Stress granule formation, disassembly, and composition are regulated by alphavirus ADP-ribosylhydrolase activity

Aravinth Kumar Jayabalana, Srivathsan Adivarahanb, Aakash Koppulac, Rachy Abrahamd, Mona Batishc,e, Daniel Zenklusenb, Diane E. Griffind, and Anthony K. L. Leunga,f,g,1

aDepartment of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205; bDépartment de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC H3T 1J4, Canada; cDepartment of Biological Sciences, University of Delaware, Newark, DE 19716; dW. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205; eDepartment of Medical and Molecular Sciences, University of Delaware, Newark, DE 19716; fDepartment of Molecular Biology and Genetics, School of Medicine, Johns Hopkins University, Baltimore, MD 21205; and gDepartment of Oncology, School of Medicine, Johns Hopkins University, Baltimore, MD 21205

Edited by Thomas Shenk, Princeton University, Princeton, NJ, and approved December 31, 2020 (received for review October 22, 2020) While biomolecular condensates have emerged as an important later infection stages, many viruses instead suppress SG forma- biological phenomenon, mechanisms regulating their composition tion or disassemble SGs altogether. The mechanisms underlying and the ways that viruses hijack these mechanisms remain unclear. this switch, and its physiological function, remain unclear. The mosquito-borne alphaviruses cause a range of diseases from SG formation and disassembly are regulated by posttransla- rashes and arthritis to encephalitis, and no licensed drugs are tional modifications of , including those that conjugate available for treatment or vaccines for prevention. The alphavirus simple chemical groups, attach polypeptides, and add nucleotides virulence factor nonstructural 3 (nsP3) suppresses the for- as in the case of ADP-ribosylation (15–21). ADP-ribosylation re- mation of stress granules (SGs)—a class of cytoplasmic condensates fers to the addition of one or more ADP-ribose units onto proteins enriched with initiation factors and formed during the (22–24). In humans, ADP-ribosylation is accomplished primarily early stage of infection. nsP3 has a conserved N-terminal macrodo- by a family of 17 ADP-ribosyltransferases, commonly known as

main that hydrolyzes ADP-ribose from ADP-ribosylated proteins and poly(ADP-ribose) polymerases (PARPs). SG components are spe- MICROBIOLOGY a C-terminal hypervariable domain that binds the essential SG com- cifically ADP-ribosylated, and ADP-ribose polymers [i.e., poly(ADP- ponent G3BP1. Here, we show that macrodomain hydrolase activity ribose) or PAR], five PARPs and two isoforms of the degradative reduces the ADP-ribosylation of G3BP1, disassembles virus-induced enzyme PAR glycohydrolase (PARG) have been localized to these SGs, and suppresses SG formation. Expression of nsP3 results in the condensates (17, 25–27). Overexpression of these PARPs and formation of a distinct class of condensates that lack translation PARG isoforms induces and suppresses SG formation, respec- initiation factors but contain G3BP1 and other SG-associated RNA- tively, while PARG knockdown delays SG disassembly (17, 26). binding proteins. Expression of ADP-ribosylhydrolase–deficient nsP3 The noncovalent interaction between PAR and proteins facili- results in condensates that retain translation initiation factors as tates SG targeting (25–27). For example, PAR-mediated targeting well as RNA-binding proteins, similar to SGs. Therefore, our data regulates TDP-43 localization to SGs and prevents the formation reveal that ADP-ribosylation controls the composition of biomolec- of pathological aggregates in amyotrophic lateral sclerosis (26, 27). ular condensates, specifically the localization of translation initiation factors, during alphavirus infection. Significance

ADP-ribosylation | stress granules | biomolecular condensates | Alphaviruses are mosquito-borne RNA viruses that cause rash, alphavirus | macrodomain arthritis, and neurologic disease. Despite a continued risk of outbreaks, there are no licensed interventions for any alpha- iomolecular condensates are prevalent in cells and critical virus. For progress in control, an understanding of the molecular Bfor a range of cellular functions, including RNA metabolism, targets that affect virus replication and virulence is essential. embryonic cell fate specification, and neuronal activity (1–3). This paper characterizes a conserved macrodomain in the viru- While condensates often dynamically exchange components with lence factor nonstructural protein 3 (nsP3). We discovered that the surrounding milieu, the overall composition of these cellular the macrodomain ADP-ribosylhydrolase activity is critical for structures remains distinct (4). How cells control the specific controlling the composition of cellular condensates, specifically composition of these condensates remains unclear. Stress granules through regulating the localization of translation factors, during (SGs), one of the best characterized biomolecular condensates, are viral infection. Given that this macrodomain is conserved across RNA–protein assemblies formed in response to a variety of envi- alphaviruses and coronaviruses and that the associated enzy- ronmental cues (1). While SG composition can vary with the type matic activity is critical for virulence, our work may open ave- of stress cue (5), certain common components, such as Ras GTP- nues for developing a class of antiviral therapeutics. activating protein-binding proteins G3BP1/2, are essential for for- Author contributions: A.K.J., M.B., D.Z., D.E.G., and A.K.L.L. designed research; A.K.J., S.A., mation of SGs (6, 7). Dysregulation of SG formation and disas- A.K., and R.A. performed research; A.K.J. and R.A. contributed new reagents/analytic sembly is implicated in the pathogenesis of diseases, including viral tools; A.K.J., S.A., A.K., D.E.G., and A.K.L.L. analyzed data; and A.K.J. and A.K.L.L. wrote infection, cancer, and neurodegeneration (2, 8–10). the paper. SG formation and disassembly are tightly regulated during The authors declare no competing interest. viral infection, often reflecting cellular translation status (11–14). This article is a PNAS Direct Submission. In the early phase of many viral infections, the presence of Published under the PNAS license. double-stranded viral RNAs (vRNAs) activate protein kinase R 1To whom correspondence may be addressed. Email: [email protected]. α (PKR), resulting in eIF2 phosphorylation, messenger RNA This article contains supporting information online at https://www.pnas.org/lookup/suppl/ (mRNA) translation inhibition, and formation of SGs enriched doi:10.1073/pnas.2021719118/-/DCSupplemental. with translation initiation factors such as eIF3b. However, in Published February 5, 2021.

PNAS 2021 Vol. 118 No. 6 e2021719118 https://doi.org/10.1073/pnas.2021719118 | 1of11 Downloaded by guest on September 29, 2021 ADViral infection B mRNA binding Translation CHIKV (hpi) proteins factors 0246810 nsP3 nsP3 nsP3 p-eIF2α TIA1 eIF3b TIA1

eIF2α eIF3b β-actin Merge Merge Image Fig. 1. Two distinct classes of biomolecular con- acquisition nsP3 nsP3 nsP3 densates form during alphavirus infection. (A) U2OS SGs SGs condensates TIAR eIF3i cells stably expressing GFP-eIF3g were infected with eIF3i appear (I) disappear (II) appear (III) TIAR WT CHIKVmCherry at MOI of 10, subjected to live cell Merge Merge imaging with the time-lapse interval of 12 min over nsP3 nsP3 t = 120 min t = 360 min t = 540 min 14 h. Images shown are the snapshots of different HuR RACK1 infection stages. (B) U2OS cells were either mock- HuR Merge RACK1 Merge infected or infected with CHIKV. Twelve hours post- infection, cells were lysed and blotted against nsP3 nsP3 indicated antibodies. (C) Violin plot shows the dis- G3BP1 RPS6 tribution of time points (minutes) representing SG

GFP-eIF3g / mCherry-CHIKV RPS6 G3BP1 Merge Merge appearance, disappearance, and nsP3 condensate appearance. Ninety-seven cells from three indepen- dent experiments were included, and the time points C EFnsP3 G3BP1 eIF3b Merge at which SGs appeared/disappeared and nsP3 con- Biological replicate densates appeared were measured within each cell. Biological replicate Error bars correspond to SD. (D) U2OS cells were in- 1000 Unstressed ARS *** * ARS+CHX fected with CHIKV WT at MOI of 1. At 6 h postin- 800 fection (hpi), cells were fixed and immunostained for * *** nsP3 and indicated SG proteins. (E) U2OS cells 100 600 CHX transfected with GFP-tagged nsP3WT were either 400 80 unstressed, treated with 100 μg/mL cycloheximide Time (min) (CHX), 0.2 mM arsenite alone (ARS), or cotreated 60 200 **** with 0.2 mM arsenite and 100 μg/mL cycloheximide ARS 0 40 (ARS+CHX) for 30 min. Cells were then fixed and I II III immunostained for G3BP1 (red) and eIF3b (blue). 20 Sequence of events during Asterisks indicate untransfected cells. (F) Bar graph viral infection (Panel A) shows the percentage of GFP-positive cells with SGs ARS+CHX * * * * 0 % GFP-positive cells with SGs WT from three independent experiments. Error bars correspond to mean ± SD. (Scale bars, 10 μm.)

