Proximity Biotin Labeling Reveals Kaposi’s Sarcoma-Associated Herpesvirus Interferon Regulatory Factor Networks

Viral protein interaction with a host protein shows at least two sides: (i) taking host protein functions for its own benefit and (ii) disruption of existing host protein complex formation to inhibit undesirable host responses. Due to the use of affinity precipitation approaches, the majority of studies have focused on how the virus takes advantage of the newly formed protein interactions for its own replication.

KSHV genome encodes four vIRFs, vIRF-1 to -4. The N termini of vIRFs exhibit similarity to N termini of cellular IRFs; however, viral IRFs lack a key tryptophan residue, which is required for binding to DNA (19). The vIRF-1, vIRF-2, and vIRF-4 genes are inducible lytic genes, although vIRF-1 can also be found in a small portion of latently infected cells. In contrast, vIRF-3 (also known as LANA2) was discovered as a latent protein, and its expression remains unchanged during reactivation (20). Studies have shown that vIRFs counteract the host IFN response by interacting with cellular proteins. For example, vIRF-1 suppresses cellular IRF-3mediated transcription by binding to p300, thereby preventing p300/CBP-IRF-3 complex formation (21,22). vIRF-1 also promotes KSHV lytic replication by recruitment of USP7 (23). vIRF-2 was found to inhibit KSHV lytic gene expression by increasing the expression of cellular antiviral factors like promyelocytic leukemia nuclear bodies (PML) (24). Similarly, vIRF-3 suppresses KSHV reactivation by interacting with USP7, and the interaction also supports PEL cell growth (23). vIRF-4 also associates with IRF-7 and inhibits IRF-7 dimerization to suppress IFN production (25). These studies demonstrated different outcomes in different cell lines, suggesting the significance of implementing proteomic approaches that can reveal vIRF interaction networks more comprehensively.
Dynamic and stable protein-protein interactions are key to cellular processes and biological pathways. Affinity purification coupled with mass spectrometry (AP-MS) has been an invaluable method used to identify protein-protein interactions. However, AP-MS often fails to identify weakly or transiently interacting proteins. To overcome this drawback, enzyme-based proximity-based labeling (PL) approaches have been developed (26,27). The approach provides sensitivity and specificity required to study dynamic protein-protein interaction. BirA R118G (BirID) was the first proximity-based labeling enzyme identified in Escherichia coli which conjugates biotin to lysine residues of neighboring proteins (28). However, original BirID required the presence of biotin for several hours to be able to biotinylate a sufficient amount of proteins for analysis, thereby restricting its use for dynamic processes. Recently, two variants of BirID have been developed by directed evolution, named mini-TurboID (28 kDa) and TurboID (35 kDa), which allow proximity labeling in less than 10 min without significant toxicity (27). The TurboID-based approach has already been successfully employed in a wide variety of species, including mammalian cells (27,(29)(30)(31)(32), Drosophila (33), plants (34)(35)(36)(37), yeasts (38), and flies and worms (27).
In this study, we prepared recombinant 3ÂFlag-mini-TurboID-vIRF-1 and 3ÂFlagmini-TurboID-vIRF-4 KSHV to biotinylate host and viral proteins in the vicinity of these two viral proteins. The proximity-labeling approach combined with mass spectrometry identified both previously identified cellular proteins and new host proteins as their possible interacting partners. Small interfering RNA (siRNA) screening of these interacting proteins showed that selective splicing factors function to suppress KSHV reactivation and are associated with antiviral responses. and unicellular organisms (27,32,33,35,37,38). We thought that applying this technique to virology would be particularly beneficial, because viruses completely depend on host cell machinery for their replication. Indeed, many key cellular proteins, such as p53, were identified as virus-interacting proteins (39)(40)(41). Our major goal is thus to report utility of PL in conjunction with the recombinant KSHV bacterial artificial chromosome (BAC) system.
