Alternative splicing coupled with transcript degradation modulates OAS1g antiviral activity

Alternative splicing coupled with transcript degradation

modulates OAS1g antiviral activity

LUKE FRANKIW,

MATI MANN,

GUIDENG LI,

1,2,3

ALOK JOGLEKAR,

1,4,5

and DAVID BALTIMORE

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

Center of Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College,

Beijing 100005, China

Suzhou Institute of Systems Medicine, Suzhou 215123, China

Center for Systems Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA

Department of Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA

ABSTRACT

At the heart of an innate immune response lies a tightly regulated gene expression program. This precise regulation is cru-

cial because small changes can shift the balance from protective to destructive immunity. Here we identify a frequently

used alternative splice site in the gene oligoadenylate synthetase 1g (

Oas1g

), a key component of the 2

–

5A antiviral sys-

tem. Usage of this splice site leads to the generation of a transcript subject to decay, and removal of the site leads to in-

creased expression of

Oas1g

and an improved antiviral response. However, removal of the splice site also leads to an

increase in apoptotic cell death, suggesting this splicing event exists as a compromise between the pathogen protective

benefits and collateral damage associated with OAS1g activity. Across the innate immune response, we show that a mul-

titude of alternative splicing events predicted to lead to decay exist, and thus have the potential to play a significant role in

the regulation of gene expression in innate immunity.

Keywords: AS-NMD; alternative splicing; antiviral response; oligoadenylate synthetase; posttranscriptional regulation

INTRODUCTION

Central to an inflammatory response is a robust and coor-

dinated gene expression program. Precise regulation of

this gene expression program is crucial to avoid immune-

mediated collateral damage (Kontoyiannis et al. 1999).

Though transcription and protein turnover are the best-ex-

amined areas of gene expression regulation (Gautier et al.

2012; Chen and Chen 2013; Smale and Natoli 2014; Smale

et al. 2014), a variety of posttranscriptional mechanisms

have emerged that play a role in the fine-tuning of an in-

flammatory response. Well-studied examples include

mRNA stabilization (Hao and Baltimore 2009), mRNA

deadenylation (Leppek et al. 2013), and microRNA regula-

tion (O

’

Connell et al. 2012).

The wealth of transcriptomic data generated over the

last decade has shed light on the widespread nature of al-

ternative mRNA splicing of mammalian genes. While most

mammalian genes exhibit alternative splicing (AS) (Pan

et al. 2008; Wang et al. 2008), not all of the produced tran-

scripts encode functional proteins. Though AS can act to

increase proteomic diversity, it can also generate unpro-

ductive isoforms subject to either cytoplasmic or nuclear

decay (Lareau et al. 2007a; Bitton et al. 2015). In cases

where a splicing event leads to the introduction of a pre-

mature termination codon (PTC), degradation is believed

to occur via the nonsense-mediated mRNA decay (NMD)

machinery in the cytoplasm through a process called alter-

native splicing-coupled NMD (AS-NMD) (Lareau et al.

2007a; Kervestin and Jacobson 2012; Jangi and Sharp

2014). Coupling of AS to NMD provides cells with a poten-

tial mode of down-regulation of expression of a given

gene. It has been estimated that 10%

–

30% of mammalian

genes may be regulated posttranscriptionally through AS-

NMD (Lewis et al. 2003; Mendell et al. 2004; Weischen-

feldt et al. 2012; Jangi and Sharp 2014). However, beyond

splicing factor regulation (Lareau et al. 2007b; Jangi and

Sharp 2014), the extent to which AS-NMD represents post-

transcriptional gene expression control as opposed to

noise in the splicing process is a contentious issue, and

Corresponding author: baltimo@caltech.edu

Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.

073825.119.

RNA Society for the first 12 months after the full-issue publica-

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REPORT

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the role it plays in the finely tuned innate immune response

has yet to be explored (McGlincy and Smith 2008).

Here we identify a frequently used unproductive splicing

event in oligoadenylate synthetase 1g (

Oas1g

), an impor-

tant murine antiviral response factor. Upon binding viral

dsRNA, OAS1g acts to convert ATP into 2

′

–

′

linked oli-

goadenylates (2

–

5A), which in turn activate RNase

L. Activated RNase L degrades viral RNA, in turn inhibiting

viral replication and propagation (Silverman 2007).

