Lytic infection with murine gammaherpesvirus 68 activates host and viral RNA polymerase III-dependent promoters to enhance non-coding RNA expression

RNA polymerase III (pol III) transcribes multiple non-coding (nc) RNAs that are essential for cellular function. Pol III-dependent transcription is also engaged during certain viral infections, including the gammaherpesviruses (γHVs), where pol III-dependent viral ncRNAs promote pathogenesis. Additionally, several host ncRNAs are upregulated during γHV infection and play integral roles in pathogenesis by facilitating viral establishment and gene expression. Here we sought to investigate how pol III-dependent transcriptional activity was regulated during gammaherpesvirus infection, using the murine gammaherpesvirus 68 (γHV68) system. To compare the transcriptional regulation of host and viral pol III-dependent ncRNAs, we analyzed a series of pol III-dependent promoters using a newly-generated luciferase reporter optimized to measure pol III activity. We measured promoter activity from these constructs at the translation level via luciferase activity and at the transcription level via RT-qPCR. We further measured endogenous ncRNA expression at single cell-resolution by flow cytometry. These studies demonstrated that lytic infection with γHV68 increased the transcriptional activity of multiple host and viral pol III-dependent promoters, and further identified the ability of accessory sequences to influence both baseline and inducible promoter activity after infection. These studies highlight how lytic gammaherpesvirus infection alters the transcriptional landscape of host cells to increase pol III-derived transcription, a process that may further modify cellular function and enhance viral gene expression and pathogenesis. IMPORTANCE Gammaherpesviruses are a prime example of how viruses can alter the host transcriptional landscape to establish infection. Despite major insights into how these viruses modify RNA polymerase II-dependent generation of messenger RNAs, how these viruses influence the activity of host RNA polymerase III remains much less clear. Small non-coding RNAs produced by RNA polymerase III are increasingly recognized to play critical regulatory roles in cell biology and virus infection. Studies of RNA polymerase III dependent transcription are complicated by its use of multiple promoter types and diverse RNAs with variable stability and processing requirements. Here, we established a reporter system to directly study RNA polymerase III-dependent promoter responses during gammaherpesvirus infection and utilized single-cell flow cytometry-based methods to reveal that gammaherpesvirus lytic replication broadly induces pol III activity to enhance host and viral non-coding RNA expression within the infected cell.


INTRODUCTION 28
Gammaherpesviruses (γHVs) are large, lymphotropic viruses that establish a life-long infection in their 29 hosts, with long-term latency in lymphocytes (1, 2). The γHVs include the human pathogens, Epstein-Barr virus 30 (EBV) and Kaposi's sarcoma-associated herpesvirus , and murine gammaherpesvirus 68 31 (γHV68 or MHV-68; ICTV nomenclature Murid herpesvirus 4, MuHV-4) (3). These viruses establish a primary 32 lytic infection in their host that is followed by a prolonged quiescent infection termed latency. Latency is 33 maintained in healthy individuals by a homeostatic relationship between the virus and the host immune response; 34 if this balance is disrupted (e.g., by immunosuppression), γHVs can reactivate from latency and actively replicate. 35 Disruption between the balance of γHV infection and host immune control is associated with HV multiple 36 pathologies, including a range of malignancies (4). 37 The γHVs contain several types of non-coding (nc) RNAs, including nuclear ncRNAs and functional 38 miRNAs; these diverse RNAs including ncRNAs transcribed by RNA polymerase II (e.g. the KSHV PAN RNA 39 and the KSHV and EBV miRNAs) or by RNA pol III (e.g. the EBV-encoded small RNAs (EBERs) and the HV68 40 tRNA-miRNA-encoded RNAs (TMERs)) (5-13). Viral ncRNAs are considered to have important host-modulatory 41 functions, interacting with host proteins and regulating host and viral gene expression. For example, the EBV 42 EBERs are highly expressed during latency, and were discovered through their interaction with the host lupus-43 associated antigen (La) protein, which putatively mediates EBER interaction with TLR3 (14-17).The EBERs have 44 further been shown to interact with several host proteins including ribosomal protein L22, protein kinase R (PKR), 45 and retinoic-acid inducible gene I (RIG-I) (18). These interactions can trigger sustained host innate