2.1 Generation of iBAC

CRISPR-Cas9-mediated gene editing has become a prevalent method for knocking out individual or multiple cellular genes using lentiviruses, followed by antibiotic selection in various research domains [33]. The knockout of cellular genes within the KSHV infection cell model is pivotal for elucidating their roles in KSHV replication and pathogenesis. However, the iSLK-BAC16 system's reliance on three common antibiotic selection markers—puromycin for rtTA, hygromycin for BAC16, and G418 for doxycycline-inducible RTA—poses challenges for easily generating cellular gene knockouts through antibiotic selection [27]. To address this issue, we modify BAC16 by incorporating doxycycline-inducible elements into the RTA promoter and simplifying SLK cell model to include only one hygromycin selection marker. Initially, seven consecutive Tet response elements were introduced into the RTA promoter region of BAC16. Subsequently, the coding region of EGFP under the EF1α promoter was replaced with Tet transactivator (TET3G), which responds structurally to doxycycline [34], resulting in the creation of a new BAC termed iBAC, featuring Tet-on RTA but devoid of EGFP (Figure 1A). Digestion of iBAC with EcoRV and XhoI revealed KSHV DNA fragments of various sizes, which matched well with the predicted digestion pattern according to the published KSHV genome sequence (NC_009333) (Figure 1B). The migration of the digested KSHV DNA fragments was also comparable to most of the fragments from BAC16 (Figure 1B) [16]. Verification of the modified sequence and its surrounding sequence was done through DNA sequencing (Figure 1C). In addition to the modified regions containing the RTA promoter and EF1-GFP, there are only 5 base mutations and 18 base insertions in the TR region (between the second and the third repeat), while the remaining sequence is identical to that of BAC16 (Figure 1D). The sequence information of the KSHV iBAC clone has been stored in GenBank (GenBank access number: PV360714). Following transfection of iBAC into SLK cells, the SLK-iBAC stable cell line was established using hygromycin selection, maintaining KSHV latent infection while refraining from expressing EGFP. With the use of a hygromycin selection marker only in SLK-iBAC cells, it becomes more convenient to produce knockouts or establish stable expression of target genes through lentiviruses and antibiotic selection.

2.2 Induction of KSHV Lytic Replication in SLK-iBAC Cells

Next, we aimed to investigate the efficiency of KSHV in undergoing lytic replication upon doxycycline treatment. Both SLK-iBAC and iSLK-BAC16 cells were subjected to increasing concentrations of doxycycline, and the viral gene expressions were assessed via immunoblotting. It was observed that RTA expression was induced by doxycycline at 0.01 μg/mL and markedly enhanced at 0.1 μg/mL in SLK-iBAC cells, significantly surpassing the expression level in iSLK-BAC16 cells at equivalent doxycycline concentration, suggesting that the Tet response elements in BAC exhibit a more efficient response (Figure 2A). As a consequence, iSLK-BAC16 cells exhibited inadequate reactivation by doxycycline at 0.1 μg/mL, whereas SLK-iBAC cells displayed high reactivation even at 0.01 μg/mL, as evidenced by the expression of K8 and ORF45 via specific antibodies (Figure 2A,B). Additionally, a comparison of the KSHV viral gene expression at the RNA levels between SLK-iBAC and iSLK-BAC16 cells at 72 h post-transactivation by doxycycline at 0.01 μg/mL revealed markedly higher levels of immediate early genes (K4, K8, ORF50, and ORF71), early genes (K3, ORF56, ORF59, and ORF68), and late genes (K11, ORF40, ORF52, and ORF65) in SLK-iBAC cells compared to iSLK-BAC16 cells (Figure 2C). Similarly, the viral DNA copy number was substantially elevated in SLK-iBAC cells but not in iSLK-BAC16 cells (Figure 2D). Furthermore, the progeny virus level in SLK-iBAC was approximately 10 times higher than that in iSLK-BAC16 cells, as determined by quantifying the viral genomic DNA levels in the culture supernatant at 72 h post-transactivation (Figure 2E). Notably, the baseline viral genome copy number of SLK-iBAC cells is approximately 1.4-fold higher than that in iSLK-BAC16 cells, which is likely attributed to the 5-base mutations and 18-base insertions in the TR region of iBAC. Furthermore, we normalized the genome amplification in SLK-iBAC and iSLK-BAC16 cell lines separately to their respective starting points. The results indicate that viral DNA amplification is significantly higher in SLK-iBAC compared to iSLK-BAC16 (Figure S1A,B), suggesting that KSHV lytic replication is more efficient in SLK-iBAC cells.

