Autophagy Stimulation Abrogates Herpes simplex Virus-1 Infection
SCIENTIFIC REPORTS |
Autophagy Stimulation Abrogates Herpes simplex Virus-1 Infection
Abraam M. Yakoub 0 1
Deepak Shukla dshukla@uic 0 1
0 Department of Microbiology and Immunology, University of Illinois , Chicago, IL USA, 60612 , USA
1 Department of Ophthalmology and Visual Sciences, University of Illinois Medical Center , Chicago, IL USA, 60612 , USA
Herpes simplex virus-1 (HSV-1) is a double-stranded DNA virus that causes life-long infections. HSV-1 infections may lead to herpetic stromal keratitis that may advance to corneal blindness. HSV-1 infections can also cause fatal conditions, such as herpes encephalitis, or neonatal disease. A major virulence mechanism of HSV-1 is the control of autophagy, an innate immune defense strategy that could otherwise Adepgrirla2d0e1v5iral particles. Here, to investigate a new mechanism for antiviral therapy, we tested the effect of various autophagy inducers (physiological and pharmacological) on infection. Autophagy stimulation was confirmed to significantly suppress HSV-1 infection in various cell types, without affecting cell viability. This study establishes the importance of autophagy for regulating HSV-1 infection, and provides a proof-of-principle evidence for a novel antiviral mechanism.
requests for materials
should be addressed to
H infectious cause of corneal blindness globally2, while central nervous system dissemination of the infection
SV-1 infects most humans worldwide, and causes significant healthcare concerns1. HSV-1 is the leading
may result in fatal encephalitis3. Current HSV-1 therapy, mainly comprising nucleoside analogs such as
acyclovir, suffers the significant drawback of emergence of resistant virus strains4 causing failure of treatment1,4,
which emphasizes the need for investigating new mechanisms to control HSV-1 infections.
Macroautophagy (or, simply, autophagy) is a cellular process that degrades certain cytoplasmic components of
the cell, or intracellular pathogens5. Autophagy involves sequestration of a part of the cytosol within isolation
membranes, which then mature into double-membrane vesicles (autophagosomes) that eventually fuse with the
lysosomes for lysosomal destruction of the cargo6. Autophagy plays an important role to combat bacterial or viral
infections5?7. It was shown to limit the replication, or enhance the degradation, of various viruses8?10, in addition
to its role in assisting processing and presentation of pathogen antigens, boosting the host adaptive immunity to
HSV-1 is a double-stranded DNA virus that controls host?s autophagic responses through binding of the viral
protein ICP34.5 to the host protein beclin113, leading to inhibition of autophagy. Mutations of ICP34.5 lower
virulence in mice14 and enhance viral degradation by autophagy15. Since control of autophagy is a robust virulence
mechanism of the virus, we reasoned that enabling autophagy activation in infection may suppress the infection,
and thus provide an unprecedented antiviral therapeutic tool. In this study, we investigate this novel concept.
Results and Discussion
To investigate the effect of autophagy induction on HSV-1 infection, we induced autophagy in mouse embryonic
fibroblasts (MEFs) via starvation. The cells were cultured in starvation medium for 3 hours, and then successful
induction of autophagy was validated by multiple assays. Starved MEFs transiently expressing LC3-GFP (Ref. 16)
were assessed for autophagy induction after starvation, using confocal microscopy. After treatment, the cells were
fixed in paraformaldehyde, and imaged microscopically. While unstarved cells showed diffuse LC3 presence in
the cell and only few LC3-GFP punctae (autophagosomes), starved cells showed enhanced autophagosomal
development, as manifested by the increase in number, size and fluorescence intensity of LC3-GFP punctae
which accumulated and clustered mostly in the cell cytoplasm (Figure 1A, B, and C). To further confirm
persistent autophagy upregulation at later points in starved cells, we determined the levels of sequestosome1
(SQSTM1/p62), a protein degraded mainly by autophagy, using immunoblotting. Starved cells showed
significantly decreased p62 levels, consistent with autophagy activation in the cells (Figure 1D).
