Mechanism of herpesvirus protein kinase UL13 in immune escape and viral replication

Upon infection, the herpes viruses create a cellular environment suitable for survival, but innate immunity plays a vital role in cellular resistance to viral infection. The UL13 protein of herpesviruses is conserved among all herpesviruses and is a serine/threonine protein kinase, which plays a vital role in escaping innate immunity and promoting viral replication. On the one hand, it can target various immune signaling pathways in vivo, such as the cGAS-STING pathway and the NF-κB pathway. On the other hand, it phosphorylates regulatory many cellular and viral proteins for promoting the lytic cycle. This paper reviews the research progress of the conserved herpesvirus protein kinase UL13 in immune escape and viral replication to provide a basis for elucidating the pathogenic mechanism of herpesviruses, as well as providing insights into the potential means of immune escape and viral replication of other herpesviruses that have not yet resolved the function of it.


Introduction
Herpes virus is a virus of double-stranded DNA that can be divided into three subfamilies: a-, b-, and g-herpesvirus. For instance, Herpes simplex virus type 1/2 (HSV1/2), Varicella-zoster virus (VZV), Pseudorabies virus (PRV), Epstein-Barr virus (EBV), Human cytomegalovirus (HCMV), Kaposi's sarcoma-associated herpesvirus (KSHV), Murine gamma-herpesvirus 68(MHV-68), Marek's disease virus (MDV) and Duck plague virus (DPV) (1)(2)(3). Herpes virus infections severely impact the health of humans and animals. The host's innate immune system is the first line of defense against invading pathogens, it relies on the mutual recognition of various pathogen recognition receptors (PRR) and pathogen-associated molecular patterns (PAMP) on the surface of the pathogenic organism. The interaction between PRR and PAMP on the surface of pathogenic organisms induces the production of Type I interferon (IFN-I) and other antiviral factors, promoting cellular antiviral immunity and activating the corresponding immune system (4). cyclic GMP-AMP synthase (cGAS) is a nucleotidyltransferase, as a member of the PRR family, which is activated by binding viral double-stranded DNA to induce the production of IFN-ß (5, 6). The nuclear factor kappa-B (NF-kB) regulates the production of inflammatory and immune responses to protect the host from pathogens (7,8). Similarly, the JAK-STAT signaling pathway, the PKR-eIF2a signaling pathway, the Sterile alpha motif and HD domaincontaining protein 1 (SAMHD1), and the CD8+ T cell play a critical role in antiviral response.
The HSV pUL13 and its homologs (e.g., EBV pBGLF4, HCMV pUL97, KSHV pORF36, MHV-68 pORF36, and VZV pORF47) are serine/threonine protein kinase belonging to the conserved herpesvirus protein kinase family (CHPK), which is a tegument protein of herpes virions (9-11). Their catalytic core consists of 12 conserved subdomains (12-15) ( Table 1), which can catalyze the transfer of the g-phosphate of a nucleoside triphosphate to amino acid residues of protein substrates to affect their function. CHPK from different herpesvirus subfamilies has considerable amino acid variation, and there is no consensus phosphorylation sequence for all CHPKs (16-18). Moreover, the herpesvirus protein kinases have very low homology with known cell kinases.
With the continuous discovery of UL13 protein kinase substrates (Table 2), pUL13 has been shown to play an important role in the physiological activity of the herpes virus.
For example, VZV pORF47 and KSHV pORF36 are essential for virus proliferation in T and B cells (19-21); PRV pUL13 affects IFN-b by inhibiting zinc finger CCHC-type containing protein 3 (ZCCHC3) expression (22); EBV pBGLF4 is a regulator of the EBV immune genes BCRF1 and BPLF1 (23). Moreover, HSV-2 pUL13 Ser18 was significantly crucial for the HSV-2 capacity of replication and cell-to-cell spread in U2OS cells (24); the deletion of pUL13 reduced the size and number of Viral plaques of DPV (25); CHPK of b and g herpes viruses promotes DNA virus replication by mimicking cyclindependent kinases1/2 (CDK1/2) phosphorylation of cyclin (26,27). These suggest that pUL13 plays an essential role in immune escape and viral replication of herpes viruses.