ORF49 coding region is located on the opposite strand of RTA (ORF50) and Tet element insertion does not overlap with the coding sequence of ORF49 in KSHV iBAC (Figure S2A). To determine whether Tet element insertion affects ORF49 expression, we evaluated the ORF49 transcription kinetics in both SLK-iBAC and iSLK-BAC16 cells upon lytic reactivation. We treated both SLK-iBAC and iSLK-BAC16 cells with doxycycline alone or with a combination of doxycycline and sodium butyrate for 48 h, followed by quantitative RT-PCR analysis to measure ORF49 expression. The results indicated that ORF49 expression is induced by low concentrations of doxycycline (0.01−0.1 μg/mL) in SLK-iBAC cells, but not in iSLK-BAC16 cells. Treatment with a higher concentration of doxycycline (1 μg/mL) can induce ORF49 expression in iSLK-BAC16 cells; however, ORF49 expression increases approximately 10-fold in SLK-iBAC cells under the same conditions (Figure S2B). Additionally, co-treatment with sodium butyrate and doxycycline further enhanced the ORF49 levels in both iSLK-BAC16 and SLK-iBAC cells, with similar trends in ORF49 expression observed in both cell lines during these treatments. These findings underscore that SLK-iBAC cells exhibit a more efficient response to low levels of doxycycline, leading to significantly higher levels of KSHV lytic replication compared to iSLK-BAC16 cells.

2.3 Knockout of Cellular Genes in SLK-iBAC Cells by CRISPR-Cas9

Genetic knockout of cellular genes in KSHV-infected cell model is critical for studying KSHV replication and pathogenesis. To evaluate the knockout efficiency of this novel SLK-iBAC system, we opted to concurrently knockout two cellular genes using CRISPR-Cas9 technology. Previously, we found that ORF45 interacts with and persistently activates cellular p90 ribosomal protein S6 kinase (RSK)1 and RSK2, which is critical for KSHV lytic replication and progeny virus production [35, 36]. KSHV ORF45 forms high molecular mass protein complexes with RSK and ERK. The complexes protect RSK and ERK from dephosphorylation, leading to their persistent activation [37]. The ORF45-RSK axis enhances transcription and translation of a subset of viral and cellular genes during KSHV lytic replication. This is achieved at least partially through the phosphorylation of c-Fos, an immediate early transcription factor, and eIF4B, a regulatory translation initiation factor, respectively, to enable efficient lytic replication [38, 39]. Additionally, ORF45 promotes the SUMOylation of RSK1, facilitating the association between RSK1 and eIF4B via SUMO−SIM interactions, which is required for efficient lytic replication [26, 30].

To verify the role of RSKs for KSHV lytic replication in SLK-iBAC cells, we generated the double knockout of RSK1 and RSK2 in SLK-iBAC cells and characterized KSHV lytic replication. Specific sgRNAs targeting RSK1 or RSK2 were designed and incorporated into the Lenti-CRISPR V2-puro or Lenti-CRISPR V2-Blast vectors, respectively. These vectors contain the coding sequence of Cas9 and can deliver both Cas9 and sgRNA into target cells through lentivirus infection. Lentiviruses carrying both Cas9 and sgRNA directed against RSK1 or RSK2, or nontargeting control sgRNA (sgNTC) were generated from HEK293T cells and utilized to infect SLK-iBAC cells, followed by the antibiotic selection with both puromycin and blasticidin (Figure 3A). After 14 days of selection, the knockout of RSK1 and RSK2 was validated by immunoblotting with specific antibodies. The expression of both RSK1 and RSK2 was completely abolished in SLK^{RSK1/2 DKO}-iBAC cells compared to the wild-type SLK-iBAC cells (Figure 3B). Subsequently, we investigated whether the double knockout of RSK1 and RSK2 impacted KSHV lytic replication in the SLK-iBAC system. Consistent with previous findings in HEK293, iSLK-BAC16, or BCBL1 cells [35, 40], the dual knockout of RSK1 and RSK2 significantly impeded viral gene expression at both protein and mRNA levels during KSHV lytic replication (Figure 3C,D). Notably, viral DNA replication and progeny virus production were also markedly impeded in SLK^{RSK1/2 DKO}-iBAC cells compared to the wild-type SLK-iBAC cells (Figure 3E,F). These results highlight the efficient knockout of cellular genes in SLK-iBAC system using CRISPR-Cas9 combined with antibiotic selection, establishing it as a more convenient platform for investigating cellular gene functions in the KSHV life cycle compared to the iSLK-BAC16 system.

