Viral pseudo-enzyme facilitates KSHV lytic replication via suppressing PFAS-mediated RTA deamidation

doi:10.21203/rs.3.rs-4860884/v1

Download PDF

Research Article

Viral pseudo-enzyme facilitates KSHV lytic replication via suppressing PFAS-mediated RTA deamidation

https://doi.org/10.21203/rs.3.rs-4860884/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Background

Deamidation, a type of post-translational modification commonly considered a hallmark of protein “aging” and function decay, is increasingly recognized for its pivotal role in regulating biological processes and viral infection. Our previous study has demonstrated that the deamidation of replication and transcription activator (RTA), a master regulator of ubiquitous and oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV), mediated by phosphoribosylformylglycinamidine synthetase (PFAS), hinders its nuclear import and transcriptional activity.

Results

Here we report that the viral glutamine amidotransferase (vGAT) pseudo-enzyme was exploited to promote KSHV lytic infection through inhibition of PFAS-mediated RTA deamidation, RTA nuclear localization, and NF-κB activation. To be more specific, vGAT competitively interacted with RTA and cellular PFAS, thereby suppressing the deamidation and nuclear localization of RTA, while concomitantly augmenting RTA-mediated transcriptional activation. Consistently, the RTA deamidation-resistant mutant demonstrated elevated levels of transcriptional activity and lytic replication. Lastly, vGAT appeared to regulate the deamidation process of multiple viral proteins in KSHV.

Conclusions

Collectively, these findings unveil that a viral pseudo-enzyme was exploited to enhance viral infection via deamidation regulation.

KSHV

vGAT

RTA

deamidation

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic gamma-herpesvirus intimately involved in human diseases, with a life cycle that consists of a latent infection phase and a lytic replication phase [1–3]. During KSHV lytic replication, viral open reading frames (ORFs), such as polyadenylated nuclear (PAN), ORF45, ORF50, ORFK8, ORFK9, ORF57, ORF59, ORF25, and ORF65, are expressed in a cascade manner [3–5]. As an immediate-early gene product, replication and transcription activator (RTA), encoded by ORF50, initiates the expression of multiple genes associated with replication and pathogenesis of gamma-herpesvirus like KSHV, as well as mediating the switch of the viral cycle from latency to lytic replication [6]. Besides transcriptional activation, RTA also regulates cytokine activity and modulates immunity. For example, RTA facilitates ubiquitination and degradation of interferon regulatory factor (IRF), thereby perturbing the production of type Ⅰ interferons (IFNs) [7]. RTA also interacts with myeloid differentiation factor 88 (MyD88) and degrades MyD88 via the ubiquitin-proteasome pathway, eliciting blockade of toll-like receptor (TLR) signaling and associated antiviral dysfunction [8].

Protein function is generally regulated by a variety of post-translational modifications, such as acetylation, methylation, ubiquitination, and deamidation. Extensive studies have established potential relationships between gain- or loss- of-function and RTA modifications. For example, manipulated N⁶-adenosine methylation (m⁶A) mediates the splicing of the pre-mRNA encoding the RTA, thereby modulating KSHV lytic replication [9]. Besides, RTA promoter histone acetylation rather than demethylation regulates murine gamma-herpesvirus 68 (MHV68) reactivation [10]. Deubiquitination and stabilization of RTA by recruiting host deubiquitinases (OTUD4 and USP7) facilitate KSHV lytic reactivation [11]. Conversely, ubiquitination and degradation of RTA by interacting with RNF213 inhibits the de novo infection and lytic reactivation of KSHV [12]. Even though deamidation of proteins is no longer an emerging topic, it remains widely recognized as protein “aging” or functional decay, and our insight into the exact functions of deamidation remains confined [13]. To the best of our knowledge, no attention has been focused on the modulation of RTA function by deamidation beyond our previous study [14].

Our previous study has illustrated that phosphoribosylformylglycinamidine synthetase (PFAS) deamidates RTA and diminishes RTA nuclear localization and transcriptional activation, thereby inhibiting KSHV lytic replication [14]. As a member of the glutamine amidotransferase (GAT) family, PFAS is generally recognized as a deamidase widely involved in the biosynthesis of nucleotides, amino acids, and lipids [15]. Interestingly, the gamma-herpesvirus genome also encodes viral GAT (vGAT), sharing homology with cellular PFAS but deficient in intrinsic amidotransferase activity. As a pseudo-enzyme, vGAT exerts crucial roles in mediating innate immune evasion of gamma-herpesviruses. It has been demonstrated that vGAT is exploited to recruit cellular PFAS to deamidate retinoic acid-induced gene I (RIG-I) and negate cytokine production during MHV68 infection [13].