The mosquito-borne alphaviruses, which cause a range of dis- cells and specifically regulates translation factor localization. To- eases from rashes and arthritis to encephalitis, induce SG forma- gether, these data argue that nsP3 ADP-ribosylhydrolase activity tion early in infection and later initiate SG disassembly (11, 14, 28, modulates SG formation, disassembly, and composition. 29). Previous studies have identified the alphaviral nonstructural protein 3 (nsP3), a key factor for virus replication and virulence Results (30–32), as able to suppress SG formation (28, 33–35). The Two Distinct Classes of Biomolecular Condensates Formed during alphaviral nsP3 is a tripartite protein composed of a highly Alphavirus Infection. In the early phase of alphaviral infection, conserved macrodomain (MD) in the N terminus, a central zinc- the presence of double-stranded vRNAs activates PKR, resulting binding domain (ZBD), and a C-terminal hypervariable domain in eIF2α phosphorylation, mRNA translation inhibition, and SG (HVD; ref. 30). Recent studies indicate that the HVD, which is formation (11–14). SGs are aggregates of stalled translation ini- of low complexity, directs alphaviral nsP3 binding to host SG tiation complexes, indicated by the presence of translation initia- proteins (30, 36). For example, the HVD of chikungunya virus tion factors as condensates (5). To monitor SG kinetics during (CHIKV) binds the essential SG components G3BP1 and G3BP2 viral infection, U2OS cells stably expressing GFP-tagged transla- (33, 37). Given that nsP3 expression increases over the course of tion eIF3g (U2OSGFP-eIF3g) were infected with viral infection, it has been proposed that nsP3 sequesters G3BP1/2, CHIKV with mCherry-tagged nsP3. SGs were induced at the early resulting in the suppression of SG formation during the late phase stage of infection, but these translation factor-enriched conden- of infection (28, 29, 34). sates disassembled as the infection progressed (Fig. 1A and Movie Here, we report that the expression of the G3BP-binding HVD S1) despite persistent eIF2α phosphorylation (Fig. 1B). These data alone does not suppress SG formation; rather, expression of the suggest an active mechanism to suppress the formation of SGs. N-terminal MD alone can trigger the suppression of this biomo- Live cell imaging further revealed that another class of con- lecular condensate. The structural integrity of SGs is dependent densates containing the viral protein nsP3 form after SG disap- on ADP-ribosylation (17), and we and others recently found that pearance, as signified by the absence of any eIF3g-containing the viral MD can remove single ADP-ribose groups, and possibly condensates (Fig. 1 A and C and Movie S1). Immunofluorescence PAR, from ADP-ribosylated proteins (31, 38–40). We therefore data demonstrated that these nsP3-containing condensates hypothesized that MD ADP-ribosylhydrolase activity is required (hereby termed nsP3 condensates) possess SG-associated RNA- to suppress SG formation across stress conditions, with G3BP1 binding proteins such as G3BP1, TIA1, TIAR, and HuR, but not being a key target substrate. Indeed, we find that MD ADP- SG-associated translation factors, including eIF3b, eIF3i, RACK1, ribosylhydrolase activity is critical for disassembling SGs formed and RPS6 (Fig. 1D). Notably, these nsP3 condensates can be by G3BP1 expression and during viral infection. Consistent with formed by the expression of this alphaviral nsP3 protein alone in this premise, live cell imaging revealed that SGs persist in cells unstressed and stressed cells, and their formation is not sensitive infected with a hydrolase-deficient recombinant CHIKV. ADP- to cycloheximide—a translation elongation inhibitor (5) (Fig. 1E). ribosylhydrolase activity is required for altering the composition of Cycloheximide traps mRNAs along with translation factors in biomolecular condensates in nsP3-expressing or virus-infected polysomes, thus decreasing the availability of mRNAs for SG

2of11 | PNAS Jayabalan et al. https://doi.org/10.1073/pnas.2021719118 Stress granule formation, disassembly, and composition are regulated by alphavirus ADP-ribosylhydrolase activity Downloaded by guest on September 29, 2021 ARS A B Biological replicate nsP3 domain structure ARS+CHX 100 162 318 530 *** *** FGDFFGDF 80

NH2 COOH 60 Macrodomain Zinc binding Hypervariable (MD) domain (ZBD) domain (HVD) 40

with SGs 20 Arsenite

% GFP-positive cells 0 nsP3 G3BP1 eIF3b Merge MD ZBD HVD * * * * C Arsenite MD GFP G3BP1 eIF3b Merge

GFP * * * * * * * * Vector ZBD Fig. 2. nsP3 macrodomain suppresses SG formation. (A) Schematic representation of the nsP3 domain * * * * WT * * * * architecture. U2OS cells transfected with GFP-tagged HVD nsP3MD, nsP3ZBD, or nsP3HVD were either treated with 0.2 mM arsenite alone or cotreated with 100 μg/mL * * * * GFP-nsP3 cycloheximide for 30 min. Cells were then processed AGDA Arsenite+Cycloheximide and immunostained for SG markers G3BP1 (red) and eIF3b (blue). Asterisks indicate untransfected cells. nsP3 G3BP1 eIF3b Merge (B) Bar graph shows the percentage of GFP-positive * * * * D Biological replicate cells with SGs. ***P < 0.005, two-tailed, unpaired MD Student’s t test. Error bars correspond to mean ± SD 100 *** with three independent experiments. (C) U2OS cells 80 transfected with GFP vector, GFP-tagged nsP3WT,or 60 * nsP3AGDA were treated with 0.2 mM arsenite for ZBD 40 30 min and immunostained for G3BP1 (red) and with SGs 20 * * * * eIF3b (blue). Asterisks indicate untransfected cells. MICROBIOLOGY 0 (D) Bar graph shows the percentage of GFP-positive % GFP-positive cells HVD * * * * cells with SGs. *P < 0.05, ***P < 0.001, two-tailed, ’ WT unpaired Student s t test. Error bars correspond to GFP VectorGFP-nsP3 AGDA mean ± SD with n = 3. (Scale bars, 10 μm.)

formation and, thereby, promoting SG disassembly (5). Therefore, treatment, the nsP3HVD no longer colocalized with the transla- bona fide SGs disappear upon cycloheximide treatment. However, tion factor eIF3b, suggesting that the nsP3HVD-containing con- nsP3 condensates persisted in the presence of cycloheximide densates formed upon arsenite treatment were indeed SGs (Fig. 1E, CHX). In addition, these nsP3 condensates formed in the (Fig. 2A). Notably, nsP3HVD can still form G3BP1-positive but absence of the essential SG components G3BP1/2 (SI Appendix, eIF3b-negative condensates in the absence of arsenite. Unlike Fig. S1A). These nsP3 condensates are, therefore, distinct in SGs, these condensates were cycloheximide-insensitive (SI Ap- composition and properties from SGs. pendix, Fig. S1D). These data indicate that the low-complexity Notably, nsP3 expression also suppresses the formation of HVD alone is insufficient to suppress SG formation, but this SGs, even in the presence of the classic SG inducer arsenite (28, domain possesses the ability to form condensates. 33–35). In untransfected cells, eIF3b and G3BP1 colocalized as Unlike its stress-specific colocalization with eIF3b, nsP3HVD SGs after arsenite treatment (Fig. 1E, asterisk, ARS). In nsP3- colocalized with G3BP1 to form condensates in both unstressed transfected cells, only 27% of cells have SGs (i.e., condensates and arsenite-stressed cells likely through direct interaction stained both positive with eIF3b and G3BP1; Fig. 1F). The (Fig. 2A and SI Appendix, Fig. S1D). The nsP3 C-terminal HVD suppression of SGs is thought to result from sequestration of the has two FGDF motifs that bind strongly to the NTF2-like essential SG component G3BP1 and its paralog G3BP2 by nsP3 (NTF2L) domain of G3BP1. To further test whether G3BP1 binding through its C-terminal HVD (33, 34, 37, 41–43). How- binding to the nsP3 HVD is critical for suppressing SG forma- ever, it is unclear whether G3BP sequestration by the HVD is tion, we mutated the two FGDF motifs to AGDA and analyzed sufficient to suppress SGs because other host cell factors also the ability of full-length nsP3 to suppress SG formation. Consistent bind the nsP3 HVD (30, 41, 44). In addition, cellular G3BP1/2 with previous reports, nsP3AGDA no longer colocalized with G3BP1 are highly abundant, challenging a model based on complete and G3BP2 in either unstressed or stressed cells (Fig. 2C and SI sequestration (SI Appendix, Table S1). Appendix,Fig.S1E). Yet, nsP3AGDA still formed condensates in both conditions, further indicating that this class of condensates is nsP3 Macrodomain Suppresses SG Formation and Hypervariable distinct from SGs. Upon arsenite treatment, SG formation was still Domain Facilitates Condensate Formation. To determine whether suppressed in nsP3AGDA-expressing cells (36%) (Fig. 2 C and D). the HVD alone is sufficient to suppress SG formation, we Taken together, these data indicated that regions(s) other than the overexpressed a GFP-tagged HVD fragment (318–530) in U2OS nsP3 HVD are responsible for suppressing SG formation. cells (Fig. 2A and SI Appendix, Fig. S1B) and HeLa cells (SI To examine which domains were linked to SG suppression, we Appendix, Fig. S1C), exposed the cells to arsenite, and examined next examined the central ZBD (163-317) of nsP3, which posi- the colocalization of nsP3HVD with SG components G3BP1 and tively regulates alphaviral replication and transcription (45). eIF3b. In contrast to cells expressing full-length nsP3 (Fig. 1E), Overexpressed nsP3ZBD formed filament-like structures in cells eIF3b and G3BP1 colocalized as SGs in 85% of cells transfected and did not suppress SG formation (Fig. 2 A and B) (28). Given with nsP3HVD and these nsP3HVD-containing condensates were that the N-terminal MD can catalyze removal of ADP-ribosyla- sensitive to cycloheximide (Fig. 2 A and B). After cycloheximide tion (31, 38), a modification essential for structural integrity of

