20 1 (Figure 7B). GEMIN5 impacts protein synthesis at the translation elongation step through its ....

1

Understanding RNP remodelling uncovers RBPs functionally required for viral 1

replication 2

Manuel Garcia-Moreno*1, Marko Noerenberg*1, Shuai Ni*3,4, Aino I Järvelin1, Esther 3

González-Almela5, Caroline Lenz1, Marcel Bach-Pages1, Victoria Cox1, Rosario Avolio1,6, 4

Thomas Davis1, Svenja Hester1, Thibault J.M Sohier7, Bingnan Li8, Miguel A Sanz5, Luis 5

Carrasco5, Emiliano P. Ricci7, Vicent Pelechano8, Bernd Fischer3,9, Shabaz Mohammed1,2 6

and Alfredo Castello1 7

* equally contributed to this work 8

1. Department of Biochemistry, University of Oxford, OX1 3QU Oxford, UK 9

2. Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 10

Mansfield Road, Oxford OX1 3TA, UK 11

3. German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany 12

4. Faculty of Biosciences, Heidelberg University, Heidelberg, Germany 13

5. Centro de Biologia Molecular “Severo Ochoa”, Universidad Autonoma de Madrid, 14

28049 Madrid, Spain 15

6. Department of Molecular Medicine and Medical Biotechnology, University of Naples 16

Federico II, Italy. 17

7. Université de Lyon, ENSL, UCBL, CNRS, INSERM, LBMC, 46 Allée d'Italie, 69007, 18

Lyon, France. 19

8. SciLifeLab, Department of Microbiology, Tumor, and Cell Biology, Karolinska 20

Institutet, 17165 Solna, Sweden 21

9. Deceased 22

Correspondence to: [email protected] 23

24

25

26

.CC-BY-NC-ND 4.0 International licenseavailable under anot certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (which wasthis version posted June 20, 2018. ; https://doi.org/10.1101/350686doi: bioRxiv preprint

https://doi.org/10.1101/350686

http://creativecommons.org/licenses/by-nc-nd/4.0/

2

SUMMARY 1

The compendium of RNA-binding proteins (RBPome) has been greatly expanded by the 2

development of RNA-interactome capture (RNA-IC). However, it remains unknown how 3

responsive is the RBPome and whether these responses are biologically relevant. To 4

answer these questions, we created ‘comparative RNA-IC’ to analyse cells challenged with 5

an RNA virus, called sindbis (SINV). Strikingly, the virus altered the activity of 245 RBPs, 6

many of which were newly discovered by RNA-IC. Mechanistically, alterations in RNA 7

binding upon SINV infection are caused by changes in the subcellular localisation of RBPs 8

and RNA availability. Moreover, ‘RBPome’ responses are crucial, as perturbation of dynamic 9

RBPs modulates the capacity of the virus to infect the cell. For example, ablation of XRN1 10

causes cells to be refractory to infection, while GEMIN5 moonlights as a novel antiviral 11

factor. Therefore, RBPome remodelling provides a mechanism by which cells can 12

extensively rewire gene expression in response to physiological cues. 13

14

HIGHLIGHTS 15

A quarter of the RBPome remodels upon SINV infection. 16

The remodelling is caused by changes in protein localisation and RNA availability. 17

Rewiring of the RBPome is crucial for viral infection efficacy. 18

We discover RBPs with previously unknown anti- or pro-viral activity. 19

20

21

22

23

24

25

26



https://doi.org/10.1101/350686


3

INTRODUCTION 1

RNA-binding proteins (RBPs) assemble with RNA forming ribonucleoproteins (RNPs) that 2

dictate RNA fate (Glisovic et al., 2008). Historically, most of the known RBPs were 3

characterised by the presence of well-established RNA-binding domains (RBDs), which 4

include the RNA recognition motif (RRM), K-homology domain (KH) and others (Lunde et 5

al., 2007). However, stepwise identification of unconventional RBPs (e.g. (Chang et al., 6

2013; Hentze and Argos, 1991; Sampath et al., 2004)) evoked the existence of a broader 7

universe of protein-RNA interactions than previously anticipated. Notably, in silico and in 8

cellulo system-wide approaches have greatly expanded the compendium of RBPs 9

(RBPome), adding hundreds of proteins lacking known RBDs and previous links with RNA 10

biology (Baltz et al., 2012; Brannan et al., 2016; Castello et al., 2012; Gerstberger et al., 11

2014; Hentze et al., 2018). For example, RNA-interactome capture (RNA-IC) employs 12

ultraviolet (UV) crosslinking, oligo(dT) capture under denaturing conditions and quantitative 13

mass spectrometry to identify the repertoire of proteins interacting with polyadenylated 14

[poly(A)] RNA in living cells (Baltz et al., 2012; Castello et al., 2012; Castello et al., 2013). 15

Applied to HeLa cells, RNA-IC identified 860 RBPs, 315 of which were previously unrelated 16

to RNA biology (Castello et al., 2012). Importantly, the crucial participation of several of 17

these ‘unorthodox’ RBPs in RNA life has been proven in follow up studies (Hentze et al., 18

2018). How unorthodox RBPs interact with RNA remained, nevertheless, poorly understood. 19

Several proteomic approaches tackled this question on a global scale, revealing that protein-20

protein interaction domains, enzymatic cores, DNA-binding domains and intrinsically 21

disordered regions can function as RNA-binding surfaces (Castello et al., 2016; He et al., 22

2016; Kramer et al., 2014). However, it remains unknown whether the RBPome responds 23

globally to physiological cues and if so, how these responses are triggered and what are 24

their biological consequences. Recent work approached these questions by analysing the 25

RBPome during fruit fly embryo development, and reported changes in RBP composition 26



https://doi.org/10.1101/350686


4

that could be explained by matching alterations in protein abundance. In other words, the 1

proteome of the embryo changes through development and as a consequence the scope of 2

available RBPs (Hentze et al., 2018; Sysoev et al., 2016). However, several RBPs did not 3

follow this trend, displaying protein-level independent changes in RNA binding and raising 4

the question of whether physiological perturbations can induce such responsive behaviour 5

more widely. To address this possibility, we used, here, an improved ‘comparative RNA-IC’ 6

approach to profile with high accuracy RBP dynamics in cells infected with sindbis virus 7

(SINV) (Figure 1A and 1B). 8

Viruses have been fundamental for the discovery and characterisation of important steps of 9

cellular RNA metabolism such as RNA splicing, nuclear export and translation initiation. This 10

is due to their ability to hijack the required host resources, often by interfering with the activity 11

of the master regulators of these pathways (Berget et al., 1977; Castello et al., 2011; 12

Castello et al., 2012; Chow et al., 1977; Krausslich et al., 1987; Lloyd, 2015; Sun and 13

Baltimore, 1989; von Kobbe et al., 2000). Furthermore, specialised RBPs are at the frontline 14

of cellular antiviral defences, detecting pathogen-associated molecular patterns (PAMPs) 15

such as double stranded RNA or RNAs with 5’ tri-phosphate ends (Barbalat et al., 2011; 16

Garcia et al., 2006; Rehwinkel and Reis e Sousa, 2013; Vladimer et al., 2014). Since RBPs 17

are crucial for both the viral biological cycle and the host antiviral defences (Garcia-Moreno 18

et al., 2018), virus infection thus represents an optimal scenario to assess the RBPome 19

dynamics. 20

Our data show that the complement of active cellular RBPs is strongly altered in response 21

to virus infection, revealing that the RBPome is highly dynamic. This phenomenon is not due 22

to changes in protein abundance, but to an extensive subcellular redistribution of host RBPs 23

and to alterations in the availability of RNA substrates. Importantly, RBP responses are 24

biologically crucial, as dynamic RBPs modulate the viral replication cycle or/and the ability 25



https://doi.org/10.1101/350686


5

of the cell to counteract the infection. We envision that these proteins may represent novel 1

targets for host-based antiviral therapies. 2

3

RESULTS AND DISCUSSION 4

Applying RNA-IC to cells infected with SINV 5

To study the dynamics of cellular RBPs in response to physiological cues, we challenged 6

cells with a cytoplasmic RNA virus and applied RNA-IC. We chose SINV and HEK293 cells 7

as the viral and cellular model, respectively. SINV is a highly tractable viral model that is 8

transmitted from mosquito to vertebrates, causing high fever, arthralgia, malaise and rash 9

in humans. Its biological cycle is relatively well-understood, involving a similar scope of 10

cellular factors to other pathogenic alphaviruses such as chikungunya virus (CHIKV) and 11

venezuelan equine encephalitis virus (VEEV) (Carrasco et al., 2018). SINV replicates in the 12

cytoplasm of the infected cell and produces three viral RNAs (Figure 1B and S1A): the 13

genomic (g) RNA; the subgenomic (sg)RNA and the negative stranded RNA. gRNA is 14

packaged into the viral capsid and is translated to produce the non-structural proteins (NSP) 15

that form the replication complex. The sgRNA is synthesised from an internal promoter and 16

encodes the structural proteins (SP) required to generate the viral capsids. The negative 17

strand serves as a template for replication. Both gRNA and sgRNA are capped and 18

polyadenylated. 19

HEK293 cells are an excellent cellular model to study SINV, as its infection exhibits all the 20

expected molecular signatures, including: 1) active viral replication (Figure 1C and S1B-C); 21

2) shut off of host protein synthesis while viral proteins are massively produced (Figure 1C 22

and S1B); 3) phosphorylation of the eukaryotic initiation factor 2 subunit alpha (EIF2α) due 23

to the activation of the protein kinase R (PKR) (Ventoso et al., 2006) (Figure 1D); and 4) 24

formation of cytoplasmic foci enriched in viral RNA and proteins, commonly known as viral 25

replication factories (Figure S1C). Infection of these cells with SINV caused a strong 26



https://doi.org/10.1101/350686


6

induction of the antiviral programme, including β-interferon (β-IFN) and interferon inducible 1

factors, which reflects the existence of active antiviral sensors and effectors (Figure S1D). 2

Importantly, we can achieve a synchronised and near-complete infection of the cell culture 3

with relatively low number of viral particles [multiplicity of infection (MOI)] (Figure S1E), 4

reducing cell-to-cell variability and biological noise. 5

Pilot RNA-IC experiments in uninfected (mock) and SINV-infected cells revealed the 6

isolation of a protein pool matching that previously observed for human RBPs (Baltz et al., 7

2012; Castello et al., 2012), which strongly differed from the total proteome (Figure 1E). No 8

proteins were detected in non-irradiated samples, demonstrating the UV-dependency of 9

RNA-IC. Infection did not induce major alterations in the protein pattern observed by silver 10

staining, which correspond to the most abundant, housekeeping RBPs (Figure 1E). 11

However, other less predominant bands displayed substantial differences, calling for in-12

depth proteomic analysis. Oligo(dT) capture led to the isolation of both host and SINV RNAs 13

in infected cells (Figure 1F), which is expected as gRNA and sgRNA are polyadenylated. 14

The amount of viral RNA captured increased throughout the infection, confirming the active 15

replication of SINV in HEK293 cells. 16

SINV causes a global remodelling of the cellular RBPome 17

To allow accurate quantification of RBPs associated with poly(A) RNA under different 18

physiological conditions, we developed ‘comparative RNA-IC’ by combining the original 19

protocol (Castello et al., 2013) with stable isotope labelling by amino acids in cell culture 20

(SILAC) (Figure 1A) (Ong and Mann, 2006). In brief, cells were grown in presence of light 21

(normal), medium or heavy amino acids with incorporation efficiency >98%. Labelled cells 22

were infected with SINV and irradiated with UV light at 4 and 18 hours post infection (hpi), 23

using uninfected cells as a control (Figure 1A). These times correlate with important events 24

in the SINV biological cycle; i.e. at 4 hpi, viral gene expression co-exists with host protein 25

synthesis, while the proteins synthesised at 18 hpi are almost exclusively viral (Figure 1C). 26



https://doi.org/10.1101/350686


7

SILAC labels were permutated between the three conditions (i.e. uninfected, 4 hpi and 18 1

hpi) in the three biological replicates to correct for possible isotope-dependent effects. After 2

lysis, aliquots were stored for parallel transcriptomic and whole proteome analyses (see 3

below). We combined equal amounts of the lysates from the three conditions prior to the 4

oligo(dT) capture (Figure 1A) and eluates from the oligo(dT) capture of these mixed samples 5

were analysed by quantitative proteomics. Protein intensity ratios between condition pairs 6

were computed and the significance of each protein intensity change was estimated using 7

a moderated t-test (Figure 2A-D and S2A-B). We used a previously described semi-8

quantitative method for the cases in which an intensity value was missing (‘zero’) in one of 9

the two conditions leading to ‘infinite’ or ‘zero’ ratios (Sysoev et al., 2016). 10