Herein, we report that vGAT was exploited to promote KSHV lytic infection by suppressing PFAS-mediated RTA deamidation. Mechanistically, vGAT competitively interacts with RTA and cellular PFAS, thus enhancing RTA-mediated transcriptional activation and expression of viral ORFs, promoting RTA nuclear localization, and reinforcing RTA-mediated repression of nuclear factor-kappa B (NF-κB) signaling, collectively contributing to viral lytic replication (Fig. 7).

vGAT competitively interacts with RTA and cellular PFAS

We previously reported that PFAS interacts with and deamidates RTA [14]. Given that vGAT is a glutamine amidotransferase homologous to cellular PFAS, we thus speculated that vGAT might competitively interact with RTA and cellular PFAS, and regulate the deamidation of RTA. Gel filtration also hinted at potential relationships among PFAS, RTA, and vGAT (Fig. 1A). To validate this hypothesis, we first checked the interaction of vGAT with its cellular counterpart, PFAS. Interestingly, as a pseudo-enzyme, vGAT does not possess intrinsic enzyme activity but interacted with PFAS (Fig. 1B), indicating a potential role in modulating PFAS-mediated RTA deamidation. The interaction between vGAT and RTA was then explored. In transfected 293T cells, vGAT co-precipitated with exogenous RTA (Fig. 1C), and in reactivated iSLK/iBAC16 cells, vGAT co-precipitated with endogenous RTA (Fig. 1D). Moreover, an interaction among endogenously expressed vGAT, PFAS, and RTA was detected (Fig. 1E). Together, these results suggested that vGAT competitively interacts with RTA and cellular PFAS.

vGAT inhibits the deamidation of RTA

Since we have confirmed the interactions among vGAT, PFAS, and RTA, to further verify the above hypothesis, we investigated the effect of vGAT on RTA deamidation. Reporter assays indicated that vGAT facilitated the RTA-mediated transcriptional activation of pPAN, pORF57, and pvIL-6 promoters in a dose-dependent manner (Fig. 2A). Next, we overexpressed vGAT and monitored RTA charge status. Overexpression of vGAT shifted RTA-WT toward the negative pole (Fig. 2B), suggesting a charge shift induced by inhibition of RTA deamidation. Moreover, vGAT failed to further shift RTA-DD (Fig. 2B). To determine the site(s) of deamidation, RTA was purified from transfected 293T cells without or with vGAT and analyzed by tandem mass spectrometry (MS) (Fig. 2C). Two peptides were determined that contained deamidated residues of N37 and N225, which were specifically repressed by vGAT (Fig. 2D). Together, these results suggested that vGAT inhibited the deamidation of RTA, thereby suppressing RTA-mediated transcriptional activation.

Deamidation impairs RTA-mediated transcriptional activation and expression of viral ORFs

To further investigate the impact of RTA deamidation during KSHV infection, deamidated RTA mutants (RTA-DD) were constructed and introduced. Reporter assays showed that RTA-DD greatly impaired the transcription of pORF57 and pvIL-6, and completely failed to activate the pPAN compared to RTA-WT (Fig. 3A). In addition, iSLK.RTA.219 system was introduced (Fig. 3B), which induces KSHV reactivation by doxycycline (doxy) and indicates viral latent infection and lytic replication with green and red fluorescence signals, respectively. Interestingly, despite the higher expression of RTA-DD than RTA-WT with the corresponding dose of doxycycline (Fig. 3D), the lytic replication of KSHV.219 was ~ 1.5- to 3-fold significantly lower in iSLK.DD.219 cells than that in iSLK.WT.219 cells, as indicated by normalized fluorescence intensity (Fig. 3C). The expression of PAN, ORF57, and ORF59 in iSLK.DD.219 cells was ~ 5- to 35-fold significantly lower than that in iSLK.WT.219 cells (Fig. 3E). Moreover, viral genome level was ~ 1.5- to 3-fold significantly lower in iSLK.DD.219 cells than that in iSLK.WT.219 cells (Fig. 3F). Notably, the above phenotypes of RTA-DD may not be adequately demonstrated since KSHV genome-expressed RTA-WT could compensate for RTA-DD-induced loss of function. Nevertheless, these results illustrate that deamidation impaired RTA-mediated transcriptional activation and expression of viral ORFs during KSHV lytic replication.

