key: cord-1055328-80c81r5n
authors: Ariumi, Yasuo
title: Host cellular RNA helicases regulate SARS-CoV-2 infection
date: 2021-06-30
journal: bioRxiv
DOI: 10.1101/2021.06.29.450452
sha: ed47610045eecf56dbf4ed9628bae51e99b8e361
doc_id: 1055328
cord_uid: 80c81r5n

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has largest RNA genome of approximately 30kb among RNA viruses. The DDX DEAD-box RNA helicase is a multifunctional protein involved in all aspects of RNA metabolism. Therefore, host RNA helicases may regulate and maintain such large viral RNA genome. In this study, I investigated the potential role of several host cellular RNA helicases in SARS-CoV-2 infection. Notably, DDX21 knockdown markedly accumulated intracellular viral RNA and viral production, as well as viral infectivity of SARS-CoV-2, indicating that DDX21 strongly restricts the SARS-CoV-2 infection. As well, MOV10 RNA helicase also suppressed the SARS-CoV-2 infection. In contrast, DDX1, DDX5, and DDX6 RNA helicases were required for SARS-CoV-2 replication. Indeed, SARS-CoV-2 infection dispersed the P-body formation of DDX6 and MOV10 RNA helicases as well as XRN1 exonuclease, while the viral infection did not induce stress granule formation. Accordingly, the SARS-CoV-2 nucleocapsid (N) protein interacted with DDX6, DDX21, and MOV10 and disrupted the P-body formation, suggesting that SARS-CoV-2 N hijacks DDX6 to utilize own viral replication and overcomes their anti-viral effect of DDX21 and MOV10 through as interaction with host cellular RNA helicase. Altogether, host cellular RNA helicases seem to regulate the SARS-CoV-2 infection. Importance SARS-CoV-2 has large RNA genome of approximately 30kb. To regulate and maintain such large viral RNA genome, host RNA helicases may involve in SARS-CoV-2 replication. In this study, I have demonstrated that DDX21 and MOV10 RNA helicases limit viral infection and replication. In contrast, DDX1, DDX5 and DDX6 are required for the SARS-CoV-2 infection. Interestingly, the SARS-CoV-2 infection disrupted P-body formation and attenuated or suppressed stress granule formation. Thus, SARS-CoV-2 seems to hijack host cellular RNA helicases to play a proviral role by facilitating viral infection and replication and, by suppressing host innate immune system.

SARS-CoV-2 infection. Notably, DDX21 knockdown markedly accumulated 23 intracellular viral RNA and viral production, as well as viral infectivity of SARS-CoV-2, 24

indicating that DDX21 strongly restricts the SARS-CoV-2 infection. As well, MOV10 25 RNA helicase also suppressed the SARS-CoV-2 infection. In contrast, DDX1, DDX5, 26 and DDX6 RNA helicases were required for SARS-CoV-2 replication. Indeed, 27 SARS-CoV-2 infection dispersed the P-body formation of DDX6 and MOV10 RNA 28 helicases as well as XRN1 exonuclease, while the viral infection did not induce stress 29 granule formation. Accordingly, the SARS-CoV-2 nucleocapsid (N) protein interacted 30 with DDX6, DDX21, and MOV10 and disrupted the P-body formation, suggesting that 31 SARS-CoV-2 N hijacks DDX6 to utilize own viral replication and overcomes their 32

The DDX DEAD-box RNA helicase family, which is an ATPase-dependent RNA 54 helicase, is a multifunctional protein involved in all aspects of RNA life cycle, including 55 transcription, mRNA splicing, RNA transport, translation, ribosome biogenesis, RNA 56 decay, and viral infection (1) (2) (3) (4) (5) (6) . DDX3 RNA helicase has two homologs termed 57 DDX3X and DDX3Y, which were located on X and Y chromosome. Actually, DDX3 58 involves in translation, transcription, cell cycle, tumorigenesis (oncogenic and tumor 59 suppressor function), viral infection and innate immunity (7). Indeed, DDX3 is known 60 to be a component of anti-viral innate immune signaling pathway and contributes to 61 induce anti-viral mediators, such as type I interferon and interferon regulatory factor 3 62 Uppsala, Sweden) as secondary antibodies. The proteins were detected by using 153 suggesting that DDX21 strongly restricts SARS-CoV-2 infection. In addition, MOV10 280 RNA helicases also suppressed the SARS-CoV-2 infection. Consistent with these 281 findings, Western blot analysis also showed that intracellular SARS-CoV-2 spike 282 protein expression was markedly enhanced in the DDX21 knockdown cells ( Figure 1C ). 283

