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Post-translational modification of baculovirus-encoded proteins
Journal Pre-proof Post-translational modification of baculovirus-encoded proteins Nan Chen, Xiangshuo Kong, Shudi Zhao, Xiaofeng Wu
PII:
S0168-1702(19)30888-3
DOI:
https://doi.org/10.1016/j.virusres.2020.197865
Reference:
VIRUS 197865
To appear in:
Virus Research
Received Date:
14 December 2019
Revised Date:
10 January 2020
Accepted Date:
12 January 2020
Please cite this article as: Chen N, Kong X, Zhao S, Xiaofeng W, Post-translational modification of baculovirus-encoded proteins, Virus Research (2020), doi: https://doi.org/10.1016/j.virusres.2020.197865
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Manuscript Details Manuscript number
VIRUS_2019_768_R1
Title
Post-translational modification of baculovirus-encoded proteins
Article type
Review Article
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Highlights Summarizes the post-translational modifications of baculovirus-encoded proteins as reported for well-studied AcMNPV and BmNPV. Summarizes the remarkable characteristics of PTMs in baculovirus-encoded proteins. Abstract
Post-translational modifications (PTMs) are the chemical modifications of proteins after
translation, and are very important to guarantee the proper biological functions of these
proteins. Baculoviruses are pathogenic viruses that infect invertebrates and have large circular
-p
double-stranded DNA genomes. Many proteins encoded by baculoviruses have been reported to have PTMs, including phosphorylation, glycosylation, ubiquitination, acetylation, and etc. However, up to now no overview of this information has been produced. In this review, we
re
have summarized the PTMs that have been reported in baculovirus. As a majority of the studies on baculovirus have focused on Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus(BmNPV), this review is focused primarily
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on these viruses.
BmNPV; AcMNPV; proteins; post-translational
Keywords
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modification
Corresponding Author
Xiaofeng Wu
Order of Authors
Nan Chen, Xiangshuo Kong, Shudi Zhao, Xiaofeng Wu
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Suggested reviewers
Hisanori Bando, Qingyou Xia, George Rohrmann, Shyam Kumar
Submission Files Included in this PDF
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Research Data Related to this Submission There are no linked research data sets for this submission. The following reason is given: No data was used for the research described in the article
Dear Editor: We tried our best to improve the manuscript and made essential changes in the revised version. All changes made on the text in compliance with the reviewers'
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comments were highlighted in YELLOW. And we want to confirm that the corresponding author is Xiaofeng Wu, the first author is Nan Chen.
We hope that our correction will meet with the standard for publication in Virus
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Research and really appreciate for your kind consideration.
Yours sincerely,
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Xiaofeng Wu
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Corresponding author College of Animal Sciences,
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Zhejiang University,
China
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Hangzhou 310058,
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Tel.: +86-571-88982198; Fax: +86-571-88982130 Email: [email protected]
Dear Reviewers,
We sincerely extend our deep thanks for your insightful and constructive comments which would substantially improve our manuscript. We tried our best to improve the manuscript and made essential changes in the revised version. Please check our response as attached. All changes made on the text in compliance with the reviewers' comments were highlighted in YELLOW. We hope that our correction will meet with the standard for publication in Virus Research and
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really appreciate for your kind consideration. To reviewer 1
1. Thanks for your good suggestions. The acetylation modification of AcMNPV encoded
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proteins has not been reported and we have mentioned it on the section of acetylation in the
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revised manuscript.
2. Thank you for pointing the mistakes in the use of abbreviations, grammar etc. All have
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been corrected and highlighted in YELLOW in the revised manuscript.
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3. We are sorry for the tedious descriptions in line 396. We have replaced it.
To reviewer 2
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Major concerning:
1. Thanks for the critical comments. Fig. 1 has been deleted.
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2. We are sorry for the wrong handwriting. The virus name has been corrected in the revised manuscript.
Minor concerning: 1. Thanks for your suggestion in article 1. The sentence has been deleted in the revised manuscript. 2. Thanks for your suggestion in article 2. P6.9 does not appear to be phosphorylated in
BV,
so
P6.9
phosphorylation
(including
hypophosphorylation
and
hyperphosphorylation) only occurs in ODV, thus we describe as phosphorylation
in
ODV
can
be
divided
into
hypophosphorylation
“P6.9 and
hyperphosphorylation”. 3. Thanks for your valuable suggestion in article 3. We have supplemented the explanation that this conclusion lacks experimental evidence . 4. We are sorry for our not accurate statement in article 4, it have been corrected in the revised manuscript. 5. Thanks for your suggestion in article 5. 10. 13. 14. We have supplemented the
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corresponding references in the revised manuscript. 6. The contents of articles 6 and 8 are a summary of the references have been cited in that paragraph.
7. Thanks for your valuable suggestion. The references you suggested in article 7. 11. 12.
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15. 16 have been cited and the inappropriate references have been deleted.
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8. We are sorry for the carelessness in article 9. The “Glycosylati” has been deleted.
1. Summarizes the post-translational modifications of baculovirus-encoded proteinsas reported for well-studied AcMNPV and BmNPV.
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proteins.
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2. Summarizes the remarkable characteristics of PTMs in baculovirus-encoded
Post-translational modification of baculovirus-encoded proteins
Abstract Post-translational modifications (PTMs) are the chemical modifications of proteins after translation, and are very important to guarantee the proper biological functions of these proteins. Baculoviruses are pathogenic viruses that infect invertebrates and have large circular double-stranded DNA genomes. Many proteins
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encoded by baculoviruses have been reported to have PTMs, including phosphorylation, glycosylation, ubiquitination, acetylation, and etc. However, up to now no overview of this information has been produced. In this review, we have
summarized the PTMs that have been reported in baculovirus. As a majority of the
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studies on baculovirus have focused on Autographa californica multiple
nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus(BmNPV),
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Autographa californica multiple nucleopolyhedrovirus Bombyx mori nucleopolyhedrovirus Bombyx mori budded virus baculovirus repeated ORF (bro) protein BV/ODV-C42 ecdystero UDP-glucosyltransferase fibroblast growth factor glutarnine Helicoverpa armigera nucleopolyhedrovirus homologous region (hr1)-binding protein immediate-early protein late expression factors leucine mass spectrometry mitogen-activated protein kinases nuclear pore complex nucleation promoting factor
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AcMNPV BmNPV B.mori BV BRO C42 EGT FGF Glu HearNPV hr1-BP IE1 LEFs Leu MS MAPKs NPC NPF
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Abbreviations used
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this review is focused primarily on these viruses.
occlusion-derived virus occlusion body post-translational modification polyhedron envelope serine/threonine kinase
PK
protein kinase
Thr
threonine
Tyr
throsine
UPP
ubiquitin-proteasome pathway
v-CATH
viral cathepsin
v-Ubi
virus ubiquitin
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ODV OB PTM PE PK1
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Key words: BmNPV; AcMNPV; proteins; post-translational modification
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Introduction
Baculoviruses are insect-specific pathogens containing large circular
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double-stranded DNA genomes of 80-180 kb and are pathogenic mainly for lepidopteran insects. The life cycle of lepidopteran baculoviruses is characterized by
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the production of two progeny phenotypes: the budded virus (BV) ,which primarily mediates the cell-to-cell infection, and invades cells mainly through
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receptor-mediated endocytosis, whereas the occlusion-derived virus (ODV), released from occlusion bodies (OBs) within the larval midgut, is responsible for virus transmission between insects via oral infection and initiates primary infections. Although the two viral forms differ in the composition of their envelopes, their nucleocapsid appear to be similar in structure and are composed of a rod-shaped capsid and a nucleoprotein core. Most baculoviruses encode fewer than 150 proteins, and their expression in host cells is strictly regulated via a cascade of gene expression.
