SARS-CoV-2 Disrupts Splicing, Translation, and Protein Trafficking to Suppress Host Defenses

Article

SARS-CoV-2 Disrupts Splicing, Translation, and

Protein Trafficking to Suppress Host Defenses

Graphical Abstract

Highlights

d

NSP16 binds mRNA recognition domains of U1/U2 snRNAs

and disrupts mRNA splicing

d

NSP1 binds in the mRNA entry channel of the ribosome to

disrupt protein translation

d

NSP8 and NSP9 bind the signal recognition particle and

disrupt protein trafficking

d

These disruptions of protein production suppress the

interferon response to infection

Authors

Abhik K. Banerjee, Mario R. Blanco,

Emily A. Bruce, ..., Jason W. Botten,

Devdoot Majumdar, Mitchell Guttman

Correspondence

dev.Majumdar@med.uvm.edu (D.M.),

mguttman@caltech.edu (M.G.)

In Brief

SARS-CoV-2 proteins directly engage

host RNAs to dysregulate essential steps

of protein production and suppress the

interferon response.

Banerjee et al., 2020, Cell

183

, 1325–1339

November 25, 2020

ª

2020 The Authors. Published by Elsevier Inc.

https://doi.org/10.1016/j.cell.2020.10.004

ll

Article

SARS-CoV-2 Disrupts Splicing, Translation,

and Protein Trafficking to Suppress Host Defenses

Abhik K. Banerjee,

1,2,8

Mario R. Blanco,

1,8

Emily A. Bruce,

3,8

Drew D. Honson,

1,9

Linlin M. Chen,

1,9

Amy Chow,

1,9

Prashant Bhat,

1,4

Noah Ollikainen,

Sofia A. Quinodoz,

Colin Loney,

Jasmine Thai,

Zachary D. Miller,

Aaron E. Lin,

Madaline M. Schmidt,

Douglas G. Stewart,

Daniel Goldfarb,

Giuditta De Lorenzo,

Suzannah J. Rihn,

Rebecca M. Voorhees,

Jason W. Botten,

Devdoot Majumdar,

and Mitchell Guttman

1,10,

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA

Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA

Departments of Medicine, Division of Immunobiology and Microbiology, and Molecular Genetics, Larner College of Medicine, University of

Vermont, Burlington, VT 05405, USA

David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA

MRC-University of Glasgow Centre for Virus Research (CVR), Glasgow G61 1QH, UK

Department of Surgery and University of Vermont Cancer Center, University of Vermont College of Medicine, 89 Beaumont Avenue,

Burlington, VT 05405, USA

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA

These authors contributed equally

Lead Contact

*Correspondence:

dev.Majumdar@med.uvm.edu

(D.M.),

mguttman@caltech.edu

(M.G.)

https://doi.org/10.1016/j.cell.2020.10.004

SUMMARY

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that

causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need,

we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively

define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recog-

nition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2

infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global

inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal

recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of

each of these essential cellular functions acts to suppress the interferon response to viral infection. Our re-

sults uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to

suppress host defenses.

INTRODUCTION

Coronaviruses are a family of viruses with notably large single-

stranded RNA genomes and broad species tropism among mam-

mals (

Graham and Baric, 2010

). Recently, a coronavirus, severe

acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was

discovered to cause the severe respiratory disease known as co-

ronavirus disease 2019 (COVID-19). It is highly transmissible in hu-

man populations, and its spreadhas resulted in a globalpandemic

with more than a million deaths to date (

Andersen et al., 2020

; Zou

et al., 2020

). We do not fully understand the molecular basis of

infection and pathogenesis of this virus in human cells. Accord-

ingly, there is an urgent need to understand these mechanisms

to guide the development of therapeutic agents.

SARS-CoV-2 encodes 27 proteins with diverse functional roles

invirus replication and packaging (

Bar-Onetal., 2020

; Wang etal.,

2020 ). These include 4 structural proteins: the nucleocapsid (N;

which binds the viral RNA) and the envelope (E), membrane (M),

and spike (S) proteins, which are integral membrane proteins. In

addition, there are 16 non-structural proteins (NSP1–NSP16)

that encode the RNA-directed RNA polymerase, helicase, and

other components required for virus replication (

da Silva et al.,

2020 ). Finally, there are 7 accessory proteins (ORF3a–ORF8)

whose function in virus replication or packaging remains largely

uncharacterized (

Chen and Zhong, 2020

; Finkel et al., 2020

As obligate intracellular parasites, viruses require host cell

components to translate and transport their proteins and to

assemble and secrete viral particles (

Maier et al., 2016

). The

mammalian innate immune system acts to rapidly detect and

block viral infection at all stages of the virus life cycle (

Chow

et al., 2018

; Jensen and Thomsen, 2012

; Wilkins and Gale,

2010

). The primary form of intracellular virus surveillance

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2020 The Authors. Published by Elsevier Inc.

