key: cord-0285145-1kytxz1y
authors: Rodriguez, William; Mehrmann, Timothy; Muller, Mandy
title: Shiftless Restricts Viral Gene Expression and Influences RNA Granule Formation during KSHV lytic replication
date: 2022-02-18
journal: bioRxiv
DOI: 10.1101/2022.02.18.480778
sha: e6523dde0424640c363c75f7583a575521d84160
doc_id: 285145
cord_uid: 1kytxz1y

Herpesviral infection reflects thousands of years of co-evolution and the constant struggle between virus and host for control of cellular gene expression. During Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic replication, the virus rapidly seizes control of host gene expression machinery by triggering a massive RNA decay event via a virally-encoded endoribonuclease, SOX. This virus takeover strategy decimates close to 80% of cellular transcripts, reallocating host resources toward viral replication. The host cell, however, is not entirely passive in this assault on RNA stability. A small pool of host transcripts that actively evade SOX cleavage has been identified over the years. One such “escapee”, C19ORF66 (herein referred to as Shiftless - SHFL) encodes a potent anti-viral protein capable of restricting the replication of multiple DNA, RNA, and retroviruses including KSHV. Here, we show that SHFL restricts KSHV replication by targeting the expression of critical viral early genes, including the master transactivator protein, KSHV ORF50, and thus subsequently the entire lytic gene cascade. Consistent with previous reports, we found the SHFL interactome throughout KSHV infection is dominated by RNA-binding proteins that influence both translation and protein stability, including the viral protein ORF57, a crucial regulator of viral RNA fate. We next show that SHFL affects cytoplasmic RNA granule formation, triggering the disassembly of processing bodies. Taken together, our findings provide insights into the complex relationship between RNA stability, RNA granule formation, and the anti-viral response to KSHV infection. Significance In the past five years, SHFL has emerged as a novel and integral piece of the innate immune response to viral infection. SHFL has been reported to restrict the replication of multiple viruses including several flaviviruses and the retrovirus HIV-1. However, to date, the mechanism(s) by which SHFL restricts DNA virus infection remains largely unknown. We have previously shown that following its escape from KSHV-induced RNA decay, SHFL acts as a potent anti-viral factor, restricting nearly every stage of KSHV lytic replication. In this study, we set out to determine the mechanism by which SHFL restricts KSHV infection. We demonstrate that SHFL impacts all classes of KSHV genes and found that SHFL restricts the expression of several key early genes, including KSHV ORF50 and ORF57. We then mapped the interactome of SHFL during KSHV infection and found several host and viral RNA-binding proteins that all play crucial roles in regulating RNA stability and translation. Lastly, we found that SHFL expression influences RNA granule formation both outside of and within the context of KSHV infection, highlighting its broader impact on global gene expression. Collectively, our findings highlight a novel relationship between a critical piece of the anti-viral response to KSHV infection and the regulation of RNA-protein dynamics.

protein, KSHV ORF50, and thus subsequently the entire lytic gene cascade. Consistent 23 with previous reports, we found the SHFL interactome throughout KSHV infection is 24 dominated by RNA-binding proteins that influence both translation and protein stability, 25

including the viral protein ORF57, a crucial regulator of viral RNA fate. We next show 26 that SHFL affects cytoplasmic RNA granule formation, triggering the disassembly of 27 processing bodies. Taken together, our findings provide insights into the complex 28 relationship between RNA stability, RNA granule formation, and the anti-viral response 29 to KSHV infection. 30 31 32 Introduction in our initial RT-qPCR screen represents the total amount of ORF50 expressed upon 145 induction of lytic reactivation from both promoters. To determine whether SHFL effect 146 on ORF50 was promoter-dependent, we next designed a set of primers to differentially 147 assess vORF50 vs. eORF50 expression ( Figure 1E) . We observed that SHFL ability to 148 repress ORF50 expression was active regardless of the ORF50 source, suggesting that 149 SHFL-mediated effect is promoter independent. Lastly, Since SHFL has been shown to 150

interact with viral RNAs, we next checked whether it could directly bind to ORF50 151 mRNA. To test this, we used RNA immunoprecipitation and found that SHFL does bind 152 to ORF50 mRNA during KSHV lytic replication ( Figure 1F) . We thus hypothesized that 153 SHFL-mediated repression of ORF50 could stem from a destabilization of ORF50 154 mRNA. However, using an Actinomycin D assay, we did not observe any significant 155 difference in ORF50 mRNA half-life (t 1/2 = 4h) upon SHFL expression (Supp Fig 2) . 156

Collectively, these data suggest that SHFL restricts lytic gene expression post-157 transcriptionally and in a manner independent from viral RNA stability. 158 (RIP) performed using mCherry antibody. Following reverse crosslinking, total RNA was then 174 harvested and subjected to RT-qPCR using primers as indicated. Statistics were determined 175 using students paired t-test between control and experimental groups; error bars represent 176 standard error of the mean; n=3 independent biological replicates.

