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Inhibition of selective autophagy by members of the herpesvirus ubiquitin-deconjugase family

TL;DR: In this paper, the ubiquitin deconjugases encoded in the N-terminal domain of the large tegument proteins of Epstein-Barr virus (EBV), Kaposi Sarcoma herpesvirus (KSHV) and human cytomegalovirus (HCMV), but not herpes simplex virus-1 (HSV-1), regulate selective autoophagy by inhibiting the activity of the autophagy receptor SQSTM1/p62.
Abstract: Autophagy is an important component of the innate immune response that restricts infection by different types of pathogens. Viruses have developed multiple strategies to avoid autophagy to complete their replication cycle and promote spreading to new hosts. Here we report that the ubiquitin deconjugases encoded in the N-terminal domain of the large tegument proteins of Epstein-Barr virus (EBV), Kaposi Sarcoma herpesvirus (KSHV) and human cytomegalovirus (HCMV), but not herpes simplex virus-1 (HSV-1), regulate selective autophagy by inhibiting the activity of the autophagy receptor SQSTM1/p62. We found that all the homologs bind to and deubiquitinate SQSTM1/p62 but with variable efficiency, which correlates with their capacity to prevent the colocalization of LC3 with SQSTM1/p62 aggregates and promote the accumulation of a model autophagy substrate. The findings highlight important differences in the strategies by which herpesviruses interfere with selective autophagy.

Summary (2 min read)

Introduction

  • Autophagy is a protein degradation and recycling machinery that regulates cellular homeostasis and participates in the host defense against infection by capturing and destroying invading microorganisms (1, 2).
  • The strategies by which pathogens capture autophagy are beginning to be elucidated at the molecular level, which provides new insight on pathogenesis and highlights potential targets for therapeutic intervention (19).
  • The authors found that similar to BPLF1, the catalytic domains of HCMV-UL48, KSHV-ORF64 and HSV1-UL36 bind to and deubiquitinate SQSTM1/p62 but the efficiency and ubiquitin chain .

Immunoblotting and immunoprecipitation

  • It is The copyright holder for this preprintthis version posted March 30, 2021.
  • For co-immunoprecipitation, the cell lysates were incubated for 4 h with anti-FLAG agarose affinity gel (Sigma, A-2220).
  • Precipitated complexes were washed with lysis buffer and eluted with the FLAG peptide (Sigma, F4799) at a concentration of 400 g/ml.
  • Equal amounts of proteins were fractionated in a polyacrylamide Bis-Tris 4– 12% gradient gels (Invitrogen, NP0321PK2).

Filter-trap assay

  • Cells were harvested 48 h after transfection, resuspended in PBS (Biowest, X0515-500) containing protease inhibitor cocktail and samples were snap frozen and stored at -20°C.
  • Lysates were sonicated with a QSonica Q125 sonicator with settings 20% amplitude, pulsating 1s on/1s off total time of 30 sec.
  • After sonication protein amounts were measured with Bio-Rad .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
  • It is The copyright holder for this preprintthis version posted March 30, 2021.
  • Images were analyzed with the ImageJ software.

Immunofluorescence and confocal microscopy

  • HeLa cells were grown to semi-confluence on glass cover slips in Dulbecco’s modified Eagle’s medium (Sigma, D6429) containing 10% fetal calf serum and 10 g/ml ciprofloxacin and transfected with the indicated plasmids using the JetPEI or Lipofectamine 2000 kit as recommended by the manufacturers.
  • The cells were labeled in 3% BSA-PBS using rabbit anti-LC3, mouse antiSQSTM1/p62 and goat anti-FLAG or mouse anti-FLAG antibodies followed by the appropriate Alexa Fluor 488, 555, 594 or 647 conjugated secondary antibodies.
  • Image analysis .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
  • It is The copyright holder for this preprintthis version posted March 30, 2021.
  • The size of SQSTM1/p62 structures and colocalization of LC3 and SQSTM1/p62 labels was analyzed by creating a mask based on thresholding the SQSTM1/p62 fluorescence.

Results

  • The herpesvirus DUBs interact with SQSTM1/p62 and regulate its ubiquitination.
  • It is The copyright holder for this preprintthis version posted March 30, 2021.
  • To investigate whether HCMVUL48, KSHV-ORF64 and HSV1-UL36 share with BPLF1 the capacity to prevent the colocalization of SQSTM1/p62 with LC3, HeLa cells transfected with plasmids expressing the viral enzymes were co-stained with antibodies specific for LC3 and SQSTM1/p62.
  • .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
  • The authors have previously shown that overexpression of a SQSTM1/p62 mutant where the Lys and Glu residues are substituted with Arg and Ala, respectively (SQSTM1/p62-E409A, K420R) can override the capacity of BPLF1 to inhibit selective autophagy, suggesting that deubiquitination of SQSTM1/p62 Lys420 may be critical for the effect of the viral enzyme (20).

