D, L-lysine acetylsalicylate + glycine Impairs Coronavirus Replication

Abstract

Coronaviruses (CoV) belong to the large family Coronaviridae within the order of Nidovirales. Among them, several human pathogenic strains (HCoV) are known to mainly cause respiratory diseases. While most strains contribute to common cold-like illnesses others lead to severe infections. Most prominent representatives are SARS-CoV and MERS-CoV, which can lead to fatal infections with around 10% and 39% mortality, respectively. This resulted in 8098 casualties in the 2002/2003 SARS-CoV outbreak and in 1806 documented human infections (September 2016) during the recent ongoing MERS-CoV outbreak in Saudi Arabia. Currently patients receive treatment focusing on the symptoms connected to the disease rather than addressing the virus as the cause. Therefore, additional treatment options are urgently needed which would ideally be widely available and show a broad affectivity against different human CoVs. Here we show that D, L-lysine acetylsalicylate + glycine sold as " Asprin i.v. 500mg® " (LASAG), which is an approved drug inter alia in the treatment of acute pain, migraine and fever, impairs propagation of different CoV including the highly-pathogenic MERS-CoV in vitro. We demonstrate that the LASAG-dependent impact on virus-induced NF-κB activity coincides with (i) reduced viral titres, (ii) decreased viral protein accumulation and viral RNA synthesis and (iii) impaired formation of viral replication transcription complexes.

Full text

D, L-lysine acetylsalicylate + glycine Impairs Coronavirus Replication
Christin Müller, Nadja Karl, John Ziebuhr and Stephan Pleschka*
Institute of Medical Virology, Justus Liebig University Giessen, Schubertstr 81, 35392 Giessen, Germany
*Corresponding author: Stephan Pleschka, Institute of Medical Virology, Schubertstr. 81, 35392 Giessen, Germany, Tel: 0049 (0)641-99-47750; Fax: 0049
(0)641-99-41209; E-mail: stephan.pleschka@viro.med.uni-giessen.de
Received date: December 05, 2016, Accepted date: December 20, 2016, Published date: December 30, 2016
Copyright: © 2016 Müller C, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Coronaviruses (CoV) belong to the large family Coronaviridae within the order of Nidovirales. Among them,
several human pathogenic strains (HCoV) are known to mainly cause respiratory diseases. While most strains
contribute to common cold-like illnesses others lead to severe infections. Most prominent representatives are SARS-
CoV and MERS-CoV, which can lead to fatal infections with around 10% and 39% mortality, respectively. This
resulted in 8098 casualties in the 2002/2003 SARS-CoV outbreak and in 1806 documented human infections
(September 2016) during the recent on-going MERS-CoV outbreak in Saudi Arabia. Currently patients receive
treatment focusing on the symptoms connected to the disease rather than addressing the virus as the cause.
Therefore, additional treatment options are urgently needed which would ideally be widely available and show a
broad affectivity against different human CoVs. Here we show that D, L-lysine acetylsalicylate + glycine sold as
“Asprin i.v. 500mg®” (LASAG), which is an approved drug inter alia in the treatment of acute pain, migraine and
fever, impairs propagation of different CoV including the highly-pathogenic MERS-CoV in vitro. We demonstrate that
the LASAG-dependent impact on virus-induced NF-κB activity coincides with (i) reduced viral titres, (ii) decreased
viral protein accumulation and viral RNA synthesis and (iii) impaired formation of viral replication transcription
complexes.
Keywords: Coronavirus replication; D, L-lysine acetylsalicylate
+glycine; NF-κB inhibition; Replication/Transcription Complexes
Introduction
Coronaviruses (CoV) are enveloped, positive-strand RNA viruses
belonging to the family of
Coronaviridae
[1,2] and are known to infect
mammals and birds. Prior to 2003 it was believed that circulating
human coronaviruses (HCoV), such as HCoV-OC43 [3,4] and
HCoV-229E [5], cause low-pathogenic infections of the upper
respiratory tract and were not recognized as a signicant threat to
human health. is changed, aer the emerging Severe Acute
Respiratory Syndrome-Coronavirus (SARS-CoV) caused an epidemic
outbreak in 2002 in Asia, Canada, the U.S. and Europe [6], which
resulted in 8098 laboratory-conrmed cases, including 774 deaths
(average mortality rate 10%) [6-9]. In 2013, a new human pathogenic
CoV with zoonotic origin was discovered in Saudi Arabia: the Middle
East Respiratory Syndrome coronavirus (MERS-CoV) causing SARS-
like symptoms including lethal pneumonia [10] and renal dysfunction
up to complete failure [11-13]. Since September 2012 1806 conrmed
cases have been reported of whom 643 died [14].