immune 46 responses that are implicated in the development of EBV-associated malignancies (16,(19)(20)(21). γHV68, a highly 47 tractable small animal model of HV infection, also encodes several pol III-transcribed ncRNAs known as the 48 tRNA-miRNA-encoded RNAs (TMERs) (22,23). The TMERs are dispensable for lytic replication and 49 establishment of latency; however, these transcripts are required for pathogenesis during acute infection of an 50 immunocompromised host (7,(24)(25)(26). The TMERs contain bi-functional elements with a tRNA-like structure on 51 the 5' end and hairpin loops that are processed into biologically-active miRNAs (7), capable of targeting a number 52 of RNAs for post-transcriptional regulation (27). Our lab has previously shown that the tRNA-like structure is 53 sufficient to rescue pathogenesis of a TMER deficient viral recombinant, suggesting that like the EBERs, the 54 TMERs may contribute to pathogenesis through their interactions with host proteins (26). Though TMER-host 55 protein interactions have yet to be fully explored, it is notable that several characteristics of the EBERs, such as 56 a 5'-triphosphate and 3'-polyU, are imparted by RNA polymerase III (pol III) transcription (28).These motifs can 57 be recognized by host RNA-binding proteins, such as RIG-I or La, to trigger an innate immune response (16, 20, 58 28, 29). 59 Pol III is often considered to perform "house-keeping" functions, as it transcribes host genes required for 60 cell growth and maintenance (e.g. U6 snRNA, tRNAs, and 5S rRNA) (30). Despite this, it is clear that the HVs 61 relative to mock-treated samples was compared after 24 h of infection (Fig 4). When we compared the relative 147 inducibility of EBER "full" versus "minimal" promoters, "minimal" promoters showed greater virus-inducibility. 148 This enhanced inducibility of the "minimal" EBER promoters likely reflects the reduced baseline luminescence 149 from these promoters (unpublished data). Conversely, TMER minimal promoters displayed a weaker induction 150 during infection than their "full" counterparts, suggesting the sequence surrounded the TMER minimal 151 promoters drives stronger expression during infection. These results indicate that the sequence surrounding 152 minimal pol III promoter elements impacts both the baseline activity and inducibility of these promoters during 153

infection. 154
Luciferase readouts of pol III promoter activity allowed us to uniformly analyze pol III promoter activity. 155 This assay does not directly measure the level of RNAs, however, instead relying on an enzymatic readout of 156 luciferase protein activity. To ensure that HV68 infection was inducing pol III activity transcriptionally, we used 157 the same NanoLuc constructs to measure promoter activity at the RNA level by performing RT-qPCR for the 158 NanoLuc transcript. Following the same protocol as used for the luciferase assays, HEK 293 cells were 159 transfected with pGL3 and the pNLP3 vector expressed by pol III promoters of interest (as outlined in Fig 2). 160 Cells were then infected with γHV68 and RNA was purified from cells 16 or 24 h post-infection. Primers 161 targeting the NanoLuc gene were used for qPCR following reverse transcription of the RNA. Infection 162 increased the NanoLuc RNA expression from the U6 and TMER1 promoters, with more modest induction from 163 the EBER promoters ( Fig 5A). These results indicate that HV68 infection stimulates pol III-promoter activity 164 from multiple host and viral promoters, measured at both the transcriptional and translational level. To extend 165 these findings, we further measured NanoLuc RNA expression from "minimal" or "full" TMER promoters. These 166 studies demonstrated the HV68 infection increased NanoLuc RNA from the "minimal" promoter relative to 167 mock infected samples, with further RNA induction from the "full" TMER promoter. These results strongly 168 suggest that the NanoLuc reporter assay serves as a faithful readout for pol III-dependent transcription, 169 quantified at both the RNA and protein level. These findings also emphasize that sequences outside of the 170 minimal TMER promoters contribute to increased expression during infection. 171 HV lytic replication critically depends on viral DNA replication and late gene transcription, processes 172 that are inhibited by phosphonoacetic acid (PAA) (40, 41). We therefore tested the impact of PAA on virus-173 induced pol III induction. To do this, HEK 293 cells were transfected with pNLP3-TMER1 and infected as 174 before, with one set of samples receiving PAA treatment (200μg/mL) after 1 h of viral inoculation. RNA was 175 isolated 16 h post-infection and RT-qPCR was performed to detect the NanoLuc transcript. Notably, treatment 176 with PAA during infection had no impact on the induction of NanoLuc RNA compared to HV68 infected cells, 177 indicating that viral DNA replication and late gene synthesis was not required for pol III induction ( Fig 6A). 