Many baculovirus-encoded proteins have been reported to undergo post-translational modifications (PTMs). PTM of proteins refers to the chemical changes proteins undergo after translation. Such modifications come in a wide variety of types, and are mostly catalyzed by enzymes that recognize specific target sequences in specific proteins [1,2]. The most common modifications are the specific cleavage of precursor proteins; formation of disulfide bonds; covalent addition or removal of low-molecular-weight groups, thus leading to modifications such as acetylation, glycosylation (enzymatic
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conjugation with carbohydrates), phosphorylation, amidation, methylation, biotinylation, and etc. PTMs play a fundamental role in regulating the folding of
proteins, their targeting to specific subcellular compartments, their interaction with ligands or other proteins, and their functional state, such as catalytic activity in the
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case of enzymes or the function of proteins involved in signal transduction pathways. Some PTMs (e.g, phosphorylation) are readily reversible by the action of specific
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deconjugating enzymes. Many PTM sites have been shown to occur in disordered regions [3].
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In baculovirus, PTMs are indispensable throughout the entire process of infection in which baculovirus uses host factors to complete the viral life cycle and
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produce infectious progeny viruses. Simultaneously, PTMs of host proteins are involved in the immune regulatory pathway attempting to inhibit viral infection. Interestingly, the proteases mediating baculovirus PTMs include proteases encoded by
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both the virus and host. The study of PTM mechanisms will assist to better understand the structure and function of the baculovirus gene products as well as the host
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response to viral infection. In this review, we summarize the post-translational modifications of baculovirus-encoded proteins as reported for well-studied AcMNPV and BmNPV.
Phosphorylation of baculovirus-encoded proteins Phosphorylation regulates protein function without changing the level of protein synthesis, and can involve site-specific phosphorylation at single or multiple sites [4].
Conformational changes resulting from reversible phosphorylation affect regulatory processes such as enzyme inhibition and activation, protein interaction recognized by domains, and protein degradation[5]. AcMNPV-P6.9, pp78/83, PP31, IE1, P10 and PP34, and BmNPV- P6.9, 39K, BRO, LEF-6, pp78/83, IE1 and PP34, have been reported to undergo phosphorylation during infection. The proteins of BV and ODV may have differing phosphorylation profiles[6,7]. Most of the proteins phosphorylated in baculovirus were found to be
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DNA-binding proteins, including P6.9, BRO protein, IE1 and pp31. P6.9 is a protamine-like DNA binding protein, which is involved in a variety of processes in
the infection cycle[8]. Phosphorylation of P6.9 has been intensively investigated and is closely related to its function. In BV, P6.9 does not appear to be phosphorylated,
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whereas P6.9 phosphorylation in ODV can be divided into hypophosphorylation and hyperphosphorylation, depending on the protein kinases through which
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phosphorylation was mediated. Hypophosphorylation may be dependent on host-encoded protein kinases (PK), while hyperphosphorylation of P6.9 relies on
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virus-encoded protein serine/threonine kinase (PK1)[9]. P6.9 hyperphosphorylation is a prerequisite for hyperexpression of very late genes[9]. Phosphorylation can
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neutralize part of the positive charge of P6.9, attenuate the interaction between P6.9 and DNA, and facilitate the release of viral DNA during the nucleocapsid uncoating process. However, viral DNA assembly into nucleocapsids requires P6.9
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dephosphorylation and is mediated by a virus-encoded phosphatase 38K. 38K is a nucleocapsid-associated protein, and the absence or mutation of the HAD motif in
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38K leads to the disruption of nucleocapsid assembly [10]. Whereas the phosphorylation of P6.9 results in nucleocapsid uncoating, the dephosphorylation of P6.9 is involved in viral DNA encapsidation during nucleocapsid assembly. Phosphorylation and dephosphorylation of P6.9 occur at different stages of viral infection, dynamically regulating late gene expression and nucleocapsid assembly. In addition to phosphorylation, there were also potential methylation sites on P6.9, intricately mixed modifications remain to be elucidated.
Phosphorylation of BRO may regulate its binding activity to DNA and RNA, but it lacks experimental evidence. They could block cellular replication and/or transcription and switch host machinery to viral DNA or RNA synthesis by binding to host chromosomal DNA. BRO also undergoes ubiquitination followed by proteasome degradation, which may protect other viral proteins from degradation and increase the efficacy of infection[11]. IE1 is a phosphorylated protein that specifically binds to DNA and acts as a transcriptional regulator to activate viral gene expression[12]. Hyperphosphorylation
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of IE1 coincides exclusively with DNA replication in AcMNPV[13]. AcMNPV PP31 and BmNPV 39k are homologous. PP3l is a DNA-binding
protein and a constituent of the virogenic stroma[14]. Like P6.9, PP3l also undergoes
reversible phosphorylationa by both cell-induced and virus-encoded kinases[15]. The
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deletion of pp31 gene did not affect the replication of viral DNA, but the expression of some early and late genes were significantly down-regulated[16].
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Hyperphosphorylation of 39K plays an important role in the transcription of late genes. Knockout of BmNPV 39k leads to a decrease in BV and polyhedron
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production, a loss of oral infectivity, and deficiency in virogenic stroma formation [17].
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There are other phosphorylated proteins in baculovirus, including P10, nucleocapsid protein P78/83 and PP34 but they do not appear to bind to DNA. During AcMNPV infection, the C-terminus of P10 is phosphorylated and inducing
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filamentous aggregation to form tubular structures [18]. Two types of phosphorylated P10 tubular structures have been described: a cage-like structure surrounding the
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nucleus and a filamentous structure within the nucleus [19]. Hyperexpression of P10 contributes to nuclear lysis and OB release in cultured cells. And P10 appears to be required for the formation of the polyhedron envelope (PE)[20]. Phosphorylated PP34 is also required to form polyhedron envelope(PE)[21,22]. P78/83 is a phosphorylated protein located at the end of the nucleocapsid. The genes pp78/83 and pk1 overlap and are reverse transcribed[23,24]. PK1 is also a component of a very late gene transcription complex. It binds to the promoter of polh and promotes overexpression
of late genes[25], which further explain the dual role of PK1 in promoting late genes transcription. Collectively, phosphorylation and dephosphorylation dynamically regulate the conformation and function of baculovirus proteins. It is necessary to make clear the mechanism why the phosphorylation and dephosphorylation of viral proteins are involved in the virogenic stroma formation and late genes hyperexpression, as well as protein-protein interactions.