Although humans have a single

Oas1

gene, in mice the

Oas1

gene locus underwent a series of duplication events

leading to the existence of eight

Oas1

paralogs. Of the

paralogs, only OAS1a and OAS1g have been shown to

be enzymatically active, with OAS1g producing signifi-

cantly more oligoadenylates as compared to OAS1a

(Kakuta et al. 2002; Elkhateeb et al. 2016). We show that

removal of the

Oas1g

alternative splice site in a murine

macrophage cell line leads to increased expression of

Oas1g

, both in stimulated and unstimulated conditions.

Further, this increased expression of

Oas1g

improves the

ability of macrophages that lack the unproductive splice

site to withstand infection by encephalomyocarditis virus

(EMCV). However, removal of the

Oas1g

alternative splice

site also leads to an increase in apoptotic cell death in un-

infected cells, a finding consistent with the idea that activa-

tion of the 2

–

5A antiviral system can be detrimental to host

fitness (Zhou et al. 1997; Andersen et al. 2007; Carey et al.

2019). Beyond

Oas1g

, we find AS-NMD events in a num-

ber of other crucial innate immune response transcripts,

suggesting this is a common mechanism of mitigation for

responses that might otherwise be unchecked or inappro-

priately scaled.

RESULTS

Oas1g

has a frequent AS-NMD event

AS events have the potential to generate both productive

isoforms coding for functional proteins, as well as unpro-

ductiveisoformssubjecttodegradation(Fig.1A).Thelatter

allows for the use of AS as a posttranscriptional mechanism

of gene-expression regulation. To investigate the extent to

which unproductive splicing acts as a posttranscriptional

regulator of gene expression during inflammation, we ana-

lyzed nuclear fractionation RNA-sequencing data from

mouse bone marrow-derived macrophages (BMDMs) stim-

ulated with the TLR3 agonist poly(I:C) for up to 12 h

(Frankiw et al. 2019). Activation of TLR3 leads to activation

of interferon regulator factors, production of interferon-

and

(IFN-

), and induction of a type I interferon re-

sponse (Perales-Linares and Navas-Martin 2013). RNA-

seq samples were derived from total nuclear RNA so as to

avoid underrepresentation of isoforms subjected to NMD

decay, which occurs rapidly in the cytoplasm. From

this data, we identified frequent usage of an alternative

′

splice site at the third splice junction in the

Oas1g

gene(Fig.1B).Ateachtime-pointweanalyzed,thisalterna-

tive

“

unproductive

”

splice site was frequently selected

over the consensus

“

productive

”

splice site (Fig. 1C, left).

This is evident by comparing the number of reads that

map across the two different junctions, as well as through

the use of the computational program MISO, which utilizes

a probabilistic framework to estimate the expression of al-

ternatively spliced isoforms (Fig. 1C, right; Katz et al.

2010). The metric percent spliced in (PSI;

) is an estimate

of the fraction of transcripts that utilize the alternative

unproductive splice site, and the associated histograms

represent the posterior distributions over

. For each indi-

vidual time-point, derived from a single RNA-seq sample,

the mean

is depicted by the red line on the histogram,

and its value is labeled along with the 95% confidence in-

tervals of the distribution. Higher

values indicate in-

creased usage of the unproductive splice isoform. The

unproductive splice isoform of

Oas1g

represents an ideal

NMD substrate as it contains a termination codon located

>50 nt upstream of a downstream exon

–

exon junction. As

might be expected, depletion of the NMD factor

Upf1

leadsto increased expression levels of

Oas1g

(Supplemen-

tal Fig. S1A,B

). Of interest was the strength of the produc-

tive and unproductive splice site, which can be quantified

using a maximum entropy model (Yeo and Burge 2004).

We find the productive and unproductive 5

′

splice sites

are similar in strength, and are fairly strong with respect

to all expressed junctions (

Supplemental Fig. S1C

Next, we looked at this AS event in the context of all ex-

pressed junctions. To do this, we calculated the alternative

junction usage at each expressed junction from the BMDM

data-set stimulated with poly(I:C) for 8 h (Fig. 1D, data from

other induced time-points shown in

Supplemental Fig.