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This is an open access article under the CC BY license (

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engages the interferon pathway, which amplifies signals result-

ing from detection of intracellular viral components to induce a

systemic type I interferon response upon infection (

Stetson

and Medzhitov, 2006

). Specifically, cells contain various RNA

sensors (such as RIG-I and MDA5) that detect the presence of

viral RNAs and promote nuclear translocation of the transcription

factor IRF3, leading to transcription, translation, and secretion of

interferon (e.g., interferon [IFN]-

and IFN-

). Binding of IFN to

cognate cell-surface receptors leads to transcription and trans-

lation of hundreds of antiviral genes.

In order to successfully replicate, viruses employ a range of

strategies to counter host antiviral responses (

Beachboard and

Horner, 2016

). In addition to their essential roles in the viral life

cycle, many viral proteins also antagonize core cellular functions

in human cells to evade host immune responses. For example,

human cytomegalovirus (HCMV) encodes proteins that inhibit

major histocompatibility complex (MHC) class 1 display on the

cell surface by retaining MHC proteins in the endoplasmic retic-

ulum (

Miller et al., 1998

), polioviruses encode proteins that

degrade translation initiation factors (eIF4G) to prevent transla-

tion of 5

-capped host mRNAs (

Kempf and Barton, 2008

; Lloyd,

2006

), and influenza A encodes a protein that modulates mRNA

splicing to degrade the mRNA that encodes RIG-I (

Kochs et al.,

2007

; Zhang et al., 2018

Suppression of the IFN response has recently emerged as a

major clinical determinant of COVID-19 severity (

Zhang et al.,

2020

), with almost complete loss of secreted IFN characterizing

the most severe cases (

Hadjadj et al., 2020

). The extent to which

SARS-CoV-2 suppresses the IFN response is a key character-

istic that distinguishes COVID-19 from SARS and Middle East

respiratory syndrome (MERS) (

Lokugamage et al., 2020

). Several

strategies have been proposed for how the related SARS- and

MERS-causing viruses may hijack host cell machinery and

evade immune detection, including repression of host mRNA

transcription in the nucleus (

Canton et al., 2018

), degradation

of host mRNA in the nucleus and cytoplasm (

Kamitani et al.,

2009

; Lokugamage et al., 2015

), and inhibition of host translation

(Nakagawa et al., 2018

). Nonetheless, the extent to which SARS-

CoV-2 uses these or other strategies and how they may be

executed at a molecular level remains unclear.

Understanding the interactions between viral proteins and

components of human cells is essential for elucidating their path-

ogenic mechanisms and for development of effective therapeu-

tic agents. Because SARS-CoV-2 is an RNA virus, and many of

its encoded proteins are known to bind RNA (

Sola et al., 2011

we reasoned that these viral proteins may interact with specific

human mRNAs (critical intermediates in protein production) or

non-coding RNAs (critical structural components of diverse

cellular machines) to promote virus propagation.

Here we comprehensively define the interactions between

each SARS-CoV-2 protein and human RNA. We show that 10

viral proteins form highly specific interactions with mRNAs or

noncoding RNAs (ncRNAs), including those involved in progres-

sive steps of host cell protein production. We show that NSP16

binds to the mRNA recognition domains of the U1 and U2 RNA

components of the spliceosome and acts to suppress global

mRNA splicing in SARS-CoV-2-infected human cells. We find

that NSP1 binds to a precise region on the 18S ribosomal RNA

that resides in the mRNA entry channel of the initiating 40S ribo-

some. This interaction leads to global inhibition of mRNA trans-

lation upon SARS-CoV-2 infection of human cells. Finally, we

find that NSP8 and NSP9 bind to discrete regions on the 7SL

RNA component of the signal recognition particle (SRP) and

interfere with protein trafficking to the cell membrane upon infec-

tion. We show that disruption of each of these essential cellular

functions acts to suppress the type I IFN response to viral infec-

tion. Our results uncover a multipronged strategy utilized by

SARS-CoV-2 to antagonize essential cellular processes and

robustly suppress host immune defenses.

RESULTS

Comprehensive Mapping of SARS-CoV-2 Protein

Binding to Human RNAs

We cloned all 27 of the known SARS-CoV-2 viral proteins into

mammalian expression vectors containing an N-terminal Halo-

Tag (

Los et al., 2008

; Figure S1

A; STAR Methods

), expressed

each in HEK293T cells, and exposed them to UV light to cova-

lently crosslink proteins to their bound RNAs. We then lysed

the cells and purified each viral protein using stringent dena-

turing conditions to disrupt any non-covalent associations and

capture those with a UV-mediated interaction (

Figure 1

A; STAR

Methods

). As positive and negative controls, we purified a known

human RNA binding protein (PTBP1) and a metabolic protein

(GAPDH) (

Figures S1

A–S1E).