We identified a total of 794 proteins; 91% of which were already annotated by the gene 11

ontology term ‘RNA-binding’ or/and were previously reported to be RBPs in eukaryotic cells 12

by RNA-IC (Hentze et al., 2018). Hence, the protein composition of our dataset largely 13

resembles that of previously established RBPomes. Most cellular RBPs remained unaltered 14

at 4 hpi with the exception of 17 RBPs (~2% of the identified RBPome) (Figure 2A-B, S2A 15

and Table S1). 15 of these were detected exclusively by the semi-quantitative method due 16

to the lack of protein intensity value in one condition, reflecting possible ‘on-off’ and ‘off-on’ 17

states (Table S1). By contrast, SINV caused a pervasive remodelling of the RBPome at 18 18

hpi (Figure 2C-D and S2B), since 236 RBPs (~30%) displayed altered RNA-binding activities 19

(48 RBPs with 1% false discovery rate [FDR], 167 with 10% FDR and 21 by the semi-20

quantitative analysis) (Table S1). The 245 RBPs with differential RNA-binding activity in 21

SINV-infected cells (4 and 18 hpi) are referred to here as ‘dynamic RBPs’. Interestingly, 181 22

out of 245 dynamic RBPs lack classical RBDs (Castello et al., 2012; Lunde et al., 2007), 23

suggesting that unconventional RBPs may have biological roles in infection. 24

To validate these results, we applied RNA-IC to cells infected with SINV but, in this case, 25

the eluates were analysed by western blotting. We selected nine dynamic RBPs falling into 26



https://doi.org/10.1101/350686


8

three statistical categories (i.e. four with 1% FDR, four with 10% FDR and one with non-1

significant changes). As positive control, we monitored the SINV capsid (C), which is a viral 2

RBP that accumulates throughout the infection. We also analysed two ‘non-dynamic’ RBPs 3

(MOV10 and bifunctional glutamate/proline-tRNA ligase, EPRS) and β-actin (ACTB) as a 4

protein lacking RNA-binding activity. The RNA-binding behaviour of each protein fully 5

matched the proteomic outcome, including those classified with 10% FDR. The eukaryotic 6

initiation factor (EIF) 4G1, GEMIN5, DDX1, proliferation-associated protein 2G4 (PA2G4) 7

and peptidyl-prolyl cis-trans isomerase A (PPIA) exhibited increased interaction with RNA 8

after infection; whereas the opposite was observed for X-ray repair cross-complementing 9

protein 5 (XRCC5), XRCC6, DDX50 and polypyrimidine tract-binding protein 1 (PTBP1) 10

(Figure 2E). These changes increased progressively throughout the infection. The 11

proteomic data assigned a non-significant decrease of RNA-binding activity to HNRNPR 12

(Table S1); however, the reduced activity of this protein was apparent by western blotting 13

(Figure 2E), suggesting that our dataset may contain false negatives. Nonetheless, the 14

excellent agreement between the proteomic and western blotting data supports the high 15

quality of our results. 16

Determination of the RBP networks responding to SINV infection 17

Among the 245 dynamic RBPs, 236 were identified at 18 hpi, 133 presented reduced and 18

103 increased association with RNA and they are referred to as ‘inhibited’ and ‘stimulated’ 19

RBPs, respectively. Most of the RBPs inhibited by SINV were linked to nuclear processes 20

such as RNA processing and export (Figure 2F and S2C). Inhibition of nuclear RNA 21

metabolism by cytoplasmic viruses has been extensively reported although it is still poorly 22

understood (Akhrymuk et al., 2012; Gorchakov et al., 2005; Lloyd, 2015), and the inhibition 23

of cellular RBPs is likely contributing to this phenomenon. Conversely, most stimulated 24

RBPs are cytoplasmic and are linked to protein synthesis, 5’ to 3’ RNA degradation, RNA 25

transport, protein metabolism and antiviral response (Figure 2F and S2D). Nonetheless, 26



https://doi.org/10.1101/350686


9

several nuclear RBPs, including splicing factors, are stimulated upon infection, indicating 1

that the above statements have notable exceptions. 2

Interestingly, several RBPs involved in translation were stimulated at 18 hpi despite the shut 3

off of host protein synthesis (Figure 1C and S1B). These include eukaryotic initiation factors 4

(EIF4G1, EIF4G2, EIF4G3, EIF3C, EIF3D, EIF3E, EIF3G, EIF3J and EIF5B), elongation 5

factors (EEF1G, EEF2, EEF1A1) and ribosomal proteins (RPS27, RPS25, RPS15A, RPS12, 6

RPL36, RPL28, RPL10, RPL29, RPL39, RPL8, RPL4, RPL9 and RPL14). This 7

enhancement is likely due to the high translational activity of SINV RNAs (Figure 1C) (Frolov 8

and Schlesinger, 1996). Conversely, four ribosomal proteins (RPS3, RPS10, RPS28 and 9

RPL7L1) showed reduced RNA-binding activity (Table S1). The essential components of 10

the cap-dependent translation initiation machinery, EIF4A1 and EIF4E, were not stimulated 11

by the infection in spite of the activation of their protein partner EIF4G1 (Table S1). This 12

agrees with previous data showing that these two initiation factors do not participate in SINV 13

sgRNA translation (Carrasco et al., 2018; Castello et al., 2006; Garcia-Moreno et al., 2015; 14

Garcia-Moreno et al., 2013). A recent report showed that EIF3D is a cap-binding protein that 15

substitutes EIF4F in the translation of specific mRNA pools (Lee et al., 2016). EIF3D is 16

stimulated by SINV, and thus its potential contribution to SINV RNA translation deserves 17

further consideration. Moreover, 87 dynamic RBPs were recently reported to associate with 18

ribosomes in a proteomic analysis using Flag-tagged ribosomal proteins in mouse cells 19

(Table S2) (Simsek et al., 2017). The existence of ‘specialised ribosomes’ in infected cells 20

has been proposed; however, experimental evidence is currently sparse (Au and Jan, 2014). 21

Our results indicate that the composition of ribosomes and the scope of proteins associated 22

with them may strongly differ between infected and uninfected cells, possibly resulting in 23

differential translational properties. 24

RNA-IC uncovered 16 dynamic RNA helicases (Table S2); 13 of which were inhibited upon 25

infection. RNA helicases are fundamental at virtually every stage of RNA metabolism, since 26



https://doi.org/10.1101/350686


10

they mediate the remodelling of RNPs (Chen and Shyu, 2014). Hence, it is expected that 1

their inhibition will have important consequences in RNA metabolism. Only 3 helicases were 2

stimulated by SINV (DDX1, DHX57 and DHX29) (Figure 2E and Table S2). DDX1 is part of 3

the tRNA ligase complex (Popow et al., 2011) and interestingly the ligase subunit, RTCB, is 4

also stimulated by SINV (Table S1), as will be discussed below. DHX29 enhances 48S 5

complex formation on SINV sgRNA in reconstituted in vitro systems (Skabkin et al., 2010), 6

and the stimulation of this helicase reported by RNA-IC supports its potential contribution to 7

viral translation in infected cells. 8

Notably, a defined subset of antiviral factors displayed increased RNA-binding activity in 9

response to SINV infection, suggesting activation through PAMP recognition. These RBPs 10

include the interferon-inducible protein 16 (IFI16), interferon-induced protein with 11

tetratricopeptide repeats 5 (IFIT5), E3 ubiquitin/ISG15 ligase TRIM25, TRIM56 and zinc 12

finger CCCH-type antiviral protein 1 (ZC3HAV1 or ZAP) (Table S1). IFI16 was previously 13

described to bind double stranded DNA in cells infected with DNA viruses (Ni et al., 2016). 14

Our data reveal that IFI16 also binds to RNA, and it is activated early after SINV infection (4 15

hpi). This agrees with the recently described ability of IFI16 to restrict RNA virus infection 16

(Thompson et al., 2014). These findings highlight the capacity of RNA-IC to identify antiviral 17

factors responding to a given RNA virus. 18

Interestingly, RNA-IC also identified viral RBPs associated with poly(A) RNA in infected 19

cells. These include the known viral RBPs (i.e. RNA helicase NSP2, the RNA polymerase 20

NSP4 and the capsid protein) and, unexpectedly, also NSP3 and E2 (Figure 2G and S2E). 21

NSP3 was only quantified in two replicates (Figure S2E), and thus its interaction with RNA 22

requires experimental confirmation. The identification of E2 in RNA-IC eluates was 23

unexpected. However, the structure of E1, E2 and C from the related VEEV provides some 24

insights into this finding. E2 interacts with C nearby cavities that communicate with the inner 25

part of the viral particle, where the gRNA density resides (Zhang et al., 2011). It is thus 26



https://doi.org/10.1101/350686


11

plausible that E2 interacts transitorily or stochastically with the viral RNA in the context of 1

the highly packed capsid. 2

RBP responses to SINV are not caused by changes in protein abundance 3

Changes detected by RNA-IC can be a consequence of matching alterations in protein 4

abundance (Sysoev et al., 2016). To assess this possibility globally, we analysed the total 5

proteome (inputs of the RNA-IC experiments, Figure 1A) by quantitative proteomics (Figure 6

3). Importantly, SINV infection did not cause noticeable alterations in host RBP levels, even 7

at 18 hpi (Figure 3A-C, S3A-C and Table S3). In agreement, coomassie staining did not 8

show major protein fluctuations in uninfected versus infected cells except for SINV C (Figure 9

3D). The lack of changes in protein levels, even for dynamic RBPs, was confirmed by 10

western blotting (Figure 3E). Taken together, these data confirm that the variations in RNA-11

binding activity of cellular RBPs are not globally caused by alterations in the host protein 12

abundance. 13

The transcriptome undergoes pervasive alterations in SINV-infected cells 14

Mechanistically, the activity of host RBPs can also be altered by changes in the availability 15

of their target RNAs. To profile the transcriptome of uninfected versus SINV-infected cells, 16

we performed RNA sequencing analysis of total RNA isolated from the RNA-IC input 17

samples (Figure 1A). 4 h of SINV infection had a relatively minor impact on the host 18

transcriptome, with only 67 and 177 up- and down-regulated RNAs, respectively (Figure 3F). 19

By contrast, profound changes in the cellular transcriptome were observed at 18 hpi, with 20

12,372 differentially expressed RNAs (p<0.1; Figure 3G and S3E-F). While 10,924 RNAs 21

had significantly lower expression in infected cells, only 1,448 RNAs were upregulated 22

(Table S4). Hence, SINV infection causes a massive loss of cellular RNAs. Upregulated 23

RNAs were enriched in genes annotated by the GO term ‘antiviral response’, reflecting the 24

activation of the host defences (Figure S1D, S3E and Table S4). To validate these results 25

by an orthogonal approach, we used RT-qPCR focusing on 20 mRNAs randomly chosen 26



https://doi.org/10.1101/350686


12

across the whole variation range. Importantly, data obtained with both techniques strongly 1

correlated (R2= 0.82) (Figure 3H). In summary, availability of cellular RNA is globally 2

reduced upon infection, correlating with the emergence of viral RNA (Figure 3G and Table 3

S4). Decreased availability of cellular RNA is expected to contribute to the inhibition of RNA-4

binding activity observed for 133 RBPs (Figure 2C-D and Table S1). 5

SINV infection induces subcellular redistribution of cellular RBPs 6

While depletion of cellular RNA can explain the reduced activity of RBPs, it cannot account 7

for the stimulation of 103 proteins. Importantly, SINV RNAs are amongst the most abundant 8

