Abstract
Studies on "hit-and-run" effects by viral proteins are difficult when using traditional affinity precipitation-based techniques under dynamic conditions, because only proteins interacting at a specific instance in time can be precipitated by affinity purification. Recent advances in proximity labeling (PL) have enabled identification of both static and dynamic protein-protein interactions. In this study, we applied a PL method by generating recombinant Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV, a gammaherpesvirus, uniquely encodes four interferon regulatory factors (IRF-1 to -4) that suppress host interferon responses, and we examined KSHV IRF-1 and IRF-4 neighbor proteins to identify cellular proteins involved in innate immune regulation. PL identified 213 and 70 proteins as neighboring proteins of viral IRF-1 (vIRF-1) and vIRF-4 during viral reactivation, and 47 proteins were shared between the two vIRFs; the list also includes three viral proteins, ORF17, thymidine kinase, and vIRF-4. Functional annotation of respective interacting proteins showed highly overlapping biological roles such as mRNA processing and transcriptional regulation by TP53. Innate immune regulation by these commonly interacting 44 cellular proteins was examined with small interfering RNAs (siRNAs), and the splicing factor 3B family proteins were found to be associated with interferon transcription and to act as suppressors of KSHV reactivation. We propose that recombinant mini-TurboID-KSHV is a powerful tool to probe key cellular proteins that play a role in KSHV replication and that selective splicing factors have a function in the regulation of innate immune responses.IMPORTANCE 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. Proximity labeling (PL), however, can also highlight transient and negative effects-those interactions which lead to dissociation from the existing protein complex. Here, we highlight the power of PL in combination with recombinant KSHV to study viral host interactions.
Highlights
Studies on “hit-and-run” effects by viral proteins are difficult when using traditional affinity precipitation-based techniques under dynamic conditions, because only proteins interacting at a specific instance in time can be precipitated by affinity purification
The wild-type (Wt) BAC16, 3ÂFlag-mini-TurboID-K9, and 3ÂFlag-mini-TurboID-K10 BAC16 were directly transfected into iSLK cells and selected with hygromycin (1 mg/ml) to generate iSLK cells harboring latent Wt BAC16 Kaposi’s sarcoma-associated herpesvirus (KSHV), 3ÂFlag-mini-TurboID-K9 KSHV, and 3ÂFlag-mini-TurboID-K10 KSHV, respectively (Fig. 1B)
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
Summary
Studies on “hit-and-run” effects by viral proteins are difficult when using traditional affinity precipitation-based techniques under dynamic conditions, because only proteins interacting at a specific instance in time can be precipitated by affinity purification. KSHV, a gammaherpesvirus, uniquely encodes four interferon regulatory factors (IRF-1 to -4) that suppress host interferon responses, and we examined KSHV IRF-1 and IRF-4 neighbor proteins to identify cellular proteins involved in innate immune regulation. Innate immune regulation by these commonly interacting 44 cellular proteins was examined with small interfering RNAs (siRNAs), and the splicing factor 3B family proteins were found to be associated with interferon transcription and to act as suppressors of KSHV reactivation. Kaposi’s sarcoma herpesvirus (KSHV) infection is associated with endothelial Kaposi’s sarcoma (KS) [1, 2], B-cell malignancies such as primary effusion lymphoma (PEL), and AIDS-related multicentric Castleman’s disease (MCD) [3,4,5,6] In these cancer cells, KSHV mostly exhibits latent infection, in which the viral genes maintain silence to avoid recognition by the host immune system. These studies demonstrated different outcomes in different cell lines, suggesting the significance of implementing proteomic approaches that can reveal vIRF interaction networks more comprehensively
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