We successfully purified 26 of the 27 viral proteins (

Figure S1

full-length S was not soluble when expressed). We found that 10

viral proteins (NSP1, NSP4, NSP8, NSP9, NSP12, NSP15,

NSP16, ORF3b, N, and E) bind to specific host RNAs (p <

0.001;

Figure 1

B; Table S1

), including 6 structural ncRNAs and

142 mRNAs (

Table S1

). These include mRNAs involved in protein

translation (e.g., COPS5, EIF1, and RPS12,), protein transport

(ATP6V1G1, SLC25A6, and TOMM20), protein folding (HSPA5,

HSPA6, and HSPA1B), transcriptional regulation (YY1, ID4, and

IER5), and immune response (JUN, AEN, and RACK1) (false dis-

very rate [FDR] < 0.05;

Figures 1

BandS1

F). Importantly, the

observed interactions are highly specific for each viral protein,

and each protein binds to a precise region within each RNA (

Fig-

ures 1

Cand

S1F).

Using these data, we identified several viral proteins that

interact with structural ncRNA components of the spliceosome

(U1 and U2 small nuclear RNA [snRNA]), the ribosome (18S

and 28S rRNA), and the SRP (7SL) (

Figure 1

B). Because these

molecular machines are essential for three essential steps of

protein production—mRNA splicing, translation, and protein

trafficking—we focused on their interactions with viral proteins

to understand their functions and mechanisms in SARS-CoV-2

pathogenesis.

NSP16 Binds to the Pre-mRNA Recognition Domains of

the U1 and U2 snRNAs

After transcription in the nucleus, nascent pre-mRNAs are spliced

to generate mature mRNAs that are translated into protein.

Splicing is mediated by a complex of ncRNAs and proteins known

asthespliceosome.Specifically,theU1snRNAhybridizestothe5

splice site at the exon-intron junction, and the U2 snRNA

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Article

hybridizes to the branchpoint site in the intron to initiate splicing of

virtually all human mRNAs (

raphin et al., 1988

We identified a highly specific interaction between the NSP16

viral protein and the U1 and U2 snRNAs (

Figure 1

B). Because U1

and U2 are small RNAs (164 and 188 nt, respectively), we noticed

strong enrichment of NSP16-associated reads across the entire

length of each. To more precisely define the binding sites, we ex-

ploited the well-described tendency of reverse transcriptase to

preferentially terminate when it encounters a UV-crosslinked pro-

tein on RNA (

nig et al., 2010

; Figures 1

Aand

S1D). We deter-

mined that NSP16 binds to the 5

splice site recognition sequence

of U1 (

Figures 2

A, 2B,

S2A, and S2B) and the branchpoint recog-

nition site of U2 (

Figures 2

C, 2D,

S2C, and S2D). These binding

sites are highly specific to NSP16 relative to all of the other viral

and human proteins (

Figures 1

B, S2A, and

2C). Consistent with

its interaction with U1/U2, we observed that NSP16 localizes in

the nucleus upon SARS-CoV-2 infection (

Figures 2

E, S2E, and

S2F) and when expressed in human cells (

Figure S2

G).

NSP16 Disrupts Global mRNA Splicing upon SARS-CoV-

2 Infection

Based on the locations of the NSP16 binding sites relative to the

mRNA recognition domains of the U1/U2 spliceosomal compo-

nents, we hypothesized that NSP16 might disrupt splicing of

newly transcribed genes (

Figure 2

F). To test this, we co-ex-

pressed NSP16 in human cells along with a splicing reporter

derived from IRF7 (an exon-intron-exon minigene) fused to

GFP (

Majumdar et al., 2018

). In this system, if the reporter is

spliced, then GFP is made; if not, then translation is terminated

(via a stop codon present in the first intron), and GFP is not pro-

duced (

Figure 3

A). We observed a more than 3-fold reduction in

GFP levels in the presence of NSP16 compared with a control

human protein (

Figures 3

B and

S3A).

To explore whether NSP16 has a global effect on splicing of

endogenous mRNAs, we measured the splicing ratio of each

gene using nascent RNA sequencing. Specifically, we metaboli-

cally labeled nascent RNA by feeding cells for 20 min with 5-ethy-

nyl uridine (5EU), purified and sequenced 5EU-labeled RNA, and

quantified the proportion of unspliced fragments spanning the 3

splice site of each gene (

Figures 3

C and

S3B). We observed a

global increase in the fraction of unspliced genes in the presence

of NSP16 compared with controls (

Figures 3

D, S3C, and S3D).

Given that NSP16 is sufficient to suppress global mRNA

splicing, we expect that its expression in SARS-CoV-2-infected

cells would result in a global mRNA splicing deficit. To test this,

we infected human lung epithelial cells (Calu3) with SARS-CoV-2

Figure 1. Global RNA Binding Maps of SARS-CoV-2 Proteins

(A) Schematic of our approach.

(B) Enrichment heatmap of each SARS-CoV-2 protein (rows) by significantly enriched 100-nt RNA bins (columns; p < 0.001 and enrichment > 3-fold;

STAR

Methods

). Shared colored bars indicate multiple bins within the same mRNA. For spacing reasons, the 82 mRNAs bound by N protein are displayed separately.

(C) Examples of sequencing reads over specific mRNAs for viral proteins (red) relative to input RNA coverage (gray) are shown. Coding regions (thick lines) and

untranslated regions (thin lines) are shown for each mRNA.