Discussion

  • While compelling evidence points to a key role of selective autophagy in the clearance of both the incoming viruses and newly synthesized viral proteins (19), different viruses have evolved distinct strategies for counteracting this cellular defense to promote their replication and spread.
  • CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
  • It is The copyright holder for this preprintthis version posted March 30, 2021.
  • A detailed mapping of the interaction site(s) of SQSTM1/p62 with the herpesvirus homologs would be required to clarify this issue.
  • Conceivably, the failure of UL36 to inhibit the formation of Lys63 linked chains on Lys7 could explain the retained formation of large SQSTM1/p62 aggregates that colocalize with LC3 in UL36 expressing cells, but differences in the ubiquitination status of this residue are not sufficient to explain the concomitant failure to inhibit selective autophagy.

Data availability

  • All data that support the findings of this study are contained within the manuscript and are available on reasonable request.
  • It is The copyright holder for this preprintthis version posted March 30, 2021.

Figure legends

  • Figure 1. EBV-BPLF1, HSV-1-UL36, HCMV-UL48 and KSHV-ORF64 interact with SQSTM1/p62 but differentially affect its ubiquitination.
  • It is The copyright holder for this preprintthis version posted March 30, 2021.
  • HeLa cells were co-transfected with plasmids expressing aggregation-prone HTTQ109-GFP and increasing amounts of the viral DUBs.
  • Blots from one representative experiment out of two are shown.
  • In addition, intramolecular blockade of the UBA by Lys63-linked chains may redirect SQSTM1/p62 to other functions.

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1
Inhibition of selective autophagy by members of the herpesvirus ubiquitin-deconjugase
family
Päivi Ylä-Anttila
1,#
and Maria G. Masucci
1,#
1
Department of Cell and Molecular Biology, Karolinska Institutet, S-17165 Stockholm, Sweden
Running Title: Herpesvirus DUBs inhibit selective autophagy
# Address correspondence to: Päivi Ylä-Anttila, Maria G. Masucci, Department of Cell and
Molecular Biology, Biomedicum A6, Karolinska Institutet, S-17165 Stockholm, Sweden.
E-mail: paivi.ylaanttila@gmail.com, maria.masucci@ki.se. Tel: +46 (0)8 52486755
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 30, 2021. ; https://doi.org/10.1101/2021.03.30.437649doi: bioRxiv preprint

2
Abstract
Autophagy is an important component of the innate immune response that restricts infection by
different types of pathogens. Viruses have developed multiple strategies to avoid autophagy to
complete their replication cycle and promote spreading to new hosts. Here we report that the
ubiquitin deconjugases encoded in the N-terminal domain of the large tegument proteins of
Epstein-Barr virus (EBV), Kaposi Sarcoma herpesvirus (KSHV) and human cytomegalovirus
(HCMV), but not herpes simplex virus-1 (HSV-1), regulate selective autophagy by inhibiting
the activity of the autophagy receptor SQSTM1/p62. We found that all the homologs bind to
and deubiquitinate SQSTM1/p62 but with variable efficiency, which correlates with their
capacity to prevent the colocalization of LC3 with SQSTM1/p62 aggregates and promote the
accumulation of a model autophagy substrate. The findings highlight important differences in
the strategies by which herpesviruses interfere with selective autophagy.
Keywords: autophagy, deubiquitinase, herpesviruses, large tegument protein, SQSTM1/p62
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 30, 2021. ; https://doi.org/10.1101/2021.03.30.437649doi: bioRxiv preprint