RNA species in the cell at 18 hpi (Figure 3G). Such abundant RNA substrate is likely to 9

influence cellular RBPs, potentially driving the remodelling of the RBPome. SINV produces 10

two overlapping mRNAs, gRNA and sgRNA (Figure 1B and S1A) and, consequently, the 11

read coverage was substantially higher in the last third of the gRNA, where both transcripts 12

overlap (Figure 4A). Both sgRNA and gRNA are substantially more abundant than the 13

negative strand (Figure 4A). As the copy number of the negative strand is low and it lacks 14

poly(A) tail, it should not contribute to the RNA-IC results. 15

SINV RNAs are known to accumulate in cytoplasmic compartments, so called viral 16

replication factories, derived from the membranes of cellular organelles (Figure 4B) 17

(Romero-Brey and Bartenschlager, 2014). These viral RNA-containing foci can explain the 18

punctuate accumulations of poly(A) RNA in the cytosol of infected cells (Figure S4A). We 19

thus reasoned that RBPs interacting with SINV RNAs should accumulate in these 20

compartments. To explore this idea systematically, we generated 26 tetracycline-inducible 21

cell lines expressing host RBPs fused to eGFP. This cell library included 16 lines expressing 22

‘stimulated RBPs’ and 8 expressing ‘inhibited RBPs’. A non-dynamic RBP, MOV10 (Figure 23

2E), and unfused eGFP were used as controls. Strikingly, 9 out of the 16 stimulated RBPs 24

(56%) accumulated at viral replication factories demarcated by SINV C, including 25

cytoplasmic and nuclear RBPs (Figure 4C-D and S4B). As SINV C is a good proxy for the 26



https://doi.org/10.1101/350686


13

localisation of viral RNAs (Figure 4B), RBPs retained at these foci are likely to interact with 1

viral RNA. To our knowledge, this is the first time that the idea of viral RNA hijacking cellular 2

RBPs in viral replication factories is assessed in a systematic way. Five additional stimulated 3

RBPs (29%) showed diffuse localisation in cytoplasm, but were also present at the SINV C-4

containing foci (Figure S4B). Amongst the stimulated RBPs, only neuroguidin (NGDN), 5

heterogeneous ribonucleoprotein A1 (HNRNPA1) and the mitochondrial Tu translation 6

elongation factor (TUFM) (3 out of 16; 17%) were absent in the viral factories. The absence 7

of these three proteins in the replication factories suggests that the stimulation of their RNA-8

binding activity may involve exclusively host RNA. Nevertheless, HNRNPA1 was shown to 9

bind SINV RNA (LaPointe et al., 2018; Lin et al., 2009), although in our analysis it displays 10

strictly nuclear localisation (Figure S4B). We cannot rule out that a small pool of HNRNPA1 11

is present in the viral factories at undetectable levels or, alternatively, that the eGFP tag is 12

affecting HNRNPA1 localisation. 13

In contrast to stimulated RBPs, only one (out of 8; 12.5%) virus-inhibited RBP was enriched 14

in the viral factories (Figure 4D and S4B). This protein, called regulator of nonsense 15

transcripts 1 (UPF1), is a helicase involved in the non-sense mediated decay (NMD) 16

pathway and is known to inhibit infection of alphaviruses (Balistreri et al., 2014). Conversely, 17

5 out of 8 (62.5%) virus-inhibited RBPs are nuclear and remained nuclear after infection 18

(Figure 4C-D and S4B). Several of the nuclear RBPs (e.g. RPS10, DKC1, NGDN or 19

HNRNPA1) display different degrees of delocalisation in infected cells, suggesting either 20

release from the nuclear compartments where they reside or partial disassembly of these 21

structures. 22

MOV10 is a helicase unaffected by SINV infection (Figure 2E) that localises in p-bodies 23

(Goodier et al., 2012). To our surprise, MOV10-containing p-bodies were surrounded by 24

viral replication factories in SINV-infected cells, in a non-overlapping fashion (Figure 4C). 25

Our results indicate that p-bodies may crosstalk with viral replication factories. 26



https://doi.org/10.1101/350686


14

In summary, global alterations of RBP localisation and differential availability of RNA 1

substrates are likely jointly driving the changes in RNA-binding activity in SINV-infected 2

cells. 3

Activation of XRN1 and RNA degradation in SINV-infected cells 4

The loss of cellular mRNAs can be an important driving force at shaping the RBPome in 5

SINV-infected cells (Figure 3G). However, it is unclear how the transcriptome remodelling 6

is originated and whether it benefits viral infection. Alterations in RNA levels can be a 7

consequence of reduced transcription and/or increased RNA degradation (Mukherjee et al., 8

2017). To explore which of these pathways contribute the most to RNA loss in SINV infected 9

cells, we compared the fold change of each mRNA in our dataset to available data compiling 10

the speed of synthesis, processing and degradation of each individual transcript (Mukherjee 11

et al., 2017) (Figure 3G and Table S4). Transcription could explain most of the differences 12

between uninfected and 4 hpi (Figure 5A and S5A). However, RNA degradation accounted 13

for more than 50% of the explained variance at 18 hpi. We reasoned that this phenomenon 14

can be a combined effect of the activation of the 5’ to 3’ RNA degradation machinery, as the 15

exonuclease XRN1 and its interactor PAT1 homolog 1 (PATL1) are stimulated at 18 hpi 16

(Figure S2D and Table S1), and a reduced transcriptional activity (Gorchakov et al., 2005). 17

The role of XRN1 in virus infection is currently controversial. Some studies reported that 18

XRN1 restricts viral replication by erasing viral RNA (Molleston and Cherry, 2017), whereas 19

others proposed that RNA fragments resistant to XRN1 degradation can interfere with the 20

antiviral response (Manokaran et al., 2015). In SINV-infected cells, XRN1-containing foci (p-21

bodies) are juxtaposed to the viral replication factories (Figure 5B), as observed for MOV10 22

(Figure 4C). To assess XRN1 effects in SINV infection, we generated XRN1 knock out cell 23

lines and studied the fitness of the chimeric SINV-mCherry (Figure 1B). To our surprise, 24

XRN1 knock out cells were completely refractory to SINV-mCherry infection, while partial 25

knock out led to an intermediate phenotype (Figure 5C). The fact that SINV fails to infect 26



https://doi.org/10.1101/350686


15

cells lacking XRN1 suggests that its activity is critical for the infection. Importantly, the 1

identification of XRN1 as an essential host RBP for SINV highlights the capacity of RNA-IC 2

to uncover in a global scale host factors implicated in virus infection. 3

RBPome responses are biologically important 4

To determine in a broader extent whether RBP responses are functionally important, we 5

sought to study the impact of dynamic RBPs on virus infection. The ligase RTCB, together 6

with DDX1, FAM98A, FAM98B, CGI-99 and Ashwin (ASHWN) form the tRNA ligase 7

complex (TRLC). Two components of this complex, RTCB and DDX1, displayed enhanced 8

RNA-binding activity (Table S1). Moreover, these two proteins and the nuclear factor 9

FAM98A accumulated in the viral replication factories (Figure 4C and S4B). TRLC ligates 10

the exons during the splicing of tRNAs (Popow et al., 2011; Popow et al., 2014) and a recent 11

study showed that it also mediates the cytoplasmic splicing of xbp1 mRNA upon unfolded 12

protein response (Jurkin et al., 2014). RTCB mediates the unusual ligation of 3´-phospate 13

or 2’, 3´-cyclic phosphate to a 5´-hydroxyl end, which are generated by a limited repertoire 14

of cellular endonucleases that includes the serine/threonine-protein 15

kinase/endoribonuclease IRE1 (IRE1α) (Jurkin et al., 2014; Popow et al., 2011; Popow et 16

al., 2014). SINV has been proposed to cause unfolded protein response (Rathore et al., 17

2013), which is compatible with the activation of IRE1α and TRLC in infected cells. To 18

assess the relevance of this post-transcriptional circuit in SINV-mCherry-infected cells, we 19

used a specific inhibitor (4µ8C) against the endonuclease IRE1α. Notably, this compound 20

strongly reduced viral fitness in low, non-cytotoxic concentrations (Figure 6A and S6A). 21

Hence, our data suggest that IRE1α and TRLC are positively contributing to SINV infection, 22

although the precise mechanism by which this occurs should be further investigated. 23

The peptidyl-prolyl cis-trans isomerase A (PPIA, also called cyclophilin A) has also been 24

classified as an RBP by RNA-IC studies, together with other six members of the PPI family 25

(Hentze et al., 2018). These enzymes switch proline conformation and modulate protein 26



https://doi.org/10.1101/350686


16

activity, with important regulatory consequences for RNA metabolism (Mesa et al., 2008). 1

PPIA is essential for hepatitis C virus as it modulates NS5A activity during replication. 2

Hence, the inhibition of PPIA has been exploited as a host-based antiviral therapy (Rupp 3

and Bartenschlager, 2014). PPIA is also important for the infection of human 4

immunodeficiency virus (HIV), influenza virus A and hepatitis B virus (Li et al., 2007; Liu et 5

al., 2016; Zhou et al., 2012). RNA-IC revealed that PPIA is the sole PPI enzyme that 6

responds to SINV infection (Table S1). In addition, PPIA is recruited to the viral replication 7

factories (Figure S4B). Interestingly, SINV-mCherry infection is delayed by both the ablation 8

of PPIA and the treatment with the PPIA inhibitor cyclosporine A at non-toxic concentrations 9

(Figure 6B and S6A). However, PPIA-eGFP overexpression had no effect in SINV-mCherry 10

fitness (Figure 6C, bottom panel). 11

The interaction of heat shock protein 90AB1 (HSP90AB1) with RNA is enhanced by SINV 12

infection (Table S1). This protein has been identified as an RBP in numerous RNA-IC 13

studies (Hentze et al., 2018) and its RBD has been located in a discrete region at its C-14

terminal domain (Figure S6B) (Castello et al., 2016). Chaperones from the HSP90 family 15

play important roles in the remodelling of RNPs and have been linked to virus infection 16

(Geller et al., 2012; Iwasaki et al., 2010). To address whether HSP90AB1 is involved in SINV 17

infection, we generated a knockout cell line. Notably, the kinetics of SINV-mCherry infection 18

was substantially delayed in cells lacking HSP90AB1 (Figure 6C, upper panel), even though 19

homologues of this protein exist (i.e. HSP90AA1, HSP90AA2, HSP90B1 and TRAP1). The 20

ability of HSP90AB1 to promote SINV infection was confirmed using the specific inhibitors 21

ganetespib and geldanamycin. Treatment with non-toxic concentrations of these 22

compounds abrogated SINV-mCherry infection (Figure 6C, middle panels, and S6A). 23

However, HSP90AB1 overexpression had no effect in SINV-mCherry fitness (Figure 6C, 24

bottom panel). HSP90AB1 is abundant in the cell and thus overexpression may have limited 25

effects. The implication of PPIA and HSP90 in the biological cycle of a variety of unrelated 26



https://doi.org/10.1101/350686


17

viruses highlights these proteins as master regulators of infection, representing potential 1

targets for broad-spectrum antivirals (Rupp and Bartenschlager, 2014; Wang et al., 2017). 2

Proliferation-associated protein 2G4 (PA2G4) RNA-binding activity was also enhanced by 3

SINV (Table S1). This protein associates with ribosomes (Table S2) (Simsek et al., 2017) 4

and regulates the cap-independent translation of footh-and-mouth disease virus (FMDV) 5

RNA (Monie et al., 2007). Treatment with the PA2G4-specific inhibitor WS6 hampered SINV-6

mCherry fitness (Figure 6D, upper panel), suggesting that this protein promotes SINV 7

infection. Overexpression did not cause any effect as with previous examples (Figure 6D, 8

bottom panel). The possibility that PA2G4 contributes to the non-canonical, cap-dependent 9

translation of SINV RNAs should be further investigated. 10

SRSF Protein Kinase 1 (SRPK1) phosphorylates the RS repeats present in SR proteins, 11

which are involved in alternative splicing regulation, RNA export and stability (Howard and 12

Sanford, 2015). SINV infection stimulates the RNA-binding activity of SRPK1 (Table S1). 13

We hypothesise that the recruitment of this protein to the viral replication factories (Figure 14

4C) might i) act as a sponging mechanism, preventing it from acting elsewhere or ii) promote 15

phosphorylation of proteins in the context of the viral replication factories. An earlier study 16

suggested that inhibition of SRPK1 hampers SINV and HIV infection (Fukuhara et al., 2006). 17