Inhibition of deamidation promotes RTA-mediated transcriptional activation and expression of viral ORFs

Our previous study has confirmed that N to Q mutations reinforce RTA resistance to PFAS-mediated deamidation [14]. Deamidation-resistant RTA mutants (RTA-N37Q) were then utilized to probe the effects of RTA deamidation during KSHV infection from a reverse perspective. As expected, RTA-N37Q significantly induced transcriptional activation at all promoters compared to RTA-WT (Fig. 4A). In addition, HOK.iBAC.RTA system was introduced (Fig. 4B). Interestingly, despite the lower expression of RTA-N37Q than RTA-WT (Fig. 4D), infectious virions in the supernatant of SLK.iBAC.N37Q cells were higher than that of HOK.iBAC.WT cells (Fig. 4C). The expression of PAN, ORF25, and ORF59 in HOK.iBAC.N37Q cells was one to two orders of magnitude higher than that in HOK.iBAC.WT cells (Fig. 4E). Furthermore, the viral genome level of HOK.iBAC.N37Q cells was ~ 5-fold significantly higher than that of HOK.iBAC.WT cells (Fig. 4F). Together, these results demonstrated in reverse that deamidation impaired RTA-mediated transcriptional activation and expression of viral ORFs during KSHV lytic replication.

Deamidation blocks the nuclear translocation of RTA

We previously reported that RIG-I bound RTA and blocked its nuclear translocation, thus repressing viral replication [16]. It was then hypothesized that deamidation might block the nuclear translocation of RTA, thus impairing RTA-mediated transcriptional activation. According to subcellular fractionation, ~ 90% of RTA-WT was localized in the nucleus, whereas only ~ 10% of RTA-DD was localized in the nucleus (Fig. 5A). In addition, RTA was predominantly localized in the nucleus at the early stage of induction, whereas extensively retained in the cytoplasm at 72 h after induction (Fig. 5B), indicating that endogenous deamidation during KSHV reactivation might block the nuclear translocation of RTA. Consistently, immunofluorescence microscopy presented intuitive results (Fig. 5C). Together, these results demonstrated that deamidation blocked the nuclear translocation of RTA.

Deamidation alleviates RTA-mediated suppression of NF-κB signaling

NF-κB signaling is a canonical pro-inflammatory pathway, whereas IFN signaling is a classical antiviral pathway. We then investigated the impact of RTA deamidation on NF-κB and IFN signaling. Immunoblotting analysis showed that deamidation alleviated RTA-induced reduction in RelA, IRF3, and IRF7 protein levels (Fig. 5D, F). Reporter assays indicated that deamidation alleviated the RTA-induced repression of NF-κB promoter transcriptional activation (Fig. 5F, G). Together, these results demonstrated that deamidation alleviated RTA-mediated suppression of NF-κB signaling.

vGAT inhibits the deamidation of KSHV lytic ORFs

Lastly, we overexpressed vGAT and monitored viral proteins, including ORF45, ORF59, ORFK9, and ORFK8 charge status. Overexpression of vGAT shifted ORF45, ORF59, and K9 toward the negative pole (Fig. 6A-C), suggesting a charge shift induced by inhibition of KSHV lytic ORFs deamidation. However, vGAT appeared to fail to alter the charge of ORFK8 (Fig. 6D). Together, these results demonstrated that besides RTA, vGAT might also inhibited the deamidation of some other lytic ORFs of KSHV.