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Glycosylation of baculovirus-encoded proteins Glycosylation usually occurs on the side chains of some amino acids and has structural diversity. N-linked glycosylation refers to the connection between the
glycan and the asparagine side chain of proteins. O-linked glycosylation is the transfer
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of a sugar chain to the oxygen atoms of serine or threonine [26].
Many viral envelope proteins are modified by glycosylation. In AcMNPV these
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include the major envelop protein GP64, ecdystero UDP-glucosyltransferase (EGT), GP41, GP37, ac23 (F-protein), cathepsin (v-CATH) and chitinase. In BmNPV,
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glycosylation has been reported in GP64, fibroblast growth factor (FGF), EGT, GP41 and v-CATH. Glycosylation in baculovirus-encoded proteins can influence their
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structures, physical and chemical properties, intracellular transport, and functions [27].
GP64 and F protein are the most well studied glycoproteins. The baculovirus
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family is subdivided into four genera: Alpbabaculovirus, Betabaculovirus Deltabaculovirus, and Gammabaculovirus. Alpbabaculovirus is divided into Group I
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and Group II. GP64 is glycosylated and is the envelope fusion protein of Group I baculovirus. In contrast, Group II baculovirus lack the gp64 gene, but employ the glycoprotein F for virion binding and membrane fusion. Glycosylation of GP64 affects BV production and membrane fusion. GP64 is a dimer connected by disulfide bonds, and the cluster formed by three histidine residues (H245, H304, and H430) located near the base of the central coiled coil act as pH sensors. Their protonation induces a low pH-induced conformational change, which mediates membrane fusion
[28]. However, the role for glycosylation sites on GP64 in conformational change remains an open question [29]. Knockout of one or more GP64 N-glycosylation sites impairs cell recognition and entry by AcMNPV BV, resulting in less infectious progeny viruses production [30]. It has also been reported that the C-terminal region of AcMNPV GP64 is fatty acid acylated and this may be involved in anchoring of GP64 in the viral envelope[31]. The F protein is cleaved by a furin-like protease to produce two disulfide-linked subunits F1 and F2[32]. Both F1 and F2 undergo N-linked glycosylation, producing N-linked glycans[33,34]. The glycosylation site on
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F protein in Helicoverpa armigera nucleopolyhedrovirus（HearNPV）is necessary for its folding and transport to the golgi apparatus[35]. No N-linked glycoprotein in the
ODV of HearNPV have been reported. All detected N-linked glycosylation occurs in BV and contain signal peptides [6]. The F protein exists as a trimer in BV. However,
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the N-linked glycosylation of the F2 subunit in HearNPV is not essential for BV
production and its fusion with host cells. The disappearance of N-linked glycans on
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the F2 subunit enhances its fusion with the membrane[33]. Not all glycosylation plays a role in promoting membrane fusion in baculovirus. Importantly, glycosylation sites,
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and N-linked glycans may play biological roles. Further study is required to explain the specific function of glycosylation sites in BV.
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EGT is a glycoprotein, and although these sugars do not appear to be required for its enzymatic activity [36]. Glycosylation of its N-terminus plays a crucial role in its secretion on of its N-terminus plays a crucial role in its secretion [37]. Although,
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AcMNPV FGF does not undergo glycosylation modification[37], BmNPV FGF is glycosylated and this processing is required for it to be secreted [38]. GP37 undergoes
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N-linked glycosylation and is associated with polyhedron [39]. GP41 is an O-glycosylated tegument protein located between the envelop and the capsid. It exists as a trimer in the BV and ODV of AcMNPV[40]. GP41 is involved in the formation of ODV morphology[41] and the budding of nucleocapsids to form BV [42]. Both baculovirus chitinase and v-CATH are glycosylated and may be involved in the release of OBs from BmNPV-infected cells[43,44]. As a summary, the glycoproteins participate in the formation of protein structures
and play a key role in the interaction with membranes, for example membrane fusion, nucleocapsid budding, the release of OB. N-linked glycosylation and protein folding are closely interrelated processes that occur in the endoplasmic reticulum. There are many enzymes mediating glycosylation in insect cells. It is unknown whether BmNPV can encode glycosyltransferases.
Ubiquitination of baculovirus-encoded proteins Ubiquitination is the covalent addition of one or more ubiquitin molecules or
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ubiquitin-like modifiers to the side chain amino acid groups of the target protein by the ubiquitin ligase [45].
Ubiquitination mostly happens in baculovirus envelop proteins and nucleocapsid proteins. The virus ubiquitin(v-Ubi) has 75% homology with ubiquitin of eukaryotic
co-expressed with nucleocapsid proteins [47].
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cells[46]. Guarino et al. showed that v-Ubi molecules of baculovirus were
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There are relatively few studies on baculovirus ubiquitination modification and viral ubiquitinated proteins are present in nucleocapsid. Compared with ODV
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nucleocapsid, BV nucleocapsid proteins are highly ubiquitinated by vUbi, and the expression level of v-Ubi in BV is about 4 times that of ODV[48].
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The ubiquitination of nucleocapsid proteins by v-Ubi is a signal determining whether the nucleocapsid will bud from the nucleus to form BV or remain in the nucleus to form ODV[48]. AC141 is an E3 ubiquitin ligase with RING-domain.
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AC141 interacts with either v-Ubi or cellular ubi and participates in ubiquitination of nucleocapsid protein AC66, which is necessary for effective BV production [48].
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However, some viral proteins inhibit ubiquitination to facilitate self-reproduction process, because the ubiquitin-proteasome pathway(UPP) is the main mechanism for protein degradation. Ac102 binds to BV/ODV-C42(C42) to suppress K48-linked ubiquitination of C42, which decreases C42 proteasomal degradation and consequently allows P78/83 to function as a stable nucleation promoting factor (NPF) to induce actin polymerization [49]. Ac102 inhibits both K48- and K63-linked ubiquitination of C42. K63-linked ubiquitination of C42 is hypothesized to modulate
P78/83-induced actin polymerization via a yet unknown mechanism (Fig.1). It was reported that many E3 ligases with RING-domains inhibit viral replication through degrading by polyubiquitination in the host. Interestingly, BmNPV also encodes 6 ring-finger proteins: IAP1, ORF35, IAP2, CG30, IE2 and PE38. Previous studies showed that IAP2, IE2 and PE38 have E3 ubiquitin ligase activity and can catalyze ubiquitination. The activity depends on the ring-finger motif[50]. As an immediate-early gene PE38 is important for the function of transcription and translation[51,52], it also interacts with proteins related to the ubiquitin-proteasome
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pathway [53], thus it may play an important role in regulating the stability and degradation of protein. This suggests that the virus may have other associated proteins
Acetylation of baculovirus-encoded proteins
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or mechanisms that evade the ubiquitin-proteasome pathway.