We first examined optimal duration of incubation time, and the amount of exogenous biotin for efficient labeling was determined. To test biotin ligase activity, Wt BAC16, vIRF-1 mTID, and vIRF-4 mTID cells were reactivated with doxycycline (Dox) and sodium butyrate (NaB) for 24 h. Subsequently, stable cells were incubated with 500 mM biotin for various periods, and Wt BAC16 cells were used as a negative control. Labeling was terminated by incubating cells at 4°C and removing excess biotin. Immunoblot analysis using horseradish peroxidase (HRP)-conjugated streptavidin showed multiple biotinylated proteins, indicating successful labeling with vIRF-1-and vIRF-4-tagged mini-TurboID. Mini-TurboID biotinylated proteins within 1 h after addition of exogenous biotin, and signal intensity gradually increased along with incubation time. Wt BAC16 cells did not show biotinylation signal even after 24 h of biotin incubation, indicating that the signal is specific to mini-TurboID (Fig. 1C, lanes 1). Next, we optimized the exogenous biotin concentration. The vIRF-1 and vIRF-4 mTID cells were reactivated for 24 h, incubated with different amounts of biotin (0, 125, 250, and 500 mM) for 1 h, and subsequently monitored for their biotinylation signal in wholecell lysates using streptavidin immunoblotting. Untreated cells in the absence of biotin were used as a negative control. Comparable levels of signal intensity were observed from 125 mM to 500 mM, suggesting that saturation of protein biotinylation occurs at 125 mM (Fig. 1D). Considering that only a small proportion of cells were reactivating in a dish, we concluded that there was a sufficient amount of biotinylation in a reactivating cell for protein identification. For the following studies, we decided to use a saturating amount of biotin (500 mM) for 1 h of incubation.
Recombinant vIRF-1 mTID and vIRF-4 mTID KSHV gene expression. We next verified the induction of viral genes to ensure that tagging K9 or K10 genes with 3ÂFlag-mini-TurboID has little effect on viral gene expression or virion production. We compared induction of viral inducible genes in vIRF-1 and vIRF-4 mTID cells with that in Wt BAC16 cells. We stimulated Wt BAC16, vIRF-1, and vIRF-4 mTID cells with Dox and NaB and performed quantitative PCR (qPCR) for selected KSHV genes. We observed induction of KSHV lytic genes, PAN RNA, ORF6, vIRF-1, and vIRF-4 in all three cell lines. The viral gene expression in vIRF-1 mTID cells was comparable to that in Wt BAC16 cells, while v-IRF-4 mTID showed approximately 4 to 5 times lower levels of gene expression ( Fig. 2A). In addition, we verified induction of selected lytic KSHV proteins at 24 h (Fig. 2B) and confirmed the production of virion particles in culture supernatants. We reactivated the Wt BAC16, vIRF-1, and v-IRF-4 mTID cells for 96 h and quantified the virion particles in the culture supernatant. The results showed comparable levels of virion particles in Wt BAC16 cells and vIRF-1 mTID cells, whereas vIRF-4 mTID cells showed approximately 2-fold-lower virion production (Fig.  2C). Finally, viral genomic copy numbers were adjusted to one viral genome copy per cell (ii) The kanamycin cassette with I-SceI recognition sequence along with 50 bp of homologous sequence was generated by PCR with pEP-Kan plasmid as a template and cloned into the AccI restriction enzyme site. (iii to v) The resulting plasmid was fully sequenced and used as a template to generate a DNA fragment for homologous recombination with BAC16 inside bacteria. (vi and vii) After confirmation of insertion at correct site by colony PCR screening, the kanamycin cassette was deleted by recombination with induction of I-SceI in bacteria by incubation with L-arabinose. Correct insertion of the mini-TurboID and integrity of BAC DNA were confirmed by sequencing of PCR-amplified fragments and restriction digestions. Primers and the DNA fragment used are listed in Table 1. (B) Generation of Wt BAC16, vIRF-1 mTID, and vIRF-4 mTID stable cells. iSLK cells were transfected with Wt BAC16, 3ÂFlag-mini-TurboID-K9, and 3ÂFlag-mini-TurboID-K10 KSHV BAC16 and stably selected with hygromycin (1 mg/ml). Green fluorescent protein (GFP) images show iSLK cells latently infected with Wt BAC16, 3ÂFlag-mini-TurboID-K9, and 3ÂFlag-mini-TurboID-K10 KSHV BAC16. BF, bright field. (C) Protein biotinylation with timescale. Wt BAC16 (Wt), vIRF-1 mini-TurboID (vIRF-1 mTID), and vIRF-4 mini-TurboID (vIRF-4 mTID) cells were stimulated with Dox (1 mg/ml) and NaB (3 mM) for 24 h, followed by incubation with D-biotin (500 mM) for the indicated periods. Activity of mini-TurboID was analyzed by immunoblotting using streptavidin (Strep)-HRP conjugate. b-Actin was used as the input control. T(h), time (hours). (D) Determination of the optimal amount of exogenous D-biotin (B) for labeling. vIRF-1 mTID and v-IRF-4 and HEK293FT cells were infected to examine infectivity by flow cytometry. The results demonstrated similar levels of infectivity with all three viruses (Fig. 2D). These results suggested that both vIRF-1 and vIRF-4 mTID viruses were able to complete the viral lytic replication cycle, although the mini-TurboID tag interferes with viral gene expression in vIRF-4 mTID virus.