The role of pUL13 in viral evasion of innate immunity Inhibition of the cGAS/STING pathway The type I interferon pathway is a significant component of innate immunity and plays an essential role in the control and clearance of pathogens. Upon infection, inhibition of interferon regulatory factor 3 (IRF3) by viral infection is a critical link for the termination of the type I interferon pathway. Here, IRF3 is an essential target for pUL13 action during herpes virus infection because many studies have shown that they can phosphorylate IRF3 and inhibit IRF3 dimerization, binding to the positive regulatory domains III-I (PRDIII-I), and interaction with the CREB-binding protein and P300 protein (CBP/P300) (28-32). Meanwhile, Lin Lv et al. showed that PRV UL13 relies on its kinase active sites of Lys49 and Lys387 to target IRF3 and promote its ubiquitination for degradation by the proteasome (33) (Figure 1). There are no conserved amino acids among the subdomains IV, V, VIA and X. "/" indicates that there is no Conserved amino acid site in "conserved amino acid" and that the Function is not clear in "function".
In addition, pUL13 promotes the ubiquitinated degradation of immunomodulatory proteins as a necessary action affecting innate immunity. For example, PRV pUL13 recruits the E3 ligase RING-finger protein 5 (RNF5) to degrade the stimulator of interferon genes protein (STING) indirectly and also participates in ubiquitination degradation of the host protein peroxidase 1 (PRDX1) to inhibit innate immunity (34, 35) ( Figure 1). Tripartite motif (TRIM) proteins play a critical role in the antiviral host response. Based on E3 ubiquitin ligases RNF5, TRIM29 and TRIM30a are responsible for the ubiquitination degradation of STING protein; TRIM18 recruit protein phosphatase 1A (PPM1A) to dephosphorylate TANK binding kinase 1 (TBK1) to suppress the innate immune response (36, 37). We believe that the TRIM family members (such as TRIM29 and TRIM18) may be essential partners of herpesvirus pUL13 in promoting the ubiquitination degradation of host immune proteins. However, there are no reports about the interaction between TRIM family members and pUL13.

Inhibition of NF-kB pathway
After virus infection, NF-kB is activated and translocated into the nucleus, which induces an inflammatory and immune response to protect the host from the pathogen (38, 39). As a vital component of the immune system, which can be regulated by the ubiquitously expressed transcript (UXT). In 2012, Chang et al. found that EBV pBGLF4 phosphorylated UXT at the Thr3, weakening interaction with p65 to inhibit NF-kB activity (40) (Figure 1). Not only did it reveal the role of the conserved herpesvirus protein kinases in evading immune clearance by NF-kB, but it also revealed its essential for promoting the lytic cycle.
Furthermore, short interspersed elements (SINEs) are noncoding retrotransposons transcribed by RNA polymerase III (RNA Pol III), which activate antiviral NF-kB signaling through a mitochondrial antiviral signaling protein (MAVS)dependent and independent mechanism pathways (41  (Figure 1). Much more interesting is that the activated SINE ncRNA can directly interact with RNA pol II to participate in the transcriptional suppression of genes (44, 45). By and large, the B2 SINEs seem to do more harm than good for viral replication. Nevertheless, the herpes virus pUL13 chose it, demonstrating that B2 SINEs have many potential mechanisms to be developed in the life cycle of herpes viruses.
Inhibition of the JAK/STAT signaling pathway JAK/STAT acts as an inflammatory signaling pathway for stress and has immunomodulatory effects, receiving multiple cytokine signals from cells, such as IFN-a and IFN-g (46, 47). In 2017, Yuka Sato et al. reported that pUL13 could phosphorylate the associated constitutive transcription factor SP1 (SP1) to induce suppressor of cytokine signaling 3(SOCS3) production, which regulates the JAK/STAT signaling pathway negatively (48) (Figure 2). That is SP1 can combine with GC-rich regions of the SOCS3 promoter to facilitate transcription and translation of SOCS3 (49, 50), and then curb the JAK/STAT signal pathway (51-53). Moreover, the phosphorylation of Sp1 by pUL13 could specifically induce the transcription of the immediate-early and early genes expression of the herpes virus (54-57), which reveals the importance of pUL13 for transcriptional regulation of herpesvirus genes.