Against both highly pathogenic emerging viruses no specic anti-
viral treatment or vaccination is currently available and treatment of
infected individuals presently only aims at relieving symptoms [15,16].
In cell culture, Type 1 Interferon (IFN-1) inhibits MERS-CoV as well
as SARS-CoV replication [17-19]. IFN-1 is also used as potential
treatment of other +ssRNA viruses [20], but beside high costs it shows
massive side eects. Several other drugs were also shown to impair
MERS-CoV replication in cell culture including cyclosporine,
chloroquine, chlorpromazine, loperamide and lopinavir [21-23].
Whether these drugs will be useful in future treatment of HCoV
infections remains to be investigated.
Targeting CoV proteins [24,25] is always prone to lead to the
emergence of resistant variants/escape-mutants due to the high
mutation rate of RNA viruses [26] including CoV [27]. As all viruses
depend on their host cell for their replication, alternative anti-viral
strategies target host functions or factors. is approach has a low
potential to lead to viral resistance and provides the possibility of
broad-spectrum anti-viral applications.
For many years acetylsalicylic acid (ASA) has been therapeutically
used as an analgesic, anti-pyretic, anti-rheumatic and also as a non-
steroidal anti-inammatory drug (NSIAD). It can also slow down the
progression of Alzheimer’s disease and prevent colon cancer [28-30].
However, ASA is only soluble to a limited extent and the rate of bio-
absorption is thus limited. By using salts of ASA with basic amino
acids, the dissolution rate of the active compound itself can be
increased and high blood concentrations of the active compound can
be achieved [31]. D, L-lysine Acetylsalicylate (LASA) is the water
soluble salt of ASA and the amino acid lysine and due to its improved
solubility in water (compared to ASA) it is faster acting and can not
only be applied orally, but also intravenously. A further disadvantage of
the o-acetylsalicylate was their inadequate stability. e addition of
glycine in the production process of D, L-lysine acetylsalicylated +
glycine (LASAG) resulted in a stable active compound complex of salts
of o-acetylsalicylic acid with basic amino acids and glycine [31].
ASA as well as other salicylates are able to block the activation of
NF-κB [32,33] via inhibition of IKK2 in a low millimolar range [34].
e transcription factor NF-κB is essential for cell responses to
infection with various pathogens. It controls expression of a variety of
anti-viral cytokines and it regulates apoptotic gene expression.
erefore, it is considered to be a main mediator of the anti-viral cell
response to viral infection [35]. Besides the wide usage of ASA in pain
therapy, it was shown that ASA-mediated inhibition of the NF-κB-
Journal of Antivirals &
Antiretrovirals Müller et al., J Antivir Antiretrovir 2016, 8:4
DOI:10.4172/jaa.1000151
Research Article Open Access
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ISSN:1948-5964 ,an open access journal Volume 8(4): 142-150 (2016) - 142
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dependent induction of TRAIL and Fas/FasL, reduces inuenza virus
propagation [36-38]. ASA also displays anti-viral activity against
cytomegalovirus [39] and human rhinoviruses [40].
As SARS-CoV was shown to activate NF-κB in the lung tissue of
infected mice and NF-κB-inhibition (using CAPE, resveratrol,
BAY11-7082 or parthenolide) improved survival rates of SARS-CoV-
infected mice [41], we speculated that NF-κB activity might be
important for ecient CoV propagation. We therefore investigated
whether NF-κB inhibition via the clinically tested and approved
LASAG could negatively aect CoV replication. is hypothesis was
analysed by LASAG treatment of cells infected with the low pathogenic
human strain HCoV-229E as well as the high pathogenic MERS-CoV.
Our results could demonstrate that LASAG interferes with virus-
induced NF-κB activation, the formation of viral replication
transcription complexes, viral RNA synthesis and viral protein
accumulation resulting in an overall impaired CoV propagation.
Materials and Methods
Cells and viruses
Huh7 cells (human hepatocellular carcinoma cells) were maintained
in complete Dulbecco’s modied Eagle’s medium (DMEM, Gibco Life
Technologies, UK) supplemented with 10% foetal calf serum (FCS)
and antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin (P/S)).
Conuent cells were infected with human coronavirus 229E
(HCoV-229E, strain collection of the Institute of Medical Virology,
Giessen, Germany) and MERS-CoV (EMC/2012, kindly provided by
Christian Drosten, Bonn, Germany) at the indicated multiplicity of
infection (MOI). Aer 1h the inoculum was aspirated, and the cells
were incubated with complete DMEM at 33°C (HCoV-229E) or 37°C
(MERS-CoV).