Having validated autophagy induction by starvation, we then tested its influence on infection. Therefore,
unstarved or starved MEFs were infected with a red fluorescent protein (RFP)-expressing HSV-1 virus. Then
we monitored viral levels throughout the course of infection with fluorescence microscopy. We observed
significant suppression of infection under starvation-induced
autophagy (Figure 2A). FACS analysis of infected cells confirmed a
significant block of HSV-1 infection upon autophagy induction
(Figure 2B, and C). To further validate the effect of autophagy
induction on viral levels, we isolated HSV-1 genomic DNA from infected
cells, and quantified it using a quantitative polymerase chain
reaction (qPCR) assay. HSV-1 genome quantification indicated that
induced autophagy strongly suppresses HSV-1 infection (Figure 2D).
Moreover, virus titer determination by plaque assay further confirmed
this result (Figure 2E).
After confirming that induction of autophagy by starvation inhibits
infection of cells, we then investigated whether this effect is cell-type
dependent, especially considering that the role of autophagy in viral
infection was once suggested to be cell type-specific17. Additionally, we
also wanted to rule out the possibility that the infection-inhibiting
effect of induced autophagy is specifically associated with a
particular autophagy induction method. To address these questions, we
employed another autophagy-inducing method (pharmacologically)
and used various cell types infectable by HSV-1, and tested whether
the effect of physiological autophagy induction on viral infection in
MEFs may be recapitulated by other cell types such epithelial or
neuronal cells. Thus, we treated human corneal epithelial (HCE) cells
with the autophagy-inducing chemical benzyl
(S)-4-methyl-1-((S)-4methyl-1-((S)-4-methyl-1-oxopentan-2-ylamino)-1-oxopentan-2ylamino)-1-oxopentan-2-ylcarbamate (known as MG132) at the
concentration of 1 mM well known to induce autophagy18,19.
Validation of autophagy stimulation was achieved via LC3-II and
SQSTM1/p62 immunoblotting (Figure 3A, B and C). Moreover, a
flow cytometry-based assay confirmed that MG132 treatment
significantly upregulates autophagy levels (data not shown). To ensure equal
viral entry and measure the effects of autophagy on viral replication,
we added the autophagy-inducing agent after incubation of the cells
with the virus (after viral entry). We then assessed viral levels in the
cells by flow cytometry. We found that inducing autophagy in host
cells led to a significant suppression of viral yields in host cells
(Figure 3D, and E), as manifested by the significant drop in both
the percentage and mean fluorescence intensity (MFI) of
HSV-1RFP-positive population in MG132-treated relative to mock-treated
cells. Then we tested the influence of autophagy induction on viral
infection in neuronal retinal ganglion (RGC) cells. We found that
activating autophagy significantly hampers HSV yields and infection in
these cells also (Figure 3F, and G). Virus titer determination by plaque
assay also confirmed the effect of the autophagy-inducing agent on
infection (Figure 3H). Taken together, these data along with starvation
data demonstrate that autophagy induced via multiple means,
physiologically (starvation) or pharmacologically, abrogates HSV-1 infections
in a cell type-independent manner, suggesting that autophagy
induction may be a new mechanism for antiviral drug development.
Having observed the high efficacy of autophagy stimulation on viral
infection, to assess the possibility of pursuing autophagy induction for
future drug development, we sought to test the effect of autophagy
induction on cell viability. Activation of autophagy was shown to guard
against protein synthesis halt, cell cycle delays, or cytotoxicity20?25.
Indeed, autophagic activity of a cell was shown to be inversely
proportional to the probability of its death26,27. Thus, we have closely monitored
and validated by multiple means the activation of autophagy in treated
cells, which should prevent cell death. To confirm that autophagy
induction does not affect cell viability, starved or drug-treated cells were
assessed for their health and viability, using microscopical observation
of cells, and by measuring cell viability using a standard cytotoxicity
assay (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide
(MTT) cytotoxicity assay), as previously described28). We found that
cell viability was not affected by either autophagy induction condition
(Figure 4A, and B). To further confirm that autophagy upregulation
does not influence cell viability or produce cell death, we performed
annexin V-propidium iodide (PI) apoptosis/cell death FACS-based
assay, a highly sensitive apoptosis assay that can also detect early
apoptotic events29,30. We found that the autophagy induction methods used
did not cause significant apoptosis or cell death (Figure 4C?F).