SOCS3 plays a significant role in modulating the outcome of infections and autoimmune diseases. And many viruses, such as HSV-1, EBV, and VZV (58-61), can activate the expression of SOCS3 because of the close relationship between SOCS3 and JAK kinase with STAT signaling factors (62-65). It was suggested that SOCS3 is induced that not only inhibits the antiviral response of the JAK-STAT signal pathway but also maintains immune homeostasis in the body under pathological conditions and physiological conditions (66), such as the expression of SOCS3 inhibits several NF-kB-regulated proapoptotic pathways to protect b-cells from IL-1 bmediated apoptosis (67). Perhaps this is more important for the production of SOCS3 induced by pUL13 during herpesvirus infection.

Effect on PKR-eIF2a-mediated antiviral effects
Protein kinase R (PKR) in host cells exerts antiviral effects by inhibiting viral mRNA translation and inducing apoptosis. Many data indicate PKR promotes NF-kB activation (68-73), promotes mRNA stability of IFN-b (74), and is involved in the tumor suppressor function of p53 protein (75-77). When dsRNA binds to the Conserved double-stranded RNA binding motif (dsRBMs) of PKR, it is activated by autophosphorylation at Thr446 (78). Next, it phosphorylates Ser 51 of eukaryotic initiation factor 2a (eIF2a) and inhibits the translation initiation activity of mRNAs which encode antiviral factors and mediate stress responses (79, 80). In the PKR-eIF2a pathway, PKR inhibition and eIF2a dephosphorylation must be used to achieve massive replication of the virus, so the virus has evolved a variety of strategies in regulating the PKR-eIF2a pathway: controlling dsRNA masking and degradation (81-84), PKR degradation (85), inhibiting PKR dimerization and autophosphorylation (86-89), dephosphorylation of eIF2a (90-92), and PKR desensitization (93, 94). In 2020, Rosamaria Pennisi et al. demonstrated that HSV-1 pUL13 inhibits the phosphorylation of cellular PKR. Although the specific pathway by which pUL13 inhibits PKR phosphorylation cannot be demonstrated (95) (Figure 2). These suggest that pUL13 inhibition of PKR can not only evade innate immunity and prevent PKR-mediated apoptosis but also use eIF2a to promote viral mRNA translation. pUL13 inhibits the cGAS-STING signaling pathway and the NF-kB signaling pathway. pUL13 can inhibit the dimerization, nuclear translocation, CBP binding, and binding to IFN-b promoter elements of interferon regulatory factor 3 (IRF3); meanwhile, it can ubiquitinally degrade stimulator of interferon genes (STING), IRF-3, and peroxidase 1 (PRDX1) to inhibit the production of IFN-b. pUL13 indirectly inhibits the NF-kB pathway by regulating ubiquitously expressed transcript (UXT), Protein kinase R (PKR), and B2 SINE ncRNA.
Especially, PKR is one of four kinases that integrate stress responses. It regulates the protein homeostasis of the cell to maintain the body's homeostasis; conversely, its abnormal activation can cause severe damage to the body, such as systemic lupus erythematosus (96, 97). Based on these results, whether the molecular mechanism of pUL13 inhibition of PKR can inspire treating diseases associated with abnormal activation of PKR remains to be further studied.

Effect on CD8+ T cells mediated antiviral effects
Compared with pICP47 and pUS3 recognizing the main histocompatibility complex (MHC I) that are distributed on the cell surface and presentation of antigen peptides to T cells to exert cellular immune clearance regulation, the effect of pUL13 on it is not apparent (98-101). However, HSV-1 pUL13 triggered viral encephalitis in mice by downregulating CXCL-9 and inhibiting the infiltration of CD8+ T cell molecules at the site of infection (102) (Figure 2). The author also points out that the HSV-1 pUL13-mediated immune evasion mechanism might be specific to the CNS. Maybe it associated with CXCL-9/10 and CD8+ T cells inhibiting the reactivation of HSV within nerve cells, further suggesting the role of pUL13 in the latent reactivation of the herpes virus (103-106). Although the molecular mechanisms underlying the downregulation of CXCL-9 by pUL13 are unclear, it is suggested that inhibition of pUL13 has a potential effect in treating encephalitis of the central nervous system caused by HSV-1 infection (107-110).

