Influenza virus inhibits RNA polymerase II elongation
Annie Y. Chan, Frank T. Vreede, Matt Smith, Othmar G. Engelhardt, Ervin Fodor⁎
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
Received 9 February 2006; accepted 7 March 2006
Available online 19 April 2006
The influenza virus RNA-dependent RNA polymerase interacts with the serine-5 phosphorylated carboxy-terminal domain (CTD) of the large
subunit of RNA polymerase II (Pol II). It was proposed that this interaction allows the viral RNA polymerase to gain access to host mRNA-
derived capped RNA fragments required as primers for the initiation of viral mRNA synthesis. Here, we show, using a chromatin
immunoprecipitation (ChIP) analysis, that similar amounts of Pol II associate with Pol II promoter DNAs in influenza virus-infected and mock-
infected cells. However, there is a statistically significant reduction in Pol II densities in the coding region of Pol II genes in infected cells. Thus,
influenza virus specifically interferes with Pol II elongation, but not Pol II initiation. We propose that influenza virus RNA polymerase, by binding
to the CTD of initiating Pol II and subsequent cleavage of the capped 5′ end of the nascent transcript, triggers premature Pol II termination.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Influenza A virus; RNA-dependent RNA polymerase; RNA polymerase II; Carboxy-terminal domain (CTD); Transcription
The synthesis of eukaryotic mRNAs is performed by RNA
polymerase II (Pol II) in the cell nucleus. There is increasing
evidence suggesting that mRNA processing events, i.e.,
capping, the removal of intron sequences by splicing, and the
addition of a poly(A) tail at the 3′ end, are performed
cotranscriptionally on the nascent transcript (Bentley, 2005;
Proudfoot et al., 2002). The addition of the cap structure is
known to occur soon after transcription initiation, around the
time the transcript reaches a length of 25–30 nucleotides (Jove
and Manley, 1984; Rasmussen and Lis, 1993). The components
of the capping apparatus are recruited by binding to the
phosphorylated carboxy-terminal domain (CTD) of the large
subunit of Pol II (McCracken et al., 1997). The CTD, which is
unique to Pol II, is composed in mammalian cells of 52 heptad
repeats (YSPTSPS consensus sequence), ending with a unique
10 amino acid sequence. The CTD plays a key role in coupling
Pol II transcription and RNA processing events. It binds not
only the capping apparatus but also factors required for splicing
and 3′ end processing. During the transcription cycle, the CTD
undergoes extensive phosphorylation and dephosphorylation
events (Palancade and Bensaude, 2003). Phosphorylation of
serine-5 within the heptad repeat is associated with transcription
initiation, and it plays a role in recruitment and stimulation of
capping enzymes. As Pol II enters elongation, serine-2
phosphorylation increases. This change in the phosphorylation
pattern is coupled with the release of 5′ end processing factors
and the recruitment of 3′ end processing factors.
The influenza virus RNA-dependent RNA polymerase
(RdRp), a complex of three subunits, PB1, PB2, and PA, is
dependent on activities associated with the Pol II transcription
apparatus (Fodor and Brownlee, 2002; Lamb and Krug, 2001).
For the initiation of viral mRNA synthesis, it requires capped
RNA fragments about 9–17 nucleotides in length that are
derived from host mRNAs. These fragments are generated by an
endonucleolytic activity associated with the viral RdRp. The
capped fragments act as primers for the viral RdRp, and they
become incorporated at the 5′ end of the viral mRNA
transcripts. For this reason, viral mRNA synthesis is blocked
in the presence of the Pol II inhibitor α-amanitin (Lamb and
Choppin, 1977; Mark et al., 1979). Furthermore, two of the viral
mRNAs are processed by the cellular splicing apparatus that is
also known to be associated with Pol II transcription (Lamb and
Horvath, 1991). Therefore, it was proposed that viral transcrip-
tion might be coupled to Pol II transcription (Fodor et al., 2000).
In fact, it was shown that the trimeric viral RdRp complex binds
Virology 351 (2006) 210–217
⁎Corresponding author. Fax: +44 1865 275556.
E-mail address: firstname.lastname@example.org (E. Fodor).
0042-6822/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
to the CTD of Pol II (Engelhardt et al., 2005). Here, we were
interested to further elucidate the details of this interaction with
an emphasis on the effects this interaction might have on the
activities of Pol II.