In this study, using information available on viral virulence
mechanism and pathogenesis, we proposed and investigated a novel
mechanism for viral killing (boosting a host defense mechanism,
autophagy). Our results confirmed that inducing autophagic activity
of host cells suppresses HSV-1 infection. These results give further
evidence on the importance of autophagy for regulating HSV-1
infection, and suggest that autophagy induction may be a powerful means
for suppressing viral infections by novel antiviral therapies. The strong
efficacy (Figures 2 and 3), and the insignificant toxicity (Figure 4)
associated with autophagy induction recommend such a mechanism
as a highly efficient mechanism for new drug development.
Importantly, autophagy regulates multiple viral infections, and many viruses
tend to suppress or neutralize autophagy to achieve virulence5?7,9?15.
In addition to HSV-1, other herpesviruses such as human
cytomegalovirus (CMV) are known to suppress autophagy via multiple
mechanisms31,32. Thus, it is possible to propose that autophagy
stimulation may provide a broad-spectrum therapy against many viruses
known to be regulated by autophagy. To conclude, we investigated a
novel mechanism to control HSV-1 infections, and give evidence
supporting the new concept of therapeutic utilization of autophagy,
a currently emerging concept in medicine33,34.
Cells and cell culture. Human corneal epithelial (HCE) cells were from K. Hayashi
(National Eye Institute, Bethesda, MD) and were cultured in Minimum Essential
Media, MEM (Gibco) supplemented with antibiotics and 10% fetal bovine serum
(FBS) (Sigma). Retinal ganglion cell line (RGC5) was provided by B. Yue (University
of Illinois at Chicago), and was cultured in Dulbecco?s modified Eagle?s medium,
DMEM (Gibco), supplemented with serum, antibiotics and amino acids. Mouse
embryonic fibroblasts (MEFs) were a kind gift from C-A A. Hu (University of New
Mexico, Albuquerque, NM). African green monkey foetal kidney epithelial (Vero)
cells were from P. Spear (Northwestern University, Chicago, IL). MEFs and Vero cells
were grown in DMEM (Gibco) supplemented with antibiotics and serum.
Viruses. HSV-1 virus strain KOS was provided by P. Spear (Northwestern University,
Chicago, IL). HSV-1(KOS)-RFP (HSV-1 KOS virus that expresses RFP conjugated to
the capsid protein VP26) is a kind gift from P. Desai (The Johns Hopkins University,
Baltimore, MD). Viruses were propagated and purified as previously described28.
Plasmids. The plasmid pEX-GFP-hLC3WT (simply referred to as GFP-LC3)
previously described16 was obtained from Addgene (plasmid number 24987).
Antibodies. Polyclonal antibodies against LC3 were from Novus Biologicals (Catalog
number NB100-2220). Polyclonal antibodies against GAPDH were purchased from
Santa Cruz (Catalog number sc-25778). Polyclonal antibodies against SQSTM1/p62
were from Santa Cruz (Catalog number sc-25575). Horseradish
peroxidaseconjugated secondary (anti-rabbit) antibodies were from Jackson Immunoresearch
(Catalog number 111-005-144).
Starvation. To induce autophagy by starvation, the cells were washed in
Phosphatebuffered Saline (PBS) for three times, to remove residual medium. The cells were then
cultured in Hanks? Balanced Salt Solution (Gibco).
Infection. The cells were incubated with the virus in PBS containing 0.1% glucose and
1% serum at 37uC?5% CO2 conditions. After 2 hrs, the virus was removed and fresh
medium was added to the cells (or Hanks? Balanced Salt Solution in case of
Transfection. Transfections were performed using Lipofectamine2000 (Invitrogen)
according to the manufacturer?s protocols.