PFAS is a highly conserved enzyme belonging to the GAT family, and catalyzes the purine synthesis pathway [13]. Several studies have demonstrated that PFAS mediates the deamidation of RIG-I, RTA, and Phosphoglycerate dehydrogenase (PHGDH) [13–15]. Although traditionally recognized as a signature of protein “aging” and functional decay, emerging evidence has been proposed to illustrate that deamidation regulates host antiviral immunity during pathogenic infection [17, 18]. For example, deamination of RIG-I and cGAS mediated by the herpes simplex virus 1 (HSV-1) UL37 tegument protein arrests downstream innate immune activation [19, 20]. CTP synthase 1 (CTPS1) deamidates IRF3 to mute IFN induction [21]. The initiation of glycolysis and the shutdown of inflammation mediated by deamination of RelA, a subunit of NF-κB, facilitates viral replication and persistent infection [22–24]. vGAT, homologous to cellular PFAS, is regarded as a viral pseudo-enzyme due to the absent catalytic activity [13]. Several studies have demonstrated that pseudo-enzymes are widely involved in viral infection and host immunity [25]. For example, vaccinia virus B1 kinase and B12 pseudo-kinase constitute a paired kinase/pseudo-kinase system to regulate the phosphorylation of cellular barrier to autointegration factor (BAF) and related viral DNA replication [26–28]. The competition between cellular superoxide dismutase (SOD) and viral pseudo-dismutase impairs cellular dismutase activity, causing superoxide overload, which represses Fas-mediated apoptosis and encourages the proliferation of infected cells [29]. Our previous study has also revealed that MHV68 vGAT binds and recruits cellular PFAS to trigger the deamidation and subsequent activation of RIG-I [13]. In the current study, the interaction of KSHV vGAT with RTA and cellular PFAS was defined, which diminishes PFAS-mediated deamidation. Since PFAS interacts with and deamidates RTA [14], the competitive binding of vGAT with RTA and PFAS might account for the attenuated deamidation of PFAS.

RTA, the initiator of the lytic replication program of gamma herpesviruses, determines the fate of the infected cell. Accumulating studies have pointed to RTA as a critical regulatory node in the process of gamma herpesvirus infection. For example, RTA interacts with and degrades inhibitor of DNA binding protein 2 (ID2), structural maintenance of chromosome (SMC) complex 5/6, and tripartite motif 32 (TRIM32) via hijacking the ubiquitin-proteasome system, allowing for the reactivation of KSHV lytic replication and virion production [30–32]. RTA binds and recruits the E3 ubiquitin-protein ligase RNF20/40 complex for the persistent expression of RTA, thus pushing forward the lytic cycle and the transactivation of viral genes [33]. The function of proteins is generally subject to an array of post-translational modifications, which is also the case for RTA. Extensive studies have defined that post-translational modifications regulate the gain or loss of RTA function. For example, KSHV RTA-induced m⁶A mediates its pre-mRNA splicing, thus facilitating viral lytic replication [9]. Histone acetylation modification at RTA promoter strongly reactivated MHV-68 from latency [10], and SUMOylation increases the transactivation activity of RTA [34]. In the current study, deamidation impaired RTA-mediated transcriptional expression and activation of viral ORFs, blocked RTA nuclear translocation, and thus inactivated KSHV lytic replication. Inhibition of deamidation, in contrast, yields the opposite result. This result was consistent with our previous findings [14], indicating that deamidation modification resulted in loss of function, whereas inhibition of deamidation led to gain of function. Notably, deamidation also alleviated RTA-suppressed NF-κB signaling, which inhibits gamma-herpesvirus lytic replication [35]. However, deamidation failed to attenuate RTA-mediated depression of IFN signaling. Hence, RTA deamidation might regulate host antiviral immunity only to a certain extent during KSHV infection.

In the current study, it was identified that vGAT might inhibit the deamidation of other viral ORFs, such as ORF45, ORF59, and ORFK9. ORF45 is a multifunctional immediate early and tegument protein of KSHV that modulates host immune responses and remodels signaling through phosphorylation, SUMOylation, and ubiquitination [36–39]. ORF59 is the KSHV DNA polymerase processivity factor that is vital for viral reactivation and lytic replication. Several studies have revealed that ORF59 binds to oriLyt and mediates KSHV DNA synthesis in an RTA-dependent [40, 41], and the interaction between ORF59 and RTA is regulated by phosphorylation [42]. ORFK9 encodes viral IRF1 (vIRF1), sharing homology with cellular IRF1, involved in the modulation of host immunity. The acetylation of vIRF1 could suppress host IFN signaling [43], and the inhibition of vIRF1-induced p53 acetylation and Hdm2 phosphorylation could impair the cellular p53-mediated antiviral response [44]. It was speculated that the inhibition of the deamidation of these viral ORFs by vGAT might also facilitate KSHV lytic replication. However, further investigations are warranted to validate this hypothesis.