To complement these experiments and distinguish between the proposed mechanisms of 18

action, we overexpressed SRPK1-eGFP reasoning that by increasing the levels of this 19

protein we would compromise the ‘sponge’ mechanism. Notably, SRPK1-eGFP 20

overexpression enhanced SINV fitness (Figure 6E), suggesting that the ‘sponge’ 21

mechanism is improbable and that it is more plausible that SRPK1 is actively contributing to 22

viral replication in the viral factories. Future work should determine the molecular targets of 23

SRPK1 in the viral replication factories, and whether its kinase activity also contributes to 24

the differential RNA-binding activity of dynamic RBPs. 25



https://doi.org/10.1101/350686


18

We tested the effects of the overexpression of nine additional responsive or inhibited RBPs 1

fused to eGFP (Figure S6C and D). Phenotypes in viral fitness ranged from non-existent 2

(ALDOA, XRCC6, RPS10, MOV10 and NGDN), to mild (RPS27, NONO and DKC1). The 3

lack of phenotypic effects in overexpression experiments does not discard that the protein 4

actually participates in SINV infection, as shown before with HSP90AB1, PPIA and PA2G4. 5

Nevertheless, RBPs for which overexpression caused mild effects in infection have potential 6

for future in detail analyses. 7

The family of tripartite motif-containing (TRIM) proteins comprises more than 75 members 8

endowed with E3 ubiquitin ligase activity that are known to modulate the host antiviral 9

response (Versteeg et al., 2013). RNA-IC studies have discovered RNA-binding activities in 10

few TRIM proteins, including TRIM25, TRIM56, TRIM28 and TRIM71 (Hentze et al., 2018). 11

Notably, SINV infection enhanced TRIM25 interaction with RNA (Table S1), correlating with 12

its relocalisation to the viral replication factories (Figure 4C). TRIM25 was proposed to 13

interact with dengue virus RNA (Manokaran et al., 2015); however, this analysis employed 14

native immunoprecipitation (IP) that cannot distinguish between direct and indirect protein-15

RNA interactions (e.g. through protein-protein interactions). To prove experimentally that 16

TRIM25 interacts directly with SINV RNA, cells expressing TRIM25-eGFP were infected with 17

SINV and irradiated with UV light to induce protein-RNA crosslinks. TRIM25-eGFP was IP 18

under very stringent conditions (500 mM NaCl and 0.1% SDS) and co-isolated RNA was 19

analysed by RT-PCR using specific primers against SINV RNA. A band matching the 20

expected PCR product was amplified from TRIM25-eGFP IPs only in infected cells (Figure 21

7A), confirming its interaction with viral RNA. No PCR product was observed in the negative 22

controls, i.e. unfused eGFP or XRCC6-eGFP. TRIM25 RNA-binding activity is mediated by 23

a short peptide present in its PRY domain and interaction with RNA enhances its E3 ubiquitin 24

ligase activity (Choudhury et al., 2017). Thus, the enhanced RNA-binding activity of TRIM25 25

in SINV-infected cells is expected to increase its E3 ubiquitin ligase activity, which is known 26



https://doi.org/10.1101/350686


19

to stimulate key effectors of the antiviral response such as RIGI and ZC3HAV1 (Gack et al., 1

2007; Li et al., 2017; Zheng et al., 2017). Hence, RBP ubiquitination may also affect the 2

RNA-binding activity of dynamic RBPs in the context of the viral factory. Another SINV-3

stimulated RBP is TRIM56, which is known to bind double stranded DNA. However, it 4

enhances the antiviral response not only in cells infected with DNA viruses but also with 5

RNA viruses (Seo et al., 2018; Shen et al., 2012; Tsuchida et al., 2010; Versteeg et al., 6

2013). RNA-IC thus complements these results revealing that TRIM56 interacts directly with 7

RNA in SINV-infected cells (Table S1). Moreover, TRIM56 also re-localises to the viral 8

replication factories (Figure 4C). Strikingly, overexpression of both TRIM25-eGFP and 9

TRIM56-eGFP strongly reduced SINV fitness (Figure 7B), confirming that these RBPs are 10

part of the cell’s molecular arsenal to restrict virus infection. 160 out of the 245 SINV-11

responsive RBPs lack previous connections to virus infection, based on our recent analysis 12

of protein annotation and automated literature mining (Garcia-Moreno et al., 2018). Hence, 13

our dataset likely contains numerous pro- and antiviral RBPs to be discovered. 14

GEMIN5 binds to key regulatory sequences in viral RNAs and regulates SINV protein 15

expression 16

GEMIN5 is a member of the survival motor neuron (SMN) complex, which mediates the 17

assembly of the small nuclear (sn)RNPs (Gubitz et al., 2002). The RNA-binding activity of 18

this protein is strongly stimulated by SINV infection and it redistributed to the viral replication 19

factories (Figure 2E and 4C). To our surprise, neither the other GEMIN proteins nor the SMN 20

proteins, known molecular partners of GEMIN5, were stimulated by SINV (Table S1), 21

suggesting a GEMIN5-specific response. This agrees with the earlier discovery of a free 22

pool of GEMIN5 (Battle et al., 2007). Further work reported that GEMIN5 is a global regulator 23

of protein synthesis and is cleaved upon FMDV infection (Francisco-Velilla et al., 2016; 24

Pacheco et al., 2009; Pineiro et al., 2015). Overexpression of GEMIN5-eGFP caused a slight 25

but highly reproducible delay of mCherry production and strongly inhibited capsid synthesis 26



https://doi.org/10.1101/350686


20

(Figure 7B). GEMIN5 impacts protein synthesis at the translation elongation step through its 1

direct interaction with the 60S ribosomal subunit and, in particular, with RPL3 and RPL4 2

(Francisco-Velilla et al., 2016). In agreement, GEMIN5 co-precipitates with purified 3

ribosomes (Table S2) (Simsek et al., 2017). Protein-protein interaction analysis of GEMIN5-4

eGFP followed by proteomics confirmed that, in our experimental settings, it interacts with 5

RPL3 and RPL4 as well as with other components of the ribosome, especially from the 60S 6

subunit (Figure 7C, pink dots in left panel, S7A-B and Table S5). Moreover, the interaction 7

with the ribosomes is maintained in SINV-infected cells (Figure 7C, pink dots in middle and 8

right panels). We noticed that GEMIN5 is by far the most enriched protein in the IPs and that 9

its iBAQ score is significantly higher than that of eGFP, suggesting that GEMIN5-eGFP 10

interacts with the endogenous GEMIN5 likely forming oligomers through its C-terminus 11

coiled-coil structure, as previously proposed (Xu et al., 2016). Moreover, our data showed 12

that GEMIN5 may interact with various viral proteins, chiefly with NSP1, NSP2, NSP3 and 13

SINV C (Figure 7C, middle panel). The biological implications of these interactions in the 14

modulation of the RNA-binding activity of GEMIN5 deserve future considerations. 15

To test whether GEMIN5 binds SINV RNA directly, we performed IP and RT-PCR analysis 16

as outlined above. A PCR product of the expected size was amplified in GEMIN5-eGFP 17

eluates, while the negative controls were remarkably clean (Figure 7A). To determine the 18

exact positions within the viral RNA bound by GEMIN5, we employed single-nucleotide 19

resolution crosslinking and immunoprecipitation (iCLIP) followed by sequencing (König et 20

al., 2010). The binding peaks with highest coverage mapped to the 5’ region of the gRNA 21

and sgRNA as well as to the 3’ untranslated region (UTR), which is shared by the two viral 22

RNAs (Figure 7D). Indeed, reads mapping to the beginning of the gRNA and sgRNA often 23

presented an additional guanosine at the 5’ end, likely reflecting binding to the cap structure. 24

This agrees with previous data reporting that GEMIN5 is a cap-binding protein (Bradrick and 25

Gromeier, 2009; Xu et al., 2016). Additional peaks overlap with the downstream loop (DLP), 26



https://doi.org/10.1101/350686


21

which is a hairpin structure that stimulates the translation of the sgRNA (Frolov and 1

Schlesinger, 1996). Interaction with the cap, 5’ UTR, 3’ UTR and DLP of viral RNAs aligns 2

well with the proposed role as translational regulator and the observed inhibition of C 3

expression. Strikingly, another GEMIN5 binding peak overlapped with the package signal 4

recognised by SINV C, suggesting a potential interference with morphogenesis of viral 5

particles. Our data strongly supports the model in which GEMIN5 recognises different 6

regulatory regions of the viral RNA and prevents translation by interfering with ribosomal 7

function. 8

Taken together, these results highlight the capacity of RNA-IC at identifying RBPs 9

participating in SINV infection either as pro- or anti-viral factors. 10

11

OUTLOOK 12

RNA-IC, and methods built on it, have been used in the past to compile the repertoire of 13

cellular RBPs and their RBDs (Hentze et al., 2018). Nevertheless, we still poorly understand 14

whether the RBPome globally responds to physiological cues and if so, how. Here, we show 15

that the RBPome is highly dynamic and has the potential to adapt to the cellular state, in 16

this case, infection with an RNA virus. SINV infection induced responses in a quarter of the 17

identified RBPome, including both well-established and unconventional RBPs newly 18

discovered by RNA-IC, evoking still unknown biological roles in infected cells. RBP 19

dynamics highlight the post-transcriptional circuits modulated by the infection, which include 20

the activation of the protein synthesis apparatus, the 5’ to 3’ RNA degradation machinery 21

and a defined scope of antiviral factors as well as the inhibition of several RBPs involved in 22

RNA processing and export. The differential RNA-binding activity of near ninety ribosomal 23

and ribosome-associated proteins (Table S2) (Simsek et al., 2017), aligns well with the 24

proposed, although not yet demonstrated, existence of specialised ribosomes in infected 25

cells (Au and Jan, 2014). RNA helicases also emerge as responsive nodes in RBP networks. 26



https://doi.org/10.1101/350686


22

Their RNP remodelling activity are linked to central regulatory roles in RNA metabolism and 1

their ability to respond to biological cues likely contributes to their regulatory function. Both 2

the existence of a specialised translation machinery and the role of dynamic RNA helicases 3

in the post-transcriptional regulation of gene expression deserve further investigation. 4

Mechanistically, RBPome response to SINV infection is mostly driven by both deep 5

alterations of the transcriptome and a global subcellular redistribution of relevant RBPs. In 6

particular, a large proportion of stimulated RBPs are relocalised to the viral replication 7

factories. Apart from these major alterations, RBP activity can be modulated in individual 8

basis by more subtle mechanisms. For example, it is known that virus infection triggers 9

signalling pathways involving kinases and E3 ubiquitin ligases (Figure 1D) (Gack et al., 10

2007; Greenwood et al., 2016; Li et al., 2017; Ventoso et al., 2006). This idea is strengthen 11

here by the increased RNA-binding activity of the kinase SRPK1 and the E3 ubiquitin ligases 12

TRIM25 and TRIM56. Hence, it is plausible that post-translational modifications also 13

contribute to RBP regulation in SINV-infected cells. Furthermore, the interaction of GEMIN5 14

with viral proteins suggests that differential complex assembly might also regulate RBP 15

activity (Figure 7C). 16

Importantly, RBP responses are biologically important as perturbation of dynamic RBPs 17

strongly affect either the viral fitness or the capacity of the cell to counteract the infection. 18

Therefore, every protein reported here to respond to SINV infection may offer new avenues 19

of research due to their potential as anti- or pro-viral factors. We believe that these regulatory 20

RBPs will represent excellent targets for novel therapeutic approaches. Moreover, viruses 21

hijack master regulators of cellular pathways (Berget et al., 1977; Castello et al., 2011; 22

Castello et al., 2012; Chow et al., 1977; Krausslich et al., 1987; Lloyd, 2015; Sun and 23

Baltimore, 1989; von Kobbe et al., 2000), suggesting that responsive RBPs are likely 24

involved in regulatory steps of RNA metabolism. 25



https://doi.org/10.1101/350686


23

Some of the outstanding questions derived from this work include why the lack of the 1

exonuclease XRN1 makes the cells refractory to SINV, what triggers the degradation of host 2

RNA and why the transcripts induced by the antiviral response are resistant to degradation. 3

Moreover, GEMIN5 emerges as a highly responsive RBP that impairs SINV infection. The 4

extra-SMN complex activity of GEMIN5 in translational control has recently been reported 5