Pharmacological induction of autophagy. For induction of autophagy, the
proteasomal inhibitor benzyl
(known as MG132; Selleckchem., Catalog number S2619) was used at a concentration
of 1 mM.
Flow cytometry. After infection or treatment, the cells were washed in FACS buffer
(PBS, 1% BSA), and analyzed cytofluorimetrically on LSRFortessa cytometer (BD).
Data analysis was performed using Summit software (Beckman Coulter).
Fluorescence microscopy. Following the infection, the cells were washed in PBS, and
imaged using Axiovert 100 M fluorescence microscope (Zeiss). Image acquisition
and analysis were carried out using MetaMorph software (Zeiss).
Confocal fluorescence microscopy. For monitoring of LC3-GFP autophagosomal
punctae, confocal microscopy (Zeiss 710 microscope, Zeiss) was used. After
treatment or infection, the cells were washed, fixed in paraformaldehyde, and used in
imaging. Image acquisition was performed using ZEN software (Zeiss), and image
analysis was performed using MetaMorph software (Zeiss).
HSV genome quantification. Infected cells were washed, and centrifuged. Cell
pellets were suspended in buffer containing 1% SDS, 50 mM Tris (pH 7.5), and
10 mM EDTA, and the cell extract was incubated with proteinase K (50 mg/mL) at
37uC for 1 hr. DNA extraction then followed, via phenol/chloroform
extractionethanol precipitation. Viral DNA was quantified using quantitative PCR (qPCR) on
an ABI 7500 Fast thermocycler (Applied Biosystems), using HSV-specific primers.
Primers used are: Forward (59-TAC AAC CTG ACC ATC GCT TG-39), and Reverse
(59-GCC CCC AGA GAC TTG TTG TA-39), which detect the HSV glycoprotein D
Immunoblotting. Immunoblotting of proteins was performed as previously
described28. Briefly, cells were harvested, lysed in RIPA buffer (Sigma, Catalog
number R0278) containing protease-phosphatase inhibitors, and the lysates were
electrophoresed through denaturing 4?12% SDS-polyacrylamide gel (Novex).
Proteins were transferred onto a PVDF membrane, followed by block of non-specific
binding with 5% non-fat milk in Tris-buffered saline (TBS). Membrane was then
incubated with primary antibody and then with HRP-conjugated secondary
antibody, followed by incubation with Femto-Sensitivity ECL (Thermo).
Chemiluminescence was detected with ImageQuant LAS4000 digital image system
Virus titer (plaque formation) Assay. Confluent monolayers of Vero cells were
infected with serially diluted supernatants in PBS containing 0.1% glucose and 1%
heat-inactivated serum for 2 hrs. After incubation, the cells were washed and
methylcellulose (Sigma)-containing DMEM was added onto the monolayer. The cells
were incubated at 37 C-5% CO2 conditions for 72 hrs, then fixed with methanol for
10 min at room temperature and stained with crystal violet. 30 min later, crystal
violet was removed, monolayers were allowed to dry, and plaques were counted.
Plaque counts were used to calculate viral titers.
Cytotoxicity and apoptosis assays. Cytotoxicity was assessed via MTT (3-[4,5
dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. MTT was purchased
from Sigma and the assay was performed according to the manufacturer?s protocols
and as previously described28. Apoptosis was assayed using FITC Annexin V/Dead
Cell Apoptosis Kit (Molecular Probes, Invitrogen). The assay was performed
according to the manufacturer?s guidelines. Flow cytometry for annexin V and PI
levels was then performed as described above, and data analysis was performed using
Summit software (Beckman Coulter). Apoptosis analysis was performed in
accordance with standardized guidelines29,30, and as previously described35,36.
Statistical analyses. Experiments were independently performed for at least three
times. Quantification shown in figures represents mean values; error bars represent
standard error of the mean. Statistical significance was determined via Student?s t-test
(minimum p-value for significance 0.05). Unless otherwise indicated, the data were
statistically significant (p-values less than 0.05).