Jayabalan et al. PNAS | 3of11 Stress granule formation, disassembly, and composition are regulated by alphavirus https://doi.org/10.1073/pnas.2021719118 ADP-ribosylhydrolase activity Downloaded by guest on September 29, 2021 ADArsenite Arsenite+Cycloheximide nsP3 G3BP1 eIF3b Merge nsP3 G3BP1 eIF3b Merge * * * * * * * * Fig. 3. ADP-ribosylhydrolase activity of nsP3 sup- WT WT presses SG formation. (A) U2OS cells transfected with GFP-tagged nsP3WT or different nsP3 point mutants * * * * (D10A, G32E, G32S, G112E, and Y114A) were treated D10A G32E with 0.2 mM arsenite for 30 min and immunostained * * * * for G3BP1 (red) and eIF3b (blue). Asterisks indicate untransfected cells. (B) 293F cells were transfected with either GFP vector or GFP-tagged nsP3WT,

G112E E F WT G32E G32S Y114A * * * * GFP-nsP3 WT G32E nsP3 , nsP3 , or nsP3 . After 24-h transfec- Arsenite – + – + tion, cells were pelleted, lysed, immunoprecipitated using anti-GFP antibodies, and the immunoprecipi- * * * * TIA-1 √ √ √ √ G32E TIAR √ √ √ √ tates were blotted against G3BP1 and GFP anti- Arsenite HuR √ √ √ √ bodies. (C) Bar graph shows the percentage of GFP- √ √ √ √ positive cells with SGs in A.(Lower) ADP-ribose proteins PABPC1 binding and hydrolase activity of the mutants tested nsP3 mRNA binding mRNA FMRP √ √ √ √ G32S (31). **P < 0.01, two-tailed, unpaired Student’s t test. eIF3e X X X √ * * * * Error bars correspond to mean ± SD with n = 3. (D) eIF3j X X X √ WT G32E Poly(A) Cells transfected with GFP-tagged nsP3 or nsP3 RACK1 X X X √ * * * * were cotreated with 0.2 mM arsenite and 100 μg/mL Y114A factors eIF4G X X X √ Merge Translation cycloheximide for 30 min. Cells were then immu- RPS6 X X X √ G32E nostained for G3BP1 (red) and eIF3b (blue). Asterisks indicate untransfected cells. (E) Table summarizes B C Biological replicate 100 the colocalization of nsP3 (WT or G32E) with differ- GFP-nsP3 ent mRNA binding proteins and translation factors as 80 ** ** ** ** ** shown in SI Appendix, Fig. S3A.(F) Superresolution Arsenite – WT G32E G32SY114A 60 microscopy indicates how the distribution of poly(A)+ GFP 40 mRNA signal overlaps with nsP3 WT or G32E mutant.

G3BP1 with SGs WT nsP3 U2OS cells transfected with GFP-tagged nsP3 or 20 IP : GFP nsP3G32E were with 0.2 mM arsenite for 30 min and

GFP % GFP-positive cells 0 hybridized with oligo(dT) probes, followed by staining G3BP1 WT D10A G32E G112E G32S Y114A Poly(A) for GFP. Green and red boxes indicate the condensates ADP-ribose binding +++ – – – + ++++ β-actin from nsP3-transfected and untransfected cells, re- Input Hydrolase activity +++ –––+ + Merge spectively. (Scale bars, 10 μm.)

SGs (17), we tested whether the MD of nsP3 (nsP3MD) is re- eIF3b, nsP3MD plays a critical role in regulating association with sponsible for loss of SGs. Similar to full-length nsP3, over- the SG components such as G3BP1 and eIF3b. expression of the MD fragment (1–162) inhibited SG formation upon arsenite treatment (Fig. 2 A and B), as well as after exposure ADP-Ribosylhydrolase Activity Controls Cellular Condensate Composition. MD to the mitochondrial stressor clotrimazole or endoplasmic reticu- To dissect which functions of nsP3 are responsible for suppressing lum stressor thapsigargin (SI Appendix,Fig.S1F and G;ref.5). SG formation (Fig. 3), we examined three classes of MD mutants However, unlike full-length nsP3, nsP3MD did not colocalize with (31): 1) no ADP-ribose–binding and negligible hydrolase activity G3BP1 (Fig. 2A), presumably due to lack of the G3BP-binding (D10A, G32E, and G112E); 2) weak ADP-ribose binding and weak HVD region. These data suggest that while the nsP3HVD is suf- hydrolase activity (G32S); and 3) weak hydrolase activity, but stronger ficient for forming condensates with SG components G3BP1 and ADP-ribose–binding than WT (Y114A). Similar to WT nsP3, all of

Table 1. Composition and property of biomolecular condensates observed in this study Example Condensates Formation figure Tested condensate components Properties

Stress granules Induced by stress (e.g., arsenite, 1A RBPs (e.g., G3BP) and translation Integrity can be disrupted by clotrimazole, thapsigargin), factors (e.g., eIF3b) cycloheximide or nsP3 viral infection, or G3BP1 expression ADP-ribosyhydrolase Expression of nsP3 HVD domain in 2A nsP3 HVD domain, RBPs (e.g., G3BP) Integrity can be disrupted by the presence of stress (HVD) and translation factors (e.g., eIF3b) cycloheximide Expression of hydrolase-deficient 3A Hydrolase-deficient nsP3, RBPs (e.g., Integrity can be disrupted by nsP3 in the presence of stress G3BP) and translation factors cycloheximide (e.g., eIF3b)

nsP3 condensates Expression of WT nsP3 in the 1E WT nsP3 and RBPs (e.g., G3BP) Insensitive to cycloheximide presence or absence of stress Expression of hydrolase-deficient S2A Hydrolase-deficient nsP3 and RBPs Insensitive to cycloheximide mutant nsP3 in the absence (e.g., G3BP) of stress Expression of nsP3 HVD domain S1D nsP3 HVD domain, G3BP1 Insensitive to cycloheximide in the absence of stress

4of11 | PNAS Jayabalan et al. https://doi.org/10.1073/pnas.2021719118 Stress granule formation, disassembly, and composition are regulated by alphavirus ADP-ribosylhydrolase activity Downloaded by guest on September 29, 2021 ABGFP-tagged GFP-tagged associated RNA-binding proteins from translation factors during stress.

