key: cord-0294888-dhmy4mol
authors: Hirai, Yuya; Tomonaga, Keizo; Horie, Masayuki
title: The mechanism underlying the organization of Borna disease virus inclusion bodies is unique among mononegaviruses
date: 2021-05-24
journal: bioRxiv
DOI: 10.1101/2021.05.24.445377
sha: 0f4dc4985343fdfb609d268839a049a5f3550720
doc_id: 294888
cord_uid: dhmy4mol

Inclusion bodies (IBs) are characteristic biomolecular condensates organized by mononegaviruses. Here, we characterize the IBs of Borna disease virus 1 (BoDV-1), a unique mononegavirus that forms IBs in the nucleus, in terms of liquid-liquid phase separation (LLPS). The BoDV-1 phosphoprotein (P) alone induces LLPS and the nucleoprotein (N) is incorporated into the P droplet in vitro. In contrast, co-expression of N and P is required for the formation of IB-like structure in cells. Furthermore, while BoDV-1 P binds to RNA, an excess amount of RNA dissolves the liquid droplets formed by N and P. Notably, the N-terminal intrinsically disordered region of BoDV-1 P is essential to drive LLPS and bind to RNA, suggesting that both abilities could compete with one another. These features are unique among mononegaviruses, and thus this study will contribute to a deeper understanding of LLPS-driven organization and RNA-mediated regulation of biomolecular condensates.

The order Mononegavirales contains many pathogens of great importance to public health, 51 such as measles virus, Ebola virus, rabies virus, and human respiratory syncytial virus 52 (RSV) 1 . These mononegaviruses form various types of inclusion bodies (IBs) in infected 53 cells, which are thought to be the sites of viral replication 2 . The IBs of mononegaviruses 54 are membraneless organelles, also called biomolecular condensates 3-6 . Growing 55 evidence has shown that the IBs of several mononegaviruses, such as rabies virus 7 , 56 vesicular stomatitis virus (VSV) 8 , measles virus 9,10 , and RSV 11 , are organized by liquid-57 liquid phase separation (LLPS). 58 The viral ribonucleoprotein (vRNP) complexes of mononegaviruses, which function 59 as the fundamental units of transcription and replication, consist of viral genomic RNA, 60 nucleoprotein (N), phosphoprotein (P), and large RNA-dependent RNA polymerase (L). 61 Among the components of vRNP complexes, the expression of N and P is sufficient for 62 the formation of IBs of rabies virus 7 , measles virus 9 , RSV 11 , human metapneumovirus 63 12 , and human parainfluenza virus 3 13 . Previous studies of these IBs have provided 64 insights into the viral replication strategies, thereby contributing to the control of viral 65 infectious diseases. Additionally, since viruses exploit the host machinery for replication, 66 further studies would be helpful to gain a deeper understanding of the molecular 67 condensates in cells. 68 Borna disease virus 1 (BoDV-1) is a prototype virus of the family Bornaviridae of 69 the order Mononegavirales, which causes fatal encephalitis in mammals, including 70 humans 14 . BoDV-1 is a unique mononegavirus as replication occurs in the nucleus of the 71 infected cell, whereas that of most mononegaviruses occurs in the cytoplasm 15 . 72 Consequently, the IBs of cytoplasmic mononegaviruses are formed in the cytoplasm, 73 5 while those of BoDV-1, termed as viral speckle of transcripts (vSPOTs), are organized in 74 the nucleus 16 . The vSPOTs are membraneless spherical structures containing the viral 75 genome, antigenome, and several viral proteins. The BoDV-1 genome encodes six genes, 76 N, P, matrix protein (M), envelope glycoprotein (G), L, and accessory protein (X), among 77 which N, P, M, L, and X are localized in the vSPOTs 17, 18 . 78 Although present in the nucleus, the vSPOTs share common properties with those of 79 the other mononegaviruses, such as membraneless structures, and their components. 80 However, the surrounding environment is important for the formation of biomolecular 81 condensates. As described above, BoDV-1 forms IBs in the cell nucleus, where the 82 surrounding environment differs from that of the cytoplasm. Thus, the IBs of BoDV-1 83 may be formed by a different mechanism from those of other cytoplasmic 84 mononegaviruses. Nonetheless, the propensities and minimal components of BoDV-1 IBs 85 remain entirely elusive. 86 In this study, to clarify the mechanism of the formation of vSPOTs, we analyzed the 87 properties of the BoDV-1 N and P proteins from the aspect of phase-separated 88 condensates. We revealed that the BoDV-1 P protein alone causes LLPS in vitro, 89 depending on the intrinsically disordered region (IDR) present at the N-terminal region. 90 Although BoDV-1 N alone is not sufficient for the induction of LLPS, it was incorporated 91 into the BoDV-1 P droplets. Also, co-expression of BoDV-1 N and P is sufficient to induce 92 the formation of IB-like structures in cells. Further, P of BoDV-1, but not VSV or RSV, 93 binds to RNA. Interestingly, the N-terminal IDR of BoDV-1 P is involved in this RNA-94 biding activity, which is overlapped with the region important for driving LLPS, while an 95 excess amount of RNA dissolved the liquid droplets consisting of BoDV-1 P alone and NEBuilder HiFi DNA Assembly Master Mix (E2621; New England Bioscience, Ipswich, 108 MA, USA), which were designated as pET-15b-BoDV-1-Nco and pET-15b-BoDV-1-Pco, 109 respectively. The synthesized oligonucleotides of His-tagged mCherry and EGFP were 110 cloned into pET-15b using the NcoI and NdeI restriction sites, which were named pET-111 15b-His-mCherry and pET-15b-His-EGFP, respectively. The codon-optimized N and P 112 sequences were amplified by PCR and then inserted into pET-15b-His-mCherry and pET-113 15b-His-EGFP, respectively, using the NcoI site. 114 For mammalian expression vectors, the coding regions of the N and P genes of 