In the current study, we report that vGAT was exploited to promote KSHV lytic infection by suppressing PFAS-mediated RTA deamidation. Mechanistically, vGAT competitively interacted with RTA and cellular PFAS, thus suppressing PFAS-mediated RTA deamidation (Fig. 7). Inhibition of deamidation enhanced RTA-mediated transcriptional activation and expression of viral ORFs, promoted RTA nuclear localization, and reinforced RTA-mediated suppression of NF-κB signaling, thus facilitating viral lytic replication (Fig. 7). Collectively, our work may provide valuable insights into potential strategies for tackling KSHV infection.

Methods

Detailed methods (Additional file 1) and reagent information (Additional file 2: Table S1) are available in the Supplementary Information.

Cell maintenance and stable cell line generation

HEK293T and SLK cells were maintained in DMEM supplemented with FBS (10%), penicillin (100 U/mL), and streptomycin (100 µg/mL). HOK cells were maintained in KGM. The establishment of iSLK.RTA-WT and iSLK.RTA-DD stable cell lines were conducted as previously described [14].

Site-directed mutagenesis

The codon encoding the asparagine residue (N) of RTA was mutated to aspartate (D) or glutamine (Q) using a commercial kit and confirmed by sequencing. Mutagenesis of iBAC16 was performed as previously described [14]. iBAC DNA was isolated using a commercial kit. All primers were listed in Additional file 2: Table S2.

DNA transfection and luciferase reporter assay

For transfection with 293T cells, polyethyleneimine (PEI) was used. 293T cells were transfected with pPAN, pORF57, or pvIL-6 promoter reporter cocktails (100 ng) and different doses of plasmids expressing vGAT (1 ng, 10 ng, and 50 ng) without or with RTA (1 ng). 293T cells were transfected with pPAN, pORF57, or pvIL-6 promoter reporter cocktails (100 ng) and different doses of plasmids expressing RTA-WT, RTA-DD, or RTA-N37Q (2 ng, 4 ng, and 8 ng). 293T cells were transfected with IFN-β or NF-κB promoter reporter cocktails (100 ng) and different doses of plasmids expressing RTA-WT or RTA-DD (0 ng, 4 ng, 8 ng, and 16 ng) with TBK1 or IKK-β (1 ng). For iBAC DNA transfection into SLK and HOK cells, Lipofectamine 3000 was used. SLK.iBAC and HOK.iBAC-positive cells were selected with hygromycin B. At 30 h post-transfection, cells were harvested and luciferase activity was determined using a commercial kit.

Two-dimensional gel electrophoresis

Sample proteins were isolated and dissolved in 150 µL rehydration buffer. After centrifugation (20,000×g, 30 min), the supernatant was subjected to isoelectric focusing, and incubated with SDS equilibration buffer containing dithiothreitol (70 mM) and 2-iodoacetamide (25 µg/mL) for 15 min, respectively. Washed with SDS-PAGE buffer, strips were resolved by SDS-PAGE and analyzed by immunoblotting.

Subcellular fractionation and immunofluorescence microscopy

Subcellular fractionation was performed as previously described [14]. Proteins obtained from cytoplasmic and nuclear fractions were analyzed by SDS-PAGE and immunoblotting with indicated antibodies, respectively. Immunofluorescence analysis was performed as previously described [14]. The treated slides were directly analyzed with a confocal microscope, and representative views were presented for all analysis.

MS analysis

RTA was purified from 293T cells transfected with a plasmid expressing RTA without or with vGAT by SDS-PAGE and Coomassie staining. Mass spectrometry (MS) analysis was performed by the Taplin Mass Spectrometry Facility.

Coimmunoprecipitation (CO-IP) and immunoblotting

CO-IP were performed as previously described [14]. Treated samples were subjected to SDS-PAGE and immunoblotting. All immunoblotting analysis were performed using the indicated primary antibodies and secondary antibodies and visualized.

Quantitative real-time PCR (qPCR)

Reactivated iSLK.219 and HOK.iBAC cells were extracted using TRIzol reagent and transcripted into cDNA using a commercial kit. qPCR was performed using a commercial kit. The relative mRNA expressions of targeted genes were calculated using the 2^−ΔΔCt method and normalized to the expression levels of β-Actin. All primers were listed in Additional file 2: Table S2.

Statistical analysis

All data were presented as mean ± standard deviations (SD). Statistical analysis was performed using Student’s t-test or one-way analysis of variance (ANOVA). Statistical significance was set at *P < 0.05, **P < 0.01, ***P < 0.001, while “ns” indicated “not significant”.

