The comprehensive phosphoproteome quantification has been extensively studied in several species attributable to its pivotal role leading to the altered gene expression through conformational changes in some functional proteins.
B. mori and BmNPV were a topic of intense focus among several groups due to their correlation in the interaction of insect host and baculovirus pathogen with respect to the mechanism of virus invasion of the host [
16] and the corresponding host immune system responses [
17]. The present study analyzed the impact of infection of BmNPV to the global pattern of proteomic phosphorylation in silkworm cells. However, to our knowledge, this is the first study determining the phosphoproteome of
B. mori after BmNPV infection resulting in a significant amount of candidate target phosphorylated proteins for deeper insight into the mechanism of phosphorylation to control the alterations on the host cells during viral infection.
Overall, the results showed that BmNPV infection greatly influenced the phosphorylation of diverse proteins related to several functions (Table
1). The notable alterations on the phosphorylation of proteins were involved in binding activity, protein synthesis, viral replication, and apoptosis through kinase activity and are further discussed below.
Phosphorylation may have a pivotal function on the regulation of binding activity
As shown by the GO annotation, the most significant altered phosphoproteins (65%) were found in the binding activity for molecular function, either up-regulated or down-regulated (Fig.
2c). Among those modulated proteins, 34% were involved in nucleic acid binding; 10 proteins were up-regulated and 5 proteins were down-regulated. Although the phosphorylation ratio was little, more than 1/3 of the total number of proteins in the binding activity were involved in nucleic acid binding activity, indicating that phosphorylation may have a significant role in either the DNA or RNA binding activity in the silkworm cells after BmNPV infection. For instance, DNA topoisomerase II, one of the up-regulated proteins with 1.43 ratio phosphorylation, is a well-known enzyme in the process of transcription, replication, chromatin remodeling, and segregation [
18]. This enzyme has been reported to have the ability to simplify the DNA topology by relaxing supercoiled DNA and unlinking knotted or catenated DNA which is in accordance with its physiological role in DNA replication and chromosome segregation [
19,
20]. Moreover, the enzyme is also a predominant component of the nuclear scaffold and matrix [
18,
21]. The matrix associated with the DNA regions was also reported to consist of topoisomerase II binding sites [
22]. According to a search of the BmNPV genome sequence, it does not encode an ortholog of topoisomerase II. Furthermore, a recent report has proposed a plausible hypothesis that the intertwined baculovirus DNA was decatenated by topoisomerase 2 during viral replication [
23]. Hence we hypothesize that the virus employs this enzyme for its genome replication by activating the enzyme through up-regulation. Study about topoisomerase for baculovirus is still lack. Further studies are necessitated to substantiate our hypothesis.
Another remarkable protein is the subunitssp27, a component of the upstream activation factor (UAF) complex, which interestingly has two phosphorylation sites in the position 84th and 88th of amino acid sequence with the elevation ratio 0.57- and 0.73-fold, respectively. Although in
B. mori this protein is not yet examined, in yeast
Saccharomyces cerevisiae, the upstream activation factor (UAF) is well-known as a multi-protein complex that tightly binds to the upstream element of the rDNA promoter and also strongly stimulates the transcription of RNA polymerase I (Pol I) [
24]. Subunit spp27 of this protein is a homolog from
Schizosaccharomyces pombe which has UAF-like activity [
25] such as the ability to interact with rDNA upstream sequence binding protein; Acr1, an accumulation of condensing at rDNA 1 which has been reported to be weakly similar to RRN9, a component of
Saccharomyces cereviseae UAF complex [
26]. Subunit spp27 also has been reported to have good alignment with UAF30p, another subunit of UAF complex of
S. cereviseae [
25]. Furthermore, UAF also plays a critical role in the silencing of transcription through RNA polymerase II (Pol II) by stabilizing Pol I state [
24]. The down-regulation of this protein may cause the inhibition of the silencing, which leads to the activation of transcription by Pol II. This activation of Pol II might correspond with the BmNPV-based manipulation of the host gene by miRNA that is canonically transcribed by Pol II. By encoding a miRNA, the BmNPV represses the expression of the silkworm GTP-binding nuclear protein Ran, resulting in the decrease of the host small-RNA population [
27]. The down-regulation of UAF causing all those possible consequents which are eventually leading to the alleviation of the amount of host small-RNA might be required for the enhancement of BmNPV load in the infected cells.