(Francisco-Velilla et al., 2016; Pineiro et al., 2015). We show here that this newly discovered 6

activity can be regulated, as GEMIN5 display altered RNA-binding activity and localisation 7

in infected cells as it engages in interactions with viral proteins and RNAs. The exact 8

mechanisms underpinning GEMIN5 effects in translation requires further investigation. 9

Finally, ‘comparative RNA-IC’ has been, here, applied to cells infected with SINV. However, 10

it can now be extended to other viruses or physiological cues to improve our understanding 11

on RBP dynamics and its biological importance. 12

13

AUTHOR CONTRIBUTIONS 14

Conceptualization, M.N., L.C., B.F., S.M., A.C.; Methodology, M.G.M, M.N., N.S., A.I.J, 15

E.G.A, C.L., B.F., S.M., A.C.; Investigation, M.G.M., M.N., N.S., A.I.J., E.G.A, C.L., M.B.P, 16

V.C., R.A., T.D., S.H., T.J.M.S., B.L., M.A.S., E.P.R., V.P., B.F., S.M., A.C.; Writing, original 17

draft, M.G.M., M.N., A.I.J, A.C.; Writing, editing, M.G.M., M.N., N.S. A.I.J, C.L., S.M., A.C.; 18

Funding acquisition, M.G.M, A.C.; Resources, L.C., E.P.R., V.P., B.F., S.M., A.C.; 19

Supervision, M.G.M, M.N., A.I.J., B.F., S.M., A.C. 20

21

ACKNOWLEDGEMENTS 22

We dedicate this work to our colleagues and friends Bernd Fischer and Katrin Eichelbaum, 23

who sadly passed away during the development of this work. We thank Matthias W Hentze, 24

Encarnacion Martinez-Salas, Gracjan Michlewski, Javier Martinez, Kui Li, Ilan Davis, 25

Richard Parton and Jan Rehwinkel for reagents and advice. The support from Oxford Micron 26



https://doi.org/10.1101/350686


24

Advanced Bioimaging Unit in microscopy experiments is also acknowledged. A.C. is funded 1

by MRC Career Development Award #MR/L019434/1 and the John Fell Funds from the 2

University of Oxford. M.G.M is funded by the European Union’s Horizon 2020 research and 3

innovation programme under the Marie-Sklodowska-Curie grant agreement No. 700184. 4

V.P is funded by a SciLifeLab Fellowship, the Swedish Research Council (VR 2016-01842), 5

a Wallenberg Academy Fellowship (KAW 2016.0123) and the Ragnar Söderberg 6

Foundation. L.C. is funded by grant DGICYT SAF2015-66170-R (MINECO/FEDER). E.G.A. 7

was awarded with a short-term EMBO fellowship (ASTF 358-2015) and the FPU Fellowship 8

FPU15/05709. 9

10

FIGURE LEGENDS 11

Figure 1. Implementation of RNA-IC to HEK293 cells infected with SINV. A) Schematic 12

representation of RNA-IC combined with SILAC and virus infection. Several modifications 13

were implemented into the original protocol (Castello et al., 2012; Castello et al., 2013), 14

including the use of isotopic labelled amino acids and the combination of the samples after 15

lysis and prior to the oligo(dT) capture. B) Schematic representation of SINV (wild type) and 16

chimeric SINV-mCherry genomes. Subgenomic (sg)RNA is synthesised from the 17

‘subgenomic promoter’. C) Analysis of the proteins synthesised in uninfected and SINV-18

infected HEK293 cells by [35S]-Met/Cys labelling. D) Analysis of total and phosphorylated 19

eIF2α in cells from panel (C) by western blotting. E) Silver staining analysis of the ‘inputs’ 20

(i.e. total proteome, left) and eluates (i.e. RNA-bound proteome, right) of a representative 21

RNA-IC experiment in SINV-infected HEK293 cells. F) RT-qPCR analysis of the eluates of 22

RNA-IC experiments using specific primers against SINV RNAs, gapdh and actb mRNAs. 23

Error bars represent standard deviation of three biological replicates. hpi, hours post 24

infection. MW, molecular weight. 25



https://doi.org/10.1101/350686


25

Figure 2. Analysis of the RNA-bound proteome in SINV-infected HEK293 cells by RNA-1

IC. A) Scatter plot compiling the intensity ratio between 4 hours post infection (hpi) and 2

uninfected conditions of each protein (dots) in the eluates of two biological replicates of 3

RNA-IC. B) Volcano plot comparing the log2 fold change of each protein between 4 hpi and 4

uninfected conditions and the p-value of this change across the three biological replicates. 5

C) As in (A) but comparing 18 hpi and uninfected cells. D) As in (B) but comparing 18 hpi 6

and uninfected cells. E) Western blotting analysis with specific antibodies of the eluates of 7

a representative RNA-IC experiment in SINV-infected HEK293 cells. F) Molecular function 8

(upper panel) and cellular component (bottom panel) gene ontology (GO) term enrichment 9

analysis of the RBPs stimulated (salmon) against those inhibited (blue) by SINV at 18 hpi. 10

G) Representative scatter plot comparing the raw intensity of each protein in the eluates of 11

two RNA-IC replicates from cells infected for 18 h. FDR, false discovery rate; n.s. non-12

significant. 13

Figure 3. Proteomic and transcriptomic analyses of whole SINV-infected cell lysates. 14

A) Scatter plot compiling the intensity ratio between 4 hpi and uninfected conditions of each 15

protein (dots) in the inputs (total proteome) of two biological replicates of RNA-IC (scatter 16

plots of the three biological replicates in Figure S3). Black dots represent proteins 17

significantly enriched in either 4 hpi or uninfected conditions in the RNA-IC experiment 18

(Figure 2 and Table S1). B) As in (A) but between 18 hpi and uninfected conditions. C) 19

Scatter plot comparing the intensity of each protein in the inputs (total proteome) of two 20

RNA-IC replicates from cells infected for 18 h (scatter plots of the three biological replicates 21

in Figure S3). D) Representative Coomassie blue staining of the whole cell lysate of cells 22

infected with SINV for 2, 4, 8, 18 or 24 h. E) Western blotting analysis with specific antibodies 23

of the whole cell lysates (inputs) from SINV-infected HEK293 cells. F) MA plot comparing 24

the read coverage and the log2 fold change between 4 hpi and uninfected cells of each 25

transcript detected in the RNAseq experiment. Red dots represent RNAs enriched in one of 26



https://doi.org/10.1101/350686


26

the two conditions with p<0.1. G) As in (F) but between 18 hpi and uninfected cells. H) 1

Correlation of the RNAseq and RT-qPCR data by plotting the log2 fold change for randomly 2

selected transcripts by the two methods. Error bars represent standard error of three 3

independent experiments. 4

Figure 4. Host RBP localisation in SINV infected cells. A) Read coverage of the positive 5

and negative RNA strand of SINV in the RNAseq analysis at 4 and 18 hpi. Note that the Y 6

axis in both plots have different scales to facilitate the visualisation of the data. B) 7

Localisation analysis of SINV RNA and C protein in infected HeLa cells at 18 hpi by 8

combined in situ hybridisation and immunofluorescence. C) Localisation by 9

immunofluorescence of the eGFP-fused RBPs and SINV C in HeLa cells uninfected or 10

infected with SINV for 18 hpi. D) Summary of the observed localisation of the 26 proteins 11

tested in panel C and Figure S4B. Scale-bar represents 10 µm. 12

Figure 5. The exonuclease XRN1 in cells infected with SINV. A) Analysis of the 13

contribution of transcription, processing and degradation to the transcriptome of cells 14

infected with SINV. The RNA expression data (Figure 3F-G and Table S4) was compared 15

with the speed of RNA synthesis, processing and degradation independently measured for 16

each transcript in (Mukherjee et al., 2017). ANOVA was used to estimate the percentage of 17

variance explained by each RNA biological process (i.e. transcription, processing and 18

degradation) in the different conditions. B) Immunolocalisation of XRN1 and SINV C using 19

specific antibodies. ROI, region of interest. C) Upper panel shows the fluorescence derived 20

from mCherry in XRN1 knock out and control HEK293 cells infected with SINV-mCherry. 21

Red fluorescence was measured every 15 min in a plate reader with atmospheric control 22

(5% CO2 and 37°C). RFU, relative fluorescence units. Bottom panel shows the expression 23

of SINV C in XRN1 knock out and wild type HEK293 cells analysed by western blotting. 24



https://doi.org/10.1101/350686


27

Figure 6. Impact of stimulated-RBPs in SINV infection. A) Expression of mCherry in 1

HEK293 cells infected with SINV-mCherry and treated or not with the IRE1α inhibitor, 4µ8C. 2

Red fluorescence was measured as in Figure 5C. B) As in (A) but with PPIA knock out cells 3

(first panel), the PPIA inhibitor cyclosporine A (CysA) (second panel) and cells 4

overexpressing PPIA-eGFP (third panel). Knock out and overexpression of PPIA was 5

assessed by Western blotting with specific antibodies. Bottom panel shows SINV C 6

accumulation in cells overexpressing PPIA and infected with either SINV or SINV-mCherry, 7

analysed by Western blotting. C) mCherry expression in HSP90AB1 knock out cells (first 8

panel), in cells treated with inhibitors of HSP90 (ganetespib and geldamycin, second and 9

third panels) or in cells overexpressing HSP90AB1-eGFP (fourth panel) and infected with 10

SINV-mCherry. Red fluorescence was measured as in Figure 5C. Knock out and 11

overexpression of HSP90AB1 was assessed by Western blotting with specific antibodies. 12

Bottom panel shows SINV C accumulation in cells overexpressing HSP90AB1 and infected 13

with either SINV or SINV-mCherry, analysed by Western blotting. D) As in (A) but the PA2G4 14

inhibitor WS6 (first panel) and cells overexpressing PA2G4-eGFP (second panel). Third 15

panel shows SINV C accumulation in cells overexpressing PA2G4 and infected with either 16

SINV or SINV-mcherry, analysed by Western blotting. E) As in (A) but with cells 17

overexpressing SRPK1 (first panel). Overexpression of SRPK1 was assessed by Western 18

blotting with specific antibodies. Second panel shows SINV C accumulation in cells 19

overexpressing SRPK1 and infected with either SINV or SINV-mcherry for 8 and 18 h, 20

analysed by Western blotting. Dox, doxycycline, which induces the (over)expression of the 21

RBP-eGFP fusion protein in HEK293 Flp-In TREx cells. SINV-mChe, SINV-mCherry. 22

Figure 7. Effects of RBPs with antiviral potential in SINV infection. A) UV crosslinking 23

and immunoprecipitation of TRIM25-eGFP, GEMIN5-eGFP, XRCC6-eGFP or unfused 24

eGFP in cells infected or not with SINV. The presence SINV RNA in eluates and inputs was 25

detected by RT-PCR using specific primers against SINV RNAs (overlapping sequence). B) 26



https://doi.org/10.1101/350686


28

Relative mCherry fluorescence produced in cells overexpressing TRIM25-eGFP (left upper 1

panel), TRIM56-eGFP (middle upper panel), GEMIN5-eGFP (upper right panel) and infected 2

with SINV-mCherry. Red fluorescence was measured as in Figure 5C. Overexpression of 3

these proteins was assessed by Western blotting with specific antibodies. Bottom panel 4

shows SINV C accumulation in cells overexpressing these proteins and infected with either 5

SINV or SINV-mcherry. SINV C was analysed by Western blotting using β-actin (ACTB) as 6

loading control. C) Volcano plots comparing the intensity of proteins in GEMIN5-eGFP vs 7

unfused eGFP IP in uninfected (left panel) and infected cells (middle panel); every dot 8

represents a protein. Dark green dots are proteins enriched in either GEMIN5-eGFP or 9

unfused eGFP with p<0.01; blue dots are those enriched with p<0.1 and grey dots represent 10

non-enriched proteins. Pink dots represent ribosomal proteins. Right panel shows a volcano 11

plot comparing the intensity of proteins in GEMIN5 IPs in infected vs uninfected cells. D) 12

iCLIP analysis of GEMIN5 binding sites on SINV RNA. The upper panel shows the reads 13

mapping to SINV positive-stranded RNA. Discontinuous boxes below the plot delimit 14

relevant regulatory regions within the SINV RNA. Black boxes demark the position of gRNA 15

and sgRNA, grey boxes demark the position of viral proteins and coloured boxes delimit the 16

position of relevant regulatory regions. The reads mapping to the 5’ and 3’ regions of the 17

gRNA and sgRNA (including the downstream loop or DLP) as well as the C-binding site 18

