Strand-Specific Dual RNA Sequencing of Bronchial Epithelial Cells Infected with Influenza A/H3N2 Viruses Reveals Splicing of Gene Segment 6 and Novel Host-Virus Interactions

Strand-Specific Dual RNA Sequencing of Bronchial Epithelial

Cells Infected with Influenza A/H3N2 Viruses Reveals Splicing

of Gene Segment 6 and Novel Host-Virus Interactions

Giulia Fabozzi,

*

Andrew J. Oler,

Poching Liu,

Yong Chen,

Samuel Mindaye,

Michael A. Dolan,

Heather Kenney,

Marjan Gucek,

Jun Zhu,

Ronald L. Rabin,

Kanta Subbarao

*

a_{Laboratory of Immunobiochemistry, Division of Bacterial, Parasitic and Allergenic Products, Office of Vaccines}

Research and Review, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland, USA

b_{Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health,}

Bethesda, Maryland, USA

c_{Bioinformatics and Computational Biosciences Branch, Office of Cyber Infrastructure and Computational}

Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

d_{Systems Biology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda,}

Maryland, USA

e_{Proteomics Core, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland,}

USA

f_{Emerging Respiratory Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and}

Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

ABSTRACT

Host-influenza virus interplay at the transcript level has been

exten-sively characterized in epithelial cells. Yet, there are no studies that simultaneously

characterize human host and influenza A virus (IAV) genomes. We infected human

bronchial epithelial BEAS-2B cells with two seasonal IAV/H3N2 strains, Brisbane/

10/07 and Perth/16/09 (reference strains for past vaccine seasons) and the

well-characterized laboratory strain Udorn/307/72. Strand-specific RNA sequencing

(RNA-seq) of the infected BEAS-2B cells allowed for simultaneous analysis of host and viral

transcriptomes, in addition to pathogen genomes, to reveal changes in mRNA

ex-pression and alternative splicing (AS). In general, patterns of global and immune

gene expression induced by the three IAVs were mostly shared. However, AS of host

transcripts and small nuclear RNAs differed between the seasonal and laboratory

strains. Analysis of viral transcriptomes showed deletions of the polymerase

compo-nents (defective interfering-like RNAs) within the genome. Surprisingly, we found

that the neuraminidase gene undergoes AS and that the splicing event differs

be-tween seasonal and laboratory strains. Our findings reveal novel elements of the

host-virus interaction and highlight the importance of RNA-seq in identifying

molec-ular changes at the genome level that may contribute to shaping RNA-based innate

immunity.

IMPORTANCE

The use of massively parallel RNA sequencing (RNA-seq) has revealed

insights into human and pathogen genomes and their evolution. Dual RNA-seq

al-lows simultaneous dissection of host and pathogen genomes and strand-specific

RNA-seq provides information about the polarity of the RNA. This is important in the

case of negative-strand RNA viruses like influenza virus, which generate positive

(complementary and mRNA) and negative-strand RNAs (genome) that differ in their

potential to trigger innate immunity. Here, we characterize interactions between human

bronchial epithelial cells and three influenza A/H3N2 strains using strand-specific dual

RNA-seq. We focused on this subtype because of its epidemiological importance in

caus-ing significant morbidity and mortality durcaus-ing influenza epidemics. We report novel

ele-Received27 March 2018Accepted24 May 2018

Accepted manuscript posted online5 July 2018

CitationFabozzi G, Oler AJ, Liu P, Chen Y, Mindaye S, Dolan MA, Kenney H, Gucek M, Zhu J, Rabin RL, Subbarao K. 2018. Strand-specific dual RNA sequencing of bronchial epithelial cells infected with influenza A/H3N2 viruses reveals splicing of gene segment 6 and novel host-virus interactions. J Virol 92:e00518-18.

https://doi.org/10.1128/JVI.00518-18.

EditorAdolfo García-Sastre, Icahn School of Medicine at Mount Sinai

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

Address correspondence to Ronald L. Rabin, ronald.rabin@fda.hhs.gov, or Kanta Subbarao, KSUBBARAO@niaid.nih.gov.

*Present address: Giulia Fabozzi, Vaccine

Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA; Kanta Subbarao, WHO Collaborating Centre for Reference and Research on Influenza and the Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.

G.F. and A.J.O. contributed equally to this article.

crossm

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ments that differ between seasonal and laboratory strains highlighting the complexity of

the host-virus interplay at the RNA level.

KEYWORDS

alternative splicing, influenza A virus H3N2, neuraminidase, Udorn/72

strain, bronchial epithelial cells, dual RNA-seq, host-virus interaction, seasonal H3N2

strains, strand-specific RNA seq, viral transcriptome

I

nfluenza A virus (IAV) (family

Orthomyxoviridae

) contains a negative-sense,

single-stranded RNA genome divided into eight segments. The eight gene segments are:

polymerase basic 2 (PB2), polymerase basic 1 (PB1), polymerase acidic (PA),

hemagglu-tinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix (M), and nonstructural

protein (NS) (1). Gene segments M and NS are known to undergo splicing to generate

additional proteins (2, 3). Recently, a novel PB2 splicing product has been reported (4).

Gene segments HA and NA encode surface glycoproteins whose antigenic properties

are used to classify IAV subtypes.

When two influenza viruses coinfect a host, their gene segments can be exchanged

in a phenomenon referred to as genetic reassortment, and this can result in the

generation of novel influenza viruses. If the novel reassortant virus is able to infect

humans, cause disease, and spread from person to person, it may have the potential to

cause a pandemic. In 1968, a human IAV H2N2 virus reassorted with an avian H3N2

virus (5), giving rise to the H3N2 pandemic strain (6). Since 1968, H3N2 viruses have

caused epidemics associated with higher levels of morbidity and mortality than H1N1

viruses (7).

During the influenza life cycle, genomic viral RNA (vRNA) is transcribed into

mes-senger RNA (mRNA), and complementary RNA (cRNA); the latter serves as the template

for generation of progeny vRNA. In addition, other RNAs can be synthesized, such as

small viral RNAs that have been proposed to regulate the switch between transcription

and replication (8) and defective interfering (DI) RNA that can act as

pathogen-associated molecular patterns (PAMPs) (9). Host pattern recognition receptors (PRRs)

can discriminate between PAMPs produced during active replication (vita-PAMPs; e.g.,

viral genome) from those generated as intermediates during viral replication (e.g.,

double-stranded RNA [dsRNA]) (10, 11). Stimulation of PRRs triggers several cellular

responses, including interferon regulatory factor 3 and 7 (IRF3/IRF7)-activated

expres-sion of type I and III interferons (IFNs) (12, 13).

Influenza infection of epithelial cells (14–16) induces expression of types I and III

IFNs, which activate the STAT1/STAT2/IRF9 (ISGF3) complex, which in turn triggers

expression of more than 300 IFN-stimulated genes (ISGs) (17). Gene expression changes

in epithelial cells infected with IAV H1N1 and H5N1 subtypes and with laboratory

strains Puerto Rico/8/34 and Udorn/307/72 (UD/72) have been described (18, 19).

Deep-sequencing analysis of 284 IAV/H3N2 strains that circulated from 1968 to 2011

(20) provide insights about virus evolution and antigenicity. To date, the only example

of dual RNA sequencing (RNA-seq) study with influenza viruses is from murine lungs

infected with two avian viruses, H5N1 and H5N8 (21). However, there are no dual

RNA-seq studies that have simultaneously characterized the transcriptome of human

epithelial cells and the genome and transcriptome of clinically relevant IAV strains.

While the role of alternative splicing (AS) in human diseases has been studied

intensively (22, 23), the importance of AS and noncoding RNAs and other

posttran-scriptional regulatory mechanisms in the control of inflammatory response(s) has only

been recently reported (24). For example, murine transcriptome studies revealed novel

transcripts and AS of host genes expressed in response to mouse-adapted respiratory

viruses (25), and a well-characterized example of AS of host genes during viral infection

is the herpes simplex virus 1-induced AS of

MX1

, whose gene product favors virus

replication (26). However, whether IAV modulates AS of human host genes encoding

immune response and/or key biological functions is not known.

Here, we report host-virus interactions at the genomic level using the human

bronchial epithelial cell line BEAS-2B infected with two seasonal H3N2 viruses and the

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well-characterized H3N2 laboratory strain UD/72. We selected BEAS-2B cells because it

is a nonmalignant cell line isolated from the normal human bronchial epithelium of a

cancer-free individual (27, 28), thus representing cells that are the primary target of

influenza virus infection. By using strand-specific dual RNA-seq, the transcriptome of

BEAS-2B cells infected with seasonal or laboratory strains revealed immune response

patterns centered on a subset of IRFs, the expression of DI-like RNAs, and AS of host

transcripts. Analysis of the H3N2 transcriptome revealed that the NA gene undergoes

AS and that active splice sites differ between seasonal and laboratory strains.

