOI of 2. After incubation of cells for 1 h, cells
were washed to remove residual virus and fresh media were added. At indicated times post infection
(p.i.), DMSO or GPS491 was added to cultures at a final concentration of 1.25 µM and incubation
continued. At 24 h post-virus infection, media were harvested and the abundance of viral RNA in
media was determined by RTqPCR.
4. Discussion
Due to the high variability in polymerase/protease sequences/structures within and
among different viruses, compounds targeting these proteins may show limited activity
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against unrelated viruses. Furthermore, the high error rate of the viral polymerases provides the viruses with an ability to rapidly evolve resistant viral variants once the drug
is applied. However, the dependence of all viruses on cellular processes for their replication suggests that host factor manipulation may provide a robust target for therapies
with the additional benefit that such treatments may affect multiple viruses dependent on
the same process. One example is alternative RNA splicing, which multiple mammalian
viruses exploit to greatly expand their coding capacity without having to increase their
genome size. Examples include influenza, retroviruses, adenoviruses, polyoma, and papilloma viruses [24,40–47]. RNA splicing is a multistep process involving the assembly
of a ~1.8 megadalton RNA–protein complex known as the spliceosome [48] and is a key
point of gene expression regulation for >90% of human RNA polymerase II transcripts
that undergo alternative splicing [49]. Splice site selection is governed by the action of
multiple splicing regulatory factors [50,51]. For many viruses, balancing the extent of alternative splicing is essential for virus replication, since altering the proportion of individual
viral mRNAs leads to changes in viral protein expression and a severe reduction in virion
yield [6,7,17,19,52–54]. Consequently, altering the relative activity of key cellular factors
regulating particular stages of RNA processing could be the basis of a broad-spectrum
antiviral in contrast to existing treatment paradigms that target virus-encoded enzymes.
Such host-targeted therapies (HTT) could also provide a higher barrier to the emergence of
resistance [4,5,55], particularly in the case of splicing, where the virus would need to alter
its interaction with multiple components of the splicing apparatus to establish a new, productive equilibrium. In support of this hypothesis, studies with the anti-HIV-1 compound
ABX464 have shown a reduction in HIV-1 replication in both humanized mice and humans
without the appearance of resistant HIV-1 strains [56–58]. Other studies have identified
compounds (IDC16, 1C8, 791, 5342191, 5350150) [7,9,19,52,56,59,60] that can dramatically
reduce HIV-1 gene expression through alterations in viral RNA abundance with limited
effects on host RNA accumulation, indicating a greater sensitivity of viral RNA to minor
perturbations in the host RNA processing machinery.
Although the current data do not identify the target host factor, which mediates
GPS491′ s effect on the viruses studied, the compound’s ability to inhibit the replication of
multiple different viruses demonstrates its potential as a broad-spectrum antiviral. In the
context of HIV-1, GPS491 inhibited multiple HIV-1 variants, including those resistant to
current therapeutics. GPS491′ s reduction of HIV-1 protein expression in cell lines (HeLa
rtTA HIV∆mls, J-Lat 10.6) containing integrated HIV-1 proviruses indicates that it acts
by affecting post-integration events not targeted by current drugs. Consistent with the
observed loss of Gag and Env, GPS491 treatment significantly reduced the abundance of
their corresponding RNAs to a greater extent than seen with 5350150 [7]. Levels of HIV-1
MS RNA were slightly elevated, indicating that GPS491 has limited effect on transcription
from the viral promoter but alters the balance in the processing of the primary transcript to
favor the formation of the fully spliced form of the viral RNA. Similar to 5350150, GPS491
treatment induced changes in splice site usage within the MS RNAs to favor Tat1 RNA [7],
but also reduced Rev1/2 RNA, with the resulting reduced level of Rev protein possibly
affecting viral US and SS RNA accumulation in the cytoplasm [61,62]. Unlike 5350150,
GPS491 treatment significantly reduced Tat protein levels. The ability of proteasome
inhibition (through addition of MG132) to restore Tat p16 expression suggests that GPS491
reduces either the translation of MS RNA encoding Tat or the stability of the Tat protein.
In the case of adenovirus, treatment of cells with GPS491 did not block genome delivery to the nucleus as evidenced by the presence of E1A RNA and protein. However, the
compound affects a very early phase of virus gene expression as E1A RNA accumulation
was enhanced while the expression of other early genes was significantly reduced, indicating that the regulatory cascades driving their expression were interrupted by GPS491. One
example is the loss of E2A and E2B, whose transcription is dependent upon the induction of
the host E2F transcription factor mediated by the combined action of adenovirus E1A and
E4 proteins [63,64]). Other effects are possibly due to the altered synthesis of E1A isoforms
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produced by alternative splicing of E1A RNA. Addition of GPS491 strongly favoured the
accumulation of the 13S E1A RNA. The failure of E1A RNA processing to transition (from
predominantly 13S/12S early to 9S late) in the presence of GPS491 may be attributable to
the loss of E4 as the E4-orf4 protein interacts with the cellular PP2A phosphatase to induce
hypo-phosphorylation of cellular SR proteins [24,65,66]. With GPS491 affecting expression
of so many of the early proteins critical for viral DNA replication, the observed lack of viral
DNA amplification is expected. Similarly, the loss of late viral gene expression (i.e., hexon)
is anticipated given its dependence on viral DNA replication [27,28].