Inhibition of SAMHD1
SAMHD1 is an antiviral host limiting factor (111-116), and the virus has adopted a variety of strategies to inhibit its dNTP pUL13 inhibits PKR, CD8+ T cells, and the JAK-STAT signaling pathway to evade innate immunity. pUL13 inhibits PKR phosphorylation to evade innate immunity and promotes viral protein translation with eukaryotic initiation factor 2a (eIF2a); pUL13 phosphorylates the associated constitutive transcription factor SP1 (SP1), which induces suppressor of cytokine signaling 3 (SOCS3) expression to inhibit the JAK-STAT signaling pathway; and pUL13 can down-regulate the C-X-C motif chemokine ligand 9 (CXCL9) signaling molecules to prevent cd8+ T cell molecules from clustering at the site of infection.
enzyme activity, such as HIV-2 and SIV virus-encoded Vpx proteins, to induce SAMHD1 degradation and promote selfreplication (117)(118)(119)(120); Ribonucleotide reductase (RNR) (121) and thymidine kinase (TK) (122,123) encoded by DNA viruses can antagonize SAMHD1's dNTP enzyme activity, providing the necessary substrate for viral DNA polymerase; The intracellular CyclinA2/CDK1/CDK2 complex regulates phosphorylation of SAMHD1 Thr592 (124), and phosphorylation of Thr592 has been shown to reduce SAMHD1 antiviral activity (125), echoing IFN-I-induced dephosphorylation of SAMHD1 Thr592 (126). It has been also reported that pUL13 of the b and g herpes virus participates in phosphorylation of SAMHD1 T592, inhibiting the dNTP enzyme activity of SAMHD1 from ensuring adequate intracellular levels of dNTPs for viral replication (127,128) (Figure 3). SAMHD1 can inhibit the excessive immune and inflammatory response, possibly proving why VZV and KSHV proliferate in lymphocytes requiring pORF47 and pORF36 (129). However, whether and how pUL13 can phosphorylate SMADH1 to coordinate the immune and inflammatory response remains to be studied.
It is revealed here that pUL13 plays an essential role in inhibiting various antiviral factors from escaping innate immunity. Additionally, pUL13 also plays an important role in viral replication, latent infection, and other critical physiological activities.
The role of pUL13 in promoting viral replication pUL13 phosphorylates H2AX to promote viral replication DNA-damage response (DDR) is a mechanism by which cells protect themselves through DNA damage repair and apoptosis to resist DNA damage induced by various factors (130, 131). Micah A. Luftig has discussed the interrelationship between viruses and DDR, noting that DNA viruses require DDR activation for replication (132). The research shows that the viral infection process acts on the different nodes of the DNA damage response pathway. For example, HSV-1 infections activate ataxia telangiectasia mutated (ATM) kinase activity but inhibit the role of ataxia telangiectasia-and Rad3-related protein (ATR); EBV virus infection activates upstream regulators of the DDR pathway in the DDR pathway-histone acetyltransferase TIP60 (133)(134)(135).
H2A histone family member X (H2AX) is a substrate of ATM, ATR, and DNA-dependent protein kinase catalytic subunits in phosphatidylinositol 3-kinase-like protein kinase family (PIKKs) (136-140); it is also a substrate for pUL13 (141, 142). In H2AX knockdown cells, the replication capacity of MHV-68 and KSHV are significantly abating (143, 144), and the date of EBV pBGLF4, PRV pUL13 what suggesting that pUL13 phosphorylate H2AX to activate DDR for viral replication (145,146). Still, VZV pORF47 cannot phosphorylate H2AX and indicates the difference in the members of the CHPKs (147). An attractive hypothesis is that replication of viral DNA requires or is enhanced by the cellular DNA damage machinery (133,(148)(149)(150) (Figure 4). Generally, more evidence is needed to support whether pUL13 of the herpes virus plays a vital role in this matter.