3
Introduction
Autophagy is a protein degradation and recycling machinery that regulates cellular homeostasis
and participates in the host defense against infection by capturing and destroying invading
microorganisms (1, 2). Autophagy entails the sequestration of a portion of the cytoplasm in a
double membraned vacuole which, upon fusion with a lysosome, causes the degradation of its
content (1). The process can be either non-selective when a random portion of the cytoplasm is
degraded, such as during starvation (3), or selective, when directed to cargoes that are marked
for degradation by tags, such as ubiquitin or galectins, that are recognized by specific autophagy
receptors (4). Known autophagy receptors include optineurin (OPTN) that recognizes ubiquitin
coated protein aggregates, bacteria and mitochondria (5), calcium binding and coiled-coil
domain 2/nuclear dot protein of 52kDa (CALCOCO2/NDP52) that recognizes galectin 8 on
bacteria (6), sequestosome 1 (SQSTM1/p62) that recognizes a variety of ubiquitinated cargoes
such as protein aggregates, lipid droplets, lysosomes, midbody rings, mitochondria,
peroxisomes, zymogen granules and pathogens (7, 8, 9, 10, 11, 12, 13, 14), BCL2 and
adenovirus E1B 19-kDa-interacting protein 3-BNIP3 like/NIP-3-like protein X (BNIP3-
BNIP3L/NIX) that are mitochondrial membrane resident proteins functioning in mitophagy
(15) and neighbor of BRCA1 gene 1 protein (NBR1) that recognizes ubiquitin on protein
aggregates or peroxisomes (16, 17). A common feature of the autophagy receptors is their
capacity to interact with the microtubule associated protein 1 light chain 3 (LC3) via a binding
module known as LC3-interacting region (LIR), which promotes the wrapping of the growing
autophagosomal membrane around the cargo (18).
Although generally considered as a protective cellular defense mechanism, autophagy can have
pro-infection properties since many pathogens have evolved ways to modulate the process to
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 30, 2021. ; https://doi.org/10.1101/2021.03.30.437649doi: bioRxiv preprint

4
their advantage. The strategies by which pathogens capture autophagy are beginning to be
elucidated at the molecular level, which provides new insight on pathogenesis and highlights
potential targets for therapeutic intervention (19). We have previously reported that Epstein-
Barr virus ubiquitin-specific protease (USP or deubiquitinating enzyme, DUB) encoded in the
N-terminal domain of the large tegument protein BPLF1 regulates autophagy (20). We found
that BPLF1 binds to and deubiquitinates SQSTM1/p62, which hampers the recruitment of LC3
to SQSTM1/p62 positive structures. The deubiquitination of SQSTM1/p62 by BPLF1 is
associated with accumulation of aggregation-prone mutant huntingtin (HTT) containing
extended polyglutamine repeats (HTT-polyQ) and with failure to clear preformed aggregates,
supporting the conclusion that BPLF1 can inhibit selective autophagy. A critical role for
deubiquitination of the Lys420 residue located in the C-terminal domain of SQSTM1/p62 that
mediates the interaction with both cargo and LC3 was revealed by the finding that the inhibitory
effect could be rescued by co-expression of a mutant SQSTM1/p62 that mimics the active
conformation induced by ubiquitination.
Although many of the BPLF1 substrates and interacting partners identified to date are shared
by the BPLF1 homologs encoded by other herpesviruses, important differences have been
observed. For example, subtle differences in the amino acid composition and charge of the
otherwise relatively well conserved helix-2 were shown to prevent the interaction of the HSV-
1 encoded homolog, UL36, with the 14-3-3 molecular scaffolds, resulting in failure to modulate
the activity of the ubiquitin ligase TRIM25 (21). Here we set out to investigate whether the
ubiquitin deconjugases encoded by EBV, HCMV, KSHV and HSV-1 share the capacity to
regulate selective autophagy by interfering with the activity of the SQSTM1/p62 receptor. We
found that similar to BPLF1, the catalytic domains of HCMV-UL48, KSHV-ORF64 and HSV-
1-UL36 bind to and deubiquitinate SQSTM1/p62 but the efficiency and ubiquitin chain
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 30, 2021. ; https://doi.org/10.1101/2021.03.30.437649doi: bioRxiv preprint

5
specificity of the viral DUBs was remarkably different. Furthermore, the HSV-1 encoded UL36
failed to inhibit the recruitment of LC3 to SQSTM1/p62 positive structures and did not promote
the accumulation of mutant HTTpolyQ into insoluble aggregates. Thus, it appears that the
different lifestyles of herpesviruses may have promoted the development of distinct strategies
for interfering with selective autophagy.
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 30, 2021. ; https://doi.org/10.1101/2021.03.30.437649doi: bioRxiv preprint

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Abstract: Macroautophagy is a dynamic process involving the rearrangement of subcellular membranes to sequester cytoplasm and organelles for delivery to the lysosome or vacuole where the sequestered cargo is degraded and recycled. This process takes place in all eukaryotic cells. It is highly regulated through the action of various kinases, phosphatases, and guanosine triphosphatases (GTPases). The core protein machinery that is necessary to drive formation and consumption of intermediates in the macroautophagy pathway includes a ubiquitin-like protein conjugation system and a protein complex that directs membrane docking and fusion at the lysosome or vacuole. Macroautophagy plays an important role in developmental processes, human disease, and cellular response to nutrient deprivation.