The changes in HIV-1 and adenovirus RNA accumulation induced by GPS491 suggested that it affected the cellular RNA processing machinery. Consistent with this hypothesis, we did observe changes in SR protein abundance, modification, and function
upon GPS491 addition, in particular a reduction in SRSF10 function, which plays a critical
role in HIV-1 and hepatitis B replication [52,67]. Suppression of virus gene expression
upon GPS491 addition occurred with limited changes in abundance of several cellular SR
kinases, with only SRPK1 levels being affected (in contrast to a known SR kinase inhibitor
TG003 [68], Supplementary Figure S4). Both overexpression and depletion studies have
highlighted several members of the SR protein family that play pivotal roles in regulating
HIV-1 gene expression and virus replication [69–76]. Studies have highlighted the effects of
both SRSF5 and SRSF4 in regulating different facets of HIV-1 gene expression. In the case
of SRSF5, in vitro studies indicated that this factor weakly modulates the use of particular
splice sites within HIV-1 RNA [70,77]. Subsequent in cell SRSF5 depletion and overexpression experiments determined that, while SRSF5 depletion had limited effect on HIV-1 RNA
accumulation, its overexpression did induce changes in splice site selection and reduced
expression of HIV-1 Gag [70,71,78]. Studies have also highlighted a role for SRSF5 in
facilitating the translation of HIV-1 Gag RNA [79,80], but this effect was only documented
in the context of murine cell lines. Less information is available regarding the effects of
SRSF4 modulation on HIV-1 RNA processing. SRSF4 overexpression increases HIV-1 Gag
expression and alters the usage of specific splice sites within the viral RNA [81,82], while
its depletion results in the selective loss of RNAs encoding Vpr [78]. While SR protein
phosphorylation is known to affect the activity and localization of the protein [33,83–85]), it
is unclear how the changes in SR phosphorylation but not localization induced by GPS491
impact function. The effects of GPS491 on SR protein function (Figure 7c,d) were limited to
SRSF10, an unexpected result in light of the absence of changes in SRSF10 abundance or
apparent phosphorylation upon GPS491 addition (Figure 6). Previous studies [52] have
demonstrated a significant role for SRSF10 in regulating HIV-1 replication, with its depletion or altered phosphorylation resulting in marked alterations in HIV-1 expression
and RNA accumulation. While the splicing modulator 1C8 altered SRSF10 activity and
inhibited HIV-1 replication through alterations in viral RNA accumulation, its effects were
distinct from that of GPS491 [52]. Unlike GPS491, 1C8 addition reduced accumulation of all
HIV-1 mRNAs, with low doses (1 µM) preferentially reducing levels of spliced (SS and MS)
viral RNAs. Consequently, while both 1C8 and GPS491 affect SRSF10 function, their distinct
effects on viral RNA accumulation suggest that their mechanisms of action are different.
Evidence demonstrating a role for individual SR proteins in the control of adenovirus
replication is more limited. Studies have determined that SRSF1 overexpression blocks
adenoviral DNA replication and the cytoplasmic accumulation of late viral mRNAs [86],
while modulation of SRSF4 expression alters E1A RNA alternative splicing to favor the 12S
form [87].
SR proteins also play essential roles in replication of other viruses (i.e., hepatitis
B [67,88,89]) not known to require splicing of their RNAs. Studies of viral RNA interactomes of multiple cytoplasmic RNA viruses have revealed a common set of hostinteracting proteins [90,91] and identified roles for SR proteins and the cellular kinases
(SRPK1,2) that modify SR protein activity in the replication of several RNA viruses (polio, hepatitis C, Sindbis, SARS-CoV-1/2) [82,92–94]. Although first identified for their
role in RNA splicing [29,51,95–100], SR proteins can modulate several other stages of
Viruses 2022, 14, 60
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RNA metabolism critical for virus replication including transcription initiation/elongation,
mRNA polyadenylation, export to the cytoplasm, and translation [30,51,79,80,101–103].
Consequently, modulating SR protein activity could alter their effect at any of these stages
of gene expression.