We gratefully acknowledge all colleagues who provided reagents. We thank Ruth Zelkha
(University of Illinois Medical Center-Department of Ophthalmology, Chicago, IL) for
assistance with confocal microscopy. This study was supported by a NIH grant (EY023058)
to DS and a core grant (EY01792). The authors claim no conflicts of interest.
A.M.Y. and D.S. designed and conceived the experiments. A.M.Y. performed the
experiments. A.M.Y. and D.S. analyzed the data, and interpreted conclusions. A.M.Y. and
D.S. wrote and reviewed the manuscript. D.S. entirely supervised the project.
Competing financial interests: The authors declare no competing financial interests.
This work is licensed under a Creative Commons Attribution 4.0 International
License. The images or other third party material in this article are included in the
article?s Creative Commons license, unless indicated otherwise in the credit line; if
the material is not included under the Creative Commons license, users will need
to obtain permission from the license holder in order to reproduce the material. To
view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
1. Whitley , R. J. & Roizman , B. Herpes simplex virus infections . Lancet 357 , 1513 - 1518 ( 2001 ).
2. Liesegang , T. J. Herpes simplex virus epidemiology and ocular importance . Cornea 20 , 1 - 13 ( 2001 ).
3. Banatvala , J. E. Herpes simplex encephalitis . Lancet Infect. Dis . 11 , 80 - 81 ( 2011 ).
4. Andrei , G. & Snoeck , R. Herpes simplex virus drug-resistance: new mutations and insights . Curr. Opin. Infect. Dis . 26 , 551 - 560 ( 2013 ).
5. Deretic , V. , Saitoh , T. & Akira , S. Autophagy in infection, inflammation and immunity . Nat. Rev. Immunol . 13 , 722 - 737 ( 2013 ).
6. Kundu , M. & Thompson , C. B . Autophagy: basic principles and relevance to disease . Annu. Rev. Pathol . 3 , 427 - 455 ( 2008 ).
7. Ravikumar , B. et al. Regulation of mammalian autophagy in physiology and pathophysiology . Physiol. Rev . 90 , 1383 - 1435 ( 2010 ).
8. Campbell , G. R. & Spector , S. A. Inhibition of human immunodeficiency virus type-1 through autophagy . Curr. Opin. Microbiol . 16 , 349 - 354 ( 2013 ).
9. Moy , R. H. et al. Antiviral autophagy restricts Rift Valley fever virus infection and is conserved from flies to mammals . Immunity 40 , 51 - 65 ( 2014 ).
10. Orvedahl , A. et al. Autophagy protects against Sindbis virus infection of the central nervous system . Cell Host Microbe 7 , 115 - 127 ( 2010 ).
11. Kuballa , P. , Nolte , W. M. , Castoreno , A. B. & Xavier , R. J. Autophagy and the immune system . Annu. Rev. Immunol . 30 , 611 - 646 ( 2012 ).
12. Levine , B. & Deretic , V. Unveiling the roles of autophagy in innate and adaptive immunity . Nat. Rev. Immunol . 7 , 767 - 777 ( 2007 ).
13. Kudchodkar , S. B. & Levine , B. Viruses and autophagy . Rev. Med . Virol. 19 , 359 - 378 ( 2009 ).
14. Orvedahl , A. et al. HSV-1 ICP34 . 5 confers neurovirulence by targeting the Beclin 1 autophagy protein . Cell Host Microbe 1 , 23 - 35 ( 2007 ).
15. Tallo?czy, Z. , Virgin , H. & Levine , B. PKR-Dependent Xenophagic Degradation of Herpes Simplex Virus Type 1 . Autophagy 2 , 24 - 29 ( 2006 ).
16. Tanida , I. et al. Consideration about negative controls for LC3 and expression vectors for four colored fluorescent protein-LC3 negative controls . Autophagy 4 , 131 - 134 ( 2008 ).
