key: cord-0268287-ws6z5k9r
authors: Fang, Jingru; Pietzsch, Colette; Tsaprailis, George; Crynen, Gogce; Cho, Kelvin Frank; Ting, Alice Y.; Bukreyev, Alexander; Carlos de la Torre, Juan; Saphire, Erica Ollmann
title: Functional interactomes of the Ebola virus polymerase identified by proximity proteomics in the context of viral replication
date: 2021-07-21
journal: bioRxiv
DOI: 10.1101/2021.07.20.453153
sha: 174849604328501be53acb08ec2d371d02cbfdf6
doc_id: 268287
cord_uid: ws6z5k9r

Ebola virus (EBOV) critically depends on the viral polymerase to replicate and transcribe the viral RNA genome. To examine whether interactions between EBOV polymerase and cellular and viral factors affect distinct viral RNA synthesis events, we applied proximity proteomics to define the cellular interactome of EBOV polymerase, under conditions that recapitulate viral transcription and replication. We engineered EBOV polymerase tagged with the split-biotin ligase split-TurboID, which successfully biotinylated the proximal proteome while retaining polymerase activity. We further analyzed the interactomes in an siRNA-based, functional screen and uncovered 35 host factors, which, when depleted, affect EBOV infection. We validated one host factor, eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), which we show physically and functionally associates with EBOV polymerase to facilitate viral transcription termination. Our work demonstrates the utility of proximity proteomics to capture the functional host-interactome of the EBOV polymerase and to illuminate host-dependent regulations of viral RNA synthesis.

turn serves as a template for synthesis of large amounts of NP-coated, progeny viral genomes (Mühlberger, 2007) .

The underlying mechanisms by which EBOV executes transcriptase and replicase activities 66 are not fully understood, partly due to a lack of structural insight for the full-length EBOV 

We confirmed that EBOV_pol split-TurboID fusion is functionally active using an established 

To assess enrichment efficiency, biotinylated material bound to SA beads (SA-pull down) from 155 equal amounts of starting material (Input) was eluted in SDS loading buffer and analyzed by 156 western blot ( Figure 2B) . We saw enrichment of biotinylated proteins specific to samples with 6 determined adjusted p-values (adjusted to False Discovery Rate of 0.05) to identify proteins 171 enriched in the TurboID_pol interactome. High-confidence hits were selected using a threshold of 172 adjusted p-value < 0.05 and log2(fold change) > 1. We found 43 high-confidence hits for cellular 173 proteins that interact with the EBOV polymerase in the presence of VP30 ( Figure 2C ). Another 28 174 high-confidence hits were found in the absence of VP30 (Figure 2D) , 7 of which were the same as 175 those seen in the presence of VP30. 

Based these results, we highlighted functional interactors identified in the proximity proteomics 

has not been implicated in viral infections. Thus, as a proof-of-principle, we focused on GSPT1, 205 which is a translation termination factor that is also termed eRF3a, eukaryotic peptide chain release 206 factor GTP-binding subunit (Zhouravleva et al., 1995) .

First, we used co-immunoprecipitation (co-IP) assays to verify whether GSPT1 indeed physically 208 interacts with EBOV polymerase (EBOV_pol). Using HEK 293T cells co-expressing an N-terminal 209 Flag-tagged GSPT1 (long isoform, 68.7KDa) and EBOV polymerase components, either 210 individually or in combination, we found that L-WT, in the presence of VP35-HA, consistently co-

IPs with Flag-GSPT1 ( Figure 5A) . However, Flag-GSPT1 did not co-IP with the VP35-HA 

Therefore, the GSPT1-EBOV protein-protein interactions we detected are likely not due to indirect 219 interactions bridged by RNA.

We next examined whether endogenous GSPT1 interacts with EBOV_pol in a cellular context 221 that recapitulates viral RNA synthesis. In HEK 293T cells transfected with the components of the 222 EBOV MG system, we observed a specific pattern of GSPT1 clusters in the cytoplasm that appear Intriguingly, GSPT1-KD initially increased EBOV titers by 2-to 4-fold relative to the non-silencing 241 control (NSC). However, at 3-and 4-days post-infection (dpi), GSPT1-KD correlated with reduced 242 EBOV titers (Figure 6A) . At 4 dpi, we observed an increase in EBOV NP mRNA accumulation, with 243 a marginal decrease in EBOV vRNA accumulation ( Figure 6B&C ). Despite the increase in viral 244 mRNA, viral protein levels, including EBOV glycoprotein subunit 2 (GP2), were reduced, and fewer 245 EBOV-infected cells were seen with GSPT1-KD ( Figure 6C&D) . Notably, we observed that EBOV 246 infection triggered a significant upregulation of GSPT1 mRNA levels (Figure 6E) , and the 247 appearance of a lower-molecular weight GSPT1 species (~40kDa) that could correspond to a 248 cleavage product of GSPT1 ( Figure 6C) . These findings suggest virus-specific, time-dependent 249 regulation of GSPT1 protein levels. 