178 Though PAA treatment had no effect on the level of NanoLuc RNA levels (i.e. transcription), PAA treatment 179 was consistently associated with increase luciferase enzymatic activity, with PAA-treated HV68-infected 180 γHV68 infection activates pol III-dependent promoters 8 cultures characterized by a greater apparent induction of luciferase activity compared to HV68-infected 181 cultures alone. This PAA-driven enhancement of luciferase activity was observed for multiple pol III promoters, 182 including U6, TMER1, 4 and 5 and EBER 1 and 2 ( Fig 6B). The increase in luciferase activity following PAA 183 treatment, with minimal impact on NanoLuc RNA, strongly suggests that PAA treatment enhanced the 184 translational output from the promoters tested. These data suggest that viral late gene expression plays an 185 additional role in translation that is not seen at the transcriptional level, a phenomenon independent of pol III 186 promoter activity. 187 Given the reported relationship between the NF-κB pathway and the expression of pol III-dependent 188 transcripts (31), we analyzed the effect of NF-κB activation or inhibition on the activity of the U6 and TMER1 189 promoters via luciferase activity. First, we measured induction of an NF-κB reporter plasmid following treatment 190 with either TNF, a known inducer of the NF-κB pathway, or following HV68 infection. Whereas TNF 191 induced NF-κB reporter activity at 4 and 24 hours post-treatment, HV68 infection had no measurable impact 192 on expression from the NF-κB reporter (Supplemental Figure 2A). Next, we analyzed the impact of NF-κB 193 manipulation on pol III promoter activity. Treating cells with TNF modestly increased U6 promoter activity, 194 albeit to a lesser extent than HV68 infection (Supplemental Figure 2B). TMER1 promoter activity was not 195 affected by TNFα treatment. Inhibition of NF-κB with the BAY 11-7082 (BAY 11) compound increased U6-196 expressed luciferase activity in virus infected conditions, yet had no significant impact on TMER1 promoter 197 activity after infection (Supplemental Figure 2C). This indicates a potential role of NF-κB in inhibiting pol III 198 promoter activity during infection; however, this effect is only observed in the case of a gene-external (i.e. type

DISCUSSION 226
While γHV infection is known to alter expression of host genes, and viral ncRNAs are integral for 227 pathogenesis, the transcriptional regulation of these ncRNAs has remained unclear. Here, we propose that 228 different pol III promoter types allow for distinct means for transcriptional regulation during infection. This study 229 focused on the impact of γHV68 lytic replication on pol III promoter activity, identifying that lytic infection drives 230 a general upregulation of promoter activity across multiple host and viral pol III-dependent transcripts. This 231 upregulation was measured by a luciferase assay optimized for pol III transcription, with increased pol III-232 dependent promoter activity quantified by luciferase activity and RNA expression, with complementary findings 233 revealed through the use of flow cytometric analysis of ncRNA expression. These findings emphasize the utility 234 of the modified NanoLuc luciferase system to analyze pol III promoter activity, and provide clear evidence for 235 pol III promoters with large differences in basal and inducible promoter activity. They further emphasize the 236 capacity of HV lytic infection to modify pol III-dependent transcriptional machinery in infected cells, a process 237 that likely facilitates productive virus replication (34). At this time, it remains unknown whether pol III machinery 238 or transcription is altered during γHV68 latent infection or reactivation from latency. 239 Our use of a luciferase reporter to measure the activity of pol III promoters allowed us to directly 240 compare the functional activity of multiple pol III promoters, while avoiding potential differences that may arise 241 due to ncRNA sequence, structure, or stability. Though pol III promoters conventionally drive expression of 242 non-coding RNAs, there is clear precedent that pol III can transcribe translation-competent RNA (42, 43), and 243 luciferase reporters have been used for high throughput and unbiased analysis of pol III promoter activity (44, 244 45). Inspired by these studies, we cloned several host and viral ncRNA promoters into a NanoLuc luciferase 245 reporter to measure their activity during lytic γHV68 infection. We chose the NanoLuc luciferase as our To 246 further enhance the robustness of this reporter, we identified and removed a pol III termination sequence within 247 the NanoLuc gene which approximately doubled luciferase reporter activity. Though pol III transcription should 248 theoretically be terminated in the original NanoLuc reporter, pol III read-through of termination signals has 249 been reported (46). In total, use of the modified NanoLuc reporter construct afforded a sensitive and robust 250 readout for assessing pol III promoter activity. 