P triggers and is necessary for the formation of liquid droplets of BoDV-1 N and P 214 We first determined the viral protein(s) that forms liquid droplets in vitro. As described 215 above, the N and (partial) P proteins are necessary for the formation of liquid droplets by 216 other mononegaviruses 10,11 . In addition, the IDR is one of several factors that initiate 217 11 phase separation 4,22,23 . Therefore, we predicted the IDRs of the BoDV-1 N and P proteins 218 ( Fig. 1A and S1). P was predicted to have a disordered amino acid sequence, especially 219 at the N-terminal region (Fig. 1A) . Notably, the BoDV-1 genome encodes two P isoforms, 220 P and P', which are translated from alternative start codons 24 . The fully disordered region 221 was predicted to be present only in P, but not P' (Fig. 1A) . 222 To investigate which proteins cause phase separation, we examined whether P, P', 223 and/or N form liquid droplets in vitro. At a near physiological salt concentration (150 mM 224 NaCl), P clearly formed liquid droplets at a protein concentration of 10 µM (Fig. 1B) , 225 while N formed aggregate-like structures but not liquid droplets, and P' formed neither 226 (Fig. 1B) . At a protein concentration of 5 µM, but not 2.5 µM, in 150 mM NaCl, P formed 227 liquid droplets (Fig. 1C) . However, in 500 mM NaCl, P failed to initiate a phase separation, 228 even at a protein concentration of 20 µM (Fig. 1C) . 229 In infected cells, N and P are colocalized within viral biomolecular condensates, 230 vSPOTs 16 . Therefore, we next investigated the effects of P on the aggregate-like 231 structures of N by mixing both N and P in vitro. Here we mixed fluorescently labeled 232 proteins (N-mCherry and P-EGFP, both were tagged at the C-terminus) to the reaction 233 mixture to visually distinguish between the N and P proteins. The fluorescent protein-234 labeled N and P were incorporated into droplets formed by unlabeled N and P (Fig. 1D) , 235 respectively, indicating that both fluorescent protein-labeled N and P exhibited the same 236 behaviors as the unlabeled proteins. When N and P were mixed, the liquid droplets were 237 observed, which include not only P but also N (Fig. 1D) . However, a mixture of N and P' 238 formed distorted aggregate-like structures with few liquid droplets. (Fig. 1E) . 239 These results suggest that P, but not P', alone can form liquid droplets via the IDR 240 at the N-terminal region and N forms liquid droplets with P, which is also dependent on Next, we investigated and compared the properties of the liquid droplets formed by 253 P alone (P droplets) and by both N and P (NP droplets) by a simplified in vitro system. 254 Fusion events were observed with the P and NP droplets ( Fig. 2B and C) , suggesting that 255 both have liquid properties. However, the NP droplets required a much longer time for 256 two droplets to form one spherical droplet as compared to the P droplets ( Fig. 2B and C) . 257 Additionally, the P droplets were completely dissolved by 1,6-hexanediol (1,6-HD), 258 which is an aliphatic alcohol that affects the formation of liquid droplets 26 , while the NP 259 droplets were relatively more resistant to 1,6-HD, although the size and solubility were 260 reduced (Fig. 2D) . FRAP analysis showed that the fluorescence recovery of P-EGFP was 261 slower within the NP droplets than the P droplets (Fig. 2E) . These results suggest that N 262 decreases the fluidity and increases the stability of droplets. (Fig. 3A) . The expression of P alone did not induce the 274 formation of nuclear condensates (Fig. 3B ). On the other hand, the expression of both N 275 and P induced the formation of nuclear condensates that were morphologically similar to 276 vSPOTs (Fig. 3C, arrowheads) . The nuclear condensates induced by the expression of N 277 and P contained HMGB1, a host factor that is known to be localized to the vSPOTs of 278 infected cells (Fig. 3C) . Note that the condensates containing both N and P were observed 279 in the nucleus as well as the cytoplasm (Fig. 3C, arrows) . The expression of N and P' did 280 not induce the formation of condensates (Fig. 3D) , which is consistent with the in vitro 281 result (Fig. 1E) . These results suggest that N and P are sufficient for the formation of 282 BoDV condensates in cells, as also reported for other mononegaviruses, and P, but not P', 283 contributes to the formation of the condensates. between P and RNA. Therefore, we investigated possible interactions between P and the 290 BoDV-1 minigenome RNA (mgRNA), which contains the leader and trailer of BoDV-1 291 and Gluc sequences (Fig. S3A) 19 , with the use of the electrophoretic mobility shift assay. 