Another intriguing result is that thymosin isoform XI (tiXI) is an up-regulated protein with four sites exhibiting high phosphorylation ratio. Those four sites were located at position 106th, 30th, 31st, and 107th on the amino acid sequence and the phosphorylation elevation ratios compared with the control group were 1.99, 2.0, 2.9, and 1.82, respectively. Although tiXI is not widely focused, the family of this protein, thymosin beta 4 (Tβ4), has been extensively studied in several species and is known to have a pivotal role in the organization of cytoskeleton by binding or sequestering to actin monomers [
28]. This protein regulates the actin cytoskeleton dynamics through retaining a large pool of actin monomers which interact with globular actin to produce a 1:1 complex for controlling the filamentous actin assembly [
29]. Tβ4 regulates the equilibrium between the globular and filamentous actin, which is vital for rapid rearrangement of the cytoskeleton [
30]. The hyperphosphorylation of this protein after BmNPV infection might activate the cellular Tβ4 protein which is responsible to the filamentous actin to globular actin ratio alteration [
31]. This regulation appears close relation with the infection process of BmNPV. The actin and microtubule have significant roles in every virus life cycle. Viruses have ability to reorganize the cytoskeleton and restructure the host transport equipment to fulfill their needs [
32], for instance, H5N1 could manipulate and exploit diverse host cytoskeletal protein to promote their infection [
33]. AcMNPV has been reported to harness actin polymerization based motility in two infection phases. In the initial phase right after cell entry, they utilize motility for cytoplasm exploration and translocation to the nucleus through nuclear periphery for initiation the viral gene. In the next phase, after early gene expression, AcMNPV required motility to accumulate the distinct set of nucleocapsid at the tips of actin-rich surface spikes [
34]. The up-regulation occurred on four sites of this tiXI protein with a high phosphorylation ratio after BmNPV infection strongly suggested that phosphorylation might have a predominant influence on cytoskeleton change through actin binding which is important for life cycle of every virus. Further studies are highly recommended to authenticate our hypothesis.
Phosphorylation may represent an obligatory regulation in protein synthesis
Similar to other viruses, baculoviruses employ the host chaperones to assist the rapid synthesis of a large quantity of their viral proteins [
35]. Baculoviruses completely rely on the host translational equipment such as ribosomes, tRNA, amino acid metabolism and transport, chaperones for protein folding and other translation factors. The presence of AT-rich regions and unstructured 5′ untranslated (UTR) regions of the virus mRNA allow the baculovirus to compete with host mRNA for translation by its higher binding activity to host translation factor [
36]. A previous study revealed some translational factors in silkworm cells that were commonly up-regulated after BmNPV infection such as eukaryotic initiation factor (eIF3–6, eIF1A, aIF3-2b) and an elongation factor (EF1d) [
16] corresponding to the present study, resulting in the hyperphosphorylation of EF1d with fold elevation ratio 1.69. Many studies have reported that the EF1d phosphorylation could alter translational efficiency [
37‐
39]. Furthermore, in mammalian cell infected by herpes viruses, EF1d also phosphorylated by conserved protein kinases encoded by these viruses suggesting that it plays an important role of efficient replication of these viruses [
40]. The up-regulation of BmNPV EF1d suggested that this virus might employ EF1d and enhance its translational efficiency for viral replication through phosphorylation.
Another notably up-regulated phosphoprotein is ATP-dependent RNA helicase DDX10. RNA helicase is well-known as an essential factor in most of the RNA metabolism processes including ribosome biogenesis, pre-mRNA splicing and translation initiation [
41,
42]. DDX10 is categorized in the group of DEAD-box family, the largest group of helicases [
43]. Although this protein from silkworm is yet less studied, the other proteins in the family from different species have been elucidated for the correlation with the viral infection. DDX1 is the common helicase from DEAD-box family, which has been reported as a cellular co-factor for viral replication by several viruses such as coronavirus [
44], JC virus [
45], hepatitis C virus [
46], and also HIV-1 for replication and nuclear export [
47]. With a high fold elevation ratio (2.31), the hyperphosphorylation of ATP-dependent RNA helicase DDX10 protein is designated to play a major role in protein synthesis in silkworm cells after BmNPV infection. Thus, further studies are imperative to better understand the role of hyperphosphorylation in the ATP-dependent RNA helicase DDX10 for protein synthesis of
B. mori cells after BmNPV infection.
Phosphorylation may be responsible for viral replication during infection
During infection, while the host DNA replication is detained, viral replication is rapidly conducted harnessing the host replication machinery and concentrating in a virogenic stroma, which is a separate compartment within the nucleus [
48,
49]. DNA ligase 1-like with the fold elevation ratio 1.44, is one of the up-regulated proteins that is associated with the replication of DNA. DNA ligase has been highlighted as a crucial factor in the linkage of Okazaki fragment on the lagging strand [
50]. Interestingly, the Viral DNA ligase of vaccinia virus has been reported to harness cellular Topoisomerase 2 to site the viral replication and assembly [
51]. However, BmNPV apparently does not encode DNA ligase, only some baculoviruses do, such as LdMNPV (
Lymantria dispar multicapsid nuclear polyhedrosis virus), according to the BLAST result, its DNA ligase is most optimally aligned with the
B. mori DNA ligase 3 isoform X1 after other lepidopteran suggesting that the DNA ligase of LdMNPV was probably acquired from host. The fact that BmNPV genome does not have the ortholog of DNA ligase suggested that the up-regulation of cellular DNA ligase may be important either for viral replication or assembly.