251 Through use of this pol III reporter assay, we found that HV68 infection increased promoter activity 252 across a range of host and viral pol III promoters. This suggests that γHV68 infection generally upregulates pol 253 III activity. Interestingly, the consequence/magnitude of induction elicited by infection varied between 254 promoters. For example, the U6 promoter conveyed high basal activity with infection resulting in a modest 255 induction of U6 promoter activity. Conversely, the TMER promoters exhibited extremely low basal activity in 256 mock-infected conditions, with dramatic induction after HV68 infection. The inducibility of the TMERs was 257 further enhanced by accessory sequences outside of the minimal A and B box elements. One explanation for 258 this enhanced induction is that these extended sequences may contain additional transcriptional elements that 259 are integral to the promoter itself. While formally possible, it is notable that accessory sequences across the 260 TMERs are not conserved (Supplemental Fig 4A). An alternate explanation for the enhanced activity of the full 261 TMER promoters is that inclusion of the extended sequence includes the full tRNA-like structure of the TMER 262 genes (Supplemental Fig 4B). It is interesting to speculate that this tRNA-like structure could either lend 263 greater stability to transcripts, or protect the transcripts from degradation by host exonucleases or the viral 264 endonuclease, muSOX (47). Although pol III transcription is frequently associated with the transcription of 265 housekeeping ncRNAs, there is clear precedent that pol III can also participate in inducible gene expression 266 (e.g. HV68-induced expression of SINEs) (34, 35). Whether the TMERs have conserved regulatory 267 mechanisms with host inducible ncRNAs is currently unknown, however, the TMERs share more promoter 268 similarity to the SINEs (type 2, gene internal) than to the vault RNAs (type 3, gene external). 269 Our studies demonstrated that infection increased NanoLuc expression at both the RNA and protein 270 level, indicating that virus infection increased pol III promoter activity and not some secondary measurement. In total, our studies revealed a γHV68-dependent induction in the activity of host and viral pol III 296 promoters. This induction was seen in the expression of a reporter gene, as well as in the endogenous 297 expression of pol III-dependent transcripts. Though previous reports have focused on the virus-mediated 298 upregulation of specific host ncRNAs, these experiments suggest a broader effect of lytic γHV68 infection on 299 pol III activity. This suggests that γHV68 modulation of the host transcriptional landscape goes beyond mRNA 300 regulation, and that pol III-dependent transcripts are likely to play a wider role in γHV68 pathogenesis than 301 previously appreciated. 302 γHV68 infection activates pol III-dependent promoters

Mutagenesis of pNL1.1 to create the pNLP3 NanoLuc luciferase reporter. The promoterless 313
NanoLuc luciferase reporter vector pNL1.1[Nluc] was obtained from Promega, and primers were designed to 314 introduce silent mutations to remove the pol III termination signal in the NanoLuc coding sequence; these 315 primers are listed in Supplementary Table 1. Mutagenesis PCR was performed with the following cycles: (i) 95° 316 for 30s, (ii) 12 cycles of 95°C for 30s, 55°C for 1 min, 68°C for 3 m. The resulting DNA was digested with DpnI 317

(New England Biolabs Inc) and transformed into XL1-Blue super-competent cells (Agilent). Bacterial colonies 318