RESULTS

RNA-seq of H3N2 viruses.

We infected BEAS-2B cells with seasonal A/Brisbane/10/

2007 (BR10/07) and A/Perth/16/2009 (PER16/09), reference strains for IAV/H3N2

vac-cines in the 2008 –2010, and 2010 –2011 seasons, respectively, and with the laboratory

strain UD/72. Total RNA was extracted from mock- and virus-infected cells at 1, 6, or 24

h postinfection (hpi) and subjected to a dual strand-specific RNA-seq strategy using

Illumina HiSeq. This strategy helps to differentiate the negative-strand RNA (vRNAs)

from positive-strand RNAs (cRNAs and mRNAs), which are synthesized during viral

transcription/replication. A schematic of the experiment is shown in Fig. 1A.

Figure 1B shows the distribution of host and viral read counts for each sample. By

normalizing viral and host read coverage, an RNA-based growth curve of IAV/H3N2 was

FIG 1Strand-specific dual RNA-seq and IAV/H3N2 genomics. (A) Schematic of the RNA-seq approach. RNA samples extracted from mock- and

IAV/H3N2-infected cells at time points 1, 6, and 24 h were subjected to a strand-specific RNA-seq assay which maintains the polarity of host and virus RNA. (B) Total

RNA-seq read counts were mapped by sample (xaxis) and read fraction distribution (yaxis). IAV/H3N2 viral growth curves obtained by the following means:

normalized viral reads, expressed as reads per million mapped reads (RPM) for IAV/H3N2-BR10/07 (blue), PER16/09 (red), and laboratory strain UD/72 (green) (C) and viral titer in BEAS-2B cells (D). (E) Principal component analysis demonstrating similarity between samples and biological replicates for each sample. (F) Temporal expression of influenza viral RNAs. Negative (top)- and positive (bottom)-stranded RNAs of each viral segment (gray boxes) expressed as reads per kilobase of sequence per million mapped reads (RPKM) normalized to total host reads. Viral segments are ordered according to size.

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generated (Fig. 1C). Growth curves of the three viruses were confirmed by virus titration

in BEAS-2B cells (Fig. 1D). Principal component analysis (Fig. 1E) shows that all the 1-hpi

time points clustered with the 1-h controls and that the seasonal IAVs at 6 hpi clustered

with the 6-h controls. However, UD/72 6-hpi samples cluster with BR10/07 and

PER16/09 24-hpi samples, consistent with the rapid growth kinetics of UD/72 compared

to the seasonal strains (Fig. 1D).

Viral genomics of seasonal and laboratory H3N2 strains.

The relative temporal

expression of vRNA versus cRNA/mRNA from each gene segment was tracked by

normalization of read coverage by viral gene segment to total host reads (Fig. 1F).

vRNAs of the two seasonal strains were relatively constant between 6 and 24 hpi, i.e.,

after transcription initiation (29), whereas vRNAs PB2, PB1, PA, and M of UD/72 were

upregulated from 6 to 24 hpi; at the latter point primary transcription is expected to be

complete.

In accordance with published literature (30, 31), positive-strand RNAs for the

poly-merase segments (PB2, PB1, and PA) of all three viruses were expressed at lower

abundance than other segments at each time point. Although we are not able to

discriminate between cRNA and mRNA, our results indicate higher relative levels of

mRNAs or cRNAs of gene segments HA, NP, M, and NS in the laboratory strain UD/72

at 6 hpi compared to seasonal strains, confirming the growth kinetics of UD/72 shown

in Fig. 1D.

DI-like RNA expression during H3N2 infection.

Viral coverage plots at the base

pair resolution revealed prominent edges for the PB2, PB1, and PA segments for all

viruses (see Fig. S1 in the supplemental material), but most noticeably for PER16/09

(Fig. 2). The valley between these “edges” corresponds to genomic deletions consistent

with reads mapping to vRNAs that are commonly associated with DI RNAs (32). DI RNAs

were reported mostly during

in vitro

infection at a high multiplicity of infection (MOI)

(33). However, DI-like RNAs were also described in clinical specimens following

infec-tion with the pandemic strain A/California07/2009 (H1N1pdm09) (34). We use the term

DI RNA to refer to reports that particles have a defective interfering effect in a

functional assay, and the term DI-like RNA when referring to our data and/or previous

reports in which the RNA appear to be similar to those previously reported as DI RNA

but interference has not been confirmed in a functional assay. As the fraction of DI-like

RNA to vRNA seems to be important for the outcome of an infection (33), we measured

this fraction and found it to be noticeably higher for all three viral polymerase

segments of PER16/09 than either BR10/07 or UD/72 (Fig. 2G). To determine whether

the high ratio in PER16/09 was due to presence of DI particles in viral stocks, we

calculated the particle/infectivity ratio of the viral stocks used to infect BEAS-2B cells by

HA and plaque titers. Each IAV/H3N2 viral stock had a similar HA titer, but infectivity

varied modestly (30-fold), with PER16/09 virus having the lowest infectivity titer and

UD/72 having the highest. Based on analysis of the start and end coordinates of

genomic deletions found in PER16/09, it appears that DI-like RNAs originate from

preferred locations (“hot spots”) (Fig. 2B, D, and F).

Alternative splicing of gene segment 6 (NA) of IAV/H3N2 viruses.

Influenza

mRNA of segments 1 (PB2), 7 (M), and 8 (NS) are known to undergo AS to generate

additional products (2–4, 35, 36). We found that segment 6 (NA) of all three IAV/H3N2

viruses underwent splicing (Fig. 3) based on two criteria: (i) the splicing event was

observed exclusively in the positive-strand RNA, thereby excluding a genomic deletion

in the vRNA, and (ii) conserved canonical or noncanonical splice site signals were

present (37), including donor and acceptor splice sites (referred to as 5=ss and 3=ss) and

the branch point sequence. The known spliced isoform of the NS mRNA segment was

confirmed as a positive control (see Fig. S2 in the supplemental material).

To quantitatively compare expression of spliced transcripts between samples, we

filtered the reads to keep all non-split positive-strand reads and all split reads with

junctions that were exclusive to the positive strand (Fig. 3A). In addition to the

unspliced NA transcript (termed NA1), we predicted seven AS transcripts (NA2-8;

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PER16/09_6h Reads

Coverage

[0-100000]

p b 0 0 0 , 2 p

b 0 0 5 , 1 p

b 0 0 0 , 1 p

b 0 0 5 p

b 0

FIG 2Deletions of the polymerase gene segments of PER16/09. By strand-specific RNA-seq analysis, deletions of gene segment PB2, PB1, and

PA are mapped on the negative-strand RNA. (The 6-hpi time point is displayed as representative.) RNA polarity is color-coded (pink,

(Continued on next page)

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Fig. 3B). Predicted NA spliced transcripts were validated by reverse transcription-PCR

(Fig. 3C) using the primer pairs shown in Fig. 3B, followed by cloning and Sanger

sequencing to validate the splice junctions. By using the primer pair NAFw26 and

NARv1272 designed to detect NA2-7, amplicons generated from the cDNA of UD/72

NAdel NA6 NA5 NA4 NA3 NA8 NA7 NA1 NA2

72 509 947 1385

[0 - 14000]

264

59 122

[0 - 14000] UD/72_6h

3162 172

121 57 10730

[0 - 28000] PER16/09_24h

4405 407

[0 - 28000] PER16/09_6h

2376 198

[0 - 28000] BR10/07_6h

127

[0 - 28000] BR10/07_24h

323 4015 39 148 UD/72_24h

B

10 160 39 5 8 6 0 95 972 11311254

149 5 9 F 6 2 F NA Rv1272 NA Rv1326

NA

N 3

N 5

NA

N 7

NA

N 8

V rus

R A s q

Po itive clones

(% o t t l)

Size

(bp

415

401

461

6 3

8 9

901

1153

UD 72

+

+

+

+

+

+

-PER16 0

BR10/07

-+

PER16 0

BR10 0

-38

80

UD 7

3

38

3

2

1

n.d.