292 A band shift was observed with an increasing concentration of P, suggesting interactions 293 with RNA (Fig. 4A) . On the other hand, P' did not interact with RNA. Note that a band 294 shift was also observed when using RNA lacking the 3' leader and 5' trailer regions (Fig.   295 S3B), suggesting that P interacts with RNA in a sequence-independent manner. No 296 apparent shift was observed with the P proteins of other mononegaviruses (i.e., VSV and 297 RSV) (Fig. 4A) . 298 We also investigated the possible incorporation of mgRNA into the droplets. We 299 observed that mgRNA was incorporated into both the P and NP droplets ( Fig. 4B and C) , 300 consistent with the RNA-binding activity of BoDV-1 P (Fig. 4A) . However, the signal 301 intensity of RNA was much stronger in the NP than P droplets, which probably reflects 302 the role of BoDV-1 N in encapsidating viral genomic RNA. 303 RNA regulates the formation of liquid droplets formed by RNA-binding proteins 28-304 31 . Therefore, we analyzed the effects of adding RNA to the P and NP droplets. The results 305 showed that mgRNA inhibited the formation of not only P but also NP droplets (Fig. 4B   306 and C, S1C). These results suggest that RNA is incorporated into the viral condensates in reconstruct the viral IB-like structures in cells (Fig. 3) . On the other hand, BoDV-1 P 320 alone drove the formation of liquid droplets in vitro, which was dependent on the IDR 321 ( Fig. 1) . This feature is distinct from other mononegaviruses that needs both N and P to 322 form liquid droplets. Further, P of BoDV-1, but not other mononegaviruses (i.e., VSV and 323 RSV), binds to RNA and high concentrations of RNA dissolved droplets formed by 324 BoDV-1 N and P in vitro (Fig. 4 and S3) . These findings revealed that although BoDV-1 325 shares common strategies with other mononegaviruses for the formation of IBs, at least 326 to a certain extent, BoDV-1 apparently adopted strategies different from those of other 327 mononegaviruses. 328 The unique feature of BoDV-1 P to form liquid droplets without N may be attributed 329 to the overlap of the region important for driving LLPS with the RNA-binding region. 330 We revealed that the N-terminal IDR is important for the RNA-binding activity of BoDV-331 1 P (Fig. 4) , which is overlapped with the region necessary to drive LLPS in vitro (Fig.   332 1). Further, an excess amount of RNA dissolved the BoDV-1 P droplets (Fig. 4) . These The transcription and replication of BoDV-1 might be regulated by the competitive 338 feature of BoDV-1 P. We revealed that an excess amount of RNA also dissolved the NP 339 droplets, even though RNA was efficiently incorporated into the NP droplet, which was 340 probably due to the ability of BoDV-1 N to encapsidate RNA (Fig. 4) . Considering that in cells (Fig. 3) . Importantly, we could compare the properties of the P and NP droplets 352 in vitro because BoDV-1 P alone can form liquid droplets, which makes it possible to 353 speculate the effect of BoDV-1 N on viral condensates. Notably, the NP droplets had 354 lower fluidity and were more resistant to treatment with 1,6-HD than the P droplets ( Fig.   355 2). Our previous studies revealed that BoDV-1 IBs had insoluble properties and that 356 BoDV-1 N has an immobile property 16,32 . Thus, besides the function as a nucleocapsid 357 protein (i.e., encapsidation of viral genomic RNA), BoDV-1 N might also convey the 358 properties of insolubility and/or viscosity to viral IBs, which might lead to stable 359 transcription and replication. 360 In this study, we did not test the effects of viral proteins other than BoDV-1 N and P. 

