Studies on "HIT&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 to identify both static and dynamic protein-protein interactions. Here we applied a PL method by generating recombinant Kaposi’s sarcoma-associated herpesvirus (KSHV). KSHV, a gamma-herpesvirus, uniquely encodes four interferon regulatory factors (IRFs 1-4) that suppress host interferon responses, and we examined KSHV vIRF-1 and vIRF-4 neighboring proteins to identify cellular proteins involved in innate immune regulation. PL identified 213 and 70 proteins as neighboring proteins of 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 were examined by siRNAs and the splicing factor 3B family proteins were found to be associated with interferon transcription and 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 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 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.
Studies on "HIT&RUN" effects by viral protein 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 study of both static and dynamic protein-protein interactions. Here we applied PL method with recombinant Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV, a gamma-herpesvirus, uniquely encodes four interferon regulatory factors (IRFs 1-4) in the genome, and we identified KSHV vIRF-1 and vIRF-4 interacting proteins during reactivation. Fusion of mini-TurboID with vIRF-1 or vIRF-4 did not interfere with KSHV gene expression, DNA replication, or de novo infections. PL identified 213 and 70 proteins for vIRF-1 and vIRF-4 respectively, which possibly interact during KSHV reactivation, and 47 of those 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 functions such as mRNA processing and transcriptional regulation by TP53. Involvement of commonly interacting 44 cellular proteins in innate immune regulation were examined by siRNAs, and we identified that splicing factor 3B (SF3B) family proteins were clearly involved in interferons transcription and suppressed KSHV reactivation. We propose that recombinant TurboID-KSHV is a powerful tool to probe key cellular proteins that play a role in KSHV replication, and selective splicing factors may have a function beyond connecting two exon sequences to regulate innate immune responses.
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