PBMC isolation
Peripheral blood mononuclear cells (PBMCs) were isolated using
the standard Ficoll-Paque gradient centrifugation and maintained in
completed DMEM. Briey, human blood, diluted 1:2 in PBS, was
layered carefully on top of Ficoll-Paque PLUS solution (GE Healthcare
Life Science; USA) and centrifuged for 30 min at room temperature
(RT) at 400 x g without brake. Aerwards the PBMCs containing
Plasma-Ficoll interface was collected, the cells were washed twice with
PBS (for 10 min at 640 ×g followed by 10 min at 400 ×g) and
resuspended in complete RPMI 1640 medium (Gibco Life
Technologies, UK) with Glutamax (Gibco Life Technologies, UK)
supplemented with P/S and 10% FCS and directly seeded in 24 or 96
well plates.
Inhibitors
D, L-lysine acetylsalicylate + glycine (LASAG, C15H22N2O6, 326.3 g/
mol) was obtained as “Asprin i.v. 500mg®” (Bayer Vital GmbH,
Germany) and dissolved in dH2O to provide a stock concentration of 1
M. BAY11-7082 (Selleckchem, USA) was solved in DMSO at a stock
concentration of 50 mM and stored at -20°C until further usage.
Lornoxicam (Selleckchem, USA) was solved in DMSO for stock
concentration of 20 mM.
Virus titration
Virus titers were determined by focus forming assay [42] as
previously described [43]. Briey, infected Huh7 cells in 96-well plates
were xed and permeabilized (4% paraformaldehyde (PFA, Roth,
Germany), and 1 % Triton X-100 (Roth, Germany) in PBS++) and kept
at 4°C for 1h. ereaer, the solution was discarded and cells were
washed 3x with PBS++/0.05% Tween-20 (Roth, Germany). Aerwards,
cells were incubated with a mouse anti-coronavirus nucleoprotein
mAb (Ingenasa, Spain) primary antibody diluted 1:1,000 in PBS
containing 3% BSA (PAN Biotech, Germany) for 1h at RT. e cells
were washed again and then incubated with a goat anti-mouse HRP-
antibody (Santa Cruz, USA) secondary antibody diluted 1:1,000 in PBS
containing 3% BSA, for 1h at RT. Aer additional washing the cells
were incubated with 40 μl AEC (3-Amino-9-ethylcarbazole) staining
solution (Santa Cruz, USA). Following incubation for 40 min at 37°C,
the substrate solution was removed and cells were washed 2x with
dH2O to remove salts. To detect and quantify foci, the plates were
scanned with a resolution of 1200 dpi using an Epson Perfection V500
Photo scanner (Epson, Japan) and analysed using Photoshop soware
(Adobe, USA). Results represent the averages from three biological
replicates.
Cell viability (CC50)
To determine the median cytotoxic concentration of the compounds
at which 50% of the cells are still viable (CC50), Huh7 cells or PBMCs
were grown in 96 well micro plates. Growth medium was replaced with
DMEM containing dierent, indicated concentrations of LASAG
solved in dH20 or BAY11-7082 or Lornoxicam both solved in DMSO
and were further incubated under conditions of infection for the
indicated time periods. Subsequently, an MTT assay was performed as
previously described [37].
To determine the CC50, the MTT values were calculated in
percentage (% viability = (100/MTT value of untreated sample) x MTT
value of inhibitor treated sample) with the control set as 100 %.
Antiviral activity (EC50)
To determine the eective concentration at which virus titres are
reduced by 50% (EC50), Huh7 were infected with MOI 0.1 in 100 μl for
1h at 33°C (HCoV-229E) or 37°C (MERS-CoV). Aer removing the
inoculum, cells were incubated directly with 500 µl complete DMEM
containing dierent inhibitor concentrations. Samples of the
supernatants were collected at the indicated time points post infection
(p.i.) and the amount of infectious virus particles was determined by
focus forming assay.
In order to dene the EC50 the viral titre of the untreated virus-
infected control was set at 100% and the titres of LASAG-,
BAY11-7082- and Lornoxicam-treated samples were calculated in
relation to it.