Proximity biotin labeling with vIRF-1 and vIRF-4. For proximity protein labeling, three replicate samples were prepared for both vIRF-1 and vIRF-4 mTID cells. Cells were reactivated with Dox and NaB for 24 h, followed by addition of biotin for 1 h. Two sets of controls were also processed concurrently to rule out nonspecific precipitations. In the first set, the cells were left without triggering reactivation, followed by incubation with biotin (1Biotin) to rule out nonspecific protein binding with biotin (Ctrl 1). For the second set, cells were reactivated with Dox and NaB for 24 h and incubated for additional 1 h in the absence of biotin (2Biotin) to rule out nonspecific interaction with streptavidin beads (Ctrl 2). A schematic workflow for the experiment is presented in Fig. 3A. We confirmed the biotinylation signal by streptavidin blotting and vIRF-1 and vIRF-4 expression by using anti-Flag antibody (Fig. 3B). The streptavidin blotting showed differences in biotinylation signal between vIRF-1 and vIRF-4; this is likely due to amount of vIRF-1 and vIRF-4 protein in the cell and/or amount of neighboring cellular proteins and their stabilities. Whole-cell lysates from vIRF-1 and vIRF-4 mTID cells were further used for enrichment of biotinylated protein using magnetic streptavidin beads. The enriched proteins were eluted from the streptavidin beads using trypsin on-bead digestion overnight. The biotinylated peptides in the experimental (Expt) samples were compared with those in Ctrl 1 and Ctrl 2 independently to remove background noise. We designated proteins with P values of ,0.05 and fold change (FC) of .2 over both Ctrl1 and Ctrl2 as positive hits (Fig. 3C). Based on our criterion setting, we identified 213 and 70 as possible interacting proteins with vIRF-1 and vIRF-4 mTID cells, respectively (see Tables S1 and S2 in the supplemental material).
vIRF-1 and vIRF-4 target pathway analysis. Next, gene ontology (GO) analysis was performed for proteins possibly interacting with vIRF-1 and vIRF-4. The vIRF-1 interactome revealed significant enrichment for functions related to mRNA processing, transcription regulation by TP53, regulation of mRNA processing, and formation of RNA polymerase II (Pol II) elongation complex. The top 20 enriched GO terms are presented in Fig. 4A. Similarly, GO analysis for the vIRF-4 revealed again enrichment of mRNA processing, regulation of mRNA processing, mRNA polyadenylation, and mRNA splicing (Fig. 4B). Consistent with the fact that vIRF-1 and vIRF-4 have overlapping biological functions, we indeed found highly overlapping pathways that are associated with vIRF-1-and vIRF-4 precipitated proteins.
Regulation of KSHV reactivation by commonly targeted cellular proteins. Previous studies demonstrated that both vIRF-1 and vIRF-4 regulate interferon pathways (18,20). We thus hypothesized that commonly targeted cellular proteins by the two viral IRFs may play an important role in interferon responses. The results showed that 123 and 23 proteins interacted exclusively with vIRF-1 and vIRF-4, respectively, and 47 proteins were found to interact with both vIRF-1 and vIRF-4. Of these 47 proteins, 44 were cellular proteins, whereas 3 were viral proteins (Fig. 5A). The iSLK.219 cell line was employed to examine the role of these cellular proteins in KSHV replication. iSLK.219 cells carry a recombinant rKSHV.219 virus encoding a constitutively expressed green fluorescent protein (GFP) and a PAN RNA promoter-driven red fluorescent protein (RFP) reporter in the viral genome, allowing us to monitor lytic promoter activation. We used siRNA to knock down the 44 cellular genes, followed by KSHV reactivation by treatment with Dox and NaB to induce K-Rta expression. We found that knockdown of 17 genes enhanced KSHV PAN RNA promoter activation, while knockdown of 6 genes decreased the activation (Fig. 5B). The corresponding RFP images of FIG 1 Legend (Continued) mTID cells were stimulated with Dox (1 mg/ml) and NaB (3 mM) for 24 h, followed by incubation with the indicated concentration of D-biotin for 1 h. Activity of mini-TurboID was examined by immunoblotting using streptavidin-HRP conjugate. b-Actin was used as the input control. selected knockdown experiments are shown in Fig. 5C, and the results were further confirmed by quantifying the viral mRNAs after knockdown of selected genes, i.e., the splicing factor 3B1 (SF3B1), SF3B2, and SNW1 genes (Fig. 5D). Consistent with increased viral gene expression, the viral DNA copy number in the culture supernatant was increased by knockdown of the SF3B1, SF3B2, or SNW1 gene (Fig. 5E). Taken together, our results suggest that certain splicing factors have a role in restricting KSHV gene expression during reactivation, in addition to their biological roles in general host gene transcription.