To further confirm the anti-viral effect of DDX21, I examined the DDX21 knockdown 284 CACO-2 human colon cancer cells and HepG2 human hepatoma cells ( Figure 1D ). 285

The DDX21 knockdown demonstrated enhanced intracellular SARS-CoV-2 RNA in 287 both CACO-2 and HepG2 cells ( Figure 1D ). Thus, DDX21 seems to restrict 288

We next examined the levels of extracellular SARS-CoV-2 nucleocapsid (N) protein indicating that DDX21 interacts with SARS-CoV-2 N. Furthermore, SARS-CoV-2 N 367 colocalized with HA-tagged DDX1 when both proteins were co-expressed in 293T cells 368 ( Figure 5E ). In contrast, SARS-CoV-2 N did not colocalize with HA-tagged DDX3 369 ( Figure 5F ). Therefore, these results suggested that SARS-CoV-2 N interacts with 

Distinct DDX DEAD-box RNA 497 helicases cooperate to modulate the HIV-1 Rev function

DDX3 RNA helicase is required for 500

HIV-1 Tat function

DEAD-box RNA helicase is required for hepatitis C virus RNA replication

Hepatitis C virus hijacks P-body and stress granule components around lipid 506 droplets

A nucleolar RNA helicase recognized by autoommune antibodies 509 from a patient with watermelon stomach disease

The DEXD/H-box RNA

Gu is a co-factor for c-Jun-activated transcription

RNA 515 helicase DDX21 coordinates transcription and ribosomal RNA processing

Xp54 and related (DDX6-like) RNA helicase: 518 roles in messenger RNP assembly, translation regulation and RNA degradation

P bodies, stress granules, and viral life cycles

P bodies and the control of mRNA trabslation and 523 degradation

Suppression of HIV-1 replication by microRNA effector

DDX6 (Rck/p54) is required for efficient 528

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by 579 a clinically proven protease inhibitor

Structure, 581 function, and antigenicity of the SARS-CoV-2 spike glycoprotein

Enhanced isolation of

SARS-CoV-2 by TMPRSS2-expressing cells

Structural and functional analysis of the D614G SARS-CoV-2 spike 592

Role of the DExH

Japanese encephalitis virus and hepatitis C virus NS3 proteins in the ATPase and 611 RNA helicase activities

Host DDX 613 helicases as possible SARS-CoV-2 proviral factors: a structural overview of their 614 hijacking through multiple viral proteins

The cellular interactome of coronavirus infectious bronchitis 617 virus nucleocapsid protein and functional implications for virus biology

A SARS-CoV-2 protein interaction map reveals targets for drug 638 repurposing

MOV10 RNA helicase is a potent 640 inhibitor of retrotransposition in cells

Liu 642 YJ. 2011. DDX1, DDX21, and DHX36 helicases form a complex with the adaptor 643 molecule TRIF to sense dsRNA in dendritic cells

Cellular DDX21 RNA helicase inhibits 645 influenza A virus replication but is counteracted by the viral NS1 protein

DDX21 translocates from nucleus to cytoplasm and stimulates the innate immune 649 response due to dengue virus infection

Dissecting the role of 652 DDX21 in regulating human cytomegalovirus replication

Autogenous 654 translational regulation of the borna disease virus negative control factor X from 655 polycistronic mRNA using host RNA helicases

Modulation of 657 hepatitis C virus RNA abundance by a liver-specific MicroRNA

The lipid droplet is an important 661 organelle for hepatitis C virus production

Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory 667 mediators

of Western blot analysis of cellular lysates with anti-DDX1 (A300-521A), anti-DDX3 673 (A300-474A), anti-DDX6 (A300-460A), anti-DDX21 (A300-627A)

A301-571A), or anti-β-actin antibody are shown. (B) The level of intracellular 675

SARS-CoV-2 RNA in the cells at 72 h post-infection at an MOI of 0.5 was monitored 676 by real-time LightCycler RT-PCR. Results from three independent experiments are 677 shown. The level of SARS-CoV-2 RNA in each knockdown cells was calculated 678 relative to the level in HEK293T ACE2 cells transduced with a control lentiviral vector 679 (Con)

