To evaluate the stability and physical movement of atoms in docked
complexes, we performed molecular dynamics simulations using the iMODS
server 34. The simulations were performed in NMA. The
results of the apo-eIF4E, m7GTP-eIF4E and hesperetin-eIF4E complex are
depicted in Fig. 8. The affine-model (Fig. 8A, large
coloured arrows) representation and NMA mobility based colour scheme of
eIF4E residue were utilized to observe the mobility of eIF4E regions in
all three cases. 34. As shown in Fig. 8A
(upper panel) , the apo-eIF4E loop regions displayed a bathochromic
shift of colors in its backbone (green, yellow and red shades). Besides,
wider affine-model (colored arrows) and longer arrow fields (black
arrows) also suggested higher mobility of these residues as compared to
the eIF4E-m7GTP and eIF4E-hesperetin complexes (Fig. 8A) .
To gain further insights into the protein flexibility and mobility, we
analysed the deformability and B factor plots, respectively, which were
generated by the iMODS server (Fig 8B and Fig. 8C) . High peaks
in the deformability plot of apo-eIF4E demonstrated flexible regions
(Fig 8B, upper panel) which diminished after complexion with
m7GTP and hesperetin (Fig 8B, middle and lower panel) . This was
complimented by the B-factor plot (Fig 8C), which is
proportional to the root mean square (RMS) and demonstrate the
stabilities of m7GTP- and hesperetin-docked complexes. Additionally,
high eigenvalues of 7.542249e-05 and
7.541401e-05 were observed for m7GTP- and
hesperetin-docked complexes respectively (Fig 8D), which when
compare with the eigenvalues of apo-eIF4E
(3.114206e-04) indicated a higher energy requirement
for the deformation of docked complexes. This suggests the stability of
docked complexes . The analysis of variance of individual modes
(indicating their relative contribution to the equilibrium motions)
(Fig. 8E) , covariance map (indicating coupling between
correlated, uncorrelated and anti-correlated motions residues)
(Fig. 8F) and elastic network model (correlating to the
stiffness of the complex) (Fig. 8G ) inferred a close
resemblance between the m7GTP-eIF4E and hesperetin-eIF4E binding. In
contrast, apo-eIF4E, displayed high mobility and flexibility due to
highly uncorrelated and elastic motion between atoms.
Hesperetin provides protection to embryonated chicken eggs
against lethal BPXV infection
A wide variety of poxviruses, including BPXV, induce pock lesions on the
CAM in embryonated chicken eggs 38,39. To evaluate thein ovo antiviral efficacy of hesperetin against BPXV, we first
determined the subcytotoxic concentration of hesperetin in specific
pathogen free (SPF) embryonated chicken eggs. As shown in Fig.
9A, hesperetin at a dose rate of ≥4000 µg/egg induced mortality in the
chicken embryos, whereas no mortality was observed at a dose rate of
1000 µg/egg. The LD50 of hesperetin was determined to be
1080.74 µg/egg. For evaluation of the in ovo antiviral efficacy,
the eggs were infected with BPXV at 100 EID50 and
treated with the indicated concentrations of hesperetin. As shown inFig. 9B, hesperetin provided protection from BPXV challenge
infection in a dose-dependent manner. Moreover, the size of localized
pock lesion was also suppressed with increasing concentrations of
hesperetin (Fig. 9C) . The EC50 was calculated
to be 22.5 µg/egg and the therapeutic index 48.1.
Evaluation of antiviral drug resistance against hesperetin
We also evaluated the potential of hesperetin to select drug resistant
virus mutants under the long-term selection pressure of hesperetin,
wherein BPXV was sequentially passaged (P) in the presence of hesperetin
or an equivalent volume of DMSO. The infectious virions collected in the
supernatant were used for the next round of infection, and the process
was repeated until P40. The resulting viruses were named
BPXV-P40-hesperetin and BPXV-P40-DMSO. As shown in Fig. 10, the
levels of suppression of the virus yield by hesperetin in Vero cells
were comparable in both BPXV-P40-hesperetin and BPXV-P40-DMSO. This
suggested that hesperetin does not select drug-resistant virus mutants.
DISCUSSION
In this study, we demonstrated the in vitro antiviral efficacy of
hesperetin against multiple poxviruses, which include BPXV, VACV and
LSDV. This, together with hesperetin-induced protection in chicken
embryos against virulent BPXV, led to the conclusion that hesperetin may
potentially be developed as a broad-spectrum antiviral drug against
poxviruses. Previous studies have also identified in vitroantiviral activity of hesperetin against DENV 20,
CHIKV 21, ZIKV 21 and Sindbis virus22. The docking and molecular dynamics simulations
displayed the effect of hesperetin on the protease 3D models of CHIKV
and ZIKV 20. However, the precise mechanism of the
antiviral action of hesperetin remains unknown.
The incubation of cell free virions with hesperetin did not affect the
residual infectivity of the virions, which seems to suggest that
hesperetin may not be directly targeting viral factors in poxviruses,
although this needs further in-depth investigation. We demonstrated that
hesperetin treatment mainly suppresses BPXV protein synthesis with a
marginal inhibitory effect on the levels of viral DNA and mRNA but
without affecting other steps of the viral life cycle such as
attachment, entry, and budding. The 5’-cap of mRNA is highly conserved
and is an important structural modification critical for eIF4E-mRNA
interaction. In order to effectively synthesize viral proteins and to
circumvent the action of 5′-3′ exonucleases, viral capping mechanisms
that generated mRNA capping identical to that of the host were selected
during the co-evolution of viruses and hosts 40.
Previous studies have demonstrated that BPXV exploits cap-dependent
mechanism of protein translation 24,35,41. Therefore,
we asked whether hesperetin blocks BPXV translation initiation by
abrogating the interaction of the 5’ cap of viral mRNA and eIF4E. The
CHIP assay suggested that hesperetin disrupts binding of the 5’ cap
(m7GTP binds) of viral mRNA with eIF4E. The molecular docking and MD
simulation studies, also confirmed stable binding of the hesperetin with
the cap-binding pocket of eIF4E, in a similar conformation as m7GTP
binds. This is a novel mechanism wherein hesperetin has been shown to
exert its indirect antiviral action by targeting a cellular factor
(eIF4E). Further, in agreement with the previous studies24,41, we demonstrated that BPXV exploits
ERK-MNK1-eIF4E signalling to effectively replicate in the target cells.
Hesperetin targets this signalling axis by inhibiting the interaction of
eIF4E with viral mRNA, which results in the shut off of viral protein
translation.
One of the major limitations of antiviral drugs is that they rapidly
induce the development of drug-resistant viral mutants42. However, as compared to the drugs that directly
target viral factors, drugs based on targeting essential cellular
factors are considered to have minimal or no tendency to induce
antiviral drug resistance 24. In our study,
hesperetin-resistant BPXV mutants were not observed even when the virus
was sequentially cultured 40 times in the presence of hesperetin. This
seems to be due to the low genetic variability of the host factor
(eIF4E), thereby imposing a higher genetic barrier to the generation of
resistant viruses 24,25,41.
Since hesperetin can alter cell metabolism, its long-term use could
eventually result in cytotoxicity. Therefore, its further validation,
long-term in vivo efficacy, and clinical trials will be essential
before actually introducing it from research into clinical settings.
In conclusion, hesperetin was shown to exert a potent in vitroand in ovo antiviral efficacy against poxviruses.
Mechanistically, hesperetin was shown to competitively inhibit binding
of the viral mRNA with eIF4E (Fig. 11), thereby blocking viral
protein translation. Most importantly, hesperetin was not shown to
readily select drug-resistant viral mutants.