2.4 Generation of Virus−Host Interaction Network Using SLK-iBAC Cells

As an intracellular parasite, KSHV harnesses its viral gene products to interact with cellular targets, manipulating cellular signaling pathways to promote virus replication and establish persistent infection. While two comprehensive interactomes between KSHV-encoded proteins and cellular proteins have been established, mainly through yeast two-hybrid screening or mass spectrometry analysis of purified viral protein complexes, these interactions were largely examined without the context of KSHV infection [41, 42]. In a previous study, our team identified the binding partners of ORF45 within the context of KSHV infection using specific antibodies against ORF45 [43]. However, this method heavily relied on the availability and quality of related antibodies. To address this limitation, we aimed to investigate the utility of the SLK-iBAC system for identifying of viral genes in the context of KSHV. To achieve this, we cloned 3xFlag-tagged ORF33, ORF52, ORF55, or GFP (as a control) into Lenti-X Tet-One inducible expression vector. This vector facilitates the expression of both the Tet-On 3G transactivator from the constitutive human PGK promoter and the gene of interest from TRE3GS promoter in a single construct. Following lentivirus infection and puromycin selection, we established stable cell lines: SLK-iBAC-GFP^Tet-On, SLK-iBAC-ORF33^Tet-On, SLK-iBAC-ORF52^Tet-On, and SLK-iBAC-ORF55^Tet-On (Figure 4A). Without doxycycline treatment, no GFP was expressed in SLK-iBAC-GFP^Tet-On cells, while doxycycline treatment for 72 h led to strong GFP expression, indicating the efficient expression induction for the genes of interest in the SLK-iBAC system (Figure 4B). Notably, doxycycline treatment induced robust expression of 3xFlag-tagged GFP, ORF33, ORF52, or ORF55, alongside efficient KSHV lytic replication within the SLK-iBAC system (Figures 4C and 2C). To examine the virus−host interaction network, we purified viral protein complexes using anti-Flag antibodies during KSHV lytic replication, eluted the associated proteins with 3xFlag peptide, and subjected the co-purified proteins to liquid chromatography and tandem mass spectrometry analysis. Through statistical analysis utilizing the MiST scoring algorithm (MiST score > 0.75, fold change > 4) [17], we identified 12, 37, and 64 cellular proteins interacting with ORF33, ORF52, and ORF55, respectively (Figure 4D−F). Localization analysis indicated that many of the ORF52 binding partners were cytoskeleton-associated proteins (Figure 4E), consistent with the findings linking ORF52 with tubulin during KSHV lytic replication [31]. Similarly, numerous ORF55 binding partners were membrane-associated proteins located in the endoplasmic reticulum, Golgi apparatus, or mitochondria (Figure 4F), in agreement with prior observations of ORF55 localizing in the Golgi apparatus during KSHV lytic replication [29]. Further validation is warranted to elucidate the functions of these cellular proteins in the KSHV life cycle. These results underscore the potential of the SLK-iBAC system for unraveling virus−host interaction networks within the context of KSHV infection.

2.5 Tracking the Localization of Viral Proteins During KSHV Lytic Replication in SLK-iBAC Cells

The subcellular localization of a protein is closely tied to its physiological functions within cells. Therefore, it is critical to determine the subcellular localization of viral proteins during both KSHV latency and lytic replication. In existing KSHV-infected cell models, such as iSLK-BAC16 cells, constitutive expression of EGFP is throughout the life cycle of KSHV. Additionally, iSLK-r219 cells not only express EGFP constitutively but also induce RFP expression during lytic replication [16, 27], complicating the tracking of viral protein subcellular localization or the colocalization between viral and cellular proteins. Since the EGFP coding region was replaced by the Tet transactivator in the generation of the iBAC system, SLK-iBAC cells do not express any fluorescent proteins. To address this, we introduced the EGFP coding sequence fused to the C-terminus of ORF52, resulting in the creation of iBAC^ORF52-EGFP and subsequently establishing stable SLK-iBAC^ORF52-EGFP cells (Figure 5A). ORF52 is a lytic gene, hence no GFP expression was observable in SLK-iBAC^ORF52-EGFP cells without doxycycline treatment (Figure 5B). Following doxycycline treatment, all the cells initiated GFP expression in iBAC^ORF52-EGFP cells, indicating the expression of ORF52 during KSHV lytic replication (Figure 5B). With this approach, we have shown that ORF52 is a cytoplasmic protein and colocalizes with tubulin and microtubule-associated protein 4 (MAP4), and iBAC^ORF52-EGFP showed similar lytic replication kinetics as wild-type iBAC [31]. These results demonstrated the utility of the SLK-iBAC system for monitoring the subcellular localization of viral proteins during KSHV lytic replication.