SARS-CoV-2 spike protein expression levels in each 681 knockdown cells. The results of the Western blot analysis of cellular lysates with 682 anti-SARS-CoV-2

SARS-CoV-2-infected HEK293T ACE2 cells at 72 h post-infection at an MOI of 0.5 are 684 shown. (D) DDX21 restricts SARS-CoV-2 infection in CACO-2 and HepG2 cells

Inhibition of endogenous DDX21 protein expression by the shRNA-producing lentiviral 686 vector. The results of Western blot analysis of cellular lysates with anti-DDX21 or 687

anti-β-actin antibody in CACO2 or HepG2 cells are shown. The level of intracellular 688

SARS-CoV-2 RNA in the cells at 72 h post-infection at an MOI of 0.5 was monitored 689 by real-time LightCycler RT-PCR. Results from three independent experiments are 690 shown. The level of SARS-CoV-2 RNA in DDX21 knockdown cells was calculated 691 relative to the level in HEK293T ACE2 cells transduced with a control lentiviral vector 692 (Con)

Figure 2. Characterization of anti-viral effect of DDX21. (A) DDX21 suppresses

The levels of extracellular SARS-CoV-2 N protein in the 697 culture supernatants from the DDX21 knockdown HEK293T ACE2 cells 72 h after 698 inoculation of SARS-CoV-2 at an MOI of 0.5 were determined by ELISA

DDX21 inhibits the level of extracellular SARS-CoV-2 RNA. The level of extracellular 702

SARS-CoV-2 RNA in the culture supernatants from the DDX21 knockdown HEK293T

ACE2 cells 72 h after inoculation of SARS-CoV-2 at an MOI of 0.5 were monitored by 704 real-time LightCycler RT-PCR. Results from three independent experiments are shown

The level of SARS-CoV-2 RNA in DDX21 knockdown cells was calculated relative to 706 the level in HEK293T ACE2 cells transduced with a control lentiviral vector (Con)

Asterisk indicates significant differences compared to the control cells

The virus titer of SARS-CoV-2 in the culture supernatants from the DDX21 knockdown 709

Naïve VeroE6 TMPRSS2 710 cells were seeded in 24-well plates at 5×10 4 cells per well and then infected the next day 711 with the indicated serial 10-fold dilutions of culture supernatants. The cells were 712 stained with 0.6% Coomassie brilliant blue in 50% methanol and 10% acetate at 72 h 713 post-infection were monitored for cytopathic effect (CPE). The virus titer was 714 determined by the tissue culture infectious dose at 50% (TCID 50 /ml). (D) The infectivity 715 of SARS-CoV-2

Naïve VeroE6 TMPRSS2 cells were plated on Lab-Tek 2 well 718 chamber slide at 2×10 4 cells per well

SARS-CoV-2-infected control or DDX21 knockdown HEK293T ACE2 cells were 720 inoculated. The cells were fixed at 24 h post-infection and stained with 721 anti-SARS-CoV-2 nucleocapsid (ab273434 [6H3]). Cells were then stained with 722

Donkey anti-mouse IgG (H+L) secondary antibody, Alexa Fluor 594 conjugate. Images 723 were visualized using confocal laser scanning microscopy

Subcellular localization of SARS-CoV-2 N protein in control or 725 DDX21 knockdown HEK293T ACE2 cells 24 h after inoculation of SARS-CoV-2. The 726 cells were stained with anti-SARS-CoV-2 nucleocapsid. (F) Subcellular localization of 727 endogenous DDX21 and SARS-CoV-2 N

The cells were stained with 729 anti-SARS-CoV-2 nucleocapsid and anti-DDX21 (A300-627A) antibodies. Cells were 730 then stained with Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 488 731 conjugate and Donkey anti-mouse IgG (H+L) secondary antibody, Alexa Fluor 594 732 conjugate

SARS-CoV-2 disrupts the P-body formation

TMPRSS2 or HEK293T ACE2 cells and their SARS-CoV-2-infected cells at 24 h 737 post-infection were stained with anti-SARS-CoV-2 nucleocapsid (ab273434

anti-DDX6 (A300-460A) antibodies. The cells were also stained with anti-SARS-CoV-2 739 nucleocapsid and either anti-Xrn1 (A300-443A) or anti-MOV10