Western blot
Cells were infected in a time course experiment and treated with the
indicated concentrations of LASAG or le untreated. Aer cell lysis,
proteins were separated by 12% SDS-PAGE and blotted onto
nitrocellulose membrane (Amersham, UK) as previously described
[44]. Membranes were incubated with the respective primary antibody
(mouse anti-nucleocapsid protein mAb [Ingenasa, Spain], mouse anti-
phospho-IκB-α antibody (Cell Signaling, USA] or rabbit anti-actin
Citation: Müller C, Karl N, Ziebuhr J, Pleschka S (2016) D, L-lysine acetylsalicylate + glycine Impairs Coronavirus Replication. J Antivir
Antiretrovir 8: 142-150. doi:10.4172/jaa.1000151
J Antivir Antiretrovir
ISSN:1948-5964 ,an open access journal Volume 8(4): 142-150 (2016) - 143

antibody [abcam, USA]) in PBS containing 3% BSA). Aer washing
the membranes three times with TBS/T (20 mM Tris-HCl, pH 7.6, 140
mM NaCl, 0.05 % Tween 20), they were further incubated with
Infrared IRDye-conjugated anti-mouse and anti-rabbit monoclonal
secondary antibodies (Li-Cor, Germany) diluted 1:10,000 in PBS
containing 3% BSA and analyzed via Li-Cor Odyssey (Li-Cor,
Germany). Western Blots were further graphically analysed by
measuring the intensity of the protein bands in regard to the loading
control of four dierent experiments for nucleocapsid protein
reduction or two independent experiments for IkB reduction.
Nothern blot
Huh7 cells were infected with HCoV-229E (MOI=3) and treated
with LASAG (20, 10, 5 mM) or le untreated. Total RNA was isolated
using Trizol (Invitrogen, Germany) 24 h p.i. and 5 µg total RNA was
separated in a denaturing gel (1% agarose, 6 % formaldehyde, 1x
MOPS) and transferred onto a positively charged nitrocellulose
membrane via Vacuum Blot. Northern Blot analysis was done using a
32P-labled probe specic for the HCoV-229E genome (nt 29297 to
27273) as described previously [45].
Immunouorescence
Huh7 cells were infected with HCoV-229E (MOI=3) and treated
with LASAG (20 mM) or le untreated. 24 h p.i. the cells were xed
with ice-cold methanol and stained with mouse anti-dsRNA mAb (J2,
English & Scientic Consulting Bt., Hungary) and rabbit anti-
HCoV-229E nsp8 mAb (both diluted 1:100 in PBS containing 3%
BSA). For detection Alexa Fluor 594 goat anti-mouse IgG and Alexa
Fluor 488 F (ab’) 2 fragment of goat anti-rabbit IgG (Invitrogen, USA)
both diluted 1:500 in PBS containing 3% BSA were used. Images were
acquired using a confocal laser-scanning microscope (Leica SP05
CLSM, Leica, Germany).
Analysis of NF-κB activation
NF-κB activation was measured as previously described [37] with
the commercial available TransAM kit (Active motif, USA) according
to the manufacturers instruction (n=4). In our study, HCoV-229E-
infected (MOI=3) Huh7 cells that were either treated with LASAG (20
mM) or le untreated were lysed at 4 or 12 h p.i.. Relative NF-κB
activation was calculated as fold induction compared to mock-infected
control cells.
Temporal LASAG treatment
To determine the eect of LASAG-treatment on the early and the
late phase of the CoV replication cycle, Huh7 cells were infected with
HCoV-229E (MOI=3) and treated with LASAG (20 mM) either from
3-6 h p.i. or from 9 -12 h p.i.. For this the cells were incubated in
regular media, which was replaced with LASAG-containing media for
the indicated times. 12 h p.i. the supernatant was collected and pooled
and the virus titer was determined via focus forming assay.
Statistical analysis
e results correspond to the mean ± SD of the indicated
experiments. e statistical signicance of dierences between the
indicated groups was tested using the unpaired, two-tailed Student’s t-
test with a threshold of p:*<0.05; **<0.005; and ***<0.0005.
Results
LASAG reduces HCoV-229E and MERS-CoV titres in cell
culture as well as in primary human cells at non-toxic
concentrations
Based on improved survival rates of SARS-CoV-infected mice,
reduced lung pathology following NF-κB inhibition [41] and the fact,
that ASA can block NF-κb activation [31,32] we aimed to elucidate
whether LASAG (Figure 1) might have a negative eect on CoV
propagation
in vitro
.
Figure 1: Structural formula of D,L-lysine acetylsalicylate glycine.
Treatment was performed with commercial “Aspirin® i.v.”
containing the active compound acetylsalicylic acid (ASA). D,L-
lysine acetylsalicylate glycine (LASAG) is a salt of ASA and the two
amino acids glycine and lysine. Aer dissolving in water, LASAG
dissociates readily into ASA and the two amino acids glycine and
lysine.