KSHV Kaposi’s sarcoma-associated herpesvirus, MHV68 Murine gamma-herpesvirus 68, PFAS Phosphoribosylformylglycinamidine synthetase, vGAT Viral glutamine amidotransferase, ORF Open reading frame, RTA Replication and transcription activator, PAN Polyadenylated nuclear, IFN Interferon, NF-κB Nuclear factor-kappa B.

We thank Dr. Pinghui Feng at University of Southern California and Dr. Jun Zhao at Cleveland Clinic for providing reagents and resources.

Authors’ contributions

YX and QZ, Data curation, Formal analysis, Investigation, Software, Visualization, Writing − original draft; LH, Investigation, Data curation, Validation. TX, Resources. DL, Project administration, Funding Acquisition, Supervision; JL, Conceptualization, Methodology, Project administration, Funding acquisition, Supervision, Writing − review & editing. All authors read and approved the final manuscript

Funding

This work was funded by the National Natural Science Foundation of China (32173021), the Natural Science Foundation of Hunan Province (2024JJ5184), the Hunan Provincial Science & Technology Major Project (2022sfq30, 20231F18), the Key Projects of Hunan Provincial Education Department (23A0196), and the earmarked fund for HARS (HARS-06).

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J, Knowles DM, et al. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science. 1994;266(5192):1865–1869.
Ye F, Lei X, Gao SJ. Mechanisms of Kaposi’s sarcoma-associated herpesvirus latency and reactivation. Adv Virol. 2011;2011:193860.
Broussard G, Damania B. Regulation of KSHV latency and lytic reactivation. Viruses. 2020;12(9):1034.
Arias C, Weisburd B, Stern-Ginossar N, Mercier A, Madrid AS, Bellare P, et al. KSHV 2.0: a comprehensive annotation of the Kaposi’s sarcoma-associated herpesvirus genome using next-generation sequencing reveals novel genomic and functional features. PLoS Pathog. 2014;10(1):e1003847.
Yang X, Zhou J, Liu C, Qu Y, Wang W, Xiao MZX, et al. KSHV-encoded ORF45 activates human NLRP1 inflammasome. Nat Immunol. 2022;23(6):916–926.
Sun R, Lin SF, Gradoville L, Yuan Y, Zhu F, Miller G. A viral gene that activates lytic cycle expression of Kaposi’s sarcoma-associated herpesvirus. Proc Natl Acad Sci U S A. 1998;95(18):10866–10871.
Yu Y, Wang SE, Hayward GS. The KSHV immediate-early transcription factor RTA encodes ubiquitin E3 ligase activity that targets IRF7 for proteosome-mediated degradation. Immunity. 2005;22(1):59–70.
Zhao Q, Liang D, Sun R, Jia B, Xia T, Xiao H, et al. Kaposi’s sarcoma-associated herpesvirus-encoded replication and transcription activator impairs innate immunity via ubiquitin-mediated degradation of myeloid differentiation factor 88. J Virol. 2015;89(1):415–427.
Ye F, Chen ER, Nilsen TW. Kaposi’s Sarcoma-associated herpesvirus utilizes and manipulates RNA N⁶-adenosine methylation to promote lytic replication. J Virol. 2017;91(16):e00466-17.
Yang Z, Tang H, Huang H, Deng H. RTA promoter demethylation and histone acetylation regulation of murine gammaherpesvirus 68 reactivation. PLoS One. 2009;4(2):e4556.
Wang S, Tian X, Zhou Y, Xie J, Gao M, Zhong Y, et al. Non-canonical regulation of the reactivation of an oncogenic herpesvirus by the OTUD4-USP7 deubiquitinases. PLoS Pathog. 2024;20(1):e1011943.
Tian H, Yu K, He L, Xu H, Han C, Zhang X, et al. RNF213 modulates γ-herpesvirus infection and reactivation via targeting the viral replication and transcription activator. Proc Natl Acad Sci U S A. 2023;120(12):e2218825120.
He S, Zhao J, Song S, He X, Minassian A, Zhou Y, et al. Viral pseudo-enzymes activate RIG-I via deamidation to evade cytokine production. Mol Cell. 2015;58(1):134–146.
Li J, Zhao J, Xu S, Zhang S, Zhang J, Xiao J, et al. Antiviral activity of a purine synthesis enzyme reveals a key role of deamidation in regulating protein nuclear import. Sci Adv. 2019;5(10):eaaw7373.
Lu A, Disoma C, Zhou Y, Chen Z, Zhang L, Shen Y, et al. Protein interactome of the deamidase phosphoribosylformylglycinamidine synthetase (PFAS) by LC-MS/MS. Biochem Biophys Res Commun. 2019;513(3):746–752.
Xu Y, Hou G, Liu Q, Zhang Q, Li C, Hu L, et al. Helicase-independent function of RIG-I against murine gammaherpesvirus 68 via blocking the nuclear translocation of viral proteins. Int J Biol Macromol. 2023;250:126527.
Zhao J, Li J, Xu S, Feng P. Emerging roles of protein deamidation in innate immune signaling. J Virol. 2016;90(9):4262–4268.
Xie T, Qin C, Savas AC, Yeh WW, Feng P. The emerging roles of glutamine amidotransferases in metabolism and immune defense. Nucleosides Nucleotides Nucleic Acids. 2024;1–15.
Zhao J, Zeng Y, Xu S, Chen J, Shen G, Yu C, et al. A viral deamidase targets the helicase domain of RIG-I to block RNA-induced activation. Cell Host Microbe. 2016;20(6):770–784.