Acetylation modification refers to the process of adding acetyl groups to lysine
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residues under the action of acetyltransferase.
Proteomic analysis indicated that some late expression factors (LEFs) of
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BmNPV have significant levels of acetylation, including LEF-3, LEF-4, LEF-6 and LEF-11, but the role of acetylation is not clear. There is no gene encoding
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acetyltransferase in the BmNPV genome, so the acetylation of viral protein depends on the host-encoded acetyltransferase[54]. No acetylation modification has been
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reported in the study of AcMNPV.
Summary
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The PTMs of baculovirus-encoded proteins have remarkable characteristics and
are summarized in this review(Table 1 and Fig.2). Phosphorylation mostly occurs in DNA-binding and nucleocapsid proteins, involved in virogenic stroma formation and late gene hyperexpression. Glycosylation mostly occurs in the envelop proteins，and these envelope glycoproteins are essential for entry into cells. Ubiquitination are present in nucleocapsid proteins, functioning as a signal of the budding of nucleocapsid. However, there are relatively few studies on baculovirual acetylation
modification. Mass spectrometry (MS)-based proteomics provides an excellent opportunity to globally analyze proteins and their modifications[55]. The technique not only yields sequence information to identify the protein, but also reveals very precisely the site and nature of post-translational modifications. Although many potential PTM sites on baculovirus proteins have been predicted, most of them were not identified or confirmed. The number of viral proteins is limited, so a viral protein may play multiple roles after undergoing PTMs during infection. Therefore, it is valuable for us
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to develop some new tools used to distinguish the grading changes of viral PTMs and reconstruct the slight changes in the system in vitro, so as to better understand the effect of its interaction with host and regulation mechanism.
Interestingly, during baculovirus infection PTMs in host are also affected.
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Acetylation is possibly beneficial for BmN cells to resist virus proliferation[54]. Not only acetylation but also the phosphorylation of host proteins is triggered after the
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infection of BmNPV[5]. Bombyx mori mitogen-activated protein kinases (MAPKs) Bmp38 and BmS6K have conserved Thr-Glu-Tyr (TEY) and Thr-Leu-Tyr (TLY)
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phosphorylation motifs, respectively. Phosphorylation of BmS6K protect against BmNPV infection[56]. PTMs protect host against virus proliferation, but in this case,
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it is different. In Sf9 cells, the host homologous region-binding protein(hr1-BP) is phosphorylated and then binds to the enhancer hr1 of AcMNPV, thereby promoting transcription of early gene ie and delaying early gene 39k[57-59].
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An important characteristic of the insect-baculovirus expression system (BEVS) is that it can provide a PTM process similar to that of eukaryotes. Therefore, BEVS
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provides a reliable PTM platform for large-scale production of eukaryotic proteins[60]. Bombyx mori has a glycan synthesis pathway similar to that of mammals[41,61]. Genetic and metabolic engineering techniques have been used to reconstruct the glycosylation of insects and thus promotes the glycosylation of baculovirus-expressed proteins, making them closer to natural protein[62,63]. Thereby, it is very promising to enhance its potential use by improving the PTMs in BEVS and exploit insect host (such as silkworm) as a ideal biofactory for recombinant protein
production. Enzymes that regulate PTMs are suitable targets for drug development in the medical field. Therefore, identifying the enzymes that promote PTMs by means of genomics and proteomics is useful for the prevention and treatment of baculovirus infection. Many proteins have been observed to be modified on multiple sites and site-specific crosstalk is prevalent among different modifications[64-67]. Further experiments are needed to determine whether there are cross-talk between different
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PTMs in baculovirus.
Acknowledgments
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We sincerely extend our thanks to Prof. George Rohrmann to critically read this manuscript and provide good suggestions. This work was supported by the National Natural Science Foundation of China (grant no. 31772675 and 31972619) and Natural Science Foundation of Zhejiang Province (LZ20C170001).
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quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Mol
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ur
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lP
re
-p
Cell Proteomics (2011) 10(10):M111 013284.
Table 1 Protein PTMs identified in AcMNPV and BmNPV PTM
protein
AcMNPV or
Function of the PTM
Peptide sequence of PTM
uncoats nucleocapsid and necessary for hyperexpression of very late genes necessary for nucleocapsid
RRRSST#TT##S TR#RRSS
BmNPV both
P78/83
both
IE1
both
activates viral gene expression
PP34
both
necessary for PE
PP31
AcMNPV
P10
AcMNPV
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P6.9
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BRO
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necessary for virogenic stroma formation exists in two different forms and necessary for PE necessary for virogenic stroma formation promotes viral DNA or RNA synthesis
lP BmNPV
na
39K
-p
Phosphorylation
NQLNQRLpSN AQTQQISAK
BmNPV
LEF-6
BmNPV
unclear
GP64
both
unclear necessary for virus to enter cells via endocytosis
EGT
both
unclear
GP41
both
involved in the budding of nucleocapsids
RHpSNAGDVY SAQQVVHAM K STDRIpSEIGD WCR
Glycosylation
v-CATH
both
affects the correct folding of V-CATH and chitinase
chitinase
AcMNPV
unclear
AMLDQVQIQT N#R
necessary for PE
NNN#DSLSTSA QFGVNK
FGF
BmNPV
necessary for its
NVLVN#SSGV
secretion
HR
AcMNPV
necessary for effective production of BV
C42
AcMNPV
unclear
BRO
BmNPV
increases the efficacy of infection
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AC66
lP na ur Jo
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AcMNPV
re
Ubiquitination
GP37
SUPPLEMENTAL FIGURE LEGENDS
Fig.1 Model of a regulatory cascade that controls actin polymerization during
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AcMNPV infection. Ac102 inhibits both K48- and K63-linked ubiquitination of C42. K48-linked ubiquitination promotes the degradation of C42 by UPP, decreasing C42 availability. The reduced C42 levels then compromise its ability to protect P78/83
from ubiquitination-independent proteasomal degradation and eventually results in the
-p
attenuation of P78/83-induced actin polymerization. K63-linked ubiquitination of C42 is hypothesized to modulate P78/83-induced actin polymerization via a yet unknown
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mechanism, as indicated by the question mark.(from Yongli Zhang, 2018[46] )
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Fig.2 The distribution of structural proteins with different PTMs. Orange diamonds represent phosphorylated proteins. Blue ellipse represents ubiquitinated
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proteins. Black circles represent glycoproteins. The outermost blue column represents the envelop. The inner black column represents the nucleocapsid. Proteins with
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different modifications are placed in corresponding positions.