VectorG3BP1G3BP2eIF3deIF3geIF3i VectorG3BP1G3BP2eIF3deIF3geIF3i The function of nsP3 resembles host cell PARG, which also 150 - 100 - possesses ADP-ribosylhydrolase activity but cleaves ribose-ribose 75 - 100 - pan-ADPr bonds within the PAR polymer. Similar to nsP3, PARG isoforms 75 - (PARG99 and particularly PARG102) suppress SG formation 50 - GFP 150 - (17). nsP3-mediated suppression of SG formation is stronger, 100 - however, than PARG102, even when the latter was expressed at GFP a higher level than nsP3 (SI Appendix, Fig. S3 B–D). Also similar 25 - 75 - Pull-down: Biotin-PAR to the nsP3 MD alone, PARG does not colocalize with G3BP1 to 100 - 25 - form condensates, likely because it lacks a G3BP1-binding motif 75 - IP : GFP (Fig. 2A and SI Appendix, Fig. S3 B, E, and F). Consistent with 50 - GFP 100 - these findings, expressing only the MD carrying the G32 mutation 75 - GFP did not suppress SG formation nor form G3BP1-containing con- densates (SI Appendix, Fig. S3 E and F). These data altogether 25 - 25 - suggest that although nsP3MD does not possess the ability to β-actin β-actin Input Input form condensates, its ADP-ribosylhydrolase activity is sufficient to suppress SG formation. Fig. 4. Differential PAR-binding ability and ADP-ribosylation of SG compo- nents. (A) 293F cells were transfected with either GFP vector or GFP-tagged nsP3 Reduces ADP-Ribosylation of the Essential SG Component G3BP1, G3BP2, eIF3d, eIF3g, or eIF3i for 36 h. Cells were then pelleted, lysed, G3BP1. Next, we examined the possible targets of nsP3 ADP- and incubated with 100 pmol Biotin-PAR and streptavidin beads. The streptavi- ribosylhydrolase activity that alter SG composition and structural din pulldown was then blotted with anti-GFP antibodies. (B) 293F cells trans- integrity. Given that nsP3 ADP-ribosylhydrolase activity regu- fected with either GFP vector or GFP-tagged G3BP1, G3BP2, eIF3d, eIF3g, or eIF3i for 36 h. Cells were then pelleted, lysed, and immunoprecipitated using anti-GFP lates the association between SG-associated RNA-binding pro- antibodies. The immunoprecipitates were then blotted with pan-ADPr reagent. teins and translation factors, we wondered whether nsP3 disrupts the interaction network in SGs formed by protein–ADP- ribose–protein interactions. We therefore hypothesized that these MD mutant nsP3s colocalized with G3BP1 in condensates with some of these SG components are ADP-ribosylated while others or without arsenite (Fig. 3A and SI Appendix,Fig.S2A). Consistently, bind to ADP-ribose noncovalently. To test whether these SG MICROBIOLOGY no differences were observed in G3BP1 association with WT and components are ADP-ribose binders, we used biotinylated PAR mutant nsP3s representative of the three different classes, arguing to pull down complexes from cell lysates expressing either GFP that ADP-ribosylhydrolase activity does not regulate nsP3 associ- only, GFP-tagged G3BP1, G3BP2, eIF3d, eIF3g, or eIF3i. ation with G3BP1 (Fig. 3B). However, unlike WT, mutants colo- Consistent with previous results showing that G3BP1 binds to calized with eIF3b upon arsenite treatment (Fig. 3 A and C), indicating PAR (46), biotinylated PAR pulled down GFP-tagged G3BP1, A that these nsP3 mutants do not suppress SG formation. but not GFP alone (Fig. 4 ). In addition, G3BP2 and both Using G32E as a model, we further showed that mutant nsP3/ translation factors eIF3d and eIF3g can also bind to PAR non- A eIF3b/G3BP1 condensates were SGs because they: 1) were formed covalently (Fig. 4 ). To test whether SG-associated RNA- in response to multiple stresses, such as arsenite, clotrimazole, binding proteins and translation factors are differentially ADP- and thapsigargin (Fig. 3A and SI Appendix, Fig. S2 B and C); 2) ribosylated, we transfected GFP-tagged G3BP1, G3BP2, eIF3d, were sensitive to cycloheximide (Fig. 3D); and 3) contained all eIF3g, or eIF3i into cells, immunoprecipitated the cell lysates using anti-GFP antibodies, and probed the immunoprecipitates tested SG-associated RNA-binding proteins and translation factors B (Fig. 3E and SI Appendix,Fig.S3A). Similar to SGs, mutant con- with a reagent that detects ADP-ribosylation (Fig. 4 ). G3BP1, densates were enriched with poly(A)+ at its core based on super- G3BP2, and eIF3d, but not eIF3g and eIF3i, were ADP-ribosy- lated in cells, where prominent ADP-ribosylation signals resolution microscopy analyses (Fig. 3F). Because all tested mutants appeared at and above the expected molecular weight of the impact ADP-ribosylhydrolase activity, nsP3-mediated suppression respective proteins. The signals appeared as smears because of of SG formation likely requires this catalytic activity. the heterogeneous number of ADP-ribose units added onto these In stressed cells, WT nsP3 colocalized with all RNA-binding proteins. Therefore, these data suggest that specific SG components proteins that were tested but none of the translation factors. G32E are ADP-ribosylated while others can bind noncovalently to ADP- In contrast, nsP3 colocalized with all tested SG-associated ribose polymers. By removing ADP-ribosylation from specific SG RNA-binding proteins and translation factors in stressed cells – – A SI Appendix A substrates, nsP3 may disrupt protein ADP-ribose protein interac- (Fig. 3 and , Fig. S3 ). The observed difference tions critical for SG formation. could be due to mutant nsP3, but not WT, associating with SG We next tested whether G3BP1 is one of the nsP3 ADP- components prior to stress conditions. To test this possibility, we ribosylhydrolase targets, given that it interacts directly with nsP3 examined the colocalization of WT and mutant nsP3 with all of (33, 34, 37, 41–43), is an essential component of SG formation these SG components in unstressed cells. Both WT and G32E across stress triggers (6), and plays a critical role in alphavirus mutant nsP3 colocalized with all tested RNA-binding proteins, infection (33, 47). To test this possibility, we expressed GFP- but not translation factors, in unstressed cells (Fig. 3E and SI G3BP1 alone or with increasing amounts of FLAG-tagged nsP3 Appendix, Fig. S3A). Taken together, we observed two classes of WT. GFP-G3BP1 was then immunoprecipitated and probed for condensates with differential sensitivity to nsP3 ADP-ribosylhydrolase ADP-ribosylation. Upon coexpression of WT nsP3, the ADP- activityinthepresenceandabsenceofstress(Table1).Inunstressed ribosylation signal associated with G3BP1 was reduced in a dose- cells, nsP3 condensates form independent of its ADP-ribosylhy- dependent manner as well as in both unstressed and multiple drolase activity. Upon stress, WT nsP3 exists in condensates that lack stress conditions (Fig. 5A and SI Appendix, Fig. S4A). Reduction translation factors and its ADP-ribosylhydrolase suppresses SG of the ADP-ribose signal was not observed when the G32E formation. In contrast, hydrolase-deficient nsP3 forms SGs with mutant nsP3 was coexpressed (Fig. 5B). In vitro incubation of the canonical components such as G3BP1 and eIF3b. Therefore, the recombinant WT MD alone was sufficient to reduce G3BP1 enzymatic activity regulates the composition of cellular condensates. ADP-ribosylation (Fig. 5C). These data together suggest that Specifically, nsP3 ADP-ribosylhydrolyase activity segregates SG- ADP-ribosylhydrolase activity residing in the nsP3 MD is

Jayabalan et al. PNAS | 5of11 Stress granule formation, disassembly, and composition are regulated by alphavirus https://doi.org/10.1073/pnas.2021719118 ADP-ribosylhydrolase activity Downloaded by guest on September 29, 2021 GFP-G3BP1 A B GFP-G3BP1 C GFP-G3BP1 FLAG-nsP3 – – FLAG-nsP3 – – WT G32E CHIKV MD – +

250 - pan-ADPr 150 - pan-ADPr 250 - pan-ADPr 150 - 100 - 100 - GFP 100 - IP : GFP IP : GFP IP : GFP GFP GFP GFP FLAG CHIKV MD β-actin FLAG Input β-actin Input Input D F G G3BP1 domain structure PABP/eIF3b Biological replicate

GFP-Vector RRM only ns NTF2L Acidic PxxP RRM RGG

* 50 135 168 339 428 466 40

ive cells * 30

with SGs 20 E GFP-G3BP1 WT RGG only * % GFP-posit 10

WT 1-135 1-168 ∆1-168RRM RGG ∆RRM∆RGG∆RRM + GFP-Vector

* 0 150 - ∆RGG WT 100 - 1-135 1-168 RRM RGG ∆1-168 ∆RRM ∆RGG

GFP-Vector * 1-135 ∆RRM 50 - ∆RRM+∆RGG

37 - pan-ADPr * Biological replicate

* H * 25 - *** 150 - 50 100 - ****

1-168 ∆RGG * 40 *

50 - * 30 37 - GFP

with SGs 20 *

25 - % GFP-positive cells 10 IP : GFP ∆1-168 ∆RRM+∆RGG * β-actin 0 Flag- Vector nsP3 Vector nsP3 Input GFP-G3BP1 GFP-G3BP1