Zhang J, Zhao J, Xu S, Li J, He S, Zeng Y, et al. Species-specific deamidation of cGAS by herpes simplex virus UL37 protein facilitates viral replication. Cell Host Microbe. 2018;24(2):234–248.e5.
Rao Y, Wang TY, Qin C, Espinosa B, Liu Q, Ekanayake A, et al. Targeting CTP synthetase 1 to restore interferon induction and impede nucleotide synthesis in SARS-CoV-2 infection. bioRxiv. 2021; doi:10.1101/2021.02.05.429959
Zhao J, Tian M, Zhang S, Delfarah A, Gao R, Rao Y, et al. Deamidation shunts RelA from mediating inflammation to aerobic glycolysis. Cell Metab. 2020;31(5):937–955.e7.
Qin C, Rao Y, Yuan H, Wang TY, Zhao J, Espinosa B, et al. SARS-CoV-2 couples evasion of inflammatory response to activated nucleotide synthesis. Proc Natl Acad Sci U S A. 2022;119(26):e2122897119.
Wan Q, Tavakoli L, Wang TY, Tucker AJ, Zhou R, Liu Q, et al. Hijacking of nucleotide biosynthesis and deamidation-mediated glycolysis by an oncogenic herpesvirus. Nat Commun. 2024;15(1):1442.
Wang TY, Zhao J, Savas AC, Zhang S, Feng P. Viral pseudoenzymes in infection and immunity. FEBS J. 2020;287(19):4300–4309.
Olson AT, Wang Z, Rico AB, Wiebe MS. A poxvirus pseudokinase represses viral DNA replication via a pathway antagonized by its paralog kinase. PLoS Pathog. 2019;15(2):e1007608.
Rico AB, Wang Z, Olson AT, Linville AC, Bullard BL, Weaver EA, et al. The vaccinia virus (VACV) B1 and cellular VRK2 kinases promote VACV replication factory formation through phosphorylation-dependent inhibition of VACV B12. J Virol. 2019;93(20):e00855-19.
Rico AB, Linville AC, Olson AT, Wang Z, Wiebe MS. The vaccinia virus B12 pseudokinase represses viral replication via interaction with the cellular kinase VRK1 and activation of the antiviral effector BAF. J Virol. 2021;95(3):e02114-20.
Teoh ML, Turner PV, Evans DH. Tumorigenic poxviruses up-regulate intracellular superoxide to inhibit apoptosis and promote cell proliferation. J Virol. 2005;79(9):5799–5811.
Combs LR, Spires LM, Alonso JD, Papp B, Toth Z. KSHV RTA induces degradation of the host transcription repressor ID2 to promote the viral lytic cycle. J Virol. 2022;96(12):e0010122.
Han C, Zhang D, Gui C, Huang L, Chang S, Dong L, et al. KSHV RTA antagonizes SMC5/6 complex-induced viral chromatin compaction by hijacking the ubiquitin-proteasome system. PLoS Pathog. 2022;18(8):e1010744.
Zhang Y, Dong Z, Gu F, Xu Y, Li Y, Sun W, et al. Degradation of TRIM32 is induced by RTA for Kaposi’s sarcoma-associated herpesvirus lytic replication. J Virol. 2024;98(6):e0000524.
Spires LM, Wind E, Papp B, Toth Z. KSHV RTA utilizes the host E3 ubiquitin ligase complex RNF20/40 to drive lytic reactivation. J Virol. 2023;97(11):e0138923.
Chang LK, Lee YH, Cheng TS, Hong YR, Lu PJ, Wang JJ, et al. Post-translational modification of Rta of Epstein-Barr virus by SUMO-1. J Biol Chem. 2004;279(37):38803–38812.
Brown HJ, Song MJ, Deng H, Wu TT, Cheng G, Sun R. NF-kappaB inhibits gammaherpesvirus lytic replication. J Virol. 2003;77(15):8532–8540.
Liang Q, Zhu F. ORF45, a multifunctional immediate early and tegument protein of KSHV. J Med Virol. 2023;95(3):e28659.
Liu Z, Wang X, Liu C, Deng H, Li W, Wang X, et al. The SUMO E3 ligase activity of ORF45 determines KSHV lytic replication. PLoS Pathog. 2022;18(4):e1010504.
Alzhanova D, Meyo JO, Juarez A, Dittmer DP. The ORF45 protein of Kaposi sarcoma-associated herpesvirus is an inhibitor of p53 signaling during viral reactivation. J Virol. 2021;95(23):e0145921.
Liu Z, Liu C, Wang X, Li W, Zhou J, Dong P, et al. RSK1 SUMOylation is required for KSHV lytic replication. PLoS Pathog. 2021;17(12):e1010123.
Liu Y, Cao Y, Liang D, Gao Y, Xia T, Robertson ES, et al. Kaposi’s sarcoma-associated herpesvirus RTA activates the processivity factor ORF59 through interaction with RBP-Jkappa and a cis-acting RTA responsive element. Virology. 2008;380(2):264–275.
Rossetto CC, Susilarini NK, Pari GS. Interaction of Kaposi’s sarcoma-associated herpesvirus ORF59 with oriLyt is dependent on binding with K-Rta. J Virol. 2011;85(8):3833–3841.
McDowell ME, Purushothaman P, Rossetto CC, Pari GS, Verma SC. Phosphorylation of Kaposi’s sarcoma-associated herpesvirus processivity factor ORF59 by a viral kinase modulates its ability to associate with RTA and oriLyt. J Virol. 2013;87(14):8038–8052.
Shi J, Jia X, He Y, Ma X, Qi X, Li W, et al. Immune evasion strategy involving propionylation by the KSHV interferon regulatory factor 1 (vIRF1). PLoS Pathog. 2023;19(4):e1011324.
Chavoshi S, Egorova O, Lacdao IK, Farhadi S, Sheng Y, Saridakis V. Identification of Kaposi sarcoma herpesvirus (KSHV) vIRF1 protein as a novel interaction partner of human deubiquitinase USP7. J Biol Chem. 2016;291(12):6281–6291.