S1D,E

). From this junction-centric viewpoint, the sequenc-

ing data supports the conclusion that most expressed junc-

tions splice with high fidelity (Fig. 1E). Still, there is some

alternative junction usage, which can be attributed to

both regulated AS events as well as splicing noise. With re-

spect to the alternatively spliced junction of

Oas1g

, it ranks

near the top percentile of alternative junction usage, sup-

porting the conclusion that this AS event is among the

most frequently utilized in poly(I:C) stimulated BMDMs

(Fig. 1E).

Removal of alternative splice site alters

Oas1g

expression and host response to EMCV

In order to explore the effect of this AS event on

Oas1g

ex-

pression, and correspondingly the antiviral response, we

used clustered regularly interspaced short palindromic re-

peats (CRISPR)

–

CRISPR-associated protein-9 nuclease

(Cas9) technology to engineer murine RAW 264.7 cell lines

devoid of this unproductive splice site (Fig. 2A). In parallel,

cell lines expressing Cas9 and a nontargeting guide were

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generated. We selected seven clones that had the splice

site removed in both alleles, which we designate as

“

fixed

”

clones (Fig. 2B;

Supplemental Fig. S2

). RT-PCR with RNA

from whole-cell fractions that was stimulated with poly(I:

C) both confirmed alternative splice site usage in control

populations, and showed forced productive splicing in

these fixed clones (Fig. 2C).

To determine what effect this forced productive splicing

has on

Oas1g

expression, we monitored mRNA levels of

Oas1g

in both unstimulated and stimulated [8 h poly(I:C)]

conditions. In each case, the engineered lines lacking

the unproductive

Oas1g

splice site had significantly higher

levels of expression, presumably due to lack of NMD asso-

ciated with selection of the unproductive splice site (Fig.

2D). Of interest, levels of

Oas1g

in unstimulated

Oas1g

splice site engineered cells were similar to levels of

Oas1g

in stimulated control cells. Next, to determine the

effect of removal of the unproductive splice site with re-

spect to the antiviral response, we used EMCV to infect

both groups of macrophages. EMCV is a (+)ssRNA mem-

ber of the

Picornaviridae

family that replicate through par-

tially dsRNA intermediates (Carocci and Bakkali-Kassimi

2012). Infection has been shown to cause accumulation

of 2

–

5A, and viral replication is sensitive to the OAS/

RNase L pathway (Hearl and Johnston 1987; Zhou et al.

1997). As oligoadenylate synthetases bind viral dsRNA,

the RNA activators in EMCV-infected cells are believed

to be the viral replicative intermediates (Silverman 2007).

Upon 18 h of infection with EMCV, we again observed sig-

nificantly higher levels of

Oas1g

expression in the engi-

neered lines lacking the unproductive

Oas1g

splice site

(Fig. 2E). Using qPCR to measure levels of EMCV following

FIGURE 1.

Oas1g

has a frequent AS-NMD event. (

) A schematic depiction of an AS event leading to either a productive isoform destined for

translation or an unproductive isoform destined for degradation. (

) Sashimi plot for the entire gene body of

Oas1g

from BMDMs stimulated with

poly(I:C) for 12 h. Sequenced RNA is derived from the total nuclear fraction.

Oas1g

is a negative strand gene and is oriented such that the negative

strand runs

left

right

)(

Left

) Sashimi plots centered at the third junction of

Oas1g

from BMDMs stimulated with poly(I:C) for 0, 1, 4, 8, and

12 h. Sequenced RNA is derived from the total nuclear fraction. The

-axis represents reads per kilobase of transcript, per million mapped reads

(RPKM). (

Right

) Posterior distributions of the

value for each individual time point. The mean

is depicted by the red line. Mean and 95% con-

fidence intervals are labeled to the

right

of the posterior distribution. (

) Schematic representation of the alternative junction usage calculation.

(

) Pie chart representing alternative junction usage for all expressed junctions upon 8 h. of poly(I:C) stimulation. The slice including the alterna-

tively spliced third junction of

Oas1g

is labeled (alt. junction usage 0.21

–

0.40). Genomic coordinates represent the mm9 genome assembly.

Frankiw et al.