SF3B subunits are important for IFN gene expression. Previous reports showed that the KSHV gene transcripts are sensed by RIG-I-like receptors (11). Poly(I·C) is a synthetic double-stranded RNA (dsRNA) polymer which is recognized by RIG-I, leading to strong induction of interferons and interferon-stimulated genes (ISGs). Because KSHV vIRFs are known to counteract IFN responses, we first examined vIRF-1 and vIRF-4 functions in interferon responses that are triggered by poly(I·C). vIRF-1 and vIRF-4 overexpression in HEK293FT cells resulted in inhibition of IFN-b1, IFN-l1, and DDX58 in response to poly(I·C) transfection ( Fig. 6Aa to Ac). Next, we examined the role of SF3B1 and SNW1 in regulation of the same IFN response. For this, SF3B1 and SNW1 were knocked down in HEK293FT cells, followed by poly(I·C) transfection. Consistent with our hypothesis that vIRFs interact with SF3B1 and SNW1 to inhibit IFN pathways, knockdown of SF3B1 and SNW1 also inhibited expression of type I interferon (IFN-b1) and type III interferon (IFN-l1), as well as an interferon downstream target (DDX58) (Fig. 6Ba to Bc) but not the non-IFN target gene (Fig. 6Bd). Although we cannot rule out whether the IFN inhibition is mediated by the SF3B1 or SNW1 interactions, the results still suggested that both SF3B1 and SNW1 are involved in IFN pathway regulation. Knockdown of SF3B1 or SNW1 also led inhibition of type I interferons IFN-b1 and IFN-a1 during KSHV reactivation in iSLK.r219 cells (Fig. 6C). Together, these results may suggest that vIRF-1 and vIRF-4 interact with SF3B1 and SNW1 to modulate host antiviral immune responses.

DISCUSSION
We applied a mini-TurboID-based system for studying the virus and host protein interaction. By constructing mini-TurboID as an integral component of the KSHV BAC16 recombination system, we demonstrated the utility of a new approach to identify protein interaction networks. We believe that this approach improves the reproducibility of identifying interacting proteins, because tight interaction between biotin and streptavidin allows us to wash magnetic beads under very stringent conditions and remove nonspecific or indirect protein interactions. The high reproducibility can be seen in our biological triplicate samples (Fig. S1).
To conveniently generate mini-TurboID-tagged viruses, we first generated template plasmids, similar to the strategy used for Rainbow-KSHV (43). With a plasmid template, homology arms can be added to primer pairs and the resultant PCR product is used for recombination (Fig. 1A). The targeted parental BAC16 genome can also be wild-type or mutant BAC16, and/or Rainbow-KSHV, which allows us to examine the formation of protein complexes during viral replication stages and also the effects of specific mutations on the protein interactions. In this study, however, we used vIRF-1 and vIRF-4 as bait for validating the efficiency of PL. vIRF-1 and vIRF-4 were selected because of their known role in regulation of the innate immune response during KSHV reactivation, and multiple interacting proteins that can be used as confirmation have been identi- fied (18,23,25,44). Consistent with previous studies, vIRF-1 and vIRF-4 were found to be physically neighboring cellular proteins that function in p53 transcriptional regulation (45). A study showed that vIRF-1 interacts with p53 to inhibit its transcriptional activation (44). Although our studies did not include precipitation of p53, we identified p53BP1 (p53 binding protein 1) as a possible partner of vIRF-1. We found USP7 in both vIRF-1 and vIRF-4 samples, validating the PL approach (23). In addition to the reported cellular proteins, we also confirmed SF3B1-vIRF interactions in transiently transfected   293FT cells (Fig. S2). The studies showed that transiently expressed Flag-tagged vIRFs were coprecipitated with endogenous SF3B1 protein.