The origin recognition complex subunit 2 (orc2), with a high phosphorylation ratio (5.69), is the most hyperphosphorylated protein for DNA replication and the highest up-regulated protein among all the different classes of proteins. Although the mechanism in detail remains elusive, the viral replication of BmNPV has been reported to affect the expression of
BmORCs [
52]
. In the case of human, ORC is known as important protein which is able to initiate DNA replication by facilitating the establishment of the pre-replication complex (pre-RC) at the origin of DNA replication [
53]. ORC is also known to be associated with an EBV (Epstein-Barr virus) replication origin for the propagation of its genome by utilizing antibodies against the three different subunits of human ORC in order to precipitate the cross-linked chromatin [
54]. The up-regulation of
B. mori orc2 protein after BmNPV infection was probably important for the activation of this protein. Together with DNA ligase, we speculated that they have an essential function for the viral genome replication of BmNPV. Further research is highly recommended to ascertain this hypothesis.
Phosphorylation may regulate anti-apoptotic cells through the kinases activity after BmNPV infection
Apoptosis is a genetically programmed cell death as an emergency response to a variety of stimuli such as radiation damage, aberrant growth or viral infection [
55]. Some viruses such as Oropouche virus (OROV) induce apoptosis in the cells right after their infection that causes degeneration in cells shortly after infection [
56]. However, some viruses evolve strategies to evade early apoptosis in order to avoid the elimination of progeny virus spread and the limitation of virus production [
55]. For instance, an inhibitor of apoptosis proteins (IAPs), a family of proteins which were originally discovered in baculovirus has been reported to be involved in suppressing the host cell death in response to viral infection [
57,
58]. Protein kinases C (PKC) family members are associated with an extensive range of cellular responses including cell permeability, migration, hypertrophy, proliferation, secretion, and also apoptosis. In particular, PKC cascades are apparently essential modulators of the apoptotic response [
59]. A recent study showed a high up-regulated phosphorylation of PKC in the silkworm cells after BmNPV infection. The hyperphosphorylated PKC occurred in the epsilon isoform (PKCε). Among several members of PKC protein family, some isoforms induceapoptosis, and the others inhibit it. PKCε possesses the ability to enhance tumorigenesis that can associate with the suppression of apoptosis [
60]. PKCε has also been reported to trigger cellular survival by initiating the NF-κB pathway [
61], which leads to the activation of gene transcription encoding IAPs [
62]. With PKCε high phosphorylation ratio (3.53), we strongly suggested that the hyperphosphorylation of this protein has a great implication on the survival of host cells during BmNPV infection.
The phosphorylation of BmNPV protein
All of the quantified proteins of BmNPV are up-regulated; some of them have a notable phosphorylation ratio and are even at more than one site. 39 K protein, also known as Bmorf 27, is one of the prominent hyperphosphorylated proteins of BmNPV which has 4 phosphorylated sites and one of them has the highest phosphorylation ratio among all of BmNPV phosphorylated protein and is even higher than it of
B. mori phosphorylated protein. It has been well known that 39 K promoters have important utility for insect cell engineering such as BEV system by providing the highest level of tightly regulated protein production [
63]. It also has been reported that BmNPV 39 K plays an important role in viral late gene transcription. Moreover, the deletion of this gene resulted in very poor budded virus production, low polyhedral production, oral infectivity attenuation, and deprived virogenic stroma formation [
64]. The homolog of 39 k, AcMNPV orf 36 also called as pp31 has been reported to be phosphorylated as well [
65]. Although the function of pp31 AcMNPV phosphorylation remains elusive, the great phosphorylation ratio of 39 K phosphorylation strongly suggested that phosphorylation may have important role for this protein in promoting protein production. Further study is necessary for supporting this hypothesis.
BmNPV protamine-like protein named P6.9 underwent phosphorylation during infection; it has two phosphorylated sites with phosphorylation ratio of 2.73 and 2.07, respectively. Although in BmNPV this protein has not been well assessed but in
Autographa californica multiple nucleopolyhedrovirus (AcMNPV), P6.9 has been reported to be transiently phosphorylated in infected cells prior to nucleocapsid assembly [
66]. This protein binds and condenses baculoviral DNA for packing into capsids [
67]. By the time nucleocapsids enter nuclei through nuclear pores, P6.9 is phosphorylated which could repel the negatively charged DNA, leading to the viral genome release associated with capsid and kinase [
68,
69]. A recent study indicated that during infection there are 13 Ser/Thr phosphorylation sites on P6.9 AcNMPV which interestingly 7 of those are reported to be dependent to PK1 (protein kinase 1). Moreover, replacement of those 7 Ser/Thr phosphorylated residues with Ala in P6.9 AcNMPV significantly diminishes the transcription of the very late viral genes and viral infectivity [
70]. Further research is necessary to find whether phosphorylation of P6.9 BmNPV has an important role on DNA binding activity and if PK1 also has influence to the phosphorylation of this protein.
Another remarkable protein of BmNPV is LEF-6 with 5 phosphorylated sites; the phosphorylation ratio in every site was relatively high. In AcMNPV lef-6 has been reported to show ability in accelerating the viral lifecycle and increasing virus yields [
71]. The study on this protein phosphorylation is still lacking. However, considering its 5 hyperphosphorylated sites, we strongly suggest that phosphorylation on this protein may play significant role in the viral yielding and lifecycle.