178 179

To better understand how SHFL is regulating ORF50 expression, we next set out 181 to map its interaction network throughout KSHV infection. iSLK.WT were either left 182 latent or were reactivated for 48h with doxycycline and sodium butyrate to trigger KSHV 183 lytic cycle. After verifying SHFL pull-down efficiency (Supp Fig 3) , the SHFL 184 interactome during KSHV infection was mapped using LC-MS/MS. In total, 98 unique 185 proteins were identified as SHFL interactors, of which 9 were exclusively detected in the 186 latent cells and 12 were exclusively found in lytic cells (Supp Table 1 ), (Figure 2A) . 187

The remaining interactors span both latency and lytic replication and include the known 188 SHFL interactor PABPC1, which we also confirmed via co-immunoprecipitation (Supp 189 Table  192 2). Notably, several important cellular RNA binding proteins were identified that are 193 known constituents of cytoplasmic stress granules including PABPC1, KPNA2, DDX3X, 194 FUS, and HNRNPK (34) (35) (36) (37) . Intriguingly, we also detected three viral proteins as 195 potential SHFL interactors: ORF59, the KSHV DNA processivity factor; ORF57, the 196 master regulator of KSHV RNA fate and ORF52, a tegument protein that inhibits 197 cytosolic viral DNA sensing via cGAS/STING (38) (39) (40) . Bodies (P-Bodies), are membrane-free, phase-separated ribonucleoprotein (RNP) 215 complexes that function in the storage, translational arrest, and/or degradation of RNA 216 in the cytoplasm and nucleus (41) (42) (43) (44) (45) (46) (47) (48) . Given the enrichment of SG components in our 217 mass spectrometry data, we next set out to determine whether SHFL localizes to RNP 218 granules. First, HEK293T cells were transfected with either a mock vector or a SHFL 219 expressing vector. Cells were then fixed, permeabilized, and immunostained for known 220 RNP granule markers including DEAD-Box Helicase 6 (DDX6) and enhancer of mRNA 221 decapping 4 (EDC4) for P-bodies and G3BP Stress Granule Assembly Factor 1 222 (G3BP1) and Cytotoxic Granule Associated RNA Binding Protein (TIA-1) for Stress 223

Granules. RNP granule quantification was performed using CellProfiler to analyze 224 immunofluorescence images stained for the hallmark P-body and SG resident proteins 225 as described in the methods section. In HEK293T cells, SHFL remains diffusely 226 cytoplasmic as we observed previously (16) (Figure 3A) . However, surprisingly, we 227 observed that SHFL expression drastically restricted the number of DDX6 ( Figure 3A ) 228 and EDC4 ( Figure 3B ) puncta per cell relative to mock transfection. Furthermore, we 229 also observed that there was a simultaneous induction of "SG-like densities" co-230 localizing with SHFL in these same expressing cells ( Figure 3B) . A similar effect was 231 found with TIA-1, a second SG marker, in SHFL expressing cells (Supp Figure 4) . 232

To determine whether SHFL expression also influences P-body formation during 233 KSHV infection, we next transfected iSLK.WT cells with SHFL, left them either latent or 234 reactivated with Doxycycline and Sodium Butyrate, and stained for the same RNP Given SHFL effect on ORF50 expression and its influence over RNP granule 262 dynamics, we were particularly intrigued by its interaction with KSHV ORF57 detected in 263 our mass spectrometry screen. We first confirmed the interaction between SHFL and 264 ORF57 by immunoprecipitation and reverse immunoprecipitation ( Figure 4A ). While 265 SHFL overexpression in iSLK.WT cells resulted in lower expression of ORF57 mRNA 266 ( Figure 1A) , transient co-expression of SHFL and ORF57 in HEK293T cells seems to 267 have no effect on ORF57 mRNA ( Figure 4D ), reinforcing the idea that the effect 268 observed on viral mRNA levels in KSHV positive cells is post-transcriptional and stems 269 from a ORF50-dependent mechanism. We thus hypothesized that SHFL could be 270 influencing the expression of ORF57, which in turn could be a mechanistic underpin to 271 SHFL-mediated translational repression of ORF50. First, we showed that the interaction 272 between ORF57 and SHFL is drastically reduced when the samples are treated with 273