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Negri bodies 409 19 and other virus membrane-less replication compartments

Germline P Granules Are Liquid Droplets That Localize 412 by Controlled Dissolution/Condensation. Science (80-. )

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RNA 417 transcription modulates phase transition-driven nuclear body assembly

Liquid demixing of intrinsically disordered proteins is seeded 420 by poly(ADP-ribose)

Negri bodies are viral factories with properties of liquid 422 organelles

Phase 424 transitions drive the formation of vesicular stomatitis virus replication 425 compartments

Measles Virus Forms Inclusion 427 Bodies with Properties of Liquid Organelles

Measles virus nucleo-and phosphoproteins form liquid-like 429 phase-separated compartments that promote nucleocapsid assembly

Minimal Elements Required for the Formation of Respiratory 432

Syncytial Virus Cytoplasmic Inclusion Bodies In Vivo and In Vitro

Human metapneumovirus nucleoprotein and phosphoprotein 435 interact and provide the minimal requirements for inclusion body formation

An amino acid of human parainfluenza virus type 3 nucleoprotein 438 is critical for template function and cytoplasmic inclusion body formation

Taxonomic reorganization of the family Bornaviridae

Borna disease virus (BDV), a nonsegmented RNA 443 virus, replicates in the nuclei of infected cells where infectious BDV 444 ribonucleoproteins are present

Bornavirus closely associates and segregates with host 446 chromosomes to ensure persistent intranuclear infection

Borna disease virus assembles porous cage-like viral factories in 449 the nucleus

Active borna disease 451 virus polymerase complex requires a distinct nucleoprotein-to-phosphoprotein 452 ratio but no viral X protein

Synergistic antiviral activity of ribavirin and interferon-α against 454 parrot bornaviruses in avian cells

Isolation of Borna disease virus from human brain tissue

Modulation of Borna disease virus phosphoprotein nuclear 458 localization by the viral protein X encoded in the overlapping open reading 459 frame

Formation and Maturation 461 of Phase-Separated Liquid Droplets by RNA-Binding Proteins

Phase Transition of a Disordered Nuage Protein Generates 464

Translation initiation of a bicistronic mRNA of Borna disease virus: A 16-kDa 468 phosphoprotein is initiated at an internal start codon

Analysis of borna disease virus trafficking in live infected 471 cells by using a virus encoding a tetracysteine-tagged p protein

Toxic PR Poly-Dipeptides Encoded by the C9orf72 Repeat 474 Expansion Target LC Domain Polymers

A reverse 476 genetics system for Borna disease virus

RNA buffers the phase separation behavior of prion-like RNA 478 binding proteins

The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually 482 exclusive condensates with RNA and the membrane-associated M protein

Genomic RNA Elements Drive Phase Separation of the SARS-485

X-linked RNA-487 binding motif protein (RBMX) is required for the maintenance of Borna disease 488 virus nuclear viral factories

Nipah virus induces two inclusion body populations: 490 Identification of novel inclusions at the plasma membrane

SARS-CoV-2 nucleocapsid protein phase-separates with 493 RNA and with human hnRNPs

495 Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich 496 polymerase-containing condensates

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Protein Suggests a Biophysical Basis for its Dual Functions

The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, 501 and phase separates with RNA

GCG inhibits SARS-CoV-2 replication by disrupting the liquid 503 phase condensation of its nucleocapsid protein

Context-dependent

The authors declare no competing interests.