* ∆1-168

I Flag-Vector Flag-nsP3 GFP Flag eIF3b Merge GFP Flag eIF3b Merge * * * * GFP GFP G3BP1 G3BP1 * * * *

GFP * * * * GFP G3BP1 G3BP1 ∆1-168 ∆1-168 * * * *

Fig. 5. nsP3 reduces ADP-ribosylation of the essential SG component G3BP1. (A) 293F cells were transfected with either GFP, GFP-tagged G3BP1 alone, or cotransfected with GFP-tagged G3BP1 with increasing concentration of FLAG-tagged nsP3WT. After 24-h transfection, cells were pelleted, lysed, immuno- precipitated using anti-GFP antibodies, and immunoblotted with pan-ADPr reagent. (B) 293F cells were transfected with GFP, GFP-tagged G3BP1 alone, or cotransfected GFP-tagged G3BP1 with either FLAG-tagged nsP3WT or nsP3G32E. Twenty-four hours posttransfection, cells were pelleted, lysed, immunopre- cipitated using anti-GFP antibodies, and immunoblotted with pan-ADPr reagent. (C) 293F cells transfected with GFP-tagged G3BP1 were lysed and immuno- precipitated using anti-GFP antibodies. The immunoprecipitates were split into two halves and incubated either with buffer or 5 μg CHIKV MD for 1 h at 37 °C. After incubation, the beads were washed and blotted with pan-ADPr reagent. (D) Schematic representation of G3BP1 domain structure. (E–G) U2OS G3BP1/2 double knockout (dKO) cells were transfected with either GFP vector or GFP-tagged G3BP1 constructs for 36 h. (E) Cells were then either lysed, immuno- precipitated using anti-GFP antibodies, and blotted with pan-ADPr reagent (asterisks indicate heavy and light chain) or (F) permeabilized, fixed, and immunostained for PABP (red) and eIF3b (blue). Asterisks indicate untransfected cells. (G) Bar graph shows the percentage of GFP-positive cells with SGs. ns, nonsignificant, two-tailed, unpaired Student’s t test. Error bars correspond to mean ± SD with n = 3. (H and I) U2OS G3BP1/2 dKO cells were cotransfected in two combinations: 1) FLAG-vector with GFP-tagged G3BP1 or G3BP1 Δ1–168, and 2) FLAG-tagged nsP3 with GFP-tagged G3BP1 or G3BP1 Δ1–168. At 36 h posttransfection, cells were fixed and stained for FLAG (red) and eIF3b (blue). Asterisks indicate untransfected cells. Bar graph shows the percentage of GFP- positive cells with SGs. ***P < 0.001, ****P < 0.0001, two-tailed, unpaired Student’s t test. Error bars correspond to mean ± SD with n = 3. (Scale bars, 10 μm.)

6of11 | PNAS Jayabalan et al. https://doi.org/10.1073/pnas.2021719118 Stress granule formation, disassembly, and composition are regulated by alphavirus ADP-ribosylhydrolase activity Downloaded by guest on September 29, 2021 A D Arsenite

Viral titer [log (pfu/ml)]

10 nsP3 G3BP1 eIF3b Merge

* * * WT Y114A * U2OS 7.1±0.3 6.3±0.4 NSC34 7.6±0.1 5.6±0.1 WT Fig. 6. ADP-ribosylhydrolase activity of nsP3 sup-

B presses SG formation during alphavirus infection. (A)

Viral titers of WT and Y114A mutant in NSC34 (31)

* * * GFP-Vector + – + – – * and U2OS cells analyzed at 24 hpi. Data are plotted Y114A GFP-G3BP1 – + – + + as plaque-forming units (pfu)/mL ± SEM. (B) CHIKV – – WT WT Y114 293T cells transfected with GFP vector or GFP-G3BP1 150 - for 12 h were either mock infected or infected with pan-ADPr 100 - E Arsenite CHIKVWT or CHIKVY114A at MOI of 1 for 24 h. At 24 GFP G3BP1 vRNA eIF3b Merge hpi, cells were lysed, immunoprecipitated using anti- GFP antibodies, and blotted with pan-ADPr re- GFP * * * * agent. (C and D) U2OS cells were infected with ei- WT WT Y114A nsP3 ther CHIKV or CHIKV atMOIof1.At5.5hpi, 8 hpi IP : GFP cells were treated with 0.2 mM arsenite for 30 min GFP and immunostained for nsP3 (green), G3BP1 (red), and eIF3b (blue). Asterisks indicate untransfected GFP * * * * Y114A cells. Bar graph shows the percentage of virus- nsP3 8 hpi infected cells (nsP3-positive) with SGs. **P < 0.01, β-actin two-tailed, unpaired Student’s t test. Error bars Input correspond to mean ± SD with n = 3. (E) U2OS cells CF were infected with either CHIKVWT or CHIKVY114A Biological replicate 100 WT atMOIof5.At7.5hpi,cellsweretreatedwith 100 nsnsns ns * 0.2 mM arsenite for 30 min. Treated cells were then 80 Y114A 80 ** fixed and hybridized with vRNA probes (red), fol- 60 60 lowed by immunostaining for G3BP1 (green) and eIF3b (blue). Asterisks indicate untransfected cells. 40 40 with SGs (F) Box plot shows the percentage of cells with MICROBIOLOGY 20 20 vRNA colocalizing with eIF3b/G3BP1 in Fig. 6E and

% cells with vRNA

% nsP3-positive cells SI Appendix,Fig.S5E. About 20 cells from each time 0 0 point were quantified for colocalization. *P < 0.05,

colocalizing with eIF3b/G3BP1 WT Y114A 12468 ns, nonsignificant, two-tailed, unpaired Student’s CHIKV hpi t test. (Scale bars, 10 μm.)

responsible for reducing ADP-ribosylation associated with the ribosylation and SG formation in CHIKV-infected cells. Because essential SG component G3BP1. CHIKV carrying nsP3MD mutations (D10A, G32E, G112E) that G3BP1 has an N-terminal NTF2L domain, followed by an acidic severely compromise ADP-ribose binding and hydrolase activi- region, PxxP, RRM, and C-terminal RGG domain (Fig. 5D). To ties are not viable (31), we focused on viable CHIKV nsP3MD identify the region undergoing G3BP1 ADP-ribosylation, we tran- mutants such as G32S and Y114A. These latter mutants have siently transfected U2OS G3BP1/2 knockout cells with different diminished, but not absent, hydrolase activity and have reduced G3BP1 domain constructs tagged with GFP and then probed titers compared with WT virus in cell cultures (ref. 31; Fig. 6A). ADP-ribosylation after GFP immunoprecipitation. We found Particularly, the Y114A mutant was used as a comparison because that ADP-ribosylation was prominently associated with full- it has replication kinetics similar to WT, producing comparable Δ – length WT and 1 168 G3BP1 (NTF2L and acidic domain amounts of vRNA (50). Upon infection with WT CHIKV, G3BP1 E deleted) (Fig. 5 ). Intriguingly, SGs only formed in cells ADP-ribosylation was reduced (Fig. 6B). However, similar re- expressing G3BP1 constructs associated with ADP-ribosylation duction of G3BP1 ADP-ribosylation was not observed in cells (i.e., with full-length or Δ1–168 G3BP1, but not other domains) F G infected with the Y114A mutant virus, indicating that G3BP1 is an (Fig. 5 and ). These data suggest that NTF2L and acidic endogenous target of nsP3 ADP-ribosylhydrolase during infection. domain were not required for ADP-ribosylation of G3BP1 nor To compare the ability of WT and Y114A mutant viruses to SG formation. suppress SG formation, infected cells were examined at 5.5 h In line with previous observations that ADP-ribosylation is postinfection (hpi) when nsP3 appears as condensates (SI Ap- critical for SG formation (17, 26), SGs formed upon expression pendix, Fig. S5A). These cells were then treated with arsenite for of either GFP-G3BP1 or Δ1–168 mutant in G3BP1/2 knockout an additional 0.5 h (a time point when ∼95% of uninfected cells cells were also suppressed by nsP3 (Fig. 5 H and I). Besides sup- have developed SGs). Consistent with experiments using nsP3 pressing SGs, the nsP3 hydrolase activity can also disassemble pre- protein expression constructs (Figs. 1E and 3A), WT CHIKV formed SGs induced by expressing G3BP1 (48, 49). Compared with the vector control or G32E mutant nsP3 transfected into G3BP1- infection suppressed SG formation. Indeed, only 27% of nsP3- positive cells had canonical (G3BP1/eIF3b double-positive) SGs expressing cells, the numbers of SGs (stained positive with G3BP1 C D and eIF3b) in WT nsP3-transfected cells were reduced by approxi- (Fig. 6 and ). In contrast, 49% of Y114A nsP3-positive cells C D mately twofold (SI Appendix,Fig.S4B). These data altogether suggest contained SGs (Fig. 6 and ). The result generalized across that the nsP3 ADP-ribosylhydrolase activity reduces G3BP1 ADP- stress triggers: colocalization of mutant nsP3, eIF3b, and G3BP1 SI Appendix ribosylation, thereby regulating SG formation and disassembly. was observed with clotrimazole and thapsigargin ( , Fig. S5 B and C). The mutant nsP3/eIF3b/G3BP1 condensates nsP3 Macrodomain ADP-Ribosylhydrolase Activity Suppresses the formed during viral infection were also cycloheximide-sensitive, Formation of SGs during Viral Infection. To test the physiological underlining their similarity to SGs (SI Appendix, Fig. S5D). relevance of nsP3 ADP-ribosylhydrolase activity, we compared These data suggest that nsP3 ADP-ribosylhydrolase activity how the WT and mutant MD hydrolase affect G3BP1 ADP- suppresses the formation of SGs during viral infection.