17. Yordy , B. , Iijima , N. , Huttner , A. , Leib , D. & Iwasaki , A. A neuron-specific role for autophagy in antiviral defense against herpes simplex virus . Cell Host Microbe 12 , 334 - 345 ( 2012 ).
18. Tumbarello , D. A. et al. Autophagy receptors link myosin VI to autophagosomes to mediate Tom1-dependent autophagosome maturation and fusion with the lysosome . Nat. Cell Biol . 14 , 1024 - 1035 ( 2012 ).
19. Klionsky , D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes . Autophagy 4 , 151 - 175 ( 2008 ).
20. Mizushima , N. Autophagy: process and function . Genes Dev . 21 , 2861 - 2873 ( 2007 ).
21. Onodera , J. & Ohsumi , Y. Autophagy is required for maintenance of amino acid levels and protein synthesis under nitrogen starvation . J. Biol. Chem . 280 , 31582 - 31586 ( 2005 ).
22. Lum , J. J. , DeBerardinis , R. J. & Thompson , C. B. Autophagy in metazoans: cell survival in the land of plenty . Nat. Rev. Mol. Cell Biol . 6 , 439 - 448 ( 2005 ).
23. Kuma , A. et al. The role of autophagy during the early neonatal starvation period . Nature 432 , 1032 - 1036 ( 2004 ).
24. Matsui , A. , Kamada , Y. & Matsuura , A. The role of autophagy in genome stability through suppression of abnormal mitosis under starvation . PLoS Genet . 9 , e1003245; DOI:10.1371/journal.pgen. 1003245 ( 2013 ).
25. Matsunaga , K. et al. Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages . Nat. Cell Biol . 11 , 385 - 396 ( 2009 ).
26. Gump , J. M. et al. Autophagy variation within a cell population determines cell fate through selective degradation of Fap-1 . Nat. Cell Biol . 16 , 47 - 54 ( 2014 ).
27. Green , D. R. & Levine , B. To be or not to be? How selective autophagy and cell death govern cell fate . Cell 157 , 65 - 75 ( 2014 ).
28. Jaishankar , D. , Yakoub , A. M. , Bogdanov , A. , Valyi-Nagy , T. & Shukla , D. Characterization of a proteolytically stable D-peptide that suppresses herpes simplex virus 1 infection: implications for the development of entry-based antiviral therapy . J. Virol. 89 , 1932 - 1938 ( 2015 ).
29. Elmore , S. Apoptosis: a review of programmed cell death . Toxicol. Pathol . 35 , 495 - 516 ( 2007 ).
30. Bossy-Wetzel , E. & Green , D. R. Detection of apoptosis by annexin V labeling . Methods Enzymol . 322 , 15 - 18 ( 2000 ).
31. Chaumorcel , M. et al. The human cytomegalovirus protein TRS1 inhibits autophagy via its interaction with Beclin 1 . J. Virol . 86 , 2571 - 2584 ( 2012 ).
32. Chaumorcel , M. , Souque`re, S., Pierron , G. , Codogno , P. & Esclatine , A. Human cytomegalovirus controls a new autophagy-dependent cellular antiviral defense mechanism . Autophagy 4 , 46 - 53 ( 2008 ).
33. Rubinsztein , D. C. , Gestwicki , J. E. , Murphy , L. O. & Klionsky , D. J. Potential therapeutic applications of autophagy . Nat. Rev. Drug Discov . 6 , 304 - 312 ( 2007 ).
34. Shoji-Kawata , S. et al. Identification of a candidate therapeutic autophagyinducing peptide . Nature 494 , 201 - 206 ( 2013 ).
35. Krautkramer , K. A . et al. Tcf19 is a novel islet factor necessary for proliferation and survival in the INS-1 b-cell line . Am. J. Physiol. Endocrinol. Metab . 305 , E600 - E610 ( 2013 ).
36. Biswas , N. K. et al. Variant allele frequency enrichment analysis in vitro reveals sonic hedgehog pathway to impede sustained temozolomide response in GBM . Sci. Rep . 5 , 7915 ; DOI:10.1038/srep07915 ( 2015 ).