were sequenced to confirm the correct mutations. The resulting plasmid was named "pNLP3" to indicate that it 319 is a NanoLuc plasmid optimized for pol III. 320 Generating a pol III promoter-driven NanoLuc reporter panel. All promoters were generated to 321 include XhoI and HindIII overhang sequences on the 5' and 3' ends respectively. Several promoters were 322 constructed using ligated oligonucleotides. PCR-amplified promoters and pNLP3 were digested with XhoI and 323 HindIII, then promoters were ligated into pNLP3 using T4 DNA ligase (New England BioLabs Inc). See 324 Supplementary Table 1 for primers used to PCR-amplify promoters. Ligated constructs were transformed into 325 One Shot electro-or chemically competent TOP10 E. coli cells (Thermo Fisher Scientific, #C404052 or 326 #C404010), which were then plated at several dilutions on LB agar containing ampicillin. Resulting colonies 327 were expanded in LB broth with ampicillin and plasmid was isolated using the QIAprep Spin Miniprep Kit 328 (Qiagen). All constructs were confirmed by sequencing. of solution in 6-well plates). For all transfections, the molar ratio of NanoLuc plasmid to Firefly plasmid was 337 kept at 10:1; the total amount of DNA transfected per well was adjusted depending on the plate size 338 (approximately 10ng for 96-well, 100ng for 12-well, and 200ng for 6-well). Cells were incubated with 339 transfection solution for 24 hours prior to downstream applications, unless otherwise stated. 340 To analyze how promoter activity was affected by γHV68 infection, transfected HEK 293 cells were 341 infected with WT, TMER-TKO, or EBER-KI γHV68 at a multiplicity of infection (MOI) of 1 plaque forming unit 342 per cell. Cells were cultured for approximately 24 h prior to infection. Cell counts were determined by treating 343 with 0.05% Trypsin-EDTA (Life Tech, #25300-054) to remove and cells. These cells were mixed with Trypan 344 Blue dye (Bio-Rad, #145-0021) to obtain a live cell count using the TC20 Automated Cell Counter (Bio-Rad). 345 Virus stocks were mixed with 5% complete DMEM, then added to cells and incubated with virus for 1 hour. (Version 8.0d). Statistical significance was tested by unpaired t test (comparing two conditions) or one-way 386 ANOVA (comparing three or more conditions) and subjected to multiple corrections tests using recommended 387 settings in Prism. All flow cytometry data were analyzed in FlowJo (version 10.6.1) with flow cytometry data 388 shown as pseudo-color dot plots. 389

ACKNOWLEDGEMENTS 390
This work was supported by the NIH grants R01AI121300 to LFvD and R21 AI134084 to LFvD and ETC, 391 T32 AI052066 to ANK and CO RNA Biosciences summer internship support to AM. 392 We thank the members of the Clambey and van Dyk lab for helpful discussions, members of the Colorado 393 RNA Bioscience Initiative for their insights, the Colorado ClinImmune core for flow cytometry services and the 394 Genomics Shared Resource of the University of Colorado Cancer Center which receives direct funding support 395 from the National Cancer Institute through Cancer Center Support Grant P30CA046934. Optimization of the NanoLuc reporter vector. Mutations were introduced into the pNL1.1 vector to remove the 541 pol III termination signal (TTTT) from the Nanoluc coding region, resulting in the pNLP3 vector. The human U6 542 promoter was cloned into each of these reporters, and dual luciferase assays were performed to compare 543 luciferase output. Data shown is representative of two independent experiments with biological triplicates. 544 Additionally, RNA was isolated from cells transfected with these two constructs. Cells were mock or WT γHV68-545 infected for 16 h, then cellular RNA was used as a template for primers targeting the entire NanoLuc gene (top 546 gel, 534 nt), or targeting just the NanoLuc sequence upstream of the termination sequence (bottom gel, 234 nt). were treated as previously described and RNA was purified from cells at 16 h post-infection (U6 n = 2, TMER1, 585 n = 5) or 24hpi (EBER1 n = 1, EBER2 n = 1, TMER4 n = 2, and TMER5, n = 1). RNA was converted to cDNA, 586 then LightCycler real-time PCR using Syber Green was performed with primers targeting the NanoLuc gene and 587 a host control gene (18S). The relative difference of NanoLuc was calculated using the Pfaffl method (2001, 588 Nucleic Acids Research), where the ratio = (Etarget) ΔCP target (control-sample) / (Eref) ΔCP ref (control-sample) . Each experiment (n) 589 included biological triplicates or duplicates. Error bars = SEM. Significant differences analyzed by t-test and 590 indicated as asterisks. P-values are indicated as follows: * = P ≤ 0.05, ** = P ≤ 0.01, *** = P≤ 0.001, **** = P ≤ 591 0.0001. 592 593 Figure 6. γHV68 infection coupled with PAA treatment further increases luciferase activity, but not 594 transcript level, from pol III promoters. A) Promoter activity was measured via RT-qPCR of the NanoLuc 595 transcript expressed from the TMER1 promoter during infection and concurrent phosphonoacetic acid (PAA) 596 treatment. RT-qPCR was performed as previously described. Data is shown as NanoLuc relative difference. N 597 = 1 with biological triplpicates. 598