The accumulation of all EBOV mRNAs we measured was increased upon GSPT1-KD. However, 267 the magnitude of this increase was greater for genes located on the promoter-distal, 5' end of the 268 EBOV genome, compared to genes on the promoter-proximal, 3' end at the polymerase entry site 269 ( Figure 6F&G) . Accordingly, in GSPT1-depleted cells, we detected higher incidence of EBOV 

Here we present the first systematic analysis of the EBOV polymerase-cellular interactome in 279 the context of viral RNA synthesis. We used proximity-labeling to characterize the EBOV 280 polymerase interactomes in situ, and performed an siRNA screen in human hepatocytes infected 281 with EBOV to identify functionally important interactors with EBOV polymerase during EBOV 282 infection. We discovered 64 high-confidence EBOV polymerase interactors, 31 of which were 283 validated by our siRNA screen as functional hits. Most polymerase-interactors we identified are 284 antiviral rather than proviral, suggesting that the viral polymerase, or viral RNA products, or both, 285 are targets of host innate defense mechanisms. As a proof-of-principle, we chose one functional 286 hit, GSPT1, to further dissect the interplay between EBOV polymerase and this host factor. GSPT1 287 appeared to play antiviral role in concert with another host factor, UPF1, in mediating RNA decay.

However, our data indicated that EBOV can in turn recruit GSPT1 to assist in highly regulated viral 289 transcription.

We identified 12 EBOV polymerase interactors that were previously shown by various 

TurboID with an EBOV minigenome (MG) system that incorporates all the molecular components 301 relevant to the EBOV polymerase activity and recapitulates EBOV RNA synthesis events. This 302 system also allowed us to compare the cellular interactome of EBOV polymerase in the absence 303 or presence of the EBOV VP30, which is essential viral cofactor for transcription, but not replication,

of the viral genome.

In the absence of VP30, we identified eight functional hits that interacted with EBOV polymerase, 

Among the eight hits, we also identified the dsRNA sensor EIF2AK2/PKR, which was previously 

In the presence of VP30, we found 19 functional hits specific to EBOV polymerase. These hits Goat, anti-human-HRP Thermofisher/Invitrogen Cat#A18811

Goat, anti-mouse-AlexaFluor568 Thermofisher/Invitrogen Cat#A11004

Goat, anti-mouse-AlexaFluor647 Thermofisher/Invitrogen Cat#A21236

Goat, anti-rabbit-AlexaFluor488 Thermofisher/Invitrogen Cat#A11008

Goat, anti-rabbit-AlexaFluor647 Thermofisher/Invitrogen Cat#A27040

Anti-FLAG M2 affinity Gel Sigma-Aldrich Cat#A2220-5ML Oligo dT(20) Primer Thermofisher/invitrogen Cat#18418020 

The fragment of VP35-sNTurboID, including the C-terminal HA tag, was subcloned to pCAGGS 728 plasmid using restriction sites Kpn1 and Nhe1 and standard ligation method.

To generate pCI-empty, the coding sequence of MS2V5-eRF3a-F76a was removed from pCI- 

The sequence of all engineered plasmid DNA was confirmed by Sanger sequencing. 

The labelling scheme for three biological replicates of each condition was as follows: including critical parameters used in thresholding were presented in Table S1 .

High-confidence hits in both EBOV polymerase interactomes in the presence and absence of VP30

were displayed as protein-protein networks using Cytoscape. The web-based bioinformatic analyzer STRING was used to perform functional enrichment analysis to cluster nodes involved in 898 the same biological process (FDR< 1%). analysis. Equal amount of total RNA in each sample was used for strand-specific RT-qPCR.

Fluorescent image acquired using Blue/Green Excitation for the GFP signal and Ultraviolet

Excitation for the Hoechst stained nucleus. Images from the same field of view but different 959 channels were merge using ImageJ/FIJI.

Total RNA was extracted from each Trizol-inactivated sample (per well) using Direct-zol RNA 

GSPT1si8) samples. In Figure 6E, samples from MOCK infected cells were used as control to 980 calculate the relative fold-change of the target amplicon in EBOV infected cells