pUL13 phosphorylates EF-1б to promote viral replication Herpesvirus pUL13 can promote host cell synthesis of proteins, such as the KSHV pORF36 mimicking cellular protein S6 kinase (S6KB1) to promote cell proliferation (151). Similarly, as a substrate of pUL13, the translation extension factor -1б (EF-1б) exists in two forms in the normal state of hypophosphorylation and hyperphosphorylation, involved in the process of mRNA translation into peptide chain extension. EF-1б is mainly present in the hyperphosphorylated form in HSV-1-infected cells. Because HSV-1 pUL13, HCMV pUL97, EBV pBGLF4, and intracellular cycle-dependent kinase cdc2 are involved in EF-1б's hyperphosphorylation and work together on its Ser 133 (152)(153)(154). It shows that UL13 can synthesize its viral protein using EF-1б.
pUL13 works with SUMO proteins to promote viral replication Small Ubiquitin-related Modifier (SUMO) is a posttranslational modifier protein. The SUMO system is essential in herpes virus replication, such as KSHV replication and transcription activator (K-Rta) and HSV-1 ICP0 degrade SUMO-modified promyelocytic leukemia-nuclear bodies (PML-NBs) (155,156), inhibition of the NF-kB signaling pathway (157) and participation in degradation of IRF-3 and IRF-7 (158)(159)(160). KSHV basic region-leucine zipper (K-bZIP) is a potent transcriptional repressor that binds directly to K-Rta and attenuates K-Rta-mediated trans-activation activity, relying on SUMO modifications to regulate viral and host gene expression (161,162). Studies have shown that KSHV ORF36 phosphorylates Thr111 of K-bZIP and inhibits the SUMO level of bZIP, causing a decrease in transcriptional inhibition activity (163) (Figure 5), and appears to cooperate with K-Rta inhibition of K-bZIP to promote viral transcriptional expression (164); Also involved in the phosphorylation of the cell chromatin remodeling molecule KAP-1 inhibits SUMO level and thus inhibits chromosomal remodeling capacity (165). It is also reflected in the EBV pBGLF4 negatively regulating SUMOmodified Zta to promote the establishment of viral latency (166, 167). It suggests that although the protein kinase of the g -herpes virus cannot be modified by SUMO, its phosphorylation and SUMO can cooperate to promote viral replication.
pUL13 promotes viral replication in conjunction with ICP22 and VP22 The interaction between herpesvirus protein kinase and viral proteins to promote its replication is a complex network, such as the interaction of KSHV pORF36 and pORF45 (168), HSV-1 pUL13 and pUL41 (169). As early as 1993, Purves reported that pUL13 phosphorylation modulated ICP22 to stabilize to increase transcription of specific subpopulations of viral RNA and accumulate corresponding viral proteins (170). Subsequently, it was found that ICP22 and pUL13 were jointly involved in phosphorylation of RNA Pol II, mediating the degradation of cyclins A and B1 and activating cdc2, in which activated cdc2 and viral DNA synthesis factor pUL42 formed a complex to recruit topoisomerase II to promote the expression of advanced genes (171-179), indicating that ICP22 and pUL13 were necessary for early gene expression of herpes virus.
In HSV-1-infected cells, UL13 protein kinase promotes the dissociation of VP22 from virions and phosphorylate VP22 (169,180). VP22 released into cells can interact with Templateactivating factor I (TAF-1) proteins and histone H4 (Histone H4), inhibit the assembly of nucleosomes on DNA and H4 histone acetylation and participate in chromatin recombination, pUL13 phosphorylated the antiviral factor SAMDH1 Thr592 to promote viral replication. Inhibiting the dNTP enzyme activity of the sterile alpha motif and HD domain-containing protein 1 (SAMHD1) from ensuring adequate intracellular levels of dNTPs for viral replication.
cell cycle control, and gene regulation (181,182). The expression of VP22 can also inhibit cGAS activity and affect natural immunity (183). It can be seen that pUL13 can promote viral replication by regulating the ICP22 and VP22 proteins and collaborating.
pUL13 is involved in multiple processes of herpes virus replication, including gene replication, transcription, and translation of viruses (184); pUL13 in herpesvirus can destroy LaminA/C to promote capsid exodus from the nucleus (185)(186)(187); assembly, maturation, and release of virions (188). It is meaningful to construct pUL13 protein interaction networks to understand better the function of UL13 protein kinases in the life cycle of the herpes virus.