In a rst set of experiments the cell viability was investigated via
MTT assay aer 24 and 48 h incubation for Huh7 cells and aer 24 h
incubation for PBMCs, applying dierent amounts of LASAG. For this
we determined the cytotoxic concentration 50 (CC50) of LASAG for
Huh7 cells, which are regularly used for CoV propagation [46,47] as
20.43 mM and 58.73 mM at 48 and 24 h, respectively (Figures 2A and
2B). Furthermore, we determined the CC50 of LASAG for human
peripheral blood mononuclear cells (PBMC) at 37.95 mM at 24 h
(Figure 2C). PBMCs can be infected with HCoV-229E [48] and served
as a primary human cell system.
Next, we analysed whether LASAG aects the replication of the
human strain HCoV-229E as well as of MERS-CoV. erefor we
treated HCoV-229E- and MERS-CoV-infected Huh7 cells (MOI=0.1)
with LASAG at dierent concentrations and analysed the viral titres
via focus forming assay at 48 h and 24 h post infection (p.i.). e
results indicate an eective concentration 50 (EC50) of 1.31 mM for
HCoV-229E resulting in a selectivity index (SI: CC50/IC50) of 15.6 as
well as an EC50 of 3.69 mM for MERS-CoV, leading to an SI of 15.9,
similar to that of HCoV-229E (Figures 2D and 2E). e EC50 for
HCoV-229E-infected PBMCs at 24 h was 6.71 mM resulting in an SI of
5.7 (Figure 2F).
Citation: Müller C, Karl N, Ziebuhr J, Pleschka S (2016) D, L-lysine acetylsalicylate + glycine Impairs Coronavirus Replication. J Antivir
Antiretrovir 8: 142-150. doi:10.4172/jaa.1000151
J Antivir Antiretrovir
ISSN:1948-5964 ,an open access journal Volume 8(4): 142-150 (2016) - 144

Figure 2: LASAG impairs coronaviral replication. To investigate the eect of LASAG on coronaviral replication we determined the CC50 (A, B,
C) and the EC50 (D, E, F) of LASAG. Huh7 cells were treated with the indicated concentrations of LASAG for 48 h (A, D) and 24 h (B, E), and
PBMC were treated for 24 h (C,F). EC50 concentrations were determined for HCoV-229E on Huh7 cells (D) and PBMC (F). For MERS-CoV
the EC50 was determined aer 24 h on sHuh7 cells (E).
ese results indicate that LASAG can eectively reduce titres of
HCoV-229E and MERS-CoV with similar SI values in Huh7 cells and,
to a lesser extent, HCoV-229E titers in primary human PBMCs in a
dose-dependent manner at non-toxic concentrations. Nevertheless,
even though the SI values are distinct, increased SI values for both cell
systems, would be advantageous for a therapeutic approach.
LASAG reduces viral protein accumulation and viral RNA
synthesis of HCoV-229E
e reduction of viral titres following LASAG treatment led us to
speculate that LASAG might impair the intra-cellular replication of
CoV. In order to elucidate which step of the viral life cycle is aected by
the treatment of LASAG, we rst determined viral protein
accumulation via Western Blot and also analysed viral RNA synthesis
via Northern Blot of HCoV-229E infected Huh7 cells treated for 24 h
p.i. at non-toxic LASAG concentrations of 20, 10 and 5 mM.
As shown in Figures 3A and 3B we found that treatment with 20
mM LASAG led to a strong reduction of the viral nucleocapsid (N)
protein as well as viral RNA amount (Figure 3C). e eect on viral
protein accumulation appears to be specic, as the quantity of cellular
actin (loading control) was not aected.
Citation: Müller C, Karl N, Ziebuhr J, Pleschka S (2016) D, L-lysine acetylsalicylate + glycine Impairs Coronavirus Replication. J Antivir
Antiretrovir 8: 142-150. doi:10.4172/jaa.1000151
J Antivir Antiretrovir
ISSN:1948-5964 ,an open access journal Volume 8(4): 142-150 (2016) - 145

Figure 3: LASAG reduces coronaviral RNA and protein
accumulation. Huh7 were infected with HCoV-229E and treated
with the indicated LASAG concentrations. 24 h p.i. the amount of
viral N protein and actin as loading control in infected and un-
infected (mock) cells were detected by Western Blot using mouse
anti-nucleocapsid protein mAb and rabbit anti-actin antibody (A)
and quantied (B). e amount of viral RNAs was detected by
Northern Blot analysis (C).
Compared to the strong reduction of the HCoV-229E titre
(EC50=1.31 mM) 48 h p.i. by LASAG the eects of 5 and 10 mM
LASAG on viral protein/RNA production are less evident.