RNAse, suggesting that SHFL and ORF57 may be brought together in a complex 274 around viral and perhaps even host RNAs ( Figure 4A ). Next, we found that SHFL co-275 expression with ORF57 markedly reduced the amount of ORF57 protein, but not mRNA 276 levels, expressed in HEK293T cells (Figure 4B-D) pointing once again to a mechanism 277 beyond viral gene transcription and RNA stability. Lastly, we wondered whether SHFL 278 and ORF57 co-localize, and if so, does this co-localization coincide with SHFL induced 279 SG as shown in Figure 3 . To test this, we again co-transfected SHFL alongside ORF57 280 in HEK293T cells and performed an immunofluorescence assay. Cells were then fixed, 281 permeabilized, and immunostained for both ORF57 and the SG marker TIA-1. 282

Interestingly, while ORF57 was predominately nuclear, distinct densities of SHFL 283 appeared to co-localize with densities of ORF57 and TIA-1 in the cytoplasm ( Figure 4E -284 F). We also did not see the same level of SG marker accumulation with TIA-1 as 285 previously observed with expression of SHFL alone, indicating that ORF57 may still be 286 dispersing SHFL induced RNA granules. While TIA-1 is a SG marker, the protein itself 287 serves a primary function in orchestrating translational arrest (50, 51) . As such, these 288 data indicate that SHFL may be restricting the translation of ORF57 and therein its 289 expression. 290 retroviruses to varying degrees and is itself an Interferon Stimulated Gene (ISG) 311 (20, 27) . Fascinatingly, for each virus that SHFL restricts, a different mechanism has 312 been described. These inhibition strategies range dramatically from the targeting of viral 313 RNA and protein stability to restriction of viral protein translation, and even the 314 architecture of viral replication organelles (23) . SHFL is also the first human gene found 315 to actively restrict the -1 programmed ribosomal frameshift (-1PRF), a translation 316 strategy conserved across several eukaryotic viruses, mammals, and even prokaryotes 317 (19, 22, (52) (53) (54) . Collectively, these studies reflect the versatility of SHFL to broadly 318 influence gene expression and therein directly impact the balance between host and 319 viral gene expression during infection. Similarly, we have previously identified SHFL as 320 a transcript that actively escapes virus-induced RNA decay during KSHV infection (18) . 321

Furthermore, we also found that the SHFL protein is a potent anti-KSHV factor, 322

restricting nearly every stage of KSHV lytic replication following reactivation from 323 latency. Here, we present our recent efforts toward understanding the mechanism(s) by 324

Looking broadly at KSHV lytic gene expression, we found that transient SHFL 326 expression stringently restricts all classes of lytic genes at both the RNA and protein 327 levels. Taken together with our previous observations of SHFL knock-down (18) , this 328 restriction is likely a domino effect from a direct impact of SHFL on KSHV early gene 329 expression. In line with this, we observed a significant restriction of KSHV ORF50 330 (RTA), which encodes the master latent-to-lytic switch protein responsible for the 331 initiation of the entire lytic gene cascade (29) . Upon further investigation, we found that 332 SHFL binds to ORF50 mRNA but does not significantly impact the half-life of ORF50 333 mRNA during lytic replication. This suggests that SHFL influence over ORF50 is likely at 334 the protein level. It would thus be interesting to investigate the effect of SHFL on 335 lysosome and proteasome-mediated degradation during KSHV infection as SHFL has 336 been implicated in these pathways before (22, 25) . This restriction of ORF50 early-on 337 following lytic reactivation would undoubtedly inhibit many crucial stages of KSHV lytic 338 gene expression and therein the remainder of lytic viral replication. Whether SHFL 339 exclusively impacts ORF50 expression during KSHV infection or, more broadly, the 340 protein stability and/or translation of multiple KSHV early genes remains an important 341 future direction for us. 342

To better understand the mechanism by which SHFL restricts herpesviral 343 translation, we next mapped the interactome of SHFL during KSHV infection using an 344 IP-MS approach. We found an enrichment of cellular RNA-binding proteins (RBPs) that 345 interact with SHFL during both KSHV latency and lytic reactivation. Among these 346 include a vital host translation factor and previously identified SHFL interactor, 347

Polyadenylate-Binding Protein Cytoplasmic 1 (PABPC). We also identified several other 348