Influenza virus RdRp associates with the promoter region of
Pol II genes
It has been proposed that the viral RdRp, in order to gain
access to nascent capped Pol II transcripts, binds to the CTD of
an initiating Pol II that is serine-5 phosphorylated in its CTD
(Engelhardt et al., 2005). This proposal was based on
coimmunoprecipitation and copurification experiments, as
well as colocalization studies using immunofluorescence
microscopy. To obtain further evidence for the interaction
between the viral RdRp and Pol II, we performed a chromatin
immunoprecipitation (ChIP) assay using lysates from HeLa
cells that were either infected with influenza A/WSN/33 virus or
First, we performed ChIP using an antibody against the PA
subunit of the viral RdRp to examine whether Pol II promoter
DNA is present in complexes containing viral RdRp.
Significant levels of the dihydrofolate reductase (DHFR)
promoter DNA were immunoprecipitated from infected cells
(Fig. 1A). Similar results were observed for the promoter
regions of the β-actin and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) genes, both transcribed by Pol II
(Figs. 1B and C). In contrast, no significant levels of
promoter DNA of the 45S rRNA or the 7SK RNA genes,
transcribed by RNA Pol I or RNA Pol III, respectively, could
be immunoprecipitated (Figs. 1A–C).
We also performed ChIPwith anantibody that recognizes the
N-terminal region of the large subunit of Pol II (N20). This
antibody precipitated complexes containing promoter DNAs of
the DHFR, β-actin, and GAPDH genes, but not promoter DNAs
of the 45S rRNA and 7SK RNA genes, as expected.
Interestingly, similar amounts of Pol II promoter DNAs were
precipitated from both infected and uninfected cells, showing
that viral infection does not interfere with RNA Pol II promoter
recognition at 3 h post-infection.
Taken together, these results show that the influenza virus
RdRp is associated with promoter regions of Pol II genes, but
not with promoter regions of Pol I and Pol III genes. This is
consistent with a previous study showing that the viral RdRp
interacts with the serine-5 phosphorylated CTD of Pol II
(Engelhardt et al., 2005).
Viral infection affects Pol II distribution on Pol II genes
Next, we performed ChIP with the Pol II-specific N20
antibody, or with no antibody as a negative control, to examine
the distribution of Pol II along the β-actin gene. ChIP was
performed on both infected and uninfected cells. Statistical
analysis of five independent ChIP experiments showed that
viral infection did not affect Pol II density in the promoter
region of the β-actin gene (Fig. 2A, left panel, also see
Supplementary Table 1). In contrast, there was about a 2-fold
decrease in RNA Pol II densities in the β-actin coding regions
and 3′ UTR in infected cells compared to uninfected cells.
These differences were statistically highly significant (Fig. 2A,
Fig. 1. Influenza virus RdRp associates with Pol II promoter DNA. ChIP was
performed with an antibody against the PA subunit of the viral RNA polymerase
(PA) (Engelhardt et al., 2005) or with the N20 Pol II-specific antibody (Pol II)
(Santa Cruz) using lysates from influenza virus-infected (+) or mock-infected
(−) HeLa cells. ChIP without specific antibody served as a negative control.
Quantitation was performed by real-time PCR using primers specific for the
promoter regions of the DHFR (panel A), 45S rRNA (A), β-actin (B), GAPDH
(C), or 7SK RNA (B and C) genes. The amount of immunoprecipitated DNA
was expressed as percentage of the input DNA. The average and standard
deviationsof three PCR repeatsof a representative ChIP areshown.Atwo-tailed
unpaired Student's t test was performed.
211A.Y. Chan et al. / Virology 351 (2006) 210–217
left panel, also see Supplementary Table 1). It should be noted
that there was about 5- to 10-foldmore Pol II associated with the
promoter–proximal region than with the downstream regions in
uninfected cells (see Supplementary Table 1). This observation
is consistent with the results of a previous report showing about
an 8-fold enrichment of Pol II at the promoter region (Cheng
and Sharp, 2003).
In parallel, we also analyzed the distribution of the viral
RdRp along the β-actin gene by ChIP performed with the PA-
specific antibody or no antibody as a negative control. ChIP was
performed on both infected and uninfected cells. We found that
the viral RdRp accumulated at the promoter region of the gene,
with much lower levels of viral polymerase associated with the
downstream regions (Fig. 2A, right panel, see also Supplemen-
tary Table 1). This finding is consistent with the previous
observation that the viral RdRp preferentially binds