(packaging signal) are zoomed in in bottom panels. 19

20

REFERENCES 21

Akhrymuk, I., Kulemzin, S.V., and Frolova, E.I. (2012). Evasion of the innate immune 22

response: the Old World alphavirus nsP2 protein induces rapid degradation of Rpb1, a 23

catalytic subunit of RNA polymerase II. J Virol 86, 7180-7191. 24

Au, H.H., and Jan, E. (2014). Novel viral translation strategies. Wiley Interdiscip Rev RNA 25

5, 779-801. 26



https://doi.org/10.1101/350686


29

Balistreri, G., Horvath, P., Schweingruber, C., Zund, D., McInerney, G., Merits, A., 1

Muhlemann, O., Azzalin, C., and Helenius, A. (2014). The host nonsense-mediated mRNA 2

decay pathway restricts Mammalian RNA virus replication. Cell Host Microbe 16, 403-411. 3

Baltz, A.G., Munschauer, M., Schwanhausser, B., Vasile, A., Murakawa, Y., Schueler, M., 4

Youngs, N., Penfold-Brown, D., Drew, K., Milek, M., et al. (2012). The mRNA-Bound 5

Proteome and Its Global Occupancy Profile on Protein-Coding Transcripts. Mol Cell 46, 674-6

690. 7

Barbalat, R., Ewald, S.E., Mouchess, M.L., and Barton, G.M. (2011). Nucleic acid 8

recognition by the innate immune system. Annu Rev Immunol 29, 185-214. 9

Battle, D.J., Kasim, M., Wang, J., and Dreyfuss, G. (2007). SMN-independent subunits of 10

the SMN complex. Identification of a small nuclear ribonucleoprotein assembly intermediate. 11

J Biol Chem 282, 27953-27959. 12

Berget, S.M., Moore, C., and Sharp, P.A. (1977). Spliced segments at the 5' terminus of 13

adenovirus 2 late mRNA. Proc Natl Acad Sci U S A 74, 3171-3175. 14

Bradrick, S.S., and Gromeier, M. (2009). Identification of gemin5 as a novel 7-15

methylguanosine cap-binding protein. PLoS One 4, e7030. 16

Brannan, K.W., Jin, W., Huelga, S.C., Banks, C.A., Gilmore, J.M., Florens, L., Washburn, 17

M.P., Van Nostrand, E.L., Pratt, G.A., Schwinn, M.K., et al. (2016). SONAR Discovers RNA-18

Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes. Mol Cell 64, 19

282-293. 20

Carrasco, L., Sanz, M.A., and Gonzalez-Almela, E. (2018). The Regulation of Translation in 21

Alphavirus-Infected Cells. Viruses 10. 22

Castello, A., Alvarez, E., and Carrasco, L. (2011). The multifaceted poliovirus 2A protease: 23

regulation of gene expression by picornavirus proteases. J Biomed Biotechnol 2011, 24

369648. 25



https://doi.org/10.1101/350686


30

Castello, A., Fischer, B., Eichelbaum, K., Horos, R., Beckmann, B.M., Strein, C., Davey, 1

N.E., Humphreys, D.T., Preiss, T., Steinmetz, L.M., et al. (2012). Insights into RNA Biology 2

from an Atlas of Mammalian mRNA-Binding Proteins. Cell 149, 1393-1406. 3

Castello, A., Fischer, B., Frese, C.K., Horos, R., Alleaume, A.M., Foehr, S., Curk, T., 4

Krijgsveld, J., and Hentze, M.W. (2016). Comprehensive Identification of RNA-Binding 5

Domains in Human Cells. Mol Cell 63, 696-710. 6

Castello, A., Horos, R., Strein, C., Fischer, B., Eichelbaum, K., Steinmetz, L.M., Krijgsveld, 7

J., and Hentze, M.W. (2013). System-wide identification of RNA-binding proteins by 8

interactome capture. Nat Protoc 8, 491-500. 9

Castello, A., Sanz, M.A., Molina, S., and Carrasco, L. (2006). Translation of Sindbis virus 10

26S mRNA does not require intact eukariotic initiation factor 4G. J Mol Biol 355, 942-956. 11

Chang, C.H., Curtis, J.D., Maggi, L.B., Jr., Faubert, B., Villarino, A.V., O'Sullivan, D., Huang, 12

S.C., van der Windt, G.J., Blagih, J., Qiu, J., et al. (2013). Posttranscriptional control of T 13

cell effector function by aerobic glycolysis. Cell 153, 1239-1251. 14

Chen, C.Y., and Shyu, A.B. (2014). Emerging mechanisms of mRNP remodeling regulation. 15

Wiley Interdiscip Rev RNA 5, 713-722. 16

Choudhury, N.R., Heikel, G., Trubitsyna, M., Kubik, P., Nowak, J.S., Webb, S., Granneman, 17

S., Spanos, C., Rappsilber, J., Castello, A., et al. (2017). RNA-binding activity of TRIM25 is 18

mediated by its PRY/SPRY domain and is required for ubiquitination. BMC Biol 15, 105. 19

Chow, L.T., Gelinas, R.E., Broker, T.R., and Roberts, R.J. (1977). An amazing sequence 20

arrangement at the 5' ends of adenovirus 2 messenger RNA. Cell 12, 1-8. 21

Francisco-Velilla, R., Fernandez-Chamorro, J., Ramajo, J., and Martinez-Salas, E. (2016). 22

The RNA-binding protein Gemin5 binds directly to the ribosome and regulates global 23

translation. Nucleic Acids Res 44, 8335-8351. 24



https://doi.org/10.1101/350686


31

Frolov, I., and Schlesinger, S. (1996). Translation of Sindbis virus mRNA: analysis of 1

sequences downstream of the initiating AUG codon that enhance translation. J Virol 70, 2

1182-1190. 3

Fukuhara, T., Hosoya, T., Shimizu, S., Sumi, K., Oshiro, T., Yoshinaka, Y., Suzuki, M., 4

Yamamoto, N., Herzenberg, L.A., Herzenberg, L.A., et al. (2006). Utilization of host SR 5

protein kinases and RNA-splicing machinery during viral replication. Proc Natl Acad Sci U S 6

A 103, 11329-11333. 7

Gack, M.U., Shin, Y.C., Joo, C.H., Urano, T., Liang, C., Sun, L., Takeuchi, O., Akira, S., 8

Chen, Z., Inoue, S., et al. (2007). TRIM25 RING-finger E3 ubiquitin ligase is essential for 9

RIG-I-mediated antiviral activity. Nature 446, 916-920. 10

Garcia-Moreno, M., Jaervelin, A.I., and Castello, A. (2018). Unconventional RNA-binding 11

proteins step into the virus-host battlefront. WIREs RNA In press. 12

Garcia-Moreno, M., Sanz, M.A., and Carrasco, L. (2015). Initiation codon selection is 13

accomplished by a scanning mechanism without crucial initiation factors in Sindbis virus 14

subgenomic mRNA. RNA 21, 93-112. 15

Garcia-Moreno, M., Sanz, M.A., Pelletier, J., and Carrasco, L. (2013). Requirements for 16

eIF4A and eIF2 during translation of Sindbis virus subgenomic mRNA in vertebrate and 17

invertebrate host cells. Cell Microbiol 15, 823-840. 18

Garcia, M.A., Gil, J., Ventoso, I., Guerra, S., Domingo, E., Rivas, C., and Esteban, M. (2006). 19

Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action. 20

Microbiol Mol Biol Rev 70, 1032-1060. 21

Geller, R., Taguwa, S., and Frydman, J. (2012). Broad action of Hsp90 as a host chaperone 22

required for viral replication. Bba-Mol Cell Res 1823, 698-706. 23

Gerstberger, S., Hafner, M., and Tuschl, T. (2014). A census of human RNA-binding 24

proteins. Nat Rev Genet 15, 829-845. 25



https://doi.org/10.1101/350686


32

Glisovic, T., Bachorik, J.L., Yong, J., and Dreyfuss, G. (2008). RNA-binding proteins and 1

post-transcriptional gene regulation. FEBS Lett 582, 1977-1986. 2

Goodier, J.L., Cheung, L.E., and Kazazian, H.H., Jr. (2012). MOV10 RNA helicase is a 3

potent inhibitor of retrotransposition in cells. PLoS Genet 8, e1002941. 4

Gorchakov, R., Frolova, E., and Frolov, I. (2005). Inhibition of transcription and translation 5

in Sindbis virus-infected cells. J Virol 79, 9397-9409. 6

Greenwood, E.J., Matheson, N.J., Wals, K., van den Boomen, D.J., Antrobus, R., 7

Williamson, J.C., and Lehner, P.J. (2016). Temporal proteomic analysis of HIV infection 8

reveals remodelling of the host phosphoproteome by lentiviral Vif variants. eLife 5. 9

Gubitz, A.K., Mourelatos, Z., Abel, L., Rappsilber, J., Mann, M., and Dreyfuss, G. (2002). 10

Gemin5, a novel WD repeat protein component of the SMN complex that binds Sm proteins. 11

J Biol Chem 277, 5631-5636. 12

He, C., Sidoli, S., Warneford-Thomson, R., Tatomer, D.C., Wilusz, J.E., Garcia, B.A., and 13

Bonasio, R. (2016). High-Resolution Mapping of RNA-Binding Regions in the Nuclear 14

Proteome of Embryonic Stem Cells. Mol Cell 64, 416-430. 15

Hentze, M.W., and Argos, P. (1991). Homology between IRE-BP, a regulatory RNA-binding 16

protein, aconitase, and isopropylmalate isomerase. Nucleic Acids Res 19, 1739-1740. 17

Hentze, M.W., Castello, A., Schwarzl, T., and Preiss, T. (2018). A brave new world of RNA-18

binding proteins. Nat Rev Mol Cell Biol. 19

Howard, J.M., and Sanford, J.R. (2015). The RNAissance family: SR proteins as 20

multifaceted regulators of gene expression. Wiley Interdiscip Rev RNA 6, 93-110. 21

Iwasaki, S., Kobayashi, M., Yoda, M., Sakaguchi, Y., Katsuma, S., Suzuki, T., and Tomari, 22

Y. (2010). Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of 23

small RNA duplexes. Mol Cell 39, 292-299. 24

Jurkin, J., Henkel, T., Nielsen, A.F., Minnich, M., Popow, J., Kaufmann, T., Heindl, K., 25

Hoffmann, T., Busslinger, M., and Martinez, J. (2014). The mammalian tRNA ligase complex 26



https://doi.org/10.1101/350686


33

mediates splicing of XBP1 mRNA and controls antibody secretion in plasma cells. EMBO J 1

33, 2922-2936. 2

König, J., Zarnack, K., Rot, G., Curk, T., Kayikci, M., Zupan, B., Turner, D.J., Luscombe, 3

N.M., and Ule, J. (2010). iCLIP reveals the function of hnRNP particles in splicing at 4

individual nucleotide resolution. Nat Struct Mol Biol 17, 909-915. 5

Kramer, K., Sachsenberg, T., Beckmann, B.M., Qamar, S., Boon, K.L., Hentze, M.W., 6

Kohlbacher, O., and Urlaub, H. (2014). Photo-cross-linking and high-resolution mass 7

spectrometry for assignment of RNA-binding sites in RNA-binding proteins. Nat Methods 11, 8

1064-1070. 9

Krausslich, H.G., Nicklin, M.J., Toyoda, H., Etchison, D., and Wimmer, E. (1987). Poliovirus 10

proteinase 2A induces cleavage of eucaryotic initiation factor 4F polypeptide p220. J Virol 11

61, 2711-2718. 12

LaPointe, A.T., Gebhart, N.N., Meller, M.E., Hardy, R.W., and Sokoloski, K.J. (2018). The 13

Identification and Characterization of Sindbis Virus RNA:Host Protein Interactions. J Virol. 14

Lee, A.S., Kranzusch, P.J., Doudna, J.A., and Cate, J.H. (2016). eIF3d is an mRNA cap-15

binding protein that is required for specialized translation initiation. Nature 536, 96-99. 16