Jayabalan et al. PNAS | 7of11 Stress granule formation, disassembly, and composition are regulated by alphavirus https://doi.org/10.1073/pnas.2021719118 ADP-ribosylhydrolase activity Downloaded by guest on September 29, 2021 A BDBiological replicate Viral infection Viral infection *** 600 500 400 G3BP1 Image eIF3 eIF3 acquisition SGs SGs G3BP1 300 RPS appear disappear 200 Viral infection-induced c c c c 2 1 3 n 100 Stress Granules

SG SG residence time (min) residence 0 time WT Y114A nsP3 C CHIKV-WT nsP3 G3BP1

t (min) = -12 0 48 96 144 196 240 852 eIF3 eIF3 nsP3 G3BP1 GFP RPSnsP3 eIF3g Fig. 7. ADP-ribosylhydrolase activity of nsP3 regu- lates SG disassembly. (A) Schematic representation of mCherry the live cell imaging experiment setup. (B and C) CHIKV Inactive Active hydrolase hydrolase U2OS cells stably expressing GFP-eIF3g were infected with either WT or Y114A CHIKVmCherry at MOI of 10

nsP3 Merge G3BP1 mut nsP3 and subjected to live cell imaging with the time-lapse G3BP1 eIF3 eIF3 nsP3 interval of 12 min over 14 h. Images shown are the G3BP1 mut G3BP1 nsP3 RPS eIF3 = − mut time-points at which no SGs were observed (t 12

CHIKV-Y114A RPS eIF3 min), followed by the time-point at which SGs were t (min) = -12 0 48 96 144 196 240 852 observed (t = 0 h). White arrows indicate SGs. (Scale bar, 10 μm.) Violin plot shows the distribution of GFP average SG residence time (minutes) in cells infected eIF3g with WT and Y114A CHIKVmCherry. ***P < 0.001, two- nsP3 ’ = G3BP1 mut nsP3 tailed, unpaired Student s t test. WT (n 51) and G3BP1 mCherry eIF3 eIF3 nsP3 = G3BP1 Y114A (n 77) infected cells from three independent mut G3BP1 CHIKV nsP3 RPS mut experiments were included to measure the SG resi- Stress nsP3 dence time. Error bars correspond to SD. (D) Model Merge Granules condensates of virus infection-induced SG assembly and disassem- bly regulated by nsP3 ADP-ribosylhydrolase activity.

As nsP3 ADP-ribosylhydrolase activity alters the condensate time-course (SI Appendix, Fig. S6 A and B). Consistent with fixed localization of poly(A)+ mRNA upon stress (Fig. 3F), we char- cell analyses of other alphaviruses (51, 52), SGs disassembled at acterized localization of the viral genome (vRNA), which is pol- later stages of CHIKV infection. However, these SGs were dis- yadenylated, and tested whether the enzymatic activity affects its assembled with different kinetics in cells infected with WT versus localization. Time-course single-molecule fluorescence in situ hy- mutant viruses (Fig. 7 B and C and Movie S2). To quantify the bridization (smFISH) experiments revealed that vRNA increas- dynamics of SG formation and disassembly in a given cell E ingly associated with G3BP1-containing condensates (Fig. 6 and (i.e., SG residence time in cell c1,c2,c3...cn), we defined t = 0as SI Appendix,Fig.S5E). Some of them also contain eIF3b, indi- the first appearance of SGs (Fig. 7A). SG residence time was E F SI cating that these condensates are SGs (Fig. 6 and and shorter (152 ± 67 min) in WT virus-infected cells when com- Appendix E F ,Fig.S5 and ). For WT virus, the percentage of cells pared with Y114A (281 ± 95 min) mutant virus-infected cells with vRNA-containing SGs peaked at 4 hpi and declined there- (Fig. 7 B and C). Because reducing ADP-ribosylhydrolase activity F after (Fig. 6 ). In contrast, vRNA-containing SGs persisted at 6 increases SG residence time, we conclude that ADP-ribosylhydrolase and 8 hpi for the hydrolase-deficient mutant Y114A (Fig. 6 E and F SI Appendix E F activity is critical for regulating SG disassembly during CHIKV and ,Fig.S5 and ). Therefore, these data suggest infection. that nsP3 ADP-ribosylhydrolase activity controls the composition of these vRNA-containing SGs. Discussion Here, we report that both SG formation and disassembly can be nsP3 Macrodomain ADP-Ribosylhydrolase Activity Regulates the regulated by the MD of alphaviral protein nsP3 through its ADP- Disassembly of Virus-Induced SGs. SGs induced during early stages of alphaviral infection are disassembled as infection pro- ribosylhydrolase activity. Expression of the MD alone, but not a gresses (11–14). Because expression of nsP3 (either by trans- catalytically dead mutant MD, suppressed SG formation. Live fection or infection) suppresses SG formation (Figs. 1E and 6D) cell imaging revealed that the MD ADP-ribosylhydrolase activity and disassembles preformed G3BP1-induced SGs through ADP- determines the residence time of virus-induced SGs, with their ribosylhydrolase activity (SI Appendix, Fig. S4B), we next tested prolonged presence in cells infected with catalytically deficient whether this same mechanism regulates later SG disassembly mutant viruses. It was previously proposed that SG disassembly is during virus infection. mediated through the nsP3-mediated sequestration of G3BP1/2 To monitor SG kinetics in live cells, we used the U2OSGFP-eIF3g (28, 29, 34). Although we confirmed the association of the nsP3 stable cell line (Fig. 1A) or, as a negative control, U2OS cells HVD and G3BP1, which is central to the sequestration model, stably expressing GFP (U2OSGFP). These cells were imaged expression of the HVD domain alone did not suppress SG every 12 min for 14 h (Fig. 7A). Infection with WT and nsP3 formation. In contrast, full-length nsP3 carrying HVD FGDF mutant CHIKVmCherry resulted in SG formation, visualized as mutations (which reduce G3BP1/2 interaction) and the intact GFP-positive condensates, in U2OSGFP-eIF3g cells (Fig. 7 B and MD alone can suppress SG formation. Moreover, G3BP1 mu- C and Movie S2). GFP-positive condensates were not observed tant lacking the NTF2L domain, which binds HVD, can still form in U2OSGFP or mock-infected U2OSGFP-eIF3g cells over the same SGs, and the formation of these SGs can still be suppressed by