-Kb 1 6 1 6 1 6 1 6 24

87.9/99.3 0.5/- 9.8/- 0.5/- 0.3/- -/0.7 0.3/-% of total UD/PER

0.4

FIG 3Splicing of segment 6 (NA) in BEAS-2B cells. (A) Sashimi plots (99, 100) of RNA-seq reads from 6- and 24-hpi samples (positive-strand reads randomly

subsampled to the same number) aligned to the NA segment of BR10/07 (blue), PER16/09 (red), or UD/72 (green). (Bottom left) An annotation track shows all predicted spliced transcripts of NA (blue lines/bars), and one positive-strand deletion (NAdel). (Bottom right) The percentages of predicted spliced transcripts showing UD/72 (left side) and one of the seasonal strains, PER16/09 (right side), are shown. (B) Architecture of NA splicing. A schematic representation of the

full-length NA transcript is presented, omitting the cap and poly(A) tail (top, line) containing the alternative 5=ss (dark blue) and 3=ss (orange). Primers NAFw1

and NARv1272 were used to detect all predicted RNA isoforms; primers NAFw950 and NARv1326 were used to confirm the NA8 isoform. The expected size of the RNA species and the percentages of positive clones obtained from cloning are reported (right; n.d., not determined). (C) Electrophoretic analysis of cDNA fragments corresponding to the NA species expressed by each virus. The contrast was adjusted for NA8 in panel C, the third image.

FIG 2Legend (Continued)

negative-strand RNA; blue, positive-strand RNA). Integrative Genomics Viewer (IGV) screen shots of PB2, PB1, and PA vRNA reads (A, C, and E)

are shown. For each deletion, the start/stop coordinates are indicated by theyandxaxes and the abundance of the deletion are expressed by

the sizes of the circle (B, D, and F). (G) Percentage of DI-like genomic (negative-strand) reads as a fraction of total genomic (negative-strand) reads

of PA (red), PB1 (green), and PB2 (blue) during infection of each IAV/H3N2 (xaxis) virus. The time points are indicated at the top.

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showed a different pattern compared to those amplified from cDNAs of BR10/07 and

PER16/09. All predicted NA spliced transcripts were confirmed, except NA7. Five clones

(2.5%) represent NA2, 76 (38%) represent NA3, and the numbers of clones representing

NA4, NA5, and NA6 were 6 (3%), 4 (2%), and 2 (1%), respectively. By the use of the

primer pair NAFw950 and NARv1326, designed to detect NA1 and NA8, we amplified

the fragment containing the predicted spliced form, NA8. By sequencing the cloned

fragments, we confirmed the presence and structure of NA8. Eight clones (80%) from

PER16/09 and 5 clones (38.5%) from BR10/07 represent NA8. While NA2 to NA7 (NA2-7)

occurred during infection with UD/72, NA8 occurred only during infection with the two

seasonal strains (Fig. 3B). Similar to the AS of segment M (38), NA2-7 use five alternative

5=ss, as well as two 3=ss. The different 5=

and 3=ss between UD/72 and the two seasonal

viruses may explain strain-specific AS events (Fig. 4). For example, NA2 splice donor and

acceptor sites follow the canonical GT-AG rule in UD/72, however, the same splice

donor site in BR10/07 and PER16/09 is AT instead of GT, consistent with the lack of NA2

transcript in BR10/07 and PER16/09. Indeed, splicing prediction scores of all 5=ss

observed in different strains are consistent with our observed strain-specific transcript

variants, i.e., strains in which a spliced transcript is observed have higher scores than

strains in which that transcript variant is not observed (Fig. 4). A similar pattern is seen

with the NA8 3=ss (i.e., higher score in PER16/09 and BR10/07 than in UD/72); however,

for the 3=ss of NA2-7, the scores are similar between strains. This pattern can also

explain the differential usage of the two alternative 3=ss: the more commonly used 3=ss

at position 972 has a higher score than the 3=ss at position 958. We also identified a

Virus Effect

FIG 4Molecular features of NA splicing of IAV/H3N2. Molecular features of the predicted spliced isoforms of NA of IAV/H3N2 are shown at the nucleotide level.

The positions and sequences of the 5=ss and 3=ss and the branch point sequence are shown. Maximum entropy scores for each 5=ss and 3=ss (MaxEnt score)

are shown (frameshift, gray box; in-frame, black box). The numbering in the “Effect” column is related to the amino acid length of the predicted gene product. Other numbering refers to nucleotides within the consensus of three assembled virus NA segments.

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prominent positive-strand deletion near the N terminus in PER16/09 (NAdel; Fig. 3A).

Because no consensus donor, acceptor, or branch sequence was identified, we

con-clude this is not likely a result of alternative splicing.

Next, we used computational methods to determine the relative expression of each

spliced isoform as a fraction of all isoforms (including the unspliced or native isoform),

for NA, M, and NS. Isoforms NA2 and NA4-7 are each estimated to be expressed at levels

ranging from 0.3 to 0.7% at 6 and 24 hpi in UD/72 infected cells (Table 1). These levels

are comparable to the expression level of M2 (0.4 to 0.6%) and slightly lower than NS2

(2.1 to 5.1%) in UD/72 at the same time points. NA3 expression is higher than NA2 and

NA4-7, comprising 9.8 and 11.2% at 6 and 24 hpi, respectively. NA8 is expressed at 0.5

to 1.7%, which is also comparable to the expression of M2 (0.8 to 1.1%) and NS2 (0.7

to 1.5%) in seasonal BR10/07 and PER16/09 at 6 and 24 hpi. Because cRNA cannot be

separated from mRNA in our RNA-seq data, these fractional values for spliced isoforms

may be lower than the actual values.

To determine whether any of these splice variants are expressed in other viral

strains, we performed a BLAST search against all IAV/H3N2 RNA-seq data sets in the

Sequence Read Archive (SRA;

http://www.ncbi.nlm.nih.gov/Traces/sra

) using 30-bp

exon-exon junction sequences for NA2-8 as a query. We identified a single hit for NA8

in influenza A/Myanmar/13M035/2013 (H3N2) (read id DRR051408.279490) (39), and

this strain has a 5=ss and 3=ss identical to PER16/09 and BR10/07. No other matches for

other NA isoforms were identified. The low number of hits in SRA data sets is likely due

to library preparation optimized for amplification of genomic RNA rather than mRNA

(39). Interestingly, of 7,446 unique N2 subtype NA sequences, 4,463 (60%) have an

intact consensus splice donor/acceptor sequence combination (maximum entropy

score

⬎

5 for both 5=ss and 3=ss), suggesting that the NA8 spliced isoform might be

produced in other N2 strains as well. This isoform is likely specific to the N2 subtype

since neither the 5=ss nor the 3=ss is conserved in 7825 unique NA sequences from

non-N2 strains (all 5=ss have a maximum entropy score

⬍

0.4, and most 3=ss have a low

score; only 7 of the 7,825 sequences have a maximum entropy

⬎

5).

Detection of NA isoforms using mass spectroscopy.

To confirm expression of NA

isoforms at the protein level, we excluded classic protein detection methods (e.g.,

Western blot assay) because there are no reagents that distinguish spliced NAs from

native NA. Supernatants of BEAS-2B cells infected with strains of IAV/H3N2 collected at

24 to 48 hpi were subjected to mass spectrometry [MS], searching in particular for

peptides specific to particular spliced isoforms. For example, NA5 lacks residues

be-tween 127 and 319 of native NA therefore tryptic cleavage of the bond C-terminal to

TABLE 1Fractional expression of AS transcripts from IAV/H3N2 segments NA, M, and NSa

Segment Isoform

% of total segment expressionb

BR10/07 PER16/09 UD/72

1 hpi 6 hpi 24 hpi 1 hpi 6 hpi 24 hpi 1 hpi 6 hpi 24 hpi

NA NA1 100.00 99.49 98.26 100.00 99.35 98.41 93.75 87.94 86.05

NA2 – – – – – – 0.00 0.54 0.75

NA3 – – – – – – 6.24 9.84 11.18

NA4 – – – – – – 0.00 0.54 0.63

NA5 – – – – – – 0.00 0.31 0.38

NA6 – – – – – – 0.01 0.55 0.70

NA7 – – – – – – 0.00 0.28 0.30

NA8 0.00 0.51 1.74 0.00 0.65 1.59 – – –

M M1 99.80 98.92 99.15 98.55 98.07 99.11 96.69 99.37 99.57

M2 0.20 1.08 0.85 1.45 1.93 0.89 3.31 0.63 0.43

NS NS1 97.71 99.02 98.52 99.17 99.41 99.29 99.00 97.92 94.88

NS2 2.29 0.98 1.48 0.83 0.59 0.71 1.00 2.08 5.12

a_{The abundances of the spliced transcripts of segments NA, M, and NS were calculated from the RNA-seq data set and are expressed as percentages. The fractional}

expression of alternatively spliced transcripts from IAV/H3N2 segments NA, M, and NS was determined by Cufflinks analysis (see Materials and Methods).

b_{–, Expression was not quantified because the isoform was not identified in this virus.}

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R118 and N-terminal to K128 produces the peptide EPYVSCDPDK in native NA and the

unique peptide EPYVSCDPGLVGDTPR in NA5. As shown in Fig. 5A and Table S1 in the

supplemental material, liquid chromatography/high-resolution MS (LC-MS/MS) analysis

revealed the presence of two peptides unique to the NA5 variant (EPYVSCDPGLVGDTPR

and LSAGGDIWVTREPYVSCDPGLVGDTPR), confirming its expression in UD/72-infected

BEAS-2B cells.