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na
ur
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PII:
S0168-1702(19)30888-3
DOI:
https://doi.org/10.1016/j.virusres.2020.197865
Reference:
VIRUS 197865
To appear in:
Virus Research
Received Date:
14 December 2019
Revised Date:
10 January 2020
Accepted Date:
12 January 2020
Please cite this article as: Chen N, Kong X, Zhao S, Xiaofeng W, Post-translational modification of baculovirus-encoded proteins, Virus Research (2020), doi: https://doi.org/10.1016/j.virusres.2020.197865
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.
Manuscript Details Manuscript number
VIRUS_2019_768_R1
Title
Post-translational modification of baculovirus-encoded proteins
Article type
Review Article
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Highlights Summarizes the post-translational modifications of baculovirus-encoded proteins as reported for well-studied AcMNPV and BmNPV. Summarizes the remarkable characteristics of PTMs in baculovirus-encoded proteins. Abstract
Post-translational modifications (PTMs) are the chemical modifications of proteins after
translation, and are very important to guarantee the proper biological functions of these
proteins. Baculoviruses are pathogenic viruses that infect invertebrates and have large circular
-p
double-stranded DNA genomes. Many proteins encoded by baculoviruses have been reported to have PTMs, including phosphorylation, glycosylation, ubiquitination, acetylation, and etc. However, up to now no overview of this information has been produced. In this review, we
re
have summarized the PTMs that have been reported in baculovirus. As a majority of the studies on baculovirus have focused on Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus(BmNPV), this review is focused primarily
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on these viruses.
BmNPV; AcMNPV; proteins; post-translational
Keywords
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modification
Corresponding Author
Xiaofeng Wu
Order of Authors
Nan Chen, Xiangshuo Kong, Shudi Zhao, Xiaofeng Wu
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Suggested reviewers
Hisanori Bando, Qingyou Xia, George Rohrmann, Shyam Kumar
Submission Files Included in this PDF
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File Name [File Type]
cover letter.docx [Cover Letter] response to reviewer.pdf [Response to Reviewers] highlights.pdf [Highlights] Revised Manuscript.pdf [Manuscript File]
Submission Files Not Included in this PDF File Name [File Type] Fig1.png [Figure] Fig2.png [Figure]
To view all the submission files, including those not included in the PDF, click on the manuscript title on your EVISE Homepage, then click 'Download zip file'.
Research Data Related to this Submission There are no linked research data sets for this submission. The following reason is given: No data was used for the research described in the article
Dear Editor: We tried our best to improve the manuscript and made essential changes in the revised version. All changes made on the text in compliance with the reviewers'
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comments were highlighted in YELLOW. And we want to confirm that the corresponding author is Xiaofeng Wu, the first author is Nan Chen.
We hope that our correction will meet with the standard for publication in Virus
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Research and really appreciate for your kind consideration.
Yours sincerely,
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Xiaofeng Wu
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Corresponding author College of Animal Sciences,
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Zhejiang University,
China
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Hangzhou 310058,
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Tel.: +86-571-88982198; Fax: +86-571-88982130 Email: [email protected]
Dear Reviewers,
We sincerely extend our deep thanks for your insightful and constructive comments which would substantially improve our manuscript. We tried our best to improve the manuscript and made essential changes in the revised version. Please check our response as attached. All changes made on the text in compliance with the reviewers' comments were highlighted in YELLOW. We hope that our correction will meet with the standard for publication in Virus Research and
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really appreciate for your kind consideration. To reviewer 1
1. Thanks for your good suggestions. The acetylation modification of AcMNPV encoded
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proteins has not been reported and we have mentioned it on the section of acetylation in the
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revised manuscript.
2. Thank you for pointing the mistakes in the use of abbreviations, grammar etc. All have
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been corrected and highlighted in YELLOW in the revised manuscript.
na
3. We are sorry for the tedious descriptions in line 396. We have replaced it.
To reviewer 2
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Major concerning:
1. Thanks for the critical comments. Fig. 1 has been deleted.
Jo
2. We are sorry for the wrong handwriting. The virus name has been corrected in the revised manuscript.
Minor concerning: 1. Thanks for your suggestion in article 1. The sentence has been deleted in the revised manuscript. 2. Thanks for your suggestion in article 2. P6.9 does not appear to be phosphorylated in
BV,
so
P6.9
phosphorylation
(including
hypophosphorylation
and
hyperphosphorylation) only occurs in ODV, thus we describe as phosphorylation
in
ODV
can
be
divided
into
hypophosphorylation
“P6.9 and
hyperphosphorylation”. 3. Thanks for your valuable suggestion in article 3. We have supplemented the explanation that this conclusion lacks experimental evidence . 4. We are sorry for our not accurate statement in article 4, it have been corrected in the revised manuscript. 5. Thanks for your suggestion in article 5. 10. 13. 14. We have supplemented the
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corresponding references in the revised manuscript. 6. The contents of articles 6 and 8 are a summary of the references have been cited in that paragraph.
7. Thanks for your valuable suggestion. The references you suggested in article 7. 11. 12.
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15. 16 have been cited and the inappropriate references have been deleted.
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8. We are sorry for the carelessness in article 9. The “Glycosylati” has been deleted.
1. Summarizes the post-translational modifications of baculovirus-encoded proteinsas reported for well-studied AcMNPV and BmNPV.
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proteins.
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2. Summarizes the remarkable characteristics of PTMs in baculovirus-encoded
Post-translational modification of baculovirus-encoded proteins
Abstract Post-translational modifications (PTMs) are the chemical modifications of proteins after translation, and are very important to guarantee the proper biological functions of these proteins. Baculoviruses are pathogenic viruses that infect invertebrates and have large circular double-stranded DNA genomes. Many proteins
ro of
encoded by baculoviruses have been reported to have PTMs, including phosphorylation, glycosylation, ubiquitination, acetylation, and etc. However, up to now no overview of this information has been produced. In this review, we have
summarized the PTMs that have been reported in baculovirus. As a majority of the
-p
studies on baculovirus have focused on Autographa californica multiple
nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus(BmNPV),
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Autographa californica multiple nucleopolyhedrovirus Bombyx mori nucleopolyhedrovirus Bombyx mori budded virus baculovirus repeated ORF (bro) protein BV/ODV-C42 ecdystero UDP-glucosyltransferase fibroblast growth factor glutarnine Helicoverpa armigera nucleopolyhedrovirus homologous region (hr1)-binding protein immediate-early protein late expression factors leucine mass spectrometry mitogen-activated protein kinases nuclear pore complex nucleation promoting factor
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AcMNPV BmNPV B.mori BV BRO C42 EGT FGF Glu HearNPV hr1-BP IE1 LEFs Leu MS MAPKs NPC NPF
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Abbreviations used
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this review is focused primarily on these viruses.
occlusion-derived virus occlusion body post-translational modification polyhedron envelope serine/threonine kinase
PK
protein kinase
Thr
threonine
Tyr
throsine
UPP
ubiquitin-proteasome pathway
v-CATH
viral cathepsin
v-Ubi
virus ubiquitin
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ODV OB PTM PE PK1
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Key words: BmNPV; AcMNPV; proteins; post-translational modification
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Introduction
Baculoviruses are insect-specific pathogens containing large circular
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double-stranded DNA genomes of 80-180 kb and are pathogenic mainly for lepidopteran insects. The life cycle of lepidopteran baculoviruses is characterized by
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the production of two progeny phenotypes: the budded virus (BV) ,which primarily mediates the cell-to-cell infection, and invades cells mainly through
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receptor-mediated endocytosis, whereas the occlusion-derived virus (ODV), released from occlusion bodies (OBs) within the larval midgut, is responsible for virus transmission between insects via oral infection and initiates primary infections. Although the two viral forms differ in the composition of their envelopes, their nucleocapsid appear to be similar in structure and are composed of a rod-shaped capsid and a nucleoprotein core. Most baculoviruses encode fewer than 150 proteins, and their expression in host cells is strictly regulated via a cascade of gene expression.