8of11 | PNAS Jayabalan et al. https://doi.org/10.1073/pnas.2021719118 Stress granule formation, disassembly, and composition are regulated by alphavirus ADP-ribosylhydrolase activity Downloaded by guest on September 29, 2021 nsP3. It is therefore unlikely that nsP3 suppresses SG formation by nsP3 could be due to its blocking of the initiation of PAR in CHIKV-infected cells through G3BP1/2 sequestration alone. formation, thus preventing not only adding single but also mul- Instead, we propose that nsP3 regulates SG formation through tiple ADP-ribose units to form PAR critical for SG formation. both C-terminal HVD interactions with SG proteins and an Given that ADP-ribosylhydrolase activity is conserved across N-terminal MD-catalyzed decrease in ADP-ribosylation. As nsP3 all MD-containing RNA viruses (60, 61), including coronaviruses, it concentration increases over the course of infection, the ac- is possible that these viruses regulate SG formation and disassembly companying increase in ADP-ribosylhydrolase activity facilitates through a common mechanism by removing ADP-ribosylation. disassembly of preexisting SGs and suppresses the formation of new ones in later stages of the replication cycle (Fig. 7D). Materials and Methods What might the function of SG disassembly by nsP3 ADP- Cell Culture, Chemicals, and Transfection. U2OS and HeLa cells were obtained ribosylhydrolase during late stages of alphavirus infection be? from American Type Culture Collection and 293F cells from Invitrogen. Cells Expression of WT nsP3 results in all tested SG-associated transla- were maintained in Dulbecco’s modified Eagle medium (DMEM) (Gibco) tion factors (eIF3b, eIF3i, eIF3e, eIF3j, RACK1, eIF4G1, and containing 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Life RPS6) separating from all tested SG-associated RNA-binding pro- Technologies). In all experiments, plasmids were transfected using JetPrime teins (G3BP1, G3BP2, TIA-1, TIAR, HuR, PABP, and FMRP). from Polyplus (U2OS) or 293fectin from Gibco (293F) as per the manufac- turer’s protocol. The following drugs were used for stress induction: Arsenite Meanwhile nsP3 remains associated with the RNA-binding proteins (Sigma, S7400); Clotrimazole (Sigma, C6091); Thapsigargin (Sigma, T9033); as a class of condensates that is distinct from SGs. Yet, expression of Cycloheximide (Sigma, C7698). ADP-ribosylhydrolase–deficient nsP3 forms SG condensates with translation factors as well as RNA-binding proteins. Therefore, the Plasmid Generations. EGFP-C1-nsP3 WT, EGFP-C1-nsP3 MD, EGFP-C1-nsP3 enzymatic activity determines whether RNA-binding proteins asso- ZBD, EGFP-C1-nsP3 HVD, EGFP-C1-nsP3 D10A, EGFP-C1-nsP3 G32E, EGFP-C1- ciate with translation factors, thereby regulating the formation of two nsP3 G32S, EGFP-C1-nsP3 G112E, EGFP-C1-nsP3 Y114A, EGFP-C1-nsP3 AGDA, distinct classes of cellular condensates that have apparently similar EGFP-C1-eIF3g, pCI-neo-Flag-nsP3 WT, pCI-neo-Flag-nsP3 G32E, pCI-neo-Flag- composition but distinct properties (see Table 1 for comparisons). nsP3 MD were constructed by restriction enzyme-based method or Gibson Our data indicate that some protein–protein interactions assembly using primers listed in SI Appendix,TableS2.

within SGs are mediated by PAR, which are covalently conju- GFP GFP-eIF3g gated to components like G3BP1 and G3BP2, whereas other Generation of U2OS and U2OS Stable Cell Lines. U2OS cells trans- components (such as eIF3g and G3BP1) bind noncovalently to fected with pEGFP vector alone or pEGFP-eIF3g were maintained in G418 (neomycin) at 0.5 mg/mL for several days to eliminate untransfected pa- this polymer. nsP3 appears to alter the composition of conden- rental U2OS cells. Medium was changed every 2 d to replenish drugs and MICROBIOLOGY sates through removing ADP-ribosylation associated with the remove dead cells. Cells were then trypsinized and diluted for single-clone essential SG component G3BP1 and potentially other ADP- selection based on manual analysis of GFP expression under the microscope. ribosylated components, a mechanism that is predicted to sig- nificantly affect the function of the resulting condensate. The Viruses. CHIKV 181/25 WT and mutant strains were prepared as described observed pattern of protein retention revealed that nsP3 ADP- previously (50). Viral stocks were grown in BHK21 cells, and titers were de- ribosylhydrolase activity promotes the release of translation termined by plaque formation in Vero cells as described previously (31). components from SGs, likely needed for viral structural protein translation during late infection stages (Fig. 7D). This possibility Immunoblot Analysis. Cells were lysed in RIPA buffer (50 mM Tris-Cl pH 8.0, is consistent with recent data showing that nsP3 ADP-ribo- 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 1% Nonidet P-40, 1 mM sylhydrolase activity is critical for the switch from translation of EDTA as well as protease inhibitors 5 mM NaF and 1 mM phenylmethylsulfonyl host proteins and viral nonstructural proteins from genomic fluoride [PMSF]) for 15 min on ice, followed by centrifugation at 14,000 rpm for 15 min, 4 °C. Protein samples were acetone-precipitated for at least 1 h at −20 RNA to translation of the viral structural proteins from a sub- °C. Precipitates were centrifuged at 13,000 rpm, 4 °C for 15 min, and the air- genomic RNA during the later stages of viral replication (50, 53). dried pellets were then diluted in 1× SDS sample buffer. The samples were re- Our smFISH data further revealed that the vRNA genome is solved in polyacrylamide gel electrophoresis and blotted with appropriate pri- temporarily colocalized with SGs at an early stage of infection, mary antibodies (SI Appendix,TableS3). and that ADP-ribosylhydrolase activity is required for reducing association between vRNA genome and translation factors (cf. Immunofluorescence. U2OS (∼4 × 104) or HeLa (∼1 × 105) cells grown on Fig. 6 E and F). This redistribution of host translation factors, vRNA coverslips were treated with the indicated stressors. Following the stress genome, and ribosomal proteins may be required for efficient virus treatment, cells were washed twice with 1× PBS, fixed with 4% parafor- structural protein translation at later stages of infection (11). maldehyde for 15 min, permeabilized with ice-cold methanol for 10 min, and Compared with other protein modifications that regulate SG washed twice with 1× PBS. The cells were then blocked with 5% normal horse × dynamics, ADP-ribosylation is unique in that the polymeric form serum in 1 PBS containing 0.02% sodium azide for 1 h at room temperature (RT). All primary antibodies (SI Appendix,TableS3) were diluted in blocking (i.e., PAR) is able to seed low-complexity region-containing – buffer and incubated with cells overnight at 4 °C, followed by three washes with proteins to form protein condensates in vitro (26, 27, 54 56). 1× PBS, 10 min each. Next, appropriate secondary antibodies were diluted in The complete removal of PAR from ADP-ribosylated proteins blocking buffer (1:500), incubated with cells for 1 h at RT, washed three times requires two steps: the degradation by PARG of the polymeric with 1× PBS, and the coverslips were mounted on glass slides using Prolong Gold. chain down to single ADP-ribose units conjugated to proteins, All experiments were performed at least thrice. followed by the hydrolysis of the final, proximal ADP-ribose groups from proteins (57–59). The alphaviral nsP3 MD effi- Image Quantitation. For all quantitation, random 40× fields were chosen and ciently hydrolyzes single ADP-ribose groups from ADP-ribosy- a total of between 80 and 120 cells were counted per condition. To quantify lated proteins in vitro but inefficiently removes PAR chains (31, the number of SGs in cells transfected with various GFP-tagged nsP3 constructs, 38). Previous studies localized PARG to SGs, where its expres- cells with eIF3b foci in GFP-positive cells were scored as SG-positive. To quantify sion level controls the formation and disassembly of SGs (17). the number of SGs in virus infection, eIF3b foci in nsP3-stained cells were scored Therefore, endogenous PARG likely mediates the breakdown of as SG-positive. All experiments were repeated three independent times. PAR in SGs, followed by the action of nsP3 ADP-ribosylhy- Live Cell Imaging. U2OSGFP or U2OSGFP-eIF3g cells were seeded 1 d prior to the drolase, which removes the final ADP-ribose. Given that nsP3 experiment at a concentration of ∼1 × 104 cells/mL on a 2-well chambered has a stronger effect than PARG in suppressing SG formation cover glass (Nunc Lab-Tek) and were cultured in DMEM, 10% (vol/vol) FBS at

and nsP3 removes ADP-ribose from acidic residues (31), the 37 °C, and 5% CO2 in a humidified incubator. On the day of the experiment, removal of specific mono-ADP-ribosylation is likely a rate- cells were infected with either CHIKV WT or Y114A mutants at a multiplic- limiting step in SG disassembly. The observed SG suppression ity of infection (MOI) of 10 and incubated at 4 °C for 1 h, followed by