Nevertheless, it should be considered that these assays (Western Blot/
Northern Blot) were performed 24 h p.i.. is was done, as the CC50
for LASAG at 48 h was 20.43 mM, whereas it was 58.73 mM for 24 h
(Figure 2), indicating that a LASAG concentration of 20 mM is less
toxic at 24 h. erefore, the eect of the lower LASAG concentrations
(5, 10 mM) on viral protein/RNA production might be less evident at
this earlier time point. Also, the MOI to determine the EC50 was only
0.1, whereas the MOI to analyse viral protein/RNA production was 3.
erefore, the eect of the LASAG concentrations used to analyse the
eect on viral protein/RNA production might be weaker. Furthermore,
it should be considered that these assays are generally less sensitive
than the direct virus titration used to determine the EC50, which could
also explain the discrepancy.
Taken together, the results indicate that the reduction in the viral
titre observed under LASAG treatment could be related to the negative
eect of LASAG on the viral protein and RNA production, which
might therefore account for the reduction of infectious progeny virions
produced.
LASAG treatment results in reduction of replication
transcription complexes
e impaired viral protein accumulation and viral RNA synthesis
led us to assume that the mode of action exerted by LASAG on CoV
propagation impairs a step of the viral life cycle before viral RNA/
protein synthesis starts. CoVs replicate and transcribe their genome
via so called replication/ transcription complexes (RTCs), anchored in
virus-induced membrane alterations that consist of double membrane
vesicles (DMVs) [49].
Figure 4: e abundance of coronaviral RTCs is reduced under
LASAG treatment. HCoV-229E-infected Huh7 cells +/- LASAG (20
mM) were analysed 24 h p.i. using confocal microscopy. As a
marker for viral RTCs, the CoV replication intermediate dsRNA
(green) and the viral non-structural protein nsp8 (red) were
detected using target specic antibodies. e nuclei were stained
with DAPI (blue). Co-localization of dsRNA and nsp8 is shown in
the merge (yellow). Open boxes (marked in white) indicate the
magnied areas shown underneath the respective panel.
We therefore assessed whether LASAG may impact the formation of
these host cell-derived DMVs. To this point, Huh7 cells were infected
with HCoV-229E (MOI=3) and analysed 24 h p.i. for the appearance of
RTCs. As markers in our immunouorescence studies (Figure 4) we
assessed the presence of dsRNA, a viral replication intermediate,
indicating the site of viral genome replication [50,51] and the viral
Citation: Müller C, Karl N, Ziebuhr J, Pleschka S (2016) D, L-lysine acetylsalicylate + glycine Impairs Coronavirus Replication. J Antivir
Antiretrovir 8: 142-150. doi:10.4172/jaa.1000151
J Antivir Antiretrovir
ISSN:1948-5964 ,an open access journal Volume 8(4): 142-150 (2016) - 146

nsp8 protein as an integral part of the viral RTCs [52]. In infected,
untreated cells the characteristic peri-nuclear immunouorescence
pattern for RTCs marked by nsp8 and dsRNA [53], which was absent
in the mock-infected cells (Figure 4, mock), was clearly visible (Figure
4, infected). In infected and LASAG-treated cells the amount of cells
showing an nsp8/dsRNA signal (RTCs in DMVs) was decreased,
compared to the untreated, infected cells (Figure 4, infected+20 mM
LASAG). is result demonstrates that 24 h p.i. LASAG-treatment
severely aects the formation of viral RTCs and/or DMVs and it is
tempting to speculate that this is the cause for the decreased
production of viral proteins and RNA. It should be noted that LASAG
might also aect a step in the viral life cycle before RTC formation,
which needs to be investigated.
HCoV-229E infection triggers NF-κB activation in the early
phase of infection, which is decreased by LASAG
Activation of the transcription factor NF-κB is one of the hallmarks
of host cell response to invasion by dierent pathogens. e
phosphorylation of the NF-κB inhibitor IκBα leads to its degradation
and release of NF-κB [54], which then translocates into the nucleus to
transactivate responsive genes [55] related to host defence mechanisms
[56].
NF-κB inhibitors have been studied as potential therapeutic drugs
in anti-viral therapy [57]. Previously, we and others [36-38,58]
demonstrated that NF-κB activity is essential for ecient inuenza
virus propagation, as inhibition of NF-κB activation results in impaired
nuclear RNP export and therefore in reduced virus titers.