The NA8 isoform was not detected in supernatants of BR10/07 and PER16/09

infected-BEAS-2B cells by LC-MS/MS (Waters), possibly because it was below the

detection limit (10 fmol). Thus, we employed an Orbitrap Lumos targeted MS/MS (tMS2)

acquisition method, followed by dual search for the NA8 variant predicted in seasonal

strains. The predicted NA8-specific peptide LHQSVLLCGVDK was detected at

m/z

456.92

(

z

⫽

3) where cysteine is carbamidomethylated (

⫹

57 Da) (Fig. 5B). As shown in Fig. 5C,

the predicted NA8-specific peptide LHQSVLLCGVDK has the same fragmentation

pat-tern (Fig. 5B) and chromatographic elution profile as those of its heavy synthetic analog

(see Fig. S3 in the supplemental material), confirming the existence of AS product of

NA8 in both PER/16/09 and BR10/07 strains (Fig. 5C and D). The amount of NA8 in 1

␮

g

of supernatant is estimated at 170 and 20 amol, respectively, for PER16/09 and BR10/07

comprising about 1 to 2% of NA proteins (data not shown). Based on the intensities of

common NA peptides, the NA proteins are about nine times more abundant in

PER/16/09 strain than in BR10/07 (data not shown).

We were unable to confirm peptides unique to NA2-4 and NA6-7 in the UD/72

digests we analyzed, possibly due to their low abundance. Deeper sequencing or

antibody-based methods could potentially confirm protein expression or a lack of

expression of these remaining isoforms.

Homology modeling of AS NA isoforms.

Next, we performed homology

mod-eling to determine whether AS affects the tertiary structure of the protein, e.g.,

alters regions of the protein core (binding site) or occurs in disordered regions.

Native NA of UD/72 and PER16/09 as well as NA5 (UD/72) and NA8 (PER16/09)

sequences were submitted to the I-TASSER homology modeling program. Models

were compared to native NA tetramers to determine whether or not they are likely

to be incorporated into native multimers or form multimers with themselves. NA of

UD/72 or PER16/09 is likely to form a tetramer with itself since both interfaces (the

interface formed by the 100, 160, and 460 regions and the interface formed by the

-37 37 R P T D G V L G P D C S V Y P E E P Y V S C D P G L V G D T P R b2-H2O b2 y3 y5-H2O y7-H2O b12-H2O b11-H2O b11 y15-H2O EPYVSCDPGLVGDTPR E rro r (p p m )

A

B

D

C

27.4 27.6 27.8 28.0 28.2 28.4 Retention Time 0.0e0 5.0e3 1.0e4 1.5e4 2.0e4 Intensity

y6 - 691.3443+ y5 - 578.2603+ y4 - 418.2296+ y2 - 262.1397+

28.2 0 ppm 28.0 -2.3 ppm

27.4 27.6 27.8 28.0 28.2 28.4 Retention Time 0.0e0 1.0e6 2.0e6 3.0e6 4.0e6 5.0e6 6.0e6 Intensity

y6 - 699.3585+ (heavy) y5 - 586.2745+ (heavy) y4 - 426.2438+ (heavy) y2 - 270.1539+ (heavy)

28.6 -1.2 ppm 28.4 -1.2 ppm 27.5 -1.3 ppm 27.9 0 ppm (dotp 0.74)

27.4 27.6 27.8 28.0 28.2 28.4 Retention Time 0.0e0 1.0e3 2.0e3 3.0e3 4.0e3 5.0e3 Intensity

y6 - 691.3443+ y5 - 578.2603+ y4 - 418.2296+ y2 - 262.1397+

27.9 -1.2 ppm

27.4 27.6 27.8 28.0 28.2 28.4 Retention Time 0.0e0 1.0e6 2.0e6 3.0e6 4.0e6 5.0e6 6.0e6 7.0e6 Intensity

y6 - 699.3585+ (heavy) y5 - 586.2745+ (heavy) y4 - 426.2438+ (heavy) y2 - 270.1539+ (heavy)

28.4 -1.5 ppm 28.3 -1.2 ppm 27.4 -1.1 ppm 27.9 -0.3 ppm (dotp 0.74)

FIG 5Mass spectrometry detection of NA5 and NA8 isoforms. (A) Representative LC-MS/MS fragment spectra of NA5-specific peptide identified in

trypsin-digested supernatants from UD/72-infected BEAS-2B cells. Annotation was carried out using PLGS software (Waters). (B) Representative fragment spectra of NA8-specific peptide from PER16/09-infected BEAS-2B cells using MS2 acquisition method. (C and D) Fragment ion chromatograms of the native NA8-specific peptide (top) and its heavy synthetic analog (bottom) from PER16/09 (C) and BR10/07 (D) supernatants.

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140/150, 170, 200, and 210 regions) are fully formed and identical to other N2

structures (Fig. 6A and B). Notably, the enzyme active site in each is unchanged

relative to other N2 structures. NA5 is predicted to be unable to bind to any other

monomer because of the large portions of missing sequence and structure, and the

active site is likely only partially formed if at all (Fig. 6C). NA8 is not likely to form

a tetramer as one interface is incomplete (the 160 and 460 regions are absent) and,

although components of the second interface are present, the absence of part of

the protein likely alters the configuration of this region (Fig. 6D). A portion of the

active site is missing (the 430 loop and the 405 region) which would likely

compromise active-site binding.

Host transcriptome of BEAS-2B cells infected with seasonal H3N2 viruses.

To

date, immune responses of epithelial cells to UD/72 infection have been reported (18).

We sought to further characterize differences in induction of innate immunity-related

pathways by each of the three H3N2 viruses and between the two seasonal viruses and

UD/72 at the mRNA level. We first identified differentially expressed genes (DEGs) in

BEAS-2B cells infected with seasonal or laboratory strains compared to time-matched,

mock-infected cells. We detected a total of 81, 372, and 1,614 DEGs at 1, 6, and 24 hpi,

respectively (Data Set S1 in the supplemental material and Fig. 7A). The laboratory

strain UD/72 induced the highest number of DEGs at each time point, such that

UD/72-specific DEGs accounted for 42, 58, and 72% of all DEGs identified at 1, 6, and

24 hpi, respectively. Next, we curated a list of 804 innate immunity genes from several

publications (18, 40–45), negative and positive regulators of IFN-

␤

(46), and from HUGO

gene families that include IFN-related genes or “IFN” as a term (see Data Set S1 in the

supplemental material). Of the 804 innate immunity genes, 119 were either up- or

downregulated during infection, while 685 were not differentially expressed (Fig. 7B

and see Table S2 in the supplemental material). Figure 7C shows a heat map of the

expression of the 119 DEGs ordered by unsupervised hierarchical clustering analysis

and Table S2 reports DEGs with

⬎

2-fold change in virus-matched gene expression.

Twenty-two DEGs were upregulated by all three viruses, including ISGs that are

antiviral (e.g.,

MX1

,

OAS

,

RSAD2

[viperin], and

IFIT1-3

), induce IFN expression (

DDX58

[

RIG-I

] and

DDX60

), repress IFN expression through posttranslational modification (e.g.,

A

B

C

D

FIG 6Homology modeling of AS NA isoforms. (A to D) Superimposition of native NA monomer

homology models from UD/72 (A), PER16/09 (B), and AS isoform NA5 (C) and AS isoform NA8 (D), each

indicated in magenta, onto a tetramer of a crystal structure of 1957 pandemic N2 NA (PDB 3TIA)

(multicolored). NA of UD/72 and PER16/09 are likely to form multimers with themselves or be incorpo-rated into native multimers. NA8 and NA5 are unlikely to form multimeric structures since they are missing large portions of sequence that form all or part of the binding interfaces.

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the ubiquitin-like

ISG15

[47] and the E3 ligase

TRIM69

), or attenuate IFN signaling (e.g.,

USP18

[48] and guanylate binding protein 4 [

GBP4

], which inhibits IRF7 [49]). The

expression of

IFNAR1

, which encodes the low-affinity subunit of the type I IFN receptor,

decreased after infection with UD/72 and PER16/09, but not BR10/07 at 6 hpi. As

previously reported (18), in addition to the classic antiviral ISGs, UD/72 predominantly

induced expression of a large set of proinflammatory genes that were either not

induced (

CXCL9

,

CXCL10

,

IL-6

,

CCL2

,

CXCL1

, and

CXCL2

) or were only minimally induced

by seasonal strains compared to UD/72 (

CCL5

and

CXCL8

).