Many baculovirus-encoded proteins have been reported to undergo post-translational modifications (PTMs). PTM of proteins refers to the chemical changes proteins undergo after translation. Such modifications come in a wide variety of types, and are mostly catalyzed by enzymes that recognize specific target sequences in specific proteins [1,2]. The most common modifications are the specific cleavage of precursor proteins; formation of disulfide bonds; covalent addition or removal of low-molecular-weight groups, thus leading to modifications such as acetylation, glycosylation (enzymatic
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conjugation with carbohydrates), phosphorylation, amidation, methylation, biotinylation, and etc. PTMs play a fundamental role in regulating the folding of
proteins, their targeting to specific subcellular compartments, their interaction with ligands or other proteins, and their functional state, such as catalytic activity in the
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case of enzymes or the function of proteins involved in signal transduction pathways. Some PTMs (e.g, phosphorylation) are readily reversible by the action of specific
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deconjugating enzymes. Many PTM sites have been shown to occur in disordered regions [3].
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In baculovirus, PTMs are indispensable throughout the entire process of infection in which baculovirus uses host factors to complete the viral life cycle and
na
produce infectious progeny viruses. Simultaneously, PTMs of host proteins are involved in the immune regulatory pathway attempting to inhibit viral infection. Interestingly, the proteases mediating baculovirus PTMs include proteases encoded by
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both the virus and host. The study of PTM mechanisms will assist to better understand the structure and function of the baculovirus gene products as well as the host
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response to viral infection. In this review, we summarize the post-translational modifications of baculovirus-encoded proteins as reported for well-studied AcMNPV and BmNPV.
Phosphorylation of baculovirus-encoded proteins Phosphorylation regulates protein function without changing the level of protein synthesis, and can involve site-specific phosphorylation at single or multiple sites [4].
Conformational changes resulting from reversible phosphorylation affect regulatory processes such as enzyme inhibition and activation, protein interaction recognized by domains, and protein degradation[5]. AcMNPV-P6.9, pp78/83, PP31, IE1, P10 and PP34, and BmNPV- P6.9, 39K, BRO, LEF-6, pp78/83, IE1 and PP34, have been reported to undergo phosphorylation during infection. The proteins of BV and ODV may have differing phosphorylation profiles[6,7]. Most of the proteins phosphorylated in baculovirus were found to be
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DNA-binding proteins, including P6.9, BRO protein, IE1 and pp31. P6.9 is a protamine-like DNA binding protein, which is involved in a variety of processes in
the infection cycle[8]. Phosphorylation of P6.9 has been intensively investigated and is closely related to its function. In BV, P6.9 does not appear to be phosphorylated,
-p
whereas P6.9 phosphorylation in ODV can be divided into hypophosphorylation and hyperphosphorylation, depending on the protein kinases through which
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phosphorylation was mediated. Hypophosphorylation may be dependent on host-encoded protein kinases (PK), while hyperphosphorylation of P6.9 relies on
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virus-encoded protein serine/threonine kinase (PK1)[9]. P6.9 hyperphosphorylation is a prerequisite for hyperexpression of very late genes[9]. Phosphorylation can
na
neutralize part of the positive charge of P6.9, attenuate the interaction between P6.9 and DNA, and facilitate the release of viral DNA during the nucleocapsid uncoating process. However, viral DNA assembly into nucleocapsids requires P6.9
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dephosphorylation and is mediated by a virus-encoded phosphatase 38K. 38K is a nucleocapsid-associated protein, and the absence or mutation of the HAD motif in
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38K leads to the disruption of nucleocapsid assembly [10]. Whereas the phosphorylation of P6.9 results in nucleocapsid uncoating, the dephosphorylation of P6.9 is involved in viral DNA encapsidation during nucleocapsid assembly. Phosphorylation and dephosphorylation of P6.9 occur at different stages of viral infection, dynamically regulating late gene expression and nucleocapsid assembly. In addition to phosphorylation, there were also potential methylation sites on P6.9, intricately mixed modifications remain to be elucidated.
Phosphorylation of BRO may regulate its binding activity to DNA and RNA, but it lacks experimental evidence. They could block cellular replication and/or transcription and switch host machinery to viral DNA or RNA synthesis by binding to host chromosomal DNA. BRO also undergoes ubiquitination followed by proteasome degradation, which may protect other viral proteins from degradation and increase the efficacy of infection[11]. IE1 is a phosphorylated protein that specifically binds to DNA and acts as a transcriptional regulator to activate viral gene expression[12]. Hyperphosphorylation
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of IE1 coincides exclusively with DNA replication in AcMNPV[13]. AcMNPV PP31 and BmNPV 39k are homologous. PP3l is a DNA-binding
protein and a constituent of the virogenic stroma[14]. Like P6.9, PP3l also undergoes
reversible phosphorylationa by both cell-induced and virus-encoded kinases[15]. The
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deletion of pp31 gene did not affect the replication of viral DNA, but the expression of some early and late genes were significantly down-regulated[16].
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Hyperphosphorylation of 39K plays an important role in the transcription of late genes. Knockout of BmNPV 39k leads to a decrease in BV and polyhedron
lP
production, a loss of oral infectivity, and deficiency in virogenic stroma formation [17].
na
There are other phosphorylated proteins in baculovirus, including P10, nucleocapsid protein P78/83 and PP34 but they do not appear to bind to DNA. During AcMNPV infection, the C-terminus of P10 is phosphorylated and inducing
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filamentous aggregation to form tubular structures [18]. Two types of phosphorylated P10 tubular structures have been described: a cage-like structure surrounding the
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nucleus and a filamentous structure within the nucleus [19]. Hyperexpression of P10 contributes to nuclear lysis and OB release in cultured cells. And P10 appears to be required for the formation of the polyhedron envelope (PE)[20]. Phosphorylated PP34 is also required to form polyhedron envelope(PE)[21,22]. P78/83 is a phosphorylated protein located at the end of the nucleocapsid. The genes pp78/83 and pk1 overlap and are reverse transcribed[23,24]. PK1 is also a component of a very late gene transcription complex. It binds to the promoter of polh and promotes overexpression
of late genes[25], which further explain the dual role of PK1 in promoting late genes transcription. Collectively, phosphorylation and dephosphorylation dynamically regulate the conformation and function of baculovirus proteins. It is necessary to make clear the mechanism why the phosphorylation and dephosphorylation of viral proteins are involved in the virogenic stroma formation and late genes hyperexpression, as well as protein-protein interactions.