For coronavirus infections there are diverting reports of NF-κB
activation [41,59-66] as well as NF-κB inhibition during CoV infection
[67,68]. To assess whether HCoV-229E infection also activates NF-κB
in Huh 7 cells we determined the level of NF-κB activation in
HCoV-229E-infected Huh7 cells via TransAM assay and Western Blot
analysis. We found that HCoV-229E infection leads to a markedly
reduced amount of IκBα at 2 h p.i., which later increases again in the
virus-infected cells (Figure 5A). When analysing the activation of NF-
κB we found an early (4 h p.i.) activation (Figure 5B, le part),
coinciding with decreased amounts of phospho-IκBα early in the
infection (Figure 5A). According to the increased amounts of IκBα at
later time points of the infection, the activation of NF-kB was found to
be lower at 12 h p.i. compared to 4 h p.i. (Figure 5B, right part).
As mentioned before, ASA is known to block NF-κB activation.
erefore we tested whether LASAG treatment also has an eect on
HCoV-229E-induced NF-κB activation. e treatment of HCoV-229E-
infected cells with 20 mM LASAG resulted in a signicant 1.5 fold
reduction of NF-κB activation at early (4 h p.i.) and late (12 h p.i.) time
points of infection (Figure 5B).
Furthermore, the addition of LASAG in the early stage of infection
(3-6 h p.i.), when NF-κB activation was most prominent and viral
RTCs are being formed, resulted in a reduction of virus titres. In
contrast, the addition of LASAG in later stages of viral life cycle (9-12 h
p.i.) resulted in less pronounced titre reduction (Figure 5C). In
summary HCoV-229E infection activates NF-κB in the early stages of
viral life cycle and the LASAG-dependent reduction of virus-induced
NF-κB activation coincides with reduced viral titers.
Figure 5: Virus-induced NF-κB activation is aected by LASAG
treatment. e total amount of IκBα in HCoV-229E-infected Huh7
cells was analysed at the indicated time points p.i. by Western Blot
using mouse anti-phospho-IκB-α antibody (A upper panel) and
quantied (A lower panel). HCoV-229E-infected Huh7 cells (+/-)
LASAG-treatment (20mM) were analysed for the activation of NF-
κB at the indicated time points p.i. via detection of the amount of
activated NF-κB (B). HCoV-229E-infected Huh7 cells were treated
with LASAG (20mM) for the indicated time frames p.i. or le
untreated (control) and virus titres (FFU/ml) were determined 12 h
p.i. (C).
NF-κB plays a pivotal role in HCoV-229E infection
ASA is a multi-target compound [69] not only blocking NF-κB
activation, but also interfering with several other cellular factors, such
as cyclooxygenase 1/2 (COX1/2) a main enzyme in the synthesis of
inammation mediators [70,71] or AMPK/mTOR [72].
To highlight the role of NF-κB activity in CoV infection, we
elucidated the eect of the NF-κB-specic inhibitor (BAY11-7082) on
HCoV-229E replication. HCoV-229E-infected (MOI=0.5) Huh7 cells
were treated with the indicated concentrations of BAY11-7082 and
virus titres were analysed 24 h p.i.. Addition of BAY11-7082 at non-
toxic concentration of 50 µM (Figure 6A) resulted in a reduction of
viral titres by about 4 log10 (Figure 6B). is result supports our
assumption that inhibition of NF-κB activity by LASAG impairs virus
replication. Beside NF-κB inhibition, one of the major activities of
ASA is the inhibition of the cellular COX1/2. We further investigated
whether this activity might (also) be responsible for the anti-viral eect
Citation: Müller C, Karl N, Ziebuhr J, Pleschka S (2016) D, L-lysine acetylsalicylate + glycine Impairs Coronavirus Replication. J Antivir
Antiretrovir 8: 142-150. doi:10.4172/jaa.1000151
J Antivir Antiretrovir
ISSN:1948-5964 ,an open access journal Volume 8(4): 142-150 (2016) - 147

of LASAG against CoV. For this we employed Lornoxicam, a non-
steroidal COX1/2 inhibitor, which is used as an anti-inammatory
drug to treat pain, osteoarthritis, and rheumatoid arthritis [73,74].
However, at the indicated non-toxic concentrations (Figure 6C)
Lornoxicam had no eect on HCoV-229E propagation in infected
Huh7 cells (MOI=0.5) analysed 24 h p.i (Figure 6D). is indicates that
the inhibitory eect of ASA on COX1/2 activity does not seem to be
important for HCoV-229E propagation.
Figure 6: Eect of BAY11-7082 and Lornoxicam on HCoV-229E
replication at non-toxic concentrations. Huh7 cells were treated
with the indicated concentrations of BAY11-7082 and Lornoxicam
for 24h and cell viability was determined via MTT assay (A, C).
HCoV-229E-infected Huh7 cells were treated with the indicated
concentration of BAY11-7082 and Lornoxicam and virus titres were
determined 24 h p.i. (B, D).