Li, J., Tang, S., Hewlett, I., and Yang, M. (2007). HIV-1 capsid protein and cyclophilin a as 17

new targets for anti-AIDS therapeutic agents. Infect Disord Drug Targets 7, 238-244. 18

Li, M.M., Lau, Z., Cheung, P., Aguilar, E.G., Schneider, W.M., Bozzacco, L., Molina, H., 19

Buehler, E., Takaoka, A., Rice, C.M., et al. (2017). TRIM25 Enhances the Antiviral Action of 20

Zinc-Finger Antiviral Protein (ZAP). PLoS Pathog 13, e1006145. 21

Lin, J.Y., Shih, S.R., Pan, M., Li, C., Lue, C.F., Stollar, V., and Li, M.L. (2009). hnRNP A1 22

interacts with the 5' untranslated regions of enterovirus 71 and Sindbis virus RNA and is 23

required for viral replication. J Virol 83, 6106-6114. 24



https://doi.org/10.1101/350686


34

Liu, C., Perilla, J.R., Ning, J., Lu, M., Hou, G., Ramalho, R., Himes, B.A., Zhao, G., Bedwell, 1

G.J., Byeon, I.J., et al. (2016). Cyclophilin A stabilizes the HIV-1 capsid through a novel non-2

canonical binding site. Nat Commun 7, 10714. 3

Lloyd, R.E. (2015). Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA 4

viruses. Virology 479-480, 457-474. 5

Lunde, B.M., Moore, C., and Varani, G. (2007). RNA-binding proteins: modular design for 6

efficient function. Nat Rev Mol Cell Biol 8, 479-490. 7

Manokaran, G., Finol, E., Wang, C., Gunaratne, J., Bahl, J., Ong, E.Z., Tan, H.C., Sessions, 8

O.M., Ward, A.M., Gubler, D.J., et al. (2015). Dengue subgenomic RNA binds TRIM25 to 9

inhibit interferon expression for epidemiological fitness. Science 350, 217-221. 10

Mesa, A., Somarelli, J.A., and Herrera, R.J. (2008). Spliceosomal immunophilins. FEBS Lett 11

582, 2345-2351. 12

Molleston, J.M., and Cherry, S. (2017). Attacked from All Sides: RNA Decay in Antiviral 13

Defense. Viruses 9. 14

Monie, T.P., Perrin, A.J., Birtley, J.R., Sweeney, T.R., Karakasiliotis, I., Chaudhry, Y., 15

Roberts, L.O., Matthews, S., Goodfellow, I.G., and Curry, S. (2007). Structural insights into 16

the transcriptional and translational roles of Ebp1. EMBO J 26, 3936-3944. 17

Mukherjee, N., Calviello, L., Hirsekorn, A., de Pretis, S., Pelizzola, M., and Ohler, U. (2017). 18

Integrative classification of human coding and noncoding genes through RNA metabolism 19

profiles. Nat Struct Mol Biol 24, 86-96. 20

Ni, X., Ru, H., Ma, F., Zhao, L., Shaw, N., Feng, Y., Ding, W., Gong, W., Wang, Q., Ouyang, 21

S., et al. (2016). New insights into the structural basis of DNA recognition by HINa and HINb 22

domains of IFI16. J Mol Cell Biol 8, 51-61. 23

Ong, S.E., and Mann, M. (2006). A practical recipe for stable isotope labeling by amino acids 24

in cell culture (SILAC). Nat Protoc 1, 2650-2660. 25



https://doi.org/10.1101/350686


35

Pacheco, A., Lopez de Quinto, S., Ramajo, J., Fernandez, N., and Martinez-Salas, E. 1

(2009). A novel role for Gemin5 in mRNA translation. Nucleic Acids Res 37, 582-590. 2

Pineiro, D., Fernandez-Chamorro, J., Francisco-Velilla, R., and Martinez-Salas, E. (2015). 3

Gemin5: A Multitasking RNA-Binding Protein Involved in Translation Control. Biomolecules 4

5, 528-544. 5

Popow, J., Englert, M., Weitzer, S., Schleiffer, A., Mierzwa, B., Mechtler, K., Trowitzsch, S., 6

Will, C.L., Luhrmann, R., Soll, D., et al. (2011). HSPC117 is the essential subunit of a human 7

tRNA splicing ligase complex. Science 331, 760-764. 8

Popow, J., Jurkin, J., Schleiffer, A., and Martinez, J. (2014). Analysis of orthologous groups 9

reveals archease and DDX1 as tRNA splicing factors. Nature 511, 104-107. 10

Rathore, A.P., Ng, M.L., and Vasudevan, S.G. (2013). Differential unfolded protein response 11

during Chikungunya and Sindbis virus infection: CHIKV nsP4 suppresses eIF2alpha 12

phosphorylation. Virol J 10, 36. 13

Rehwinkel, J., and Reis e Sousa, C. (2013). Targeting the viral Achilles' heel: recognition of 14

5'-triphosphate RNA in innate anti-viral defence. Curr Opin Microbiol 16, 485-492. 15

Romero-Brey, I., and Bartenschlager, R. (2014). Membranous replication factories induced 16

by plus-strand RNA viruses. Viruses 6, 2826-2857. 17

Rupp, D., and Bartenschlager, R. (2014). Targets for antiviral therapy of hepatitis C. Semin 18

Liver Dis 34, 9-21. 19

Sampath, P., Mazumder, B., Seshadri, V., Gerber, C.A., Chavatte, L., Kinter, M., Ting, S.M., 20

Dignam, J.D., Kim, S., Driscoll, D.M., et al. (2004). Noncanonical function of glutamyl-prolyl-21

tRNA synthetase: gene-specific silencing of translation. Cell 119, 195-208. 22

Seo, G.J., Kim, C., Shin, W.J., Sklan, E.H., Eoh, H., and Jung, J.U. (2018). TRIM56-23

mediated monoubiquitination of cGAS for cytosolic DNA sensing. Nat Commun 9, 613. 24

Shen, Y., Li, N.L., Wang, J., Liu, B., Lester, S., and Li, K. (2012). TRIM56 is an essential 25

component of the TLR3 antiviral signaling pathway. J Biol Chem 287, 36404-36413. 26



https://doi.org/10.1101/350686


36

Simsek, D., Tiu, G.C., Flynn, R.A., Byeon, G.W., Leppek, K., Xu, A.F., Chang, H.Y., and 1

Barna, M. (2017). The Mammalian Ribo-interactome Reveals Ribosome Functional 2

Diversity and Heterogeneity. Cell 169, 1051-1065 e1018. 3

Skabkin, M.A., Skabkina, O.V., Dhote, V., Komar, A.A., Hellen, C.U., and Pestova, T.V. 4

(2010). Activities of Ligatin and MCT-1/DENR in eukaryotic translation initiation and 5

ribosomal recycling. Genes Dev 24, 1787-1801. 6

Sun, X.H., and Baltimore, D. (1989). Human immunodeficiency virus tat-activated 7

expression of poliovirus protein 2A inhibits mRNA translation. Proc Natl Acad Sci U S A 86, 8

2143-2146. 9

Sysoev, V.O., Fischer, B., Frese, C.K., Gupta, I., Krijgsveld, J., Hentze, M.W., Castello, A., 10

and Ephrussi, A. (2016). Global changes of the RNA-bound proteome during the maternal-11

to-zygotic transition in Drosophila. Nat Commun 7, 12128. 12

Thompson, M.R., Sharma, S., Atianand, M., Jensen, S.B., Carpenter, S., Knipe, D.M., 13

Fitzgerald, K.A., and Kurt-Jones, E.A. (2014). Interferon gamma-inducible protein (IFI) 16 14

transcriptionally regulates type i interferons and other interferon-stimulated genes and 15

controls the interferon response to both DNA and RNA viruses. J Biol Chem 289, 23568-16

23581. 17

Tsuchida, T., Zou, J., Saitoh, T., Kumar, H., Abe, T., Matsuura, Y., Kawai, T., and Akira, S. 18

(2010). The ubiquitin ligase TRIM56 regulates innate immune responses to intracellular 19

double-stranded DNA. Immunity 33, 765-776. 20

Ventoso, I., Sanz, M.A., Molina, S., Berlanga, J.J., Carrasco, L., and Esteban, M. (2006). 21

Translational resistance of late alphavirus mRNA to eIF2alpha phosphorylation: a strategy 22

to overcome the antiviral effect of protein kinase PKR. Genes Dev 20, 87-100. 23

Versteeg, G.A., Rajsbaum, R., Sanchez-Aparicio, M.T., Maestre, A.M., Valdiviezo, J., Shi, 24

M., Inn, K.S., Fernandez-Sesma, A., Jung, J., and Garcia-Sastre, A. (2013). The E3-ligase 25



https://doi.org/10.1101/350686


37

TRIM family of proteins regulates signaling pathways triggered by innate immune pattern-1

recognition receptors. Immunity 38, 384-398. 2

Vladimer, G.I., Gorna, M.W., and Superti-Furga, G. (2014). IFITs: Emerging Roles as Key 3

Anti-Viral Proteins. Front Immunol 5, 94. 4

von Kobbe, C., van Deursen, J.M., Rodrigues, J.P., Sitterlin, D., Bachi, A., Wu, X., Wilm, M., 5

Carmo-Fonseca, M., and Izaurralde, E. (2000). Vesicular stomatitis virus matrix protein 6

inhibits host cell gene expression by targeting the nucleoporin Nup98. Mol Cell 6, 1243-7

1252. 8

Wang, Y., Jin, F., Wang, R., Li, F., Wu, Y., Kitazato, K., and Wang, Y. (2017). HSP90: a 9

promising broad-spectrum antiviral drug target. Arch Virol 162, 3269-3282. 10

Xu, C., Ishikawa, H., Izumikawa, K., Li, L., He, H., Nobe, Y., Yamauchi, Y., Shahjee, H.M., 11

Wu, X.H., Yu, Y.T., et al. (2016). Structural insights into Gemin5-guided selection of pre-12

snRNAs for snRNP assembly. Genes Dev 30, 2376-2390. 13

Zhang, R., Hryc, C.F., Cong, Y., Liu, X., Jakana, J., Gorchakov, R., Baker, M.L., Weaver, 14

S.C., and Chiu, W. (2011). 4.4 A cryo-EM structure of an enveloped alphavirus Venezuelan 15

equine encephalitis virus. EMBO J 30, 3854-3863. 16

Zheng, X., Wang, X., Tu, F., Wang, Q., Fan, Z., and Gao, G. (2017). TRIM25 Is Required 17

for the Antiviral Activity of Zinc Finger Antiviral Protein. J Virol 91. 18

Zhou, D., Mei, Q., Li, J., and He, H. (2012). Cyclophilin A and viral infections. Biochem 19

Biophys Res Commun 424, 647-650. 20

21



https://doi.org/10.1101/350686


A Mocklight isotope

4 hpimedium isotope

18 hpiheavy isotope

mz

Labeling andinfection

UV crosslinking

Lysis(Input - WCL)

Mixing andoligo (dT) capture

RNase treatment

Quantitativeproteomics

Figure 1 (Garcia-Moreno et al)

B

C

P

E1E2

C

ACTB

220

150

100

75

50

37

25

E3-2

moc

k

2 4 8 18 24 36

SINV

E

MW

220150

10075

50

37

25

5 0.5 0.05 5 0.5 0.05 5 0.5 0.05 %input MW

220150

10075

50

37

25

20

- + + + + UV

SINV(hpi)- - 4 8 18

-5

0

5

10

15

20actb mRNAgapdh mRNASINV RNA

RN

A le

vels

rela

tive

to m

ock

(log2

fold

cha

nge)

F

EIF2α

P-EIF2α

4 hpi 18 hpi

Inte

nsity

up down

hpi

D 37

37

Input (total proteome) RNA-IC eluates

SINV(hpi)4 18-

MW

SINV genome

genomicpromoter

NSP1 NSP2 NSP3 NSP4cap CE3

E26K/TF

E1 AAAAn

subgenomicpromoter

genomicpromoter

NSP1 NSP2 NSP3 NSP4cap CE3

E26K/TF

E1 AAAAn

mCherry

subgenomicpromoter

subgenomicpromoter

SINV-mCherry genome



https://doi.org/10.1101/350686



−4−2

02

46

−4 −2 0 2 4 64 hpi / mock [log2-ratio]