Overall, seasonal and laboratory strains showed similar patterns of DEGs and a

similar induction of key transcription factors (TF) that either form an ISGF3 complex

(

STAT1

and

IRF9

) or are induced by ISGF3 (

IRF7

). However, the patterns of DEGs differed

in time and magnitude for the two seasonal viruses; a common set of genes was

induced earlier and to higher levels in response to PER16/09 than to BR10/07 infection.

Only BR10/07 did not significantly induce expression of

IRF1

. In addition, only UD/72

A

B

C

D

B R 10/07

Immune Genes

P E R 16/09 U D /72

CXCL11 CXCL10 IFIT1B IFNB1 IFNL1 CCL5 TRIM69 IFIH1 GBP4 CXCL1 TNFAIP3 NFKBIA IL8 IFITM3 IRF9 BST2 TNFSF10 IFITM1 MX2 RSAD2 ISG15 IFI6 IFI44L IFI27 MX1 OAS2 DDX58 IFI44 OASL IFIT2 IFIT1 OAS1 IFIT3 JUNB ATF3 SERPINE1 TLR4 IL18 AGPAT9 C1QBP EPHA2 DUSP5 MAFF MAP1LC3B ANKFY1 IFNAR1 IRF6 PLK1 IFITM2 LY6E AHNAK2 CD9 EPAS1 MT1X NFKBIZ CXCL2 CEBPD ADM SLFN5 TXNIP DDX60 XAF1 HERC6 IFI16 SAMHD1 IFI35 EPSTI1 IL17F GBP5 DHX58 TYMP OAS3 HELZ2 IRF7 PLSCR1 USP18 TRIM22 EIF2AK2 ADAR NMI APOL6 BATF2 PARP12 RTP4 FAM46A STAT1 LAP3 DTX3L TDRD7 IL6 TNFAIP6 CCL2 IFNL2 CXCL3 IRF1 CFB B2M CHMP5 TRIM21 IFIT5 PNPT1 MB21D1 NFKBID PLEKHA4 IL34 HSH2D RPL22 VMP1 NT5C3A GCA MAFB SOCS1 NCF1 IL32 G6PC IL10 CXCL9 IL17RE IFNL3

−2 −1 0 1 2 Value Color Key

FIG 7BEAS-2B transcriptome during infection with IAV/H3N2: changes at the mRNA level. (A) Venn diagrams of the global host gene responses (either

enhanced or reduced) to IAV/H3N2 strains at each time point. (B) Venn diagram of “immune-related genes” differentially expressed in response to each of the three IAV/H3N2 at 1, 6, or 24 h. (C) Hierarchical clustering of the 119 DEGs from the “immune-related genes” group. Heat map analysis of host transcriptome showing differentially up- and downregulated genes (yellow and blue, respectively) in IAV/H3N2 infected cells compared to mock-infected cells. (D) Enrichment of DNA binding motifs for selected IRFs located in the promoters of upregulated DEGs at 6 and 24 hpi. IRF and IFN-stimulated response element (ISRE) consensus motifs (109) are shown as controls.

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induced a high expression of IFNB1/proinflammatory genes and showed early

expres-sion of TF components of the

IFNB1

enhanceosome (

JUNB

and

ATF3

) or a gene whose

product induces

IL-6

(

NFKBIZ

) (Fig. 7C and see Data Set S1 in the supplemental

material).

To further characterize pathways enriched with DEGs, we performed Ingenuity

Pathway Analysis (IPA), which revealed six canonical pathways shared by the three

H3N2 viruses at 6 and 24 hpi that included IFN signaling, the activation of IRF by

cytosolic PRR, and the role of PRR in recognition of bacteria and viruses (see Data Set

S2 in the supplemental material). The IPA upstream regulators feature predicted IRF7 as

a master upstream activator based on DEGs identified in each sample (see Fig. S4 and

Data Set S3 in the supplemental material). As shown in Fig. S4 in the supplemental

material, the network of IRF7-activated genes at 24 hpi revealed similar but distinct

patterns between seasonal and UD/72 strains.

Motif enrichment in promoters of upregulated DEGs during H3N2 infection.

TFs need to act at a certain distance from the transcription start site or other regulatory

sites, and thus TF binding sites cluster within promoter regions. Because the presence

of TF binding sites contributes to the transcriptional regulation of virally induced genes,

we used an

in silico

approach to predict motifs present in the promoters of DEGs that

were upregulated after H3N2 infection. We first evaluated the enrichment of

nonre-dundant TRANSFAC motifs, each representing a family of one or more related TF

binding motifs (50). TF motifs were evaluated based on the following criteria: (i) the

motif is enriched within promoters of upregulated DEGs compared to promoters of

unchanged genes, (ii) the TF is expressed (FPKM [fragments per kilobase per million]

⬎

5), (iii) the TF is predicted to be active based on IPA upstream regulator analysis, and

(iv) known target genes contain the binding motif.

IRF was the most highly enriched motif, demonstrating up to 115-fold enrichment

above unchanged promoters (see Data Set S4 in the supplemental material). Promoter

regions of ISGs are known to contain multiple ISREs and/or a combination of motifs that

recognize ISGF3 alone, ISGF3 with IRF7, or ISGF3 with both IRF7 and IRF3 (51). Thus, we

also evaluated enrichment of each IRF motif separately. A graphical representation of

the consensus sequence TGAA, shared among IRFs and ISRE is shown in Fig. S5A in the

supplemental material. In addition to IRF3 and IRF7, we found an enrichment of binding

motifs for IRF1 and IRF2 (Fig. 7D; see also Table S2 in the supplemental material).

Several IRF genes were expressed in H3N2-infected BEAS-2B cells, either constitutively

(IRF3) or differentially upregulated (IRF1 and IRF9) or downregulated (see Data Set S1

in the supplemental material). Though further experiments are required to confirm IRF

binding in enriched promoters, both enrichment and expression data suggest the

importance of these IRFs for H3N2-induced genes.

At 6 hpi in BR10/07 samples, no significant IRF motifs were predicted in the

promoters of upregulated DEGs (Fig. 7D). At 24 hpi, however, IRF promoter enrichment

in response to BR10/07 was similar to that of PER16/09 at 6 hpi, confirming that the

difference in response to these two seasonal viruses was mostly quantitative and

kinetic. In contrast, because the response to UD/72 was not limited to ISGs, the

enrichment of IRF binding motifs after UD/72 infection was relatively modest at 6 hpi

and fell at 24 hpi.

Other nonredundant motifs that met the TF motif search criteria include two

non-IRF TFs: RelA (52, 53) and the serum response transcription factor (SRF) (54) (see

Data Set S4 and Fig. S5B and C in the supplemental material). At 24 hpi, enrichment of

binding motifs for RelA and SRF was found in promoters of upregulated DEGs after

infection with PER16/09 and UD/72.

IFNB1

, which is upregulated during infection with

all three viruses, also has a binding motif for RelA in its promoter.

Novel RNA-based innate immune DEGs during H3N2 infection.

Next, we queried

whether dsRNA and RNA interference (RNAi) pathways (referred to here as RNA-based

innate immunity pathways) were affected during H3N2 infection. Because some small

RNAs were included in the query, we first assessed our RNA-seq library insert size (see

Fig. S6A in the supplemental material) and found that the most common insert size

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across samples is approximately 110 bp and many library fragments below 100 bp are

retained. Figure 8 shows heatmaps of RNA-based innate immune DEGs ordered by

unsupervised hierarchical clustering analysis. Among these, we found several small

nucleolar RNAs (snoRNAs), a class of noncoding RNAs involved in canonical functions

such as ribosome biogenesis, modification of ribonucleotides, and cleavage of pre-rRNA

(55, 56). We also verified the size distribution of differentially expressed snoRNAs during

H3N2 infection (see Fig. S6B in the supplemental material). To reduce the technical

bias due to library size selection, we removed snoRNAs that were shorter than 100

bp in size or showed high variance between replicates in any treatment condition.

snoRNAs are classified into three groups: SNORDs, possessing the C/D box and

directing the 2=-O-ribose methylation of rRNAs; SNORAs, containing the H/ACA box

SNHG8 GAS5 SNHG15 SNORA38B SCARNA23 SNHG6 SNORA64 SNORA27 SNORD94 SNORA25 SNHG12 SNORD22 SNORD89 AGO3 SNIP1 DLEU2 SNORA51 SNORD15A SNORA33 SNHG4 SNORA40 SNORA5C SNORA5A SCARNA7 SCARNA21 SNORA8 SNORA7B SNORA10 SNHG10 SNORA32 SNHG7 SNORA21 SNORA67 SNORA16A SNORA44 SNORA71C SNORA76 SNORA81 SNORA20 SNORA74B SNORA24 SNORA2A SNORA9 SNORA17 SNORA78 SNORA18 SNORA50 SNORA14B SNORA26 SNORA3 SNORA62 SNORA4

MIR100HG HCFC1 SNORA84 EIF2AK2

FIG 8BEAS-2B transcriptome during infection with IAV/H3N2: changes in RNA-based immunity. Hierarchical clustering of the DEGs

from RNA-based immunity list. Results for up- and downregulated DEGs (yellow and blue, respectively) in UD/72-infected cells (A) or in UD/72- and PER16/09-infected cells (B) compared to mock-infected cells are shown.