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18 h of infection, we found that the engineered lines con-

trolled viral replication more efficiently than the control

lines (Fig. 2F). While this effect was relatively minor (ap-

proximately twofold), the effect was consistent across the

clones and was statistically significant. Thus, we conclude

that forced productive splicing of

Oas1g

improves the an-

tiviral defense through increased expression of

Oas1g

Finally, since activation of the 2

–

5A system has been

FIGURE 2.

Removal of alternative splice site alters

Oas1g

expression and macrophage response to EMCV. (

) Schematic representation of the

two alternative splice isoforms, and the gRNA/Cas9 targeting of the alternative splice site. (

) Sanger sequencing gDNA from a control sample

(

top

) and an

Oas1

SS KO sample (

bottom

). Sequencing is oriented such that the negative strand runs

left

right

. The alternative splice site is rep-

resentedbytheyellowhighlightedregion.(

)RT-PCRuponstimulationwithpoly(I:C)confirmingalternativesplicesiteusageincontrolpopulations

and forced productive splicing in fixed clones. (

) RT-qPCR analysis of

Oas1g

mRNA levels in unstimulated and stimulated [8 h poly(I:C)] macro-

phages. Control samples are represented in light blue, SS KO clones are represented in dark blue. (

) RT-qPCR analysis of

Oas1g

mRNA levels in

EMCV infected (18 h) macrophages. Control samples are represented in light blue, SS KO clones are represented in dark blue. (

) RT-qPCR mea-

surement of EMCV viral load following 18 h of infection at 1 MOI. Control samples are represented in light blue, SS KO clones are represented in

darkblue.(

)AnnexinVstainingforapoptoticcellsunderunstimulatedconditions.Controlsamplesarerepresentedinlightblue,SSKOclonesare

representedindarkblue.Dataisrepresentativeoftwoindependentexperiments(

–

)andisshownasmean(errorbarsindicateSEM).(

∗

)

< 0.05,

(

∗∗

)

<0.01, and (

∗∗∗

)

< 0.001 using a Student

’

-test. Results are presented relative to those of

Rpl32

(

–

AS-NMD modulates OAS1g antiviral activity

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shown to promote apoptosis in host cells (Castelli et al.

1997), we were interested in determining whether removal

of the unproductive

Oas1g

splice site altered the levels of

apoptotic cells. We observed approximately twofold in-

crease in the fraction of apoptotic cells in the engineered

lines lacking the unproductive

Oas1g

splice site as com-

pared to the control cells (Fig. 2G) in unstimulated condi-

tions. We conclude that the increased

Oas1g

observed

with the removal of the unproductive splice site leads to in-

creased levels of apoptosis in a cell population.

Of note, the other enzymatically active member of the

murine

Oas1

family,

Oas1a,

has a highly homologous junc-

tion with an identical unproductive splice site. However,

despite nearly complete similarity of sequence at and

near this splice-site (

Supplemental Fig. S3A

), it is used

less frequently than that of

Oas1g

(Supplemental Fig.

–

D). Because of this similarity, our guide targeted to

the unproductive splice site of

Oas1g

also cut at

Oas1a

(Supplemental Fig. S4), and genotyping confirmed all se-

lected clones deleted the

Oas1a

unproductive splice site

in addition to the

Oas1g

unproductive splice site. Again,

RT-PCR with RNA whole-cell fractions upon stimulation

with poly(I:C) confirmed alternative splice site usage in

control populations, and showed forced productive splic-

ing in edited clones (

Supplemental Fig. S3C

). To deter-

mine what effect this forced productive splicing has on

Oas1a

expression, we monitored

Oas1a

mRNA levels in

both unstimulated and stimulated [8 h poly(I:C)] condi-

tions. In this case, we found that while the mean expression

Oas1a

in both unstimulated and stimulated conditions

was greater in nonengineered clones, the effect lacked

significance (

Supplemental Fig. S3D

). We hypothesize

that the dampened effect with respect to

Oas1a

as com-

pared to

Oas1g

is likely due to decreased usage of the un-

productive splice site to begin with, but also note that the

small differences observed in

Oas1a

expression levels

could play a role in the aforementioned antiviral and apo-

ptosis effects.

Human

Oas1

is regulated at the posttranscriptional

level through productive and unproductive

alternative splicing

Human

Oas1

differs quite significantly from the mouse

Oas1

paralogs, a finding that is perhaps not surprising giv-

en the volatile evolutionary history of the gene (Kumar

et al. 2000; Hancks et al. 2015; Fish and Boissinot 2016).