After learning that the mini-TurboID approach efficiently biotinylated cellular proteins, we tagged various other KSHV genes with mini-TurboID using the same approach. However, we found that the efficacy of biotin labeling varies significantly among different viral proteins. For example, mini-TurboID-ORF57 robustly increased biotinylated protein in total lysates with as little as 1 h of D-biotin incubation, while  IFN-l1 (b), and DDX58 (c) mRNAs or nonrelated DDX23 mRNA (d) was measured. (C) Five picomoles of siC, siSF3B1, or siSNW1 was transfected into iSLK.r219 cells for 48 h, followed by KSHV reactivation by Dox (1 mg/ml) and NaB (3 mM). Forty-eight hours postreactivation, total RNA was harvested and IFN-related IFN-b1 (a) and IFN-a1 (b) mRNAs were measured. ***, P # 0.001; ****, P # 0.0001. siC, nontargeting siRNA. biotinylation by mini-TurboID-ORF50 was barely detectable in the same time frame. For this study, we also generated and tested vIRF-2 and vIRF-3 mini-TurboID constructs; however, the level of biotinylation was much lower using the same amount of D-biotin and incubation periods, leading us to drop these genes from our analyses. Differences in efficacy of biotinylation have also been observed in prior studies (27), and abundance of viral protein expression during reactivation and subcellular nuclear localization seemed to have strong effects on the outcome of biotinylation.
Our PL studies showed that a large portion of interacting host proteins (36%) were related to mRNA processing. Of these RNA processing proteins, SF3B1, SF3B2, and SF3B3, components of the SF3B complex, clearly stood out in our siRNA screening. The SF3B complex is a component of the functional U2 small nuclear ribonucleoprotein (snRNP), which recognizes the exon/intron junctions and facilitates spliceosome assembly (46). Even though the SF3B1 gene is one of many cellular genes involved in RNA splicing, it has been specifically identified as a commonly mutated gene in myelodysplastic syndrome (MDS), at frequencies of 25 to 30% (47)(48)(49). Recent studies also showed that SF3B1 mutations increase R-loop formation and DNA damage (50). In this study, we found that SF3B1 knockdown inhibited IFN gene expression 3-to 4-fold and also enhanced KSHV reactivation. Indeed, SF3A1 and SF3B1 were previously reported to play a role in the innate immune response to TLR ligands. The study showed that SF3A1 and SF3B1 are necessary to increase production of interleukin 6 (IL-6) and IFN-b by modulating the splicing of MyD88, an important adaptor molecule for the TLR signaling pathway (51). It remains unknown how SF3B1 family regulates selected spliced genes. Based on the reported study and ours, we think that targeting the splicing complex might be a previously uncharacterized mechanism for KSHV to modulate host immune responses. Further studies on regulation of SF3B complex formation during KSHV reactivation and/or IFN stimulation with PL will clarify underlying mechanisms of SF3B family proteins in KSHV replication and IFN regulation.
In addition to SF3 complex, several other mRNA processing factors, like XAB2, SNRPD1, SNW1, RBM10, SYMPK, and GTF2F2, were found to suppress KSHV reactivation (Fig. 5). A recent study showed that SNW1 interacts with IKKg, the regulatory subunit of the IκB kinase (IKK) complex. SNW1 increases production of IL-6, IFN-b, and MX1 by enhanced activation of NF-κB and phosphorylation of TBK1 in response to influenza A virus and poly(I·C) (52). Influenza A virus and poly(I·C) are recognized by the innate immune sensor RIG-I, and RIG-I plays an important role in suppressing KSHV reactivation by sensing KSHV DNA (11,12,53,54). We found that knockdown of SNW1 indeed enhanced KSHV replication (Fig. 5D), and we think that this effect could be due to downregulation of IFN-b (Fig. 6).
In summary, using mini-TurboID KSHV with vIRFs as bait, we could successfully probe cellular proteins that play a role in innate immune responses. We propose that combination of mini-TurboID with the recombinant KSHV BAC system is a powerful tool to identify cellular proteins that play an important role in KSHV replication.