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and guiding synthesis of ribosomal pseudouridines; and SCARNAs, forming a

complex with small nuclear proteins (SNURPs) and guiding modification of snRNA

(e.g., U1, U2, U4, U5, and U12) (57).

Compared to mock-infected, time-matched samples, UD/72 infection shows the

greatest ability to modulate snoRNA expression (Fig. 8A). In UD/72-infected cells, five of

the six H/ACA box snoRNAs (SNORA8, -18, -25, -32, and -40) that share the same host

gene TAF(I) are upregulated. TAF(I) functions in RNA polymerase I transcription, resides

at the rRNA gene promoter in the nucleolus, and copurifies with the complex SL1 (58).

In addition, snoRNAs with reported noncanonical functions (56), such as regulation of

chromatin structure (SNORD15A and SNORA3, -81, -64, -44, and -40), and miRNA-like

overlapping functions (SNORA81-miR1248) were upregulated by UD/72.

A small subset of RNA-based innate immunity genes show similar patterns of

expression in PER16/09 as in UD/72 (Fig. 8B). At 24 hpi, PER16/09 or UD/72 infection

were associated with a 2-fold increase of

EIF2AK2

, coding for PKR, a dsRNA-binding

protein component of the dsRNA/IFN and RNAi pathways, which share two additional

partners, PACT and TRBP, with Dicer (59–61). PER16/09 and UD/72 also upregulate

PIWIL2

, a member of the RNAi pathway. One target, HCFC1, is downregulated in

PER16/09 or UD/72 infection with respect to mock-infected samples.

H3N2 viruses differentially modulate AS of host genes.

We then used an

exon-centric approach (62) to determine whether H3N2 infection induced differential

expression of AS variants of host genes. Figure 9A shows the number of variants for

each type of AS event at 1, 6, and 24 hpi. We detected 775 genes showing AS in at least

one infected sample (see Data Set S1 in the supplemental material). Pathways and gene

ontologies enriched in each data set include RNA binding, nonsense-mediated decay,

cell cycle process, influenza life cycle, and influenza viral RNA transcription (see Fig. S7

in the supplemental material). Many of the genes involved in these pathways/functions

are ribosomal protein genes. For example, the genes found in the influenza life cycle

pathway, enriched in cells infected with BR10/07 or PER16/09, are

RPLP1

,

RPL4

,

RPL31

,

RPS2

,

NUP155

,

RPL13A

,

GRSF1

, and

RP24

(see Data Set S1 in the supplemental material).

Among the AS events differentially induced by seasonal and laboratory strains, we

report one example of each of exon inclusion, intron retention, and alternative donor

splice site.

TANK

-binding kinase 1 (

TBK1

) is critical to antiviral defense and regulates IKK

␧

, which

in turn phosphorylates IRF3 and IRF7 (63).

TBK1

is also known to have an AS variant in

which exons 3 to 6 of the 21 exons are skipped (64). However, we discovered a novel

AS event of inclusion of a previously unannotated

TBK1

exon between exons 15 and 16

of the reference isoform (

NM_013254

) associated with UD/72 infection (Fig. 9B). This

novel exon gives rise to a frameshift splice variant that consists of 22 exons and is

terminated by an early stop codon deleting the last 155 amino acids of TBK1,

corre-sponding to the binding domain for TBKBP1, AZI2, and TANK (65). The predicted TBK1

variant induced by UD/72 infection at 24 hpi was confirmed by quantitative PCR (qPCR)

(Fig. 9C).

At 1 hpi, BR10/07 induced an intron retention event of

DDIT3

, with inclusion of an

additional 23 amino acids at the N terminus (Fig. 9D).

DDIT3

encodes a proapoptotic TF,

which participates in the endoplasmic reticulum stress response to induce expression

of inflammatory genes such as

IL-6

and

CXCL8

(66). Intron retention of DDIT3 by

BR10/07 at 1 hpi was evaluated by qPCR (Fig. 9E).

Finally, we report an example of an alternative donor splice site (5=ss). At 24 hpi, the

two seasonal viruses BR10/07 and PER16/09 induced AS of

IFI35

(Fig. 9F), a negative

regulator of RIG-I, that associates with NMI protein after treatment with IFN-

␣

(67). This

AS 5=ss event generates an in-frame deletion of six bases that encode amino acids 126

to 127 (Val-Met) in the NMI domain that mediates interaction with IFI35. The alternative

5=ss was confirmed by qPCR (Fig. 9G). To our knowledge, this is the first report of

modulation of AS of host transcripts during IAV/H3N2 infection that may impair innate

immune responses.

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A

Alternative donor Mutually exclusive exons Retained intron Alternative acceptor Alternative last exon Skipped exon Alternative first exon

0 50 100 150 0 50 100 150 0 50 100 150

Time 1h 6h 24h

C

Number of Genes

F

41165287 41165323 41165363 41165425 41165524 41165560 41165600 41165640 41165676

23.3 46.7 70 RP K M 16 UD/72_24h 23.3 46.7 70 RP K M 26 Mock_24h

Genomic coordinate (chr17), "+" strand

0 2 4 7 Ψ 0 3 6 10 Ψ

00.2 0.4 0.6 0.81

0 4 8 13 Ψ 0 5 11 16 Ψ = 0.67 [0.50, 0.81] = 0.55 [0.43, 0.66] = 0.91 [0.71, 0.99] = 0.94 [0.82, 0.99] MISO Ψ

D

M 42 BR10/07_1h

23.3 46.7 70 RP K M 7 PER16/09_1h

57911055 57911115 57911175 57911235 57911295 57911355 57911415 57911475 57911535 23.3

46.7 70

RPKM

22 UD/72_1h

Genomic coordinate (chr12), "-" strand

0 3 7

11 _{Ψ= 0.49}

[0.40, 0.59]

0 4 8

13 Ψ= 0.89

[0.78, 0.98]

00.2 0.4 0.6 0.8 1

0 3 7 11 Ψ= 0.77 [0.66, 0.87] 0 4 9 13 _Ψ [0.73, 0.90] = 0.82 MISO Ψ Mock_1h

B

64889482 64889514 64889550 64889766 6488 9802 64889838 64889879 64890147 64890183

10 20 30 RP K M 15 5 10 UD/72_24h

Genomic coordinate (chr12), "+" strand

0 4 8 12 0 3 6 10 0 2 5 8

00.2 0.4 0.6 0.8 1

0 1 2 3 Ψ= 0.11 [0.01, 0.30] Ψ= 0.17 [0.08, 0.34] Ψ= 0.16 [0.04, 0.37] Ψ= 0.54 [0.25, 0.88] MISO Ψ 3

E

G

Relative ratio of intron retained/spliced

MOCK BR10/07 PER16/09 UD/72 0.0 0.2 0.4 0.6 0.8 1.0 TBK1

Relative ratio of exon inclusion/exon skipping

MOCK BR 10/07 PER16/09 UD/72 0.3 0.6 IFI35

Relative ratio of proximal 5’

ss/distal 5’

ss

FIG 9AS events of BEAS-2B transcriptome during infection with IAV/H3N2: changes at the level of the mRNA structure. (A)

Schematic of the host AS events (left) and the frequency of each event type during IAV/H3N2 infection (right panels). (B to

(Continued on next page)

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DISCUSSION

For many years, studies of cellular host responses to virus infection focused on

global changes at the mRNA level. More recently, RNA-seq has been used to dissect key

regulatory events at the RNA level such as splicing and noncoding RNAi. Here, we used

a strand-specific dual RNA-seq strategy to characterize novel elements of interactions

between the BEAS-2B human bronchial epithelial cell line and three strains of IAV/

H3N2: two seasonal strains and the laboratory strain, UD/72.