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Glycosylation of baculovirus-encoded proteins Glycosylation usually occurs on the side chains of some amino acids and has structural diversity. N-linked glycosylation refers to the connection between the
glycan and the asparagine side chain of proteins. O-linked glycosylation is the transfer
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of a sugar chain to the oxygen atoms of serine or threonine [26].
Many viral envelope proteins are modified by glycosylation. In AcMNPV these
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include the major envelop protein GP64, ecdystero UDP-glucosyltransferase (EGT), GP41, GP37, ac23 (F-protein), cathepsin (v-CATH) and chitinase. In BmNPV,
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glycosylation has been reported in GP64, fibroblast growth factor (FGF), EGT, GP41 and v-CATH. Glycosylation in baculovirus-encoded proteins can influence their
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structures, physical and chemical properties, intracellular transport, and functions [27].
GP64 and F protein are the most well studied glycoproteins. The baculovirus
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family is subdivided into four genera: Alpbabaculovirus, Betabaculovirus Deltabaculovirus, and Gammabaculovirus. Alpbabaculovirus is divided into Group I
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and Group II. GP64 is glycosylated and is the envelope fusion protein of Group I baculovirus. In contrast, Group II baculovirus lack the gp64 gene, but employ the glycoprotein F for virion binding and membrane fusion. Glycosylation of GP64 affects BV production and membrane fusion. GP64 is a dimer connected by disulfide bonds, and the cluster formed by three histidine residues (H245, H304, and H430) located near the base of the central coiled coil act as pH sensors. Their protonation induces a low pH-induced conformational change, which mediates membrane fusion
[28]. However, the role for glycosylation sites on GP64 in conformational change remains an open question [29]. Knockout of one or more GP64 N-glycosylation sites impairs cell recognition and entry by AcMNPV BV, resulting in less infectious progeny viruses production [30]. It has also been reported that the C-terminal region of AcMNPV GP64 is fatty acid acylated and this may be involved in anchoring of GP64 in the viral envelope[31]. The F protein is cleaved by a furin-like protease to produce two disulfide-linked subunits F1 and F2[32]. Both F1 and F2 undergo N-linked glycosylation, producing N-linked glycans[33,34]. The glycosylation site on
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F protein in Helicoverpa armigera nucleopolyhedrovirus（HearNPV）is necessary for its folding and transport to the golgi apparatus[35]. No N-linked glycoprotein in the
ODV of HearNPV have been reported. All detected N-linked glycosylation occurs in BV and contain signal peptides [6]. The F protein exists as a trimer in BV. However,
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the N-linked glycosylation of the F2 subunit in HearNPV is not essential for BV
production and its fusion with host cells. The disappearance of N-linked glycans on
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the F2 subunit enhances its fusion with the membrane[33]. Not all glycosylation plays a role in promoting membrane fusion in baculovirus. Importantly, glycosylation sites,
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and N-linked glycans may play biological roles. Further study is required to explain the specific function of glycosylation sites in BV.
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EGT is a glycoprotein, and although these sugars do not appear to be required for its enzymatic activity [36]. Glycosylation of its N-terminus plays a crucial role in its secretion on of its N-terminus plays a crucial role in its secretion [37]. Although,
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AcMNPV FGF does not undergo glycosylation modification[37], BmNPV FGF is glycosylated and this processing is required for it to be secreted [38]. GP37 undergoes
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N-linked glycosylation and is associated with polyhedron [39]. GP41 is an O-glycosylated tegument protein located between the envelop and the capsid. It exists as a trimer in the BV and ODV of AcMNPV[40]. GP41 is involved in the formation of ODV morphology[41] and the budding of nucleocapsids to form BV [42]. Both baculovirus chitinase and v-CATH are glycosylated and may be involved in the release of OBs from BmNPV-infected cells[43,44]. As a summary, the glycoproteins participate in the formation of protein structures
and play a key role in the interaction with membranes, for example membrane fusion, nucleocapsid budding, the release of OB. N-linked glycosylation and protein folding are closely interrelated processes that occur in the endoplasmic reticulum. There are many enzymes mediating glycosylation in insect cells. It is unknown whether BmNPV can encode glycosyltransferases.
Ubiquitination of baculovirus-encoded proteins Ubiquitination is the covalent addition of one or more ubiquitin molecules or
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ubiquitin-like modifiers to the side chain amino acid groups of the target protein by the ubiquitin ligase [45].
Ubiquitination mostly happens in baculovirus envelop proteins and nucleocapsid proteins. The virus ubiquitin(v-Ubi) has 75% homology with ubiquitin of eukaryotic
co-expressed with nucleocapsid proteins [47].
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cells[46]. Guarino et al. showed that v-Ubi molecules of baculovirus were
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There are relatively few studies on baculovirus ubiquitination modification and viral ubiquitinated proteins are present in nucleocapsid. Compared with ODV
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nucleocapsid, BV nucleocapsid proteins are highly ubiquitinated by vUbi, and the expression level of v-Ubi in BV is about 4 times that of ODV[48].
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The ubiquitination of nucleocapsid proteins by v-Ubi is a signal determining whether the nucleocapsid will bud from the nucleus to form BV or remain in the nucleus to form ODV[48]. AC141 is an E3 ubiquitin ligase with RING-domain.
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AC141 interacts with either v-Ubi or cellular ubi and participates in ubiquitination of nucleocapsid protein AC66, which is necessary for effective BV production [48].
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However, some viral proteins inhibit ubiquitination to facilitate self-reproduction process, because the ubiquitin-proteasome pathway(UPP) is the main mechanism for protein degradation. Ac102 binds to BV/ODV-C42(C42) to suppress K48-linked ubiquitination of C42, which decreases C42 proteasomal degradation and consequently allows P78/83 to function as a stable nucleation promoting factor (NPF) to induce actin polymerization [49]. Ac102 inhibits both K48- and K63-linked ubiquitination of C42. K63-linked ubiquitination of C42 is hypothesized to modulate
P78/83-induced actin polymerization via a yet unknown mechanism (Fig.1). It was reported that many E3 ligases with RING-domains inhibit viral replication through degrading by polyubiquitination in the host. Interestingly, BmNPV also encodes 6 ring-finger proteins: IAP1, ORF35, IAP2, CG30, IE2 and PE38. Previous studies showed that IAP2, IE2 and PE38 have E3 ubiquitin ligase activity and can catalyze ubiquitination. The activity depends on the ring-finger motif[50]. As an immediate-early gene PE38 is important for the function of transcription and translation[51,52], it also interacts with proteins related to the ubiquitin-proteasome
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pathway [53], thus it may play an important role in regulating the stability and degradation of protein. This suggests that the virus may have other associated proteins
Acetylation of baculovirus-encoded proteins
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or mechanisms that evade the ubiquitin-proteasome pathway.