Quantification of viral replication. siRNAs targeting the cellular genes were transfected in iSLK.219 cells for 48 h, followed by KSHV reactivation by doxycycline (1 mg/ml) and sodium butyrate (NaB; 3 mM). After 24 h, the RFP fluorescence intensity was quantified using ImageJ software. The RFP signal intensity was normalized relative to that of nontargeting siRNA (siC).
Construction of vIRF-1 and vIRF-4 mini-Turbo KSHV BAC16. Recombinant KSHV was prepared by following a protocol for en passant mutagenesis with a two-step markerless red recombination technique (42). Briefly, a codon-optimized mini-TurboID coding sequence (Table 1) which also encodes 3Â Flag tag was first cloned into a pBS SK vector (Thermo Fisher, Waltham, MA). The pEPkan-S plasmid was used as a source of the kanamycin cassette, which includes the I-SceI restriction enzyme site at the 59 end of the kanamycin coding region (42). The kanamycin cassette was amplified with primer pairs listed in Table 1 and cloned into the mini-TurboID coding region at a unique restriction enzyme site. The resulting plasmid was used as a template for another round of PCR to prepare a transfer DNA fragment for markerless recombination with BAC16 (57). Recombinant BAC clones with insertion and also deletion of the kanamycin cassette in the BAC16 genome were confirmed by colony PCR with appropriate primer pairs. Recombination junctions and adjacent genomic regions were amplified by PCR, and the resulting PCR products were directly sequenced with the same primers to confirm in-frame insertion of the mini-TurboID cassette into the BAC DNA. The resulting recombinant BAC was confirmed by restriction enzyme digestions (HindIII and BglII), to determine if there were any large DNA deletions. Two independent BAC clones were generated for each mini-TurboID-tagged recombinant virus as biological replicates; one of the clones was used for protein identification. BAC DNAs were subsequently sequenced in their entirety.
Quantification of viral copy number. Two hundred microliters of cell culture supernatant was treated with 12 mg/ml of DNase I for 15 min at room temperature to degrade nonencapsidated DNA. This reaction was stopped by the addition of EDTA to 5 mM, followed by heating at 70°C for 15 min. Viral genomic DNA was purified using a QIAamp DNA minikit according to the manufacturer's protocol. Viral DNA was eluted in 100 ml of buffer AE. Four microliters of eluate was used for real-time qPCR to determine viral copy number, as described previously (58).
Preparation of purified KSHV and virus infection. iSLK cells latently infected with mini-TurboID-KSHVs were seeded in 8 to 10 15-cm dishes, stimulated with 1 mg/ml of doxycycline and 3 mM sodium butyrate for 24 h, and further incubated with culture media without stimuli for 72 h. The culture supernatant was centrifuged using a Beckman SW28 rotor (25,000 rpm for 2 h) with a 25% sucrose cushion. Virus pellets were dissolved in DMEM and further purified by discontinuous sucrose gradient (25 to 60%) centrifugation using a Beckman SW40Ti rotor (21,000 rpm for 16 h). Virus pellets were dissolved in DMEM for infection studies. HEK293FT cells were infected with 1 genomic copy of virus per cell in DMEM. Twenty-four hours postinfection, cells were washed with PBS and incubated for 24 h in complete media. After 48 h postinfection, cells were trypsinized and washed twice with PBS. Cells were resuspended in PBS containing 1% bovine serum albumin (BSA) and 1 mM EDTA, and recombinant viral infection was analyzed with flow cytometry (BD Accuri) and FlowJo software.
Real-time RT-PCR. Total RNA was isolated using a Quick-RNA miniprep kit (Zymo Research, Irvine, CA). First-strand cDNA was synthesized using high-capacity cDNA reverse transcription (RT) kit (Thermo Fisher, Waltham, MA). Gene expression was analyzed by real-time qPCR using specific primers for KSHV open reading frames (ORFs) designed by Fakhari and Dittmer (59). We used 18S rRNA as an internal standard to normalize viral gene expression.