The role of pUL13 in latent infection
Induction and escape of herpesvirus genomic silencing is a biological marker of the herpes virus. Many reports suggest that pUL13 may play an essential role in the latent infection of the herpes virus. Firstly, Jolien Van Cleemput's study found that pUL13 may be indirectly involved in the latent infection reactivation of a herpesvirus by phosphorylating other cortical proteins (189); Secondly, in g herpes virus, EBV pBGLF4 and KSHV pORF36 are closely associated with latent infectionrelated proteins as such Rta, Zta, the latency-associated nuclear antigen (LANA), and TAT interacting protein 60 kD (TIP60) (190)(191)(192)(193)(194)(195); Lastly, MHV-68 pORF36 inhibits the antiviral effects of bone marrow-specific STAT1 expression and promotes the establishment of latent infection of MHV-68 in spleen B cells (196). In addition, herpesvirus CHPKs can also use CD8+ T cells and many host proteins (UXT, H2AX, small ubiquitin-related modification regulatory proteins) to promote the establishment of latent infections (197)(198)(199). Although the complex mechanism of establishment and reactivation of herpes virus latent infection is unknown, UL13 protein kinase will be an essential breakthrough for the follow-up study of latent infection of herpes virus. pUL13 phosphorylates H2AX to promote viral replication. The conserved herpesvirus Protein kinase pUL13 regulates DNA damage marker H2A histone family member X (H2AX), and pUL13-mediated H2AX phosphorylation plays a pivotal role in efficient virus replication and progeny production.

Summary and prospect
pUL13 acts as a serine/threonine kinase encoded by the herpes virus. It is retained in the continuous natural screening of the virus and plays a vital role in the physiological activity of the herpes virus.
In terms of immune escape, to evade innate immune defense line and persist in host cells, pUL13 and its homologs directly or indirectly play a role in signaling pathways, which acts on different immunoregulatory proteins and many antiviral factors. Then pUL13 use varieties transcription factors and translation factors in host cells to assist the lytic cycle, such as EF-1б, H2AX, SP1, embodied in lacking pUL13 will lead to the weakening of the replication ability and virulence of the virus. At the same time, herpesvirus can use pUL13 to assist in the establishment and reactivation of its latent infection.
pUL13 can phosphorylate many protein targets and participate in the activation and inhibition of related protein functions. It is similar to a switch in the life cycle of the herpes virus. It is committed to building a systematic protein interaction network diagram of pUL13, which is conducive to unveiling pUL13 in the life cycle of the herpes virus.
Herpesvirus pUL13 is an important target for developing anti-herpesvirus drugs. With the initial clinical application of GCV (200), followed by the anti-herpesvirus trials of compounds such as Maribavir (201), K252A (202), ISIS 1082 (203, 204), and 17-DMAG (205), as well as the continuous innovation of UL13 gene deletion vaccine (206,207) and immunotherapy (208)(209)(210). However, given that low homology among different CHPK members complicates the development of compounds targeting an entire group, further development of more broad-spectrum, efficient and safe herpesvirus protein kinase inhibitors for the treatment of herpesvirus is needed.
pUL13 undertakes a variety of functions in the life cycle of herpes virus, and exploring the mechanism of action of pUL13 can not only solve the problem of infection, transmission, and immune escape mechanism of herpes virus but also provide a theoretical basis for the research and development of clinical drugs for the anti-herpes virus. pUL13 works with SUMO proteins to promote viral replication. Gammaherpesvirus protein kinase pORF36 interacts with histone deacetylase 1 and 2 (HADC1/2) and prevents the association of these HDACs with the viral promoter driving expression of KSHV replication and transcription activator (K-Rta). pORF36 phosphorylates Thr111 of KSHV basic region-leucine zipper (K-bZIP) and inhibits the SUMO level of K-bZIP to repress the transcriptional inhibition activity.

Author contributions
LZ and AC contributed to the design of the manuscript. XO, DS, SM, JH, QY, YW, SC, SZ, and DZ provided ideas contributing to the conception of this manuscript. RJ, ML, X-XZ, QG, and BT helped to create the figures. MW modified the manuscript. All the authors reviewed the manuscript. All authors contributed to the article and approved the submitted version.