No competing interests reported.

Additionalfile1.docx
Additional file 1: Method details
Additionalfile2.docx
Additional file 2: Table S1. Reagent and resource. Table S2. Primers used in this study.

Download PDF

Reviewers invited by journal
16 Aug, 2024
Editor assigned by journal
06 Aug, 2024
Submission checks completed at journal
06 Aug, 2024
First submitted to journal
05 Aug, 2024

You are reading this latest preprint version

Viral pseudo-enzyme facilitates KSHV lytic replication via suppressing PFAS-mediated RTA deamidation

Status:

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Results

vGAT competitively interacts with RTA and cellular PFAS

vGAT inhibits the deamidation of RTA

Deamidation impairs RTA-mediated transcriptional activation and expression of viral ORFs

Inhibition of deamidation promotes RTA-mediated transcriptional activation and expression of viral ORFs

Deamidation blocks the nuclear translocation of RTA

Deamidation alleviates RTA-mediated suppression of NF-κB signaling

vGAT inhibits the deamidation of KSHV lytic ORFs

Discussion

Conclusion

Methods

Cell maintenance and stable cell line generation

Site-directed mutagenesis

DNA transfection and luciferase reporter assay

Two-dimensional gel electrophoresis

Subcellular fractionation and immunofluorescence microscopy

MS analysis

Coimmunoprecipitation (CO-IP) and immunoblotting

Quantitative real-time PCR (qPCR)

Statistical analysis

Abbreviations

Declarations

Authors’ contributions

Funding

Availability of data and materials

References

Additional Declarations

Supplementary Files

Status:

Version 1