The ability of GPS491 to inhibit multiple coronaviruses (229E, OC43, SARS-CoV-2)
was unexpected given that replication of these viruses occurs in the cytoplasm [39,104],
independent of the cellular nuclear RNA processing machinery involved in HIV-1 or adenovirus expression. Characterization of host proteins interacting with SARS-CoV2 RNA has
revealed the association of several hnRNPs and SR proteins (including SRSF10), suggesting
a possible role in regulating coronavirus replication [90,91]. Consistent with this hypothesis, parallel CRISPR screens determined that the depletion of a subset of SR proteins can
negatively impact coronavirus replication [105]. In addition, the nucleocapsid (N) protein
of multiple coronaviruses contains a highly conserved region of arginine-serine repeats
(similar to those in SR proteins) whose phosphorylation by host kinases (SRPK1, GSK-3)
regulates the protein oligomerization required for virus replication [94,106–108]. Inhibition of SRPK1 or GSK-3 function dramatically reduces coronavirus replication [94,108].
GPS491′ s ability to block coronavirus virus particle formation when added 1 or 16 h after virus infection indicates that it is a potent post-entry inhibitor of virus replication,
able to arrest virus replication even after the onset of virus structural protein production.
Whether GPS491 acts directly to affect important steps in viral RNA/protein synthesis or
indirectly through modulation of abundance or activity of a key host factor(s) remains to
be determined. In support of the latter possibility, studies of SARS-CoV2 have revealed a
requirement for RNA splicing as treatment of cells with pladienolide B [109], an inhibitor
of the spliceosome factor SF3B1, reduced virus replication.
Together, the observations presented in this report demonstrate that GPS491 is a
potential broad-spectrum, host-directed, antiviral agent displaying potent activity against
three families of unrelated viruses, from retroviruses to coronaviruses. We have shown that
the compound effects are associated with the alteration of some host factors. In addition to
the broad antiviral spectrum of GPS491, we also documented the activity of the compound
against drug-resistant strains of HIV-1, suggesting its utility as a salvage therapy in patients
who fail current virus-directed treatments. Additional studies to define the mechanism
by which GPS491 alters the cell to render it unable to support virus replication will prove
invaluable in refining the strategy to enhance antiviral potency while limiting the effect on
the cell and the host.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
.3390/v14010060/s1, Table S1: RT-qPCR primer sequences, Figure S1: Anti-HIV-1 activity of GPS491
against wild-type clade A and B and drug-resistant viruses Figure S2: GPS491 reduces HIV-1 Gag
expression in J-Lat 10.6 cells, Figure S3: GPS491 modifies the function of SRSF10, Figure S4: Effect of
GPS491 on SR kinase expression.
Author Contributions: Conceptualization, A.C., B.C., D.G., M.B. (Martha Brown) and W.A.H.; investigation and data curation, S.D., R.C. (Ran Cheng), P.K.C., T.B., R.M., M.G., S.M. (Sarah Manianis),
L.S., S.J., J.T., R.C. (Rose Chan) and S.P.; resources, A.C., M.B. (Mohan Babu), S.M. (Samira Mubareka),
K.M., A.B. and S.G.-O.; writing—original draft preparation, S.D. and A.C.; writing—review and editing, S.D., A.C., W.A.H., B.C. and M.B. (Martha Brown); supervision, A.C., B.C., W.A.H., M.B. (Martha
Brown) and M.A.B. All authors have read and agreed to the published version of the manuscript.
Funding: Research was supported by funding from the Canadian Institutes of Health Research
(MOP-13024 to AC, PJT-153057 to MAB, and PJT-165966 to BC), the CIHR COVID-19 Rapid Research
response funding opportunity to WAH and MB (VR2-172717), as well as the Toronto COVID-19
Action Initiative to AC and WAH. SD and SJ were supported by PRiME fellowships (Precision
Medicine Initiative at University of Toronto) during the course of these studies.
Viruses 2022, 14, 60
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Institutional Review Board Statement: Peripheral blood mononuclear cells (PBMCs) were obtained
from three HIV-1-negative donors following written informed consent. Study protocols were approved by the Institutional Review Boards of the University of British Columbia-Providence Health
Care Research Institute and Simon Fraser University.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Acknowledgments: We wish to thank members of the University of Toronto Virology Core Lab
for their assistance with the coronavirus studies detailed here, with particular thanks to Natasha
Christie-Holmes and Betty Poon for their patience and availability.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
ATCC: American Type Culture Collection, cART: combination antiretroviral therapy, cDNA:
complementary DNA, DMEM: Dulbecco’s Modified Eagle Medium, DMSO: dimethyl sulfoxide,
Dox: doxycycline, FBS: fetal bovine serum, HAdV-C5: human adenovirus serotype 5,HEK 293:
Human Embryonic Kidney 293, HTT: host-targeted therapeutics, IMDM: Iscove’s Modified Dulbecco’s Medium, MEM: minimum essential medium, MOI: multiplicity of infection, MS: multiply
spliced, N: nucleocapsid, PBMCs: peripheral blood mononuclear cells, p.i.: post infection, P/S:
penicillin/streptomycin, RPMI 1640: Roswell Park Memorial Institute 1640, RT: reverse transcription,
S: spike, SR: serine-arginine-rich splicing factor, SS: singly spliced, US: unspliced.
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