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

Abstract

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.

KSHV episome tethering sites on host chromosomes and regulation of latency-lytic switch by CHD4

“…We hypothesized that, by examining cellular proteins neighboring to LANA in infected cells, we may identify the repertoire of proteins important for selection of KSHV episome docking sites. To identify proteins in close proximity to LANA, we employed a proximity biotin labeling approach ( Kumar et al, 2021 ). The mini-TurboID is a biotin ligase that covalently attaches biotin to lysine residues in neighboring proteins (<10 nm) ( Branon et al, 2018 ; Kumar et al, 2021 ).…”

“…To identify proteins in close proximity to LANA, we employed a proximity biotin labeling approach ( Kumar et al, 2021 ). The mini-TurboID is a biotin ligase that covalently attaches biotin to lysine residues in neighboring proteins (<10 nm) ( Branon et al, 2018 ; Kumar et al, 2021 ). A recombinant KSHV BAC16 with LANA N-terminally tagged with mini-TurboID (referred to as KSHV LANA-mTID) was prepared; the procedure for the preparation of KSHV LANA-mTID is presented in Figure S2A .…”

“…Recombinant KSHV was prepared by following a protocol for En passant mutagenesis with a two-step markerless red recombination technique ( Tischer et al, 2010 ). Briefly, the codon optimized mini-TurboID coding sequence ( Kumar et al, 2021 ), which also encodes the 3x Flag tag, was first cloned into a pBS SK vector (Thermo Fisher, Waltham, MA USA). The pEPkan-S plasmid was used as a source of the kanamycin cassette, which includes the I-SceI restriction enzyme site at the 5′-end of the kanamycin coding region ( Tischer et al, 2010 ).…”

KSHV Episome Tethering Sites on Host Chromosomes: Regulation of Latency-Lytic Switch by the ChAHP complex

“…To identify proteins in close proximity to LANA, we used a miniTurboID based method. The miniTurboID is a biotin ligase, which covalently attaches biotin to lysine residues in neighboring proteins (<10 nm) in less than 10 minutes with no significant cell toxicity (64,65). A recombinant KSHV BAC16 with LANA N-terminal tagged with miniTurboID (referred to as KSHV LANA-mTID) was prepared; the procedure for the preparation of KSHV LANA-mTID is presented in Supplementary Fig.…”

“…Recombinant KSHV was prepared by following a protocol for En passant mutagenesis with a two-step markerless red recombination technique (107). Briefly, the codon optimized mini-TurboID coding sequence (65), which also encodes the 3x Flag tag was first cloned into a pBS SK vector (Thermo Fisher, Waltham, MA USA). The pEPkan-S plasmid was used as a source of the kanamycin cassette, which includes the I-SceI restriction enzyme site at the 5'-end of the kanamycin coding region (107).…”