Despite differences between human

Oas1

and mouse

Oas1g

, human

Oas1

is extensively regulated at the post-

transcriptional level through both productive and unpro-

ductive AS. Productive AS at the 3

′

end of human

Oas1

gives rise to six isoforms (p42, p44(a/b), p46, p48, and

p52) (Fig. 3A; Productive AS Events (Kjær et al. 2014).

Expression of these isoforms varies in humans. For exam-

ple, RNA-sequencing of IFN-

stimulated peripheral

blood mononuclear cells (PBMCs) from two different

healthy donors reveals one donor expresses primarily the

p44a isoform, while the other expresses primarily the

p46 isoform (Fig. 3B). Genetic variation at the p46 splice-

acceptor locus plays a role in this alteration of isoform

abundance. There exists a single G/A SNP (rs10774671)

in the p46 exon 6 splice-acceptor. Those with the G allele

predominantly produce p46, while the A allele leads to

production of the other isoforms (Fig. 3A). Of the isoforms,

the p46 isoform has been shown to have the greatest oli-

goadenylate synthetase activity, an effect mediated at

least in part by defects in protein accumulation of the other

isoforms (Bonnevie-Nielsen et al. 2005; Liu et al. 2017;

Carey et al. 2019). As might be expected, production of

the high activity isoform has been shown to dampen sus-

ceptibility to and/or severity of a variety of viral infections,

including those mediated by West Nile virus (Lim et al.

2009), Epstein

–

Barr virus (Liu et al. 2017) and hepatitis C

virus (Knapp et al. 2003). However, there also appear to

be costs associated with high OAS1 activity in humans,

most notably in response to dengue virus-2 infection

(Simon-Loriere et al. 2015). The G allele, which leads to

production of the high activity p46

Oas1

isoform, is associ-

ated with increased susceptibility to plasma leakage and

shock in infected individuals, indicating immune overreac-

tion could be triggered by increased OAS1 activity arising

from altered AS (Simon-Loriere et al. 2015).

In addition to the productive splicing events at the

′

end of the

Oas1

transcript that lead to the generation

of multiple isoforms, we identified another unproductive

splicing event at the third splice junction (Fig. 3A;

Unproductive AS Event). This unproductive splicing event

is mediated by a 3

′

splice site located 47 bp into exon

3. When this unproductive splice site is selected, the frame

of the transcript is shifted and a PTC is incorporated into

the transcript, making it an ideal NMD substrate. This un-

productive splice site is used frequently in a variety of

cell types in response to a number of stimulation condi-

tions (Fig. 3C

–

E). In general,

∼

10% of spliced reads at

this junction splice to the unproductive 3

′

splice site,

though it is worth noting all of the human samples were de-

rived from whole-cell RNA and thus, might underestimate

the frequency of usage of this unproductive splice site due

to efficient degradation of PTC containing transcripts by

the NMD decay machinery in the cytoplasm. In conclusion,

though human

Oas1

and mouse

Oas1g

differ significantly,

they are both regulated posttranscriptionally via AS.

AS-NMD events are common in transcripts related

to innate immunity

While

Oas1g

contained one of the most frequently used

AS-NMD events, it was not the only AS-NMD event found

in genes related to the innate immune response. For exam-

ple, in nuclear fractionation RNA-sequencing data from

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mouse BMDMs stimulated with poly(I:C), we found signifi-

cantly utilized cassette exon events that lead to a frame-

shift and incorporation of a PTC in the important innate

immune response transcripts

Mx1

IKK

ε

, and

Oasl2

(Fig.

–

C). In each case, the exclusion of the cassette exon

leads to an ideal NMD substrate. These events were also

confirmed in the RAW264.7 macrophage cell line with

RT-PCR with RNA from whole-cell fractions that had

been stimulated with poly(I:C) for 4, 8, and 12 h (Fig.

–

F).