Acetylation modification refers to the process of adding acetyl groups to lysine
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residues under the action of acetyltransferase.
Proteomic analysis indicated that some late expression factors (LEFs) of
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BmNPV have significant levels of acetylation, including LEF-3, LEF-4, LEF-6 and LEF-11, but the role of acetylation is not clear. There is no gene encoding
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acetyltransferase in the BmNPV genome, so the acetylation of viral protein depends on the host-encoded acetyltransferase[54]. No acetylation modification has been
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reported in the study of AcMNPV.
Summary
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The PTMs of baculovirus-encoded proteins have remarkable characteristics and
are summarized in this review(Table 1 and Fig.2). Phosphorylation mostly occurs in DNA-binding and nucleocapsid proteins, involved in virogenic stroma formation and late gene hyperexpression. Glycosylation mostly occurs in the envelop proteins，and these envelope glycoproteins are essential for entry into cells. Ubiquitination are present in nucleocapsid proteins, functioning as a signal of the budding of nucleocapsid. However, there are relatively few studies on baculovirual acetylation
modification. Mass spectrometry (MS)-based proteomics provides an excellent opportunity to globally analyze proteins and their modifications[55]. The technique not only yields sequence information to identify the protein, but also reveals very precisely the site and nature of post-translational modifications. Although many potential PTM sites on baculovirus proteins have been predicted, most of them were not identified or confirmed. The number of viral proteins is limited, so a viral protein may play multiple roles after undergoing PTMs during infection. Therefore, it is valuable for us
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to develop some new tools used to distinguish the grading changes of viral PTMs and reconstruct the slight changes in the system in vitro, so as to better understand the effect of its interaction with host and regulation mechanism.
Interestingly, during baculovirus infection PTMs in host are also affected.
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Acetylation is possibly beneficial for BmN cells to resist virus proliferation[54]. Not only acetylation but also the phosphorylation of host proteins is triggered after the
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infection of BmNPV[5]. Bombyx mori mitogen-activated protein kinases (MAPKs) Bmp38 and BmS6K have conserved Thr-Glu-Tyr (TEY) and Thr-Leu-Tyr (TLY)
lP
phosphorylation motifs, respectively. Phosphorylation of BmS6K protect against BmNPV infection[56]. PTMs protect host against virus proliferation, but in this case,
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it is different. In Sf9 cells, the host homologous region-binding protein(hr1-BP) is phosphorylated and then binds to the enhancer hr1 of AcMNPV, thereby promoting transcription of early gene ie and delaying early gene 39k[57-59].
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An important characteristic of the insect-baculovirus expression system (BEVS) is that it can provide a PTM process similar to that of eukaryotes. Therefore, BEVS
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provides a reliable PTM platform for large-scale production of eukaryotic proteins[60]. Bombyx mori has a glycan synthesis pathway similar to that of mammals[41,61]. Genetic and metabolic engineering techniques have been used to reconstruct the glycosylation of insects and thus promotes the glycosylation of baculovirus-expressed proteins, making them closer to natural protein[62,63]. Thereby, it is very promising to enhance its potential use by improving the PTMs in BEVS and exploit insect host (such as silkworm) as a ideal biofactory for recombinant protein
production. Enzymes that regulate PTMs are suitable targets for drug development in the medical field. Therefore, identifying the enzymes that promote PTMs by means of genomics and proteomics is useful for the prevention and treatment of baculovirus infection. Many proteins have been observed to be modified on multiple sites and site-specific crosstalk is prevalent among different modifications[64-67]. Further experiments are needed to determine whether there are cross-talk between different
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PTMs in baculovirus.
Acknowledgments
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We sincerely extend our thanks to Prof. George Rohrmann to critically read this manuscript and provide good suggestions. This work was supported by the National Natural Science Foundation of China (grant no. 31772675 and 31972619) and Natural Science Foundation of Zhejiang Province (LZ20C170001).
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Table 1 Protein PTMs identified in AcMNPV and BmNPV PTM
protein
AcMNPV or
Function of the PTM
Peptide sequence of PTM
uncoats nucleocapsid and necessary for hyperexpression of very late genes necessary for nucleocapsid
RRRSST#TT##S TR#RRSS
BmNPV both
P78/83
both
IE1
both
activates viral gene expression
PP34
both
necessary for PE
PP31
AcMNPV
P10
AcMNPV
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P6.9
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BRO
re
necessary for virogenic stroma formation exists in two different forms and necessary for PE necessary for virogenic stroma formation promotes viral DNA or RNA synthesis
lP BmNPV
na
39K
-p
Phosphorylation
NQLNQRLpSN AQTQQISAK
BmNPV
LEF-6
BmNPV
unclear
GP64
both
unclear necessary for virus to enter cells via endocytosis
EGT
both
unclear
GP41
both
involved in the budding of nucleocapsids
RHpSNAGDVY SAQQVVHAM K STDRIpSEIGD WCR
Glycosylation
v-CATH
both
affects the correct folding of V-CATH and chitinase
chitinase
AcMNPV
unclear
AMLDQVQIQT N#R
necessary for PE
NNN#DSLSTSA QFGVNK
FGF
BmNPV
necessary for its
NVLVN#SSGV
secretion
HR
AcMNPV
necessary for effective production of BV
C42
AcMNPV
unclear
BRO
BmNPV
increases the efficacy of infection
-p
AC66
lP na ur Jo
ro of
AcMNPV
re
Ubiquitination
GP37
SUPPLEMENTAL FIGURE LEGENDS
Fig.1 Model of a regulatory cascade that controls actin polymerization during
ro of
AcMNPV infection. Ac102 inhibits both K48- and K63-linked ubiquitination of C42. K48-linked ubiquitination promotes the degradation of C42 by UPP, decreasing C42 availability. The reduced C42 levels then compromise its ability to protect P78/83
from ubiquitination-independent proteasomal degradation and eventually results in the
-p
attenuation of P78/83-induced actin polymerization. K63-linked ubiquitination of C42 is hypothesized to modulate P78/83-induced actin polymerization via a yet unknown
re
mechanism, as indicated by the question mark.(from Yongli Zhang, 2018[46] )
lP
Fig.2 The distribution of structural proteins with different PTMs. Orange diamonds represent phosphorylated proteins. Blue ellipse represents ubiquitinated
na
proteins. Black circles represent glycoproteins. The outermost blue column represents the envelop. The inner black column represents the nucleocapsid. Proteins with
Jo
ur
different modifications are placed in corresponding positions.
ro of
-p
re
lP
na
ur
Jo