Discussion
Among the CoV several human pathogenic strains can cause
common cold-like illnesses, while others as SARS-CoV and MERS-
CoV lead to severe infections. Currently, no treatment focusing on the
virus as the cause is available. Here we show that LASAG, which is an
approved drug, impairs propagation of HCoV-229E and of the highly
pathogenic MERS-CoV
in vitro
. Our results demonstrate that
inhibition of virus-induced NF-κB activity early in the viral replication
cycle via LASAG coincides with (i) reduced viral titres, (ii) decreased
viral protein accumulation and viral RNA synthesis and (iii) impaired
formation of viral replication transcription complexes.
It should be mentioned that upon NF-κB inhibition (BAY11-7082)
DeDiego et al. did not observe any virus titre reduction of the beta-
coronavirus SARS-CoV [41] which was adapted to murine cell lines.
However, it cannot be excluded that this system of a mouse-adapted
SARS-CoV in a murine cell line might not reect the situation of
human CoV or wild type SARS-CoV infection of human cells.
Despite the possibility that other cellular factors, which are also
targeted by LASAG might aect CoV propagation, the results obtained
with the NF-κB inhibitor (BAY11-7082) and the COX1/2 inhibitor
(Lornoxicam) further support the notion that NF-κB inhibition is a
likely reason for the anti-CoV eect of LASAG. To elucidate the NF-
κB-specic eect, further analysis will be needed. It should be noted
that similar results were obtained for the treatment of inuenza virus
(IV) infection with ASA. Here, the COX inhibitor Indometacin also
had no eect on the virus titer in cell culture [38].
Even though cell viability is not impaired by the applied LASAG
concentrations, the anti-coronaviral action of LASAG in cell culture
lies in a millimolar range, likewise to the anti-viral action of ASA
against IV [38]. To achieve a 20 mM LASAG concentration in the
blood, 6.53 g/L would be needed, which is toxic [75]. e Cmax in the
blood aer applying 500 mg ASA i.v. is 54.25 mg/L and 4.84 mg/L aer
oral application [76]. Nevertheless, treatment of patients with a CoV-
caused severe acute respiratory syndrome via inhalation might allow
achieving locally eective LASAG concentrations. Results from a
clinical study investigating the eectiveness of inhaled lysine-
acetylsalicylate in the treatment of asthma showed, that patients that
received a dosage of 720 mg of inhaled LASAG twice a day over a two-
week period did not experience any signicant side eects [77]. Also, a
dose escalation study of inhaled LASAG in humans for the clinical
development of an antiviral treatment of IV infections demonstrated
that inhalative doses up to 750 mg LASAG were safe and well tolerated
without serious adverse events [78]. Furthermore, administration of
aerosolic ASA via intubation directly into the trachea resulted in
increased survival rates of mice infected with a lethal dose of IV [38].
In light of the fact that currently no approved anti-viral treatment
against severe CoV-infections exist, it should be mentioned that
LASAG (i) is widely available and it is tested and approved for humans,
(ii) it targets cellular functions, (iii) is so far not known to target CoV
functions, which reduces the chance that resistant virus variants
emerge and (iv) adjacent to its direct anti-viral property, patients could
also benet from the eects of LASAG on infection-related symptoms
based on the analgetic- and anti-inammatory characteristics of
LASAG.
In conclusion, we were able to demonstrate that LASAG inhibits
virus-induced NF-κB activity, which might be connected to the anti-
viral eect against CoV, including the impaired formation of RTCs
and/or DMVs in CoV-infected cells, leading to reduced viral RNA
production and consequently decreased production of viral proteins,
resulting in an overall diminished virus titre.
Acknowledgments
We want to thank C. Drosten, Bonn, Germany, for providing
MERS-CoV EMC/2012. is work was funded in part by the German
Centre for Infection Research (DZIF), partner site Giessen, Germany
(TTU Emerging Infections to S.P. and J.Z.), and the DFG-funded
Collaborative Research Centre 1021 "RNA viruses: RNA metabolism,
host response and pathogenesis" (SFB1021; projects A01 and C01 to
J.Z. and S.P., respectively). Furthermore the work was supported in
part by a fund of the Activaero GmbH (acquired by Vectura). e
funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Disclosure Statement
e authors have no conict of interest.
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Citation: Müller C, Karl N, Ziebuhr J, Pleschka S (2016) D, L-lysine acetylsalicylate + glycine Impairs Coronavirus Replication. J Antivir
Antiretrovir 8: 142-150. doi:10.4172/jaa.1000151
J Antivir Antiretrovir
ISSN:1948-5964 ,an open access journal Volume 8(4): 142-150 (2016) - 150