IC Rep 3

4 hp

i / m

ock

[log2

-ratio

]IC

Rep

2

A B4 hpi interactome capture

C

−4 −2 0 2 4 6

−4−2

02

46

18 h

pi /

moc

k [lo

g2-ra

tio]

IC R

ep 2

18 hpi / mock [log2-ratio]IC Rep 3

D18 hpi interactome capture

MW moc

k

4 8 18

SINV

Incr

ease

d R

NA

bind

ing

EIF4G1

GEMIN5

DDX1

PPIA

SINV C

220

150

220

150

75

15

25

37

Dec

reas

ed R

NA

bind

ing XRCC5

XRCC6

DDX50

HNRNPR

PTBP1

75

100

50

75

75

100

75

50

Una

ffect

edR

NA-

bind

ing

MOV10

EPRS

100150

150

Neg

ativ

eco

ntro

l

ACTB50

37

E

F

4 hpi interactome capture

Fold change [log2]

p−v

alue

[−lo

g10]

SIN

V C

0.0

0.5

1.0

1.5

−6 −3 0 3 6

0 0.4 0.8 1.2

protein metabolic process

-0.4-0.8-1.2

RNA processing

log2 odds ratio

nucleolusnuclear part

nuclear lumennucleus

0 1 2-1-2log2 odds ratio

InfectedMock G

18 hpi interactome capture

0

1

2

−6 −3 0 3 6

3 p

−val

ue [−

log1

0]

Fold change [log2]SI

NV

C

1% FDR SINV 10% FDR SINV

1% FDR mock 10% FDR mock

n.s.

hpi

cytoplasmic part

PA2G450

16 18 20 22 24 26 28

1618

2022

2426

28 SINV C

SINV NSP2SINV E2SINV NSP4

SINV NSP3

protein intensity [log 2] IC 18 hpi - Rep 2

prot

ein

inte

nsity

[log

2]

IC 1

8 hp

i - R

ep 3



https://doi.org/10.1101/350686



−4 −2 0 2 4 6

−4−2

02

4

−4 −2 0 2 4 6

−4−2

02

46

4 hpi / mock [log2-ratio]WCL Rep 3

4 hp

i / m

ock

[log2

-ratio

]W

CL

Rep

2

4 hpi whole cell lysateA 18 hpi whole cell lysate

18 h

pi /

moc

k [lo

g2-ra

tio]

WC

L R

ep 2

18 hpi / mock [log2-ratio]WCL Rep 3

Bpr

otei

n in

tens

ity [l

og 2

]W

CL

18 h

pi -

Rep

2

protein intensity [log 2] WCL 18 hpi - Rep 3

20 22 24 26 28 30 32

2022

2426

2830

32

SINV CSINV E2

SINV NSP2

SINV NSP4SINV NSP1

SINV NSP3

C D

0 2 4 8 18 24 24

SINV

moc

k

moc

k

DDX50

DDX1

EPRS

GEMIN5

EIF4G1

HNRNPQ

PTBP1

XRCC5

XRCC6

HNRNPR

SINV C

Incr

ease

d R

NA

bind

ing

Dec

reas

ed R

NA

bind

ing

Una

ffect

ed

220

150

75

2537

75

50

75

75

100

50

PA2G450

150100

7550

E

1e−01 1e+01 1e+03 1e+05 1e+07

−4−2

02

4

mean of normalized counts

SINVRNA

18hp

i / m

ock

fold

cha

nge

[log2

-ratio

]

G HR² = 0.8185

-5

-3

-1

1

3

5

7

-5 -3 -11 3 5 7

RT-

qPC

R (l

og2

fold

cha

nge)

RNAseq (log2 fold change)

-22

unaltered RBPdynamic RBP

hpiMW

18 hpi

1e−01 1e+01 1e+03 1e+05 1e+07

−4−2

02

4

mean of normalized counts

Differentialy expressedp<0.1No significant change

4hpi

/ m

ock

fold

cha

nge

[log2

-ratio

]

F 4 hpi

PPIA15

220150

10075

5037

25

MW 0 2 4 8 18 24 24

SINVmoc

kmoc

k

hpi

C



https://doi.org/10.1101/350686


SINV 18 hpi

RBP-eGFPSINV CDAPI

RBP-eGFP

MockCA

SINV RNAs

SIN

V 18

hpi

SINV C

D

B

RBP-eGFP SINV C

Hel

a G

EMIN

5-eG

FPH

ela

SRPK

1-eG

FPH

ela

TRIM

25-e

GFP

Hel

a TR

IM56

-eG

FPH

ela

XRC

C6-

eGFP

Hel

a M

OV1

0-eG

FPH

ela

eGFP

Enriched in viralfactories

RBP localisation in SINV-infected cells


Moc

k

SINV RNAsSINV CDAPI

0 2000 4000 6000 8000 10000 12000

0

2.5e+03

nucleotide position

Cov

erag

epositive strand

negative strand

4 hpi

0 2000 4000 6000 8000 10000 12000

1e+05

2e+05

3e+05

4e+05

5e+05

6e+05

2e+03

1.5e+03

1e+03

0.5e+03

0

18 hpi

Cov

erag

e

nucleotide position

positive strand

negative strandH

ela

DD

X1-e

GFP

Hel

a R

TCB-

eGFP

GEMIN5SRPK1TRIM25TRIM56DDX1RTCBUPF1PPIA

PA2G4FAM98A

XRCC6MOV10

HNRNPCNGDNNONO

HNRNPA1DKC1

DDX56TUFM

GFPENO1VARS

HSP90AB1WDR1RPS10ALDOA

Absent in viralfactories Diffused



https://doi.org/10.1101/350686


A


B

2.3%11%

86.7%

54.2%

5.3%

40.5%

0

25

50

75

100

Mock vs 4hpi Mock vs 18hpi

Perc

enta

ge o

f var

ienc

e ex

plai

ned

Time (h)

mC

herry

exp

ress

ion

(RFU

) XRN1 (knockout)SINV-mCherry

0 6 12 18 240

250050007500

100001250015000

wt

KOpartial KO

C

XRN1

Actin

Capsid

KOhpiMock 8 16

+- +- +-

SINV wt

DegradationProcessingTranscription

SIN

V 18

hpi

SINV C

10 µm

XRN1

Moc

k

1 2 3

RO

I

1 2 3

1

2

3

1 2 3

1µm

XRN1 SINV C DAPI .CC-BY-NC-ND 4.0 International licenseavailable under anot certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made


https://doi.org/10.1101/350686


A


E

C D

Time (h)

HSP90AB1 (knockout)SINV-mCherry

0 6 12 18 240

250050007500

100001250015000

mC

herry

exp

ress

ion

(RFU

)

HSP90AB1

Actin

+- KO

Actin

Capsid

+SINV mChe

- +Mock- +

SINV wt- Induced

HSP90AB1

+- InducedHSP90AB1-eGFP

HSP90AB1 (overexpression)SINV-mCherry

Time (h)0 6 12 18 24

0250050007500

100001250015000

mC

herry

exp

ress

ion

(RFU

)

Ganetespib (HSP90AB1 inhibitor)SINV-mCherry

0 6 12 18 24Time (h)

DMSO100nM

0250050007500

100001250015000

mC

herry

exp

ress

ion

(RFU

)

0 6 12 18 240

250050007500

100001250015000

Time (h)

mC

herry

exp

ress

ion

(RFU

) Geldamycin (HSP90AB1 inhibitor)SINV-mCherry

DMSO100nM250nM

PPIA

Actin

+- KO

PPIA (knockout)SINV-mCherry

0 6 12 18 24Time (h)

mC

herry

exp

ress

ion

(RFU

)

0250050007500

100001250015000

ActinCapsid

+SINV mChe

- +Mock- +

SINV wt- Induced

CysA (PPIA inhibitor)SINV-mCherry

DMSO5µM

0 6 12 18 24Time (h)

0250050007500

100001250015000

mC

herry

exp

ress

ion

(RFU

)

PPIA (overexpression)SINV-mCherry

0 6 12 18 24Time (h)

0250050007500

100001250015000

PPIA

+- InducedPPIA-eGFP

mC

herry

exp

ress

ion

(RFU

)

Actin

Capsid

+

SINVmChe

- +Mock

- +SINV wt- Induced

SRPK1 (overexpression)SINV-mCherry

0 6 12 18 24Time (h)

0250050007500

100001250015000

mC

herry

exp

ress

ion

(RFU

)

SRPK1

+- InducedSRPK1-eGFP

+SINV mChe

- +Mock- +

SINV wt- Induced

ActinCapsid

18 hpi

ActinCapsid

8 hpi

PA2G4 (overexpression)SINV-mCherry

0 6 12 18 24Time (h)

0250050007500

100001250015000

PA2G4

+- Induced

PA2G4-eGFP

mC

herry

exp

ress

ion

(RFU

)

0 6 12 18 240

250050007500

100001250015000

Time (h)

mC

herry

exp

ress

ion

(RFU

) WS6 (PA2G4 inhibitor)SINV-mCherry

DMSO1µM

B

4µ8C (IRE1α inhibitor)SINV-mCherry

DMSO

30µM20µM

40µM

0 6 12 18 24Time (h)

mC

herry

exp

ress

ion

(RFU

)

0250050007500

100001250015000



https://doi.org/10.1101/350686


B


ActinCapsid

+SINV mChe

- +Mock- +

SINV wt-Induced

GEMIN5 (overexpression)SINV-mCherry

0 6 12 18 240

250050007500

100001250015000

Time (h)

A

ActinCapsid

+

SINVmChe

- +Mock

- +SINV wt-Induced

0 6 12 18 240

250050007500

100001250015000

TRIM56 (overexpression)SINV-mCherry

Time (h)

ActinCapsid

+SINV mChe

- +Mock- +

SINV wt-Induced

TRIM25 (overexpression)SINV-mCherry

Time (h)0 6 12 18 24

0250050007500

100001250015000

Hek293 XRCC6-eGFP

SINV

Mock

Hek293 GEMIN5-eGFP

SINV

Mock

Inputs

Hek293 eGFP

SINV

Mock

Eluates

Hek293 TRIM25-eGFP

SINV

Mock

Inputs

Eluates

D

mC

herry

exp

ress

ion

(RFU

)

GEMIN5

+- InducedGEMIN5-eGFP

TRIM25

TRIM25-eGFP TRIM56TRIM56-eGFP

IP + RT-PCR

+-+- InducedInduced

C

Fold change [log2]GEMIN5-Mock over GFP-Mock

Sign

ifica

nce

−log

10(t−

test

p−v

alue

)

GEMIN5

GFP

RPL4

RPL3SMN1

0

2

4

6

−5 0 5 10

p<0.01

p<0.1

GFP GEMIN5Mock

Fold change [log2]GEMIN5+SINV over GEMIN5-Mock

GEMIN5

RPL4

RPL3

SMN10

1

2

−2 −1 0 1 2

p<0.01

p<0.1

Mock SINVGEMIN5

Fold change [log2]GEMIN5+SINV over GFP+SINV

GEMIN5

GFP

RPL4RPL3

CAPSIDNSP1

NSP2

E2

NSP40

1

2

3

4

−5 0 5 10

p<0.01

p<0.1

SMN1

NSP3

GFP GEMIN5SINV

GEM

IN5

bind

ing

sign

al

0

5

10

15

20

2000 3000 4000 5000 6000 7000 8000 9000 10000 bp

SINV

gRNAsgRNA

NSP2NSP4

NSP3NSP1

E1E2Capsid

3’UTR

E3

6K

DLPsgRNA5’UTR

SIN

VA

nnot

atio

n

50 100 200 300 bp05101520 gRNA 5’UTR

940 980 1020 1060 1100 bp02468 packaging signal

11550 11650 11750 11850 bp0

5

10

153’UTR

7580 7600 7620 7640 7660 7680 7700 7720 7740 7760 bp0

5

10

15sgRNA 5’UTR

gRNA5’UTR packaging

signal

DLP



https://doi.org/10.1101/350686


20 1 (Figure 7B). GEMIN5 impacts protein synthesis at the translation elongation step through its ....

Documents

Transcript of 20 1 (Figure 7B). GEMIN5 impacts protein synthesis at the translation elongation step through its ....