To classify AS-NMD events globally, we utilized the tool

SplAdder to predict and quantify AS events supported by

an input sample (Kahles et al. 2016). A stringent confi-

dence criterion was required to avoid including AS events

derived from splicing noise. Then, only events that lead to

frameshifts and/or PTC inclusion were selected. Among

the list of AS-NMD events, as compared to a background

of expressed genes, we observed significant enrichment

for Gene Ontology (GO) terms associated with the innate

immune response (Fig. 4G). With respect to the viral path-

ogen response, which is tasked with limiting viral replica-

tion through degradation of viral (as well as nonviral)

mRNA and establishment of a cellular antiviral state, a

host of factors involved with the response contain AS-

NMD events that are identified here or in other published

work (Fig. 4H; Frankiw et al. 2019).

DISCUSSION

The robust and coordinated gene expression program in-

volved in the defense against pathogens requires tight

regulation. In this study, we sought to shed light on the

role of AS-NMD in this regulation. We identified a fre-

quently used unproductive splicing event in

Oas1g

, an im-

portant murine antiviral response factor, and show that

forced productive splicing leads to increased

Oas1g

ex-

pression and further, an increased ability to clear virus.

Additionally, we identified a number of other examples

of unproductive splicing events in the innate immune re-

sponse which could subject the corresponding transcript

to decay via the NMD pathway.

With respect to

Oas1g

, what benefit might this AS event

offer? The alternative splice site mediating this AS-NMD

event is of comparable strength to the consensus 5

′

splice

site ( Supplemental Fig. S1C

). If possession of the greatest

pathogen defense were the only goal of an organism, it

seems unlikely this splice site would be retained.

However, while pathogen defense systems can provide a

FIGURE 3.

Human

Oas1

is regulated at the posttranscriptional level through productive and unproductive AS. (

) Depiction of the mRNA splice

isoforms found in human

Oas1

. There exists a single G/A SNP in the OAS1 exon 6 splice-acceptor (rs10774671), with the G variant producing the

more active p46 isoform. (

) Sashimi plots depicting the productive AS event at the 3

′

end of human

Oas1

. RNA is from PBMCs stimulated with

IFN-

derived from two healthy donors. Genomic coordinates represent the hg19 genome assembly. (

–

) Sashimi plots for an AS-NMD event

identified in exon 3 of human

Oas1

from healthy donor derived PBMCs either unstimulated (

top

) or stimulated with IFN-

(

), A549 cells unin-

fected (

top

) or infected with influenza virus PR8 (

), or primary macrophages from two separate donors infected with West Nile virus (

). Genomic

coordinates represent the hg19 genome assembly. All sequencing samples in Figure 3 are derived from whole-cell RNA.

AS-NMD modulates OAS1g antiviral activity

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protective benefit, they also can cause collateral damage

to a host. With respect to

Oas1

, its pathogen defense ef-

fects are repeatedly forfeited by a host due to the fact

that its activity can be so detrimental (Kjaer et al. 2009;

Carey et al. 2019). This is exemplified by the surprisingly

high frequency of loss-of-function mutations in

Oas1

in pri-

mates (Carey et al. 2019), and the fact that OAS1 activity

has been completely lost in several animal lineages, in-

cluding teleost fish and insects (Kjaer et al. 2009).

Moreover, while mice deficient for RNase L, the down-

stream effector of

Oas1

in the 2

–

5A system, exhibit sus-

ceptibility to viral infection (Zhou et al. 1997), in the

absence of infection they display significantly increased

longevity (Andersen et al. 2007). Given the fact that host

RNAs have been shown to be able to activate OAS en-

zymes, it is reasonable to hypothesize that the longevity

FIGURE 4.

AS-NMD events are common in transcripts related to innate immunity. (

) Sashimi plots for an AS-NMD event identified in

Mx1

from

BMDMs stimulated with poly(I:C) for 0, 1, 4, 8, and 12 h. Sequenced RNA was derived from the total nuclear fraction. The

-axis represents reads

per kilobase of transcript, per million mapped reads (RPKM). (

) Same as

for

IKK

ε

) Same as

for

Oasl2

) RT-PCR of

Mx1

upon stimulation

with poly(I:C) for 4, 8, and 12 h. (

) Same as

for

IKK

ε

) Same as

for

Oasl2

) GO terms enriched for AS-NMD events, as compared to a

background of expressed genes. (

) Schematic representation of major pathways in the viral pathogen response. Red arrows are shown

above

factors containing AS-NMD events. Data are representative of two independent experiments (

–

). Genomic coordinates represent the mm9

genome assembly.

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