Transcriptome analysis of BEAS-2B cells infected with seasonal IAVs BR10/07 and

PER16/09 and the laboratory strain UD/72 showed the induction of a similar pattern of

ISG expression and IRF7-centered immune responses. This pattern was representative

of the response of other epithelial cell lines to infection with respiratory viruses (18, 19),

including the highly passaged IAV laboratory strain UD/72 (18, 19). However, we only

identified a small number of immune DEGs induced by IAV/H3N2. and this might be in

line with low enhancement of ISGs in BEAS-2B cells, as reported by Seng et al. (68).

Compared to seasonal strains, UD/72 induced delayed expression of many classic ISGs

and pronounced expression of proinflammatory chemoattractants

CXCL9

and

CXCL10

,

both of which are ISGs, and the neutrophil chemoattractants

CXCL1

and

CXCL8

. Immune

DEGs were, however, more similar between UD/72 and PER16/09 than between the two

seasonal strains. One possible mechanism is the potential recruitment of TFs, including

IRFs. Based on our

in silico

analysis of TF motifs, IRF binding site enrichment in promoters

of upregulated DEGs suggests magnitude and kinetic differences between the two

sea-sonal strains (Fig. 7D). Enrichment of non-IRF TF motifs (see Fig. S4B and C and Data Set S4

in the supplemental material) such as RelA could explain the pronounced enhancement of

proinflammatory chemokines by UD/72 (Fig. 7C). Though the presence of a TF binding site

does not necessarily imply a direct effect on expression of the gene, it is striking that at 24

hpi an enrichment for the RelA motif occurs in the promoter regions of many DEGs

upregulated uniquely by UD/72 infection. Such DEGs include gene functions involved in

inflammatory response signaling,

TNFAIP3

and members of NFK-B inhibitory family (e.g.,

NFKBIA

,

NFKBID

, and

RELB

), proinflammatory molecules

IL-6

and

IL-32

, or

EGR1

, which

acts as TF to regulate

CXCL2

expression. Interestingly, the

EGR1

promoter also had the

highest number of SRF motifs (i.e., five) among genes upregulated by UD/72. Another

potential mechanism to explain the immune DEGs shared between UD/72 and

PER16/09 might be the expression of dsRNA effectors (e.g., PKR) or of other RNAs that

may participate in RNA-based innate immunity.

The BEAS-2B transcriptome revealed changes in the expression of RNA-based innate

immune genes, including effectors, such as

EIF2AK2

(PKR),

AGO3

, and

PIWI/L2

, as well as

expression of snoRNAs (Fig. 8). To date, the role of snoRNAs during RNA virus infection

has not been fully elucidated. snoRNAs guide the chemical modification of snRNA and

mRNA nucleotides and influence AS of pre-mRNAs, in addition to other noncanonical

functions (69). Murine lung cells infected with severe acute respiratory syndrome

coronavirus or IAV/Puerto Rico/8/34 strains show differential expression of 30 small

RNAs overlapping with annotated snoRNAs (70). Human HEK293T cells infected with

chikungunya virus upregulate a group of snoRNAs—SNORD3, -44, -76, and -78 —all

clustering within the host long noncoding RNA

GAS5

(small nucleolar RNA host gene 2

[

SNHG2

]) with the exception of SNORD3 (71). UD/72 upregulates

GAS5

, a member of the

5-terminal oligopyrimidine gene (5=

TOP) family (72) that regulates transcription and

translation under conditions of growth arrest. Our data show that UD/72 upregulates

other

SNHG

s associated with cellular processes that may work together with the

FIG 9Legend (Continued)

G) Examples of AS differentially regulated by IAV/H3N2. For panels B, D, and F, the left side shows the RNA-seq read density along exons, the number of reads mapped to each splice junction, and the structural difference between alternative isoforms (below genomic coordinates), and the right side shows the distribution of percent spliced-in (PSI) expression estimates, with the 95% confidence interval indicated. Panels C, E, and G show validation by qPCR. (B and C) UD/72 differentially regulates

inclusion of a newly discovered exon ofTBK1. (D and E) BR10/07 differentially regulates intron retention ofDDIT3. (F and G)

BR10/07 and PER16/09 differentially regulate 5=AS ofIFI35.

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translational repression process. Further work is required to determine whether each

SNHG

has a unique role in influenza infection.

Subcellular localization of viral components plays an important role in host innate

responses. The nucleolus is the subnuclear compartment for ribosome biogenesis and

cellular location of

SNORA

s and

SNORD

s. It is also involved in the regulation of cellular

processes such as cell cycle, apoptosis, and RNA processing (73). Thus, it is likely that

the nucleolus acts as a platform for the virus to manipulate processes such as cell

growth and stress response, whose inhibition would encourage viral pathogenesis (73).

The nucleolus is engaged by viruses that replicate in the nucleus or cytoplasm (e.g.,

chikungunya) and two non-mutually exclusive mechanisms may occur: one by which

viral products target the nucleolus to modify its function(s), and the other by which

viruses employ nucleolar components to facilitate steps of their life cycle (73, 74). The

NS1 protein of IAV/H3N2 strains isolated from 1950 to 1987, including the laboratory

strain UD/72, carries a string of residues at the C terminus (K219, R220, R224, and K229)

that targets the nucleolus by binding to nucleolar proteins such as fibrillarin and

nucleolin (75, 76). All H3N2 strains used in our study contain these residues.

Interest-ingly, Melen et al. were the first to present a working hypothesis on viral replication not

being affected by depletion of the reported residues (75), suggesting that the primary

role for nucleolar localization of H3N2 and H2N2 NS1 proteins is modulation of the host

response. Additional evidences stem from the nucleolar proteome of HEK293 cells

infected with UD/72 (77), in which NP, PB2, NS2, and M1, in addition to NS1, were found

associated with nucleoli. The NP of UD/72 interacts with several components of

nucleolar pathways, including proteins involved in RNA maturation, such as RNA

polymerase I, which is responsible for transcription of ribosomal RNAs, and RNase P,

which plays a role in tRNA maturation (77).

Ribosome biogenesis is one of the processes that is modulated during viral

infec-tion. For example, herpes simplex virus 1, induces posttranscriptional cleavage of

precursor rRNA (78). Here, we report that infection with UD/72 induces host cell

upregulation of snoRNAs (Fig. 8), which play an important role in rRNA maturation.

While the mechanism of snoRNA upregulation is unclear, it is tempting to speculate

that it may represent a novel means by which the virus can affect nucleolar function to

promote viral pathogenesis.

Strand-specific RNA-seq retains the polarity of host and virus RNAs and enabled us

to identify three novel elements of host-virus interactions that differ during infection of

BEAS-2B cells with seasonal IAV and UD/72: (i) production of DI-like RNAs, (ii) host AS

modulated during viral infection, and (iii) novel AS of gene segment 6 (NA).

DI RNAs (33) serve as PAMPs to drive RIG-I- and MDA5-mediated expression of IFN

(79). DI RNAs may serve as an immunostimulator to protect from subsequent influenza

infection (9, 34, 79). DI-like RNAs have been identified in the nasopharynx of patients

infected with A/California/07/09 (H1N1pdm09) and may be transmitted from infected

patients along with infectious virus (34). Based on reported sequences of H1N1pdm09

and our own analysis, it appears that DI-like RNAs are strain specific (Fig. 2). In addition,

the calculated ratio of DI-like RNA to vRNA showed a modestly higher DI-like RNA

fraction in PER16/09 (Fig. 2G) than BR10/07. This may offer an explanation for why

PER16/09 induces higher early expression (6 hpi) of antiviral genes than BR10/07.

More than 90% of multiexon human genes undergo AS, suggesting that pre-mRNA

processing contributes to the cellular complexity of higher eukaryotes (80). However, it

is not fully understood how virus infection affects host AS. Viruses may differentially

modulate AS of host genes in order to counter host program(s) to generate more

protein isoforms of immune mediators (22, 23). One such example is modulation of AS

transcript of

MX1

(26) by herpes simplex virus 1. Alternatively, AS may control levels of

the gene product by maintaining levels of mRNA that produce functional proteins in

equilibrium with mRNA that generate nonfunctional products (81). How virus infection

may influence one or the other hypothesized AS functions is not yet known. Here, we

report three examples of host AS modulated by each H3N2 IAV (Fig. 9B to G), including

novel AS (exon inclusion) of

TBK1

induced by UD/72, which if expressed would generate

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a truncated protein lacking the C-terminal adaptor binding motif (82). TBK1 participates

in the activation of multiple signal transduction pathways, including IFN (with

sub-strates IRF3 and -7) and NF-

␬

B (with substrates including IKK, I

␬

B

␣

, and p65/RelA). The

specificity of substrates for TBK1 is determined by its choice of adaptors (82, 83). For

example, association with the adaptor IFN-induced protein with tetratricopeptide

repeats 3 enhances IFN expression, while ubiquitination and binding to NEMO

en-hances activation of NF-

␬

B (82). Since each adaptor localizes to distinct cellular

com-partments (83), limiting TBK1 expression through the generation of a nonfunctional splice

variant may disable multiple or selected innate responses. If the protein generated by the

AS is stable, deletion of the adaptor binding motif of TBK1 might mimic an active state of

TBK1 that can bypass the regulation based on the adaptor choice or localization (83).