Jayabalan et al. PNAS | 9of11 Stress granule formation, disassembly, and composition are regulated by alphavirus https://doi.org/10.1073/pnas.2021719118 ADP-ribosylhydrolase activity Downloaded by guest on September 29, 2021 incubation at 37 °C for 1 h. Cells were then washed twice with 1× PBS, and hybridization, coverslips were washed twice with 10% formamide, 2× SSC

fresh CO2-independent DMEM containing 20% FBS was added. Infected cells for 30 min at 37 C. Thereafter, the coverslips were rinsed in 1× PBS con- were imaged using a DeltaVision Elite system (GE Healthcare) microscope taining 0.5 μg/mL DAPI for 5 min followed by two rinses with 1× PBS and equipped with ×40 (1.516 N.A. oil) immersion objectives, a high-speed CCD mounted using ProLong Gold. 2 Camera (Cool SNAP HQ ), appropriate filter sets for FITC/mCherry, and an The superresolution images were acquired using a Zeiss Elyra PS.1 system incubation chamber (37 °C and 80% humidity). Images were acquired at equipped with a 63× oil objective, an Andor EMCCD iXon3 DU-885 CSO 12-min intervals for 14 h, controlled by the SoftWorx suite (GE Healthcare). A VP461 camera (1004 × 1002 pixels), the following lasers: 50 mW 405 nm HR – total of 15 20 random fields were chosen and imaged, and the presence of diode, 100 mW 488 nm HR diode, 100 mW 561 nm HR DPSS, 150 mW 642 nm SG was monitored as microscopically visible condensates in the FITC channel. HR diode and the following filter sets: DAPI: BP420-480 + LP750 (Zeiss SR To calculate the SG residence time, the duration between the appearance cube 07), Cy2: BP495-590 + LP750 (Zeiss SR cube 13), Cy3: LP570 (Zeiss SR cube and disappearance of SG was manually analyzed for a given cell. The SG 14), Cy5: LP655 (Zeiss SR cube 10). Each image was acquired using three grid = = residence time was calculated as the summation of t1 0min,t2 12 min, rotations using a grid size of 42 mm for all channels. t3 = 24 min, tn = n min (where, t1 is the earliest frame at which SG appeared; and t is the first frame at which SG disappeared). n Immunoprecipitation. 293F cells were spun down at 400 × g for 3 min at 4 °C, washed once with cold 1× PBS, pelleted and lysed in cold lysis buffer (CLB) Simultaneous smFISH and Immunofluorescence to Detect vRNA. Immunofluo- (50 mM Hepes pH 7.4, 150 mM NaCl, 1 mM MgCl , 1 mM ethylene glycol- rescence staining for SG proteins was coupled with single-molecule imag- 2 bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 1% Triton X-100 supple- ing of vRNA using the previously published protocol (62). A set of 40 mented with 1 mM NaF, 1 mM PMSF, 1 mM adenosine 5′-diphosphate oligonucleotide probes (each 20 nucleotides [nt] long) was designed spe- (hydroxymethyl)pyrrolidinediol). Cell lysates were mixed at 4 °C for 15 min, cifically against nsP3 RNA (SI Appendix, Table S4). Each probe was designed spun down for 15 min at 13,000 rpm, and the supernatant fluid was col- to have a 3′ amino modification (LGC Biosearch Technologie). The probes lected in a new tube. The cleared lysates were added to preincubated anti- were then pooled and coupled en mass with Texas Red, and coupled probes GFP (3E6, Invitrogen)–DYNA magnetic beads (10004D, Invitrogen) complex were purified using HPLC (63). Briefly, U2OS cells grown on glass coverslips and incubated for 2 h at RT. The beads were washed once with CLB, twice were infected with CHIKV WT or Y114A mutant for 1 h, 2 h, 4 h, 6 h, 8 h, and then treated with 0.2 mM arsenite for 30 min. Following arsenite treatment, with high-salt CLB (300 mM NaCl), followed by a final wash with CLB. The × cells were washed twice with 1× PBS, fixed with 4% paraformaldehyde for precipitates were then boiled with 1 SDS sample buffer for 10 min at ∼ 15 min, and permeabilized with 70% ethanol. The cells were washed with 1× 85 °C. All experiments were performed at least thrice. For Biotin-PAR pull- PBS twice and incubated with bovine serum albumin (BSA) for 1 h at RT. down, 293F cells transfected with appropriate plasmids for 24 h were pel- Coverslips were incubated with primary antibodies for G3BP1 or eIF3b leted, lysed in CLB for 15 min. Cell lysates were cleared by centrifugation at overnight at 4 °C and then washed three times with 1× PBS, 10 min each 13,000 rpm for 15 min, and the supernatant was collected in a new tube. 100 μ followed by incubation with appropriate secondary antibodies for 1 h at RT. pmol biotin-labeled PAR (66) and 50 L of streptavidin magnetic beads were The coverslips were then washed with 1× PBS three times, and smFISH added to each sample and incubated for 2 h at 4 °C. After incubation, the protocol was followed as described previously (63). Briefly, coverslips were beads were washed once with CLB, twice with high-salt CLB (300 mM NaCl), washed with 2× SSC buffer with 20% formamide and then hybridized with followed by a final wash with CLB. The precipitates were then boiled with 1× smFISH probes in a 20% hybridization buffer overnight at 37 °C in a humidified SDS sample buffer for 10 min at ∼85 °C. chamber. After hybridization, coverslips were washed four times with 2× SSC buffer containing 20% formamide, counterstained with DAPI, and mounted Statistical Analysis. Data were presented as mean ± SD and groups compared using deoxygenated mounting media. The coverslips were imaged with 100× oil using two-tailed unpaired Student’s t test and Kolmogorov–Smirnov test. objective using a Nikon TiE Inverted epi Fluorescence microscope equipped with P < 0.05 was considered statistically significant. All statistical analyses were a pixis 1024b camera (Princeton Instruments). The images were obtained using performed using Graphpad Prism8. Metamorph imaging software and analyzed using custom-written programs in MATLAB (Mathworks Inc.) as described previously (64). Data Availability. All study data are included in the article and/or supporting information. smFISH on poly(A)+ mRNA. Poly(A) FISH was performed using 35-nt-long dT LNA probes containing amine reactive group (synthesized by Exiqon) and ACKNOWLEDGMENTS. We thank Drs. Phillip Sharp, Nancy Kedersha, Lucas labeled with far red dye Cy5 (GEPA25001) or red dye Cy3.5 (GEPA23501) Reineke, Lyle McPherson, and members of the A.K.L.L. laboratory for their from Sigma. For labeling, one dye pack was resuspended in 30 μL of freshly critiques of the manuscript. We thank Dr. Nancy Kedersha and Dr. Paul prepared 0.1 M sodium bicarbonate solution (pH 8.4) and was used to label Anderson for G3BP1/2 dKO cells and G3BP1 constructs, and Dr. Andres Merits 20 μg of dT LNA probes. The labeled probes were purified from free dyes for nsP3 antibodies. We also thank Morgan Dasovich for synthesizing the – using the QIAGEN nucleotide removal kit (catalog no. 28304). smFISH was Biotin PAR and Debra Hauer for construction of the nsP3 mCherry-tagged performed as described in ref. 65. Briefly, cells transfected with GFP-nsP3 viruses. This work was supported by a Johns Hopkins Catalyst Award (to A.K.L.L.), pilot grants from the Johns Hopkins University School of Medicine WT or G32E were fixed using 4% paraformaldehyde in 1× PBS and kept in Sherrilyn and Ken Fisher Center for Environmental Infectious Disease (to 70% ethanol at −20 °C. Before hybridization, coverslips were rehydrated in A.K.J., D.E.G., and A.K.L.L.), NIH Grants R56AI137264 (to D.E.G. and × × 1 PBS and submerged in 10% formamide/2 SSC for 10 min. Hybridization A.K.L.L.) and R01GM104135 (to A.K.L.L.), and Canadian Institutes of Health mix was prepared using 10 ng of probes and 40 mg of salmon sperm DNA/ Research Grants PJT-148932 (to D.Z.) and UDRF_SI_19A00244 (to M.B.). D.Z. is transfer RNA were resuspended in 10% formamide, 2× SSC and 10% dextran a Fonds de Recherche du Québec-Santé Chercheur-boursier Senior Research sulfate solution containing 2 mM ribonucleoside vanadyl complexes and Scholar, and S.A. holds a Fonds de Recherche du Québec-Santé Doctoral 0.1 mg/mL BSA. Hybridization was carried out for 3 h at 37 °C in dark. After Fellowship.

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Jayabalan et al. PNAS | 11 of 11 Stress granule formation, disassembly, and composition are regulated by alphavirus https://doi.org/10.1073/pnas.2021719118 ADP-ribosylhydrolase activity Downloaded by guest on September 29, 2021