RBPs that are known constituents of cytoplasmic RNP granules including both P-bodies factors regulate their stability (49, 60, 61) . However, during lytic replication, it has been 372 clearly established that KSHV actively disassembles P-bodies and restricts the 373 formation of SGs. This restriction of RNP granules during lytic replication is directly 374 facilitated by the viral protein KSHV ORF57 (49, 62) . ORF57 is a master regulator of 375 KSHV viral RNA fate with roles ranging from viral mRNA splicing, nuclear mRNA export, 376 and even facilitation of viral mRNA translation in the cytoplasm (39, 63, 64) . As such, we 377

were keenly interested in understanding the relationship between SHFL and ORF57. 378

Here, we found that SHFL does in-fact interact with ORF57 in an RNA-dependent 379 manner. Furthermore, we also observed a down regulation of ORF57 expression when 380 co-expressed alongside SHFL. Notably, there was no impact of SHFL on ORF57 mRNA 381 levels, which in combination with our observations of ORF50, further suggests that 382 SHFL also targets ORF57 expression at the protein level. Given its ability to influence 383 RNP granules, we next investigated whether ORF57 could restrict the formation of 384 SHFL induced SG-like densities. Surprisingly, we found that SG were still restricted by 385 ORF57 despite its distinct downregulation by SHFL. In their place, we observed distinct 386 accumulations of ORF57, SHFL, and the SG marker TIA-1 in the cytoplasm. These 387 SHFL-ORF57 densities could reflect sites of translational arrest by SHFL on ORF57 388 specifically. However, it may also suggest that there could be a distinct difference in the 389 RNP composition of SHFL induced SGs that could be tailored toward a response to viral 390 genes versus host genes. Further exploration is required to determine if SHFL also 391 restricts the translation of ORF57. Or, as suggested by recent SHFL studies in ZIKV, 392 PEDV, and JEV, SHFL could be coordinating with lysosomal or ubiquitinoylation 393 pathways to degrade ORF57 (22, 25, 26) . 394

In conclusion, our findings lay the foundation for a complex relationship between 395 SHFL, a potent anti-viral factor and the DNA virus, KSHV. Following the escape of 396 SHFL mRNA from SOX cleavage, SHFL protein levels climb over the course of KSHV 397 lytic replication. SHFL expression restricts both KSHV early and delayed early genes in 398 a manner that cascades out to late gene expression, an effect whose ramifications are 399 evident across every step of KSHV lytic replication. Here, we show for the first time that protocol. cDNAs were synthesized from 1 µg of total RNA using AMV reverse 438 transcriptase (Promega) and used directly for quantitative PCR (qPCR) analysis with 439 the SYBR green qPCR kit (Bio-Rad). Signals obtained by qPCR were normalized to 440 those for 18S unless otherwise noted. Primers used in the study are listed in Supp 441 Table 3 . masked with the identified nuclei to define a cytoplasm mask. The cytoplasm mask was 518 then applied to the processing body/stress granule puncta channel images (stains with 519 DDX6 and EDC4 for processing bodies) to ensure only cytoplasmic puncta were 520 quantified. Background staining was reduced in the cytoplasmic puncta channel using 521 the "Enhance Speckles" function. Using "global thresholding with robust background 522 adjustments", puncta within a defined size and intensity range were quantified. Size and 523 intensity thresholds were unchanged between experiments with identical staining 524 parameters. Intensity measurements of puncta were quantified. Quantification data was 525 exported and used for data analysis. HEK293T cells were co-transfected with both a FLAG-tagged SHFL and a 6X HIS-594 tagged ORF57. Cells were then harvested, lysed, and co-IP performed using FLAG-tag 595 affinity beads. Reverse co-IP was performed using anti-6X HIS antibody. RNAse co-IP 596 was performed similarly to FLAG co-IP with an additional RNAse A and RNAse T1 597 treatment prior to overnight co-IP (B) HEK293T cells were co-transfected with a FLAG-598 tagged ORF57 and either a mCherry only (mock) or NC-SHFL vector. Cells were then 599 harvested and subjected to immunoblot and stained with the indicated antibodies (B) or 600 total RNA extracted for RT-qPCR to ORF57 mRNA levels (D). (E) HEK293T cells were 601 transfected with either a mCherry only (mock) or NC-SHFL vector. Cells were subjected 602 to immunofluorescence assay and stained for the indicated proteins. Statistics were 603 determined using students paired t-test between control and experimental groups; error 604 bars represent standard error of the mean; n=3 independent biological replicates. 

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