Affinity purification of biotinylated proteins. Affinity purification was done with streptavidincoated magnetic beads (Thermo Fisher). Briefly, 150 ml of magnetic beads/sample were prewashed with RIPA lysis buffer (150 mM NaCl, 5 mM EDTA [pH 8], 50 mM Tris [pH 8], 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) 3 times. A total of 3 mg of whole-cell lysate was incubated with prewashed streptavidin beads at room temperature for 1 h for rotation. The beads were collected using a magnetic stand and washed three times with wash buffer according to the manufacturer's protocol. Finally, beads were resuspended in 200 ml of wash buffer and sent to the UC Davis proteomics core for on bead digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
MS sample preparation. Protein samples on magnetic beads were washed four times with 200 ml of 50 mM ammonium bicarbonate (AMBIC) with a 20-min shake time at 4°C in between each wash. Roughly 2.5 mg of trypsin was added to the beads and AMBIC, and the samples were digested overnight at a shake speed of 800 rpm. After overnight digestion, the supernatant was removed, and the beads were washed once with enough 50 mM ammonium bicarbonate to cover. After 20 min with gentle shaking, the wash was removed and combined with the initial supernatant. The peptide extracts were reduced in volume by vacuum centrifugation and a small portion of the extract was used for fluorometric peptide quantification (Thermo Scientific Pierce). One microgram of sample based on the fluorometric peptide assay was loaded for each LC-MS analysis.  Digested peptides were analyzed by LC-MS/MS on a Thermo Scientific Q Exactive Orbitrap mass spectrometer in conjunction with a Proxeon Easy-nLC II high-performance liquid chromatograph (HPLC; Thermo Scientific) and Proxeon nanospray source. The digested peptides were loaded onto a 100-mm by 25-mm Magic C 18 100-Å 5U reverse-phase trap, where they were desalted online before being separated using a 75-mm by 150-mm Magic C 18 200-Å 3U reverse-phase column. Peptides were eluted using a 60-min gradient with a flow rate of 300 nl/min. An MS survey scan was obtained for the m/z range 300 to 1,600, and MS/MS spectra were acquired using a top-15 method, where the top 15 ions in the MS spectra were subjected to high-energy collisional dissociation (HCD). An isolation mass window of 2.0 m/z was used for precursor ion selection, and a normalized collision energy of 27% was used for fragmentation. A 15-s duration was used for dynamic exclusion.
MS/MS analysis. Tandem mass spectra were extracted and charge state deconvoluted by Proteome Discoverer (Thermo Scientific). All MS/MS samples were analyzed using X!. All MS/MS samples were analyzed using X! Tandem (The Global Proteome Machine [https://thegpm.org]; version X! Tandem Alanine [2017.2.1.4]). X! Tandem was set up to search the human and Kaposi's sarcoma herpesvirus database (149,182 entries) assuming the digestion enzyme trypsin. X! Tandem was searched with a fragment ion mass tolerance of 20 ppm and a parent ion tolerance of 20 ppm. Carbamidomethyl of cysteine and selenocysteine was specified in X! Tandem as a fixed modification. Glu!pyro-Glu of the N terminus, ammonia loss of the N terminus, Gln!pyro-Glu of the N terminus, deamidation of asparagine and glutamine, oxidation of methionine and tryptophan, and dioxidation of methionine and tryptophan were specified in X! Tandem as variable modifications.
Scaffold (version Scaffold_4.8.4; Proteome Software Inc., Portland, OR) was used to validate MS/MSbased peptide and protein identifications. Peptide identifications were accepted if they could be established at more than 98.0% probability by the Scaffold Local FDR algorithm. Peptide identifications were also required to exceed specific database search engine thresholds. Protein identifications were accepted if they could be established at more than 5.0% probability to achieve a false-discovery rate (FDR) less than 5.0% and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (60). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters.
Scaffold software was used for normalization of the peptide with the following steps. (i) Calculate the total number of spectra in each BioSample. (ii) Calculate the average number of spectra across all BioSamples. (iii) Multiply each spectrum count in each sample by the average count over the BioSample's total spectrum count.
Pathway analysis. The proteins identified as interacting with vIRF-1 and vIRF-4 were used for gene ontology. The top gene ontology processes were enriched by the Metascape web-based platform, and Metascape software was used for gene ontology analysis (61).
Statistical analysis. Results are shown as means 6 standard deviations (SD) from at least three independent experiments. Data were analyzed using unpaired Student's t test or using analysis of variance (ANOVA) followed by Tukey's honestly significant difference (HSD) test. A P value of ,0.05 was considered statistically significant.