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TL;DR: In this article, the polyubiquitin-binding protein p62/SQSTM1 has been shown to be involved in linking polyUBiquitinated protein aggregates to the autophagy machinery.
Abstract: Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and coimmunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant huntingtin. We suggest that p62 may, via LC3, be involved in linking polyubiquitinated protein aggregates to the autophagy machinery.

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TL;DR: Functional links between PINK1, Parkin and the selective autophagy of mitochondria, which is implicated in the pathogenesis of Parkinson's disease, are provided.
Abstract: Parkinson's disease is the most common neurodegenerative movement disorder. Mutations in PINK1 and PARKIN are the most frequent causes of recessive Parkinson's disease. However, their molecular contribution to pathogenesis remains unclear. Here, we reveal important mechanistic steps of a PINK1/Parkin-directed pathway linking mitochondrial damage, ubiquitylation and autophagy in non-neuronal and neuronal cells. PINK1 kinase activity and its mitochondrial localization sequence are prerequisites to induce translocation of the E3 ligase Parkin to depolarized mitochondria. Subsequently, Parkin mediates the formation of two distinct poly-ubiquitin chains, linked through Lys 63 and Lys 27. In addition, the autophagic adaptor p62/SQSTM1 is recruited to mitochondrial clusters and is essential for the clearance of mitochondria. Strikingly, we identified VDAC1 (voltage-dependent anion channel 1) as a target for Parkin-mediated Lys 27 poly-ubiquitylation and mitophagy. Moreover, pathogenic Parkin mutations interfere with distinct steps of mitochondrial translocation, ubiquitylation and/or final clearance through mitophagy. Thus, our data provide functional links between PINK1, Parkin and the selective autophagy of mitochondria, which is implicated in the pathogenesis of Parkinson's disease.

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TL;DR: The findings indicate that the pathological process associated with p62 accumulation results in hyperactivation of Nrf2 and delineates unexpected roles of selective autophagy in controlling the transcription of cellular defence enzyme genes.
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Frequently Asked Questions (11)
Q1. What are the contributions mentioned in the paper "Inhibition of selective autophagy by members of the herpesvirus ubiquitin-deconjugase family" ?

In this paper, the deubiquitination of the Lys420 residue of SQSTM1/p62 by BPLF1 was found to be associated with the accumulation of aggregation-prone mutant huntingtin ( HTT ) containing extended polyglutamine repeats ( HT-polyQ ) and with failure to clear preformed aggregates. 

SQSTM1/p62 functions in the autophagic degradation of ubiquitinated cargo by physically linking the cargo to the autophagic machinery via binding to autophagosome-anchored LC3 (32). 

The interaction with the cargo is mediated by the C-terminal ubiquitin associated domain (UBA) and is dependent on the ubiquitination of critical residues, which prevents SQSTM1/p62 homodimerization and the consequent occlusion of the cargo-binding site (25). 

Autophagy is a protein degradation and recycling machinery that regulates cellular homeostasis and participates in the host defense against infection by capturing and destroying invading microorganisms (1, 2). 

The capacity of the SQSTM1/p62-E409A, K420R mutant to rescue the inhibitory effects of BPLF1, UL48, and ORF64 suggests that deubiquitination of Lys420 is critical for the activity of viral enzymes. 

The deubiquitinating enzymes encoded in the N-terminus of HSV1-UL36, HCMV-UL48, EBV-BPLF1 and KSHV-ORF64 bind to and deubiquitinate SQSTM1/p62. 

Plasmid encoding codon optimized 3xFLAG-tagged HSV-UL36 (amino acid.CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 

Several ubiquitin ligases, including the Keap1/Cullin3 ligase (35), and under ubiquitin-stress conditions the UBE2D2/UBE2D3 conjugating enzymes (25), were shown to polyubiquitinate the Lys420 residue, but the type of ubiquitin chains was not investigated. 

elimination of all types of chains may be required to stabilize the formation of homodimers that prevent cargo binding and inhibit autophagy; whereas persistent Lys63-linked ubiquitination appears to allow autophagy. 

HeLa cells were co-transfected with fixed amounts of plasmids encoding the viral DUBs and HTT109-GFP and increasing amounts of plasmids encoding the indicated SQSTM1/p62 mutants. 

The weaker inhibitory activity of KSHV-ORF64 required high amount of transfected plasmid to achieve detectable levels of HTTQ109-GFP aggregates, which did not allow testing the effect of a large excess of the SQSTM1/p62-E409A, K420R mutant.