Whether the TBK1 AS variant found in UD/72-infected cells enhances NF-

␬

B activation or

IFN production requires further investigation.

Finally, there were six novel splice events in gene segment 6 that encodes NA of the

laboratory strain UD/72 (Fig. 3) and one in NA of the seasonal strains. The UD/72 AS

events predict the generation of several frameshift products and one novel in-frame

product (Fig. 4). These events suggest two models for generation of the AS variants,

that are not mutually exclusive. The first model (proximity rule [84, 85]) proposes that

two or more 5=ss that share the same 3=ss cause stuttering of the viral polymerase, in

which the spliceosome redirects the splicing event from the distal to the proximal 5=ss.

The common 3=ss suggests its importance in the splicing event. This is consistent with

multiple predicted AS isoforms of UD/72 (Fig. 3), including the isoform NA5 that gives

rise to an in-frame product. Interestingly, one-third of AS events in mammals generate

in-frame premature termination codons, which play a role in regulation of gene

expression by controlling the abundance of splicing factors or RNA binding proteins

(80). The second model proposes that splicing efficiency is strain specific and thus may

be indicative of the ability of the virus to replicate (86, 87) or adapt to different hosts

(88). In line with this model, the highly passaged UD/72 virus acquired multiple weak

5=ss that are absent in the seasonal IAV/H3N2 strains (Fig. 4). One possible

interpreta-tion is that splicing of UD/72 NA is a result of host adaptainterpreta-tion, and it may result in

reduced viral virulence (88). The second model is consistent with the splicing of gene

segment 7 observed in the laboratory strains IAV/WSN33 (89), IAV/PR8 and in other

nonlaboratory strains (38) or splicing of the NS gene segment to produce a novel

product, NS3 in a mouse-adapted human IAV/H3N2 (88). Whether these AS events are

unique to laboratory and mouse-adapted strains of IAV and whether they depend on

specific cofactors of the splicing machinery of a specific cell line is not known.

Mass spectrometry confirmed the presence of NA5 in UD/72 supernatants and NA8

in supernatant from both seasonal strains (Fig. 5). It is also possible that the NA8

isoform is expressed in other IAV/N2 strains based on the observed conservation of the

5=ss and 3=ss. Our homology modeling indicates that NA5 and NA8 are not predicted

to assemble as the native NA does, though functional studies are needed to confirm

these predictions. In addition, these results do not preclude the possibility that the

translation product of a newly identified splicing variant could have a function

inde-pendent of the native tetrameric NA. Low abundance or protein degradation could

account for our inability to detect the other NA isoforms by mass spectrometry.

Alternatively, if NAs were expressed in the cells but not incorporated into new particles

along with HA and M, they would not be detectable in supernatants. Further

investi-gation in other cells, in additional IAV/H3N2 viruses, and

in vivo

will be of interest.

In conclusion, our data highlight the importance of RNA-seq in studying

host-pathogen interactions and identifying novel elements that may contribute to influenza

biology and to RNA-based innate immune responses.

MATERIALS AND METHODS

Cell culture, viruses, and infection.The BEAS-2B cell line (ATCC CRL-9609) was obtained from the

American Type Culture Collection (Manassas, VA) and cultured in serum-free base media (BEMB; Lonza, Walkersville, MD) supplemented with growth factors (BEGM; bullet kit catalog no. CC-3170). Cells were cryopreserved in BEGM supplemented with 1% polyvinylpyrrolidone and 7.5% dimethyl sulfoxide and

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stocks were used within five passages. Cells were grown in tissue culture plates coated at 37°C for 2 to 3 h in BEMB media containing 0.01 mg/ml fibronectin, 0.03 mg/ml bovine collagen type I, and 0.01 mg/ml bovine serum albumin (all from Sigma Chemicals, St. Louis, MO). Madin-Darby canine kidney (MDCK) cells (ATCC CCL-34) were grown in complete Dulbecco modified Eagle medium (Invitrogen, San

Diego, CA) supplemented with 7.5% fetal bovine serum (Invitrogen), 100 U/ml penicillin G, 100␮g/ml

streptomycin, and 2 mM glutamine (Invitrogen).

Two seasonal IAV/H3N2 viruses, A/Perth/16/2009 (PER16/09) and A/Brisbane/10/2007 (BR10/07), were kindly provided by Zhiping Ye and Alexander Klimov, respectively. UD/72 was a laboratory stock. Virus stocks were amplified in the allantoic cavity of 9- to 11-day-old embryonated chicken eggs for 48 h at 37°C. Viruses were titrated by plaque assay in MDCK cells. Titers were calculated using the Reed-Muench

method (90) and are expressed as log1050% tissue culture infective doses per ml.

BEAS-2B and MDCK cells were each plated (0.5⫻106_{/12-well plate), incubated at 37°C and 5% CO}

2,

and maintained until they achieved 90% confluence. Infection of BEAS-2B and MDCK cells was performed at an MOI of 1 at 37°C. After 1 h of incubation at room temperature, the inoculum was removed and replaced with fresh media containing TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone)-treated and

irradiated trypsin (1␮g/ml; Worthington Biochemical Corporation, Lakewood, NJ).

Strand-specific total RNA library preparation and sequencing.Total RNA was extracted using an

RNeasy minikit (Qiagen, Valencia, CA), followed by DNase treatment. All samples had a RNA integrity

score of⬎8 (Agilent Technologies, Santa Clara, CA). Starting from 1␮g of DNA-free total RNA extracted

from mock- and H3N2-infected BEAS-2B cells, rRNA was first removed by two rounds of selection using a Ribo-Zero rRNA removal kit (Epicentre, Madison WI). The resulting RNA was then used to construct an RNA-seq library using the Illumina Truseq strand-specific library preparation kit (Illumina, Inc., San Diego, CA). To obtain the final sequencing library, 14 cycles of PCR were performed using Phusion Hot Start high-fidelity DNA polymerase (Finnzymes, Espoo, Finland). RNA-seq libraries were size selected for the retention of insert fragments between 100 and 300 bp; mature small RNAs (including svRNAs) were not

considered in our analysis. For each sample replicate, we obtained⬃30 million paired 50-mer reads (with

three biological replicates per sample) using the Illumina Hiseq-2000 platform.

Host RNA-seq differential expression and splicing analysis. Raw reads were mapped to the

reference human genome (hg19, including chrUn_gl000220 for rRNA reads) by Tophat (v2.0.8b) (https:// ccb.jhu.edu/software/tophat/index.shtml) with the settings “– bowtie-n –no-discordant –no-mixed -r 120 –mate-std-dev 75” and using refFlat file for the -G option. Reads mapping to chrUn_gl000220 were removed as rRNA. We measured differential gene expression using DefinedRegionDifferentialSeq (USeq

8.7.6) (91;http://useq.sourceforge.net/), with the settings “-g H_sapiens_Feb_2009 -e 5 -j -t” and using a

flattened refFlat table for annotation (merged transcripts using MergeUCSCGeneTable [USeq]).

Define-dRegionDifferentialSeq calculates a negative binomialPvalue using DESeq2 (92), followed by a Benjamin

and Hochberg false discovery rate (FDR). Differentially expressed genes were those with an FDR_ⱕ10%

and an absolute log2ratio above 1. Differentially expressed snoRNA genes were those with an FDRⱕ1%,

an absolute log2ratio above 1, size⬎100 bp, and variance⬍0.8 between replicates of each sample.

Alternative splicing analysis was performed using MISO (v0.5.2;http://genes.mit.edu/burgelab/miso/)

with the settings “–paired-end 125 50 –read-len 50” and using “–num-inc 2 –num-exc 20 –num-sum-inc-exc 30 – delta-psi 0.30 – bayes-factor 20” for filtering events. Events were plotted with sashimi_plot. Events annotated to more than three genes were removed, and pathway and gene ontology enrichment

was performed using ToppGene (93) with an FDR of⬍0.05. Related pathways/ontologies (Gene

Ontol-ogy: Molecular Function, Gene OntolOntol-ogy: Biological Process, and REACTOME [94] pathways) were grouped and displayed as an Enrichment Map v.3.0.0 (95) network in Cytoscape v.3.5.1 (96) with an overlap frac