Cells were then stained with Donkey anti-rabbit IgG (H+L) secondary 741

Alexa Fluor 488 conjugate and Donkey anti-mouse IgG (H+L) secondary

Fluor 594 conjugate. Images were visualized using confocal laser 743 scanning microscopy. The two-color overlay images are also exhibited (Merged)

SARS-CoV-2 hijacks DDX6 for own viral replication. (A) Dynamic 748 redistribution of DDX6 in response to SARS-CoV-2 infection. VeroE6 TMPRSS2 cells 749 at indicated time (hrs) after inoculation of SARS-C-V-2 were stained with 750 anti-SARS-CoV-2

B) SARS-CoV-2 does not induce stress granule formation

VeroE6 TMPRSS2 or the SARS-CoV-2-infected cells at 24 h post-infection were 753 incubated at 37˚C. Uninfected cells were also incubated at 43˚C for 45 min

C) Host protein expression levels in response to SARS-CoV-2 infection

The results of the Western blot analysis of cellular lysates with anti-SARS-CoV-2 Spike 757 (GTX632604 [1A9]), anti-DDX6, anti-G3BP1, or anti-β-actin antibody in the 758

SARS-CoV-2-infected VeroE6 TMPRSS2 or the HEK293T ACE2 cells at 24 h 759 post-infection at an MOI of 0.5 as well as in the uninfected cells are shown

Inhibition of endogenous DDX6 761 protein expression by the shRNA-producing lentiviral vector. The results of Western 762 blot analysis of cellular lysates with anti-DDX6 or anti-β-actin antibody are shown. The 763 level of intracellular SARS-CoV-2 RNA in the cells at 72 h post-infection at an MOI of 764 0.5 was monitored by real-time LightCycler RT-PCR. Results from three independent 765 experiments are shown. The level of SARS-CoV-2 RNA in the DDX6 knockdown cells 766 was calculated relative to the level in HEK293T ACE2 cells transduced with a control 767 lentiviral vector (Con)

A) Disruption of P-body formation of endogenous DDX6 by 773 ectopically expressed SARS-CoV-2 N. 293T cells transfected with 200 ng of 774 pcDNA3.1-SARS-CoV-2 N (38) were stained with anti-SARS-CoV-2

P-body formation of HA-tagged DDX6 by ectopically expressed SARS-CoV-2 N. 293T 777 cells co-transfected with 200 ng of pcDNA3-HA-DDX6 (9) and either 200 ng of 778 pcDNA3.1-SARS-CoV-2 N or pcDNA3 were stained with anti-SARS-CoV

3F10) antibodies. (C) Disruption of P-body formation of 780

HA-tagged MOV10 by SARS-CoV-2 N and colocalization of SARS-CoV-2 N and 781

1-SARS-CoV-2 N or pcDNA3 were stained with anti-SARS-CoV-2 783 nucleocapsid and anti-HA antibodies. (D) Colocalization of endogenous DDX21 and 784 ectopically expressed SARS-CoV-2 N in nucleoli

SARS-CoV-2 N. 293T cells co-transfected with 200 ng of pcDNA3-HA-DDX1 (9) and 788 either 200 ng of pcDNA3.1-SARS-CoV-2 N or pcDNA3 were stained with

(F) Subcellular localization of 790

HA-tagged DDX3 and SARS-CoV-2 N. 293T cells co-transfected with 200 ng of 791 pHA-DDX3 (8-12) and either 200 ng of pcDNA3.1-SARS-CoV-2 N or pcDNA3 were 792 stained with anti-SARS-CoV-2 nucleocapsid and anti-HA antibodies

Figure 6. SARS-CoV-2 N binds to DDX21, DDX6, and MOV10. (A)

TMPRSS2 cells (5×10 5 cells/well) were infected with SARS-CoV-2 at an MOI of 0

The cell lysates were collected at 24 h post-infection. (B) 293T cells (2X10 5 cells/well) 798 were co-transfected with 4 μg of pcDNA3.1-SARS-CoV-2 N. The cell lysates were 799 immunoprecipitated with anti-SARS-CoV-2

anti-DDX21 (A300-627A), anti-DDX6 (A300-460A), or anti-MOV10

antibody, followed by immunoblotting analysis using anti-SARS-CoV-2 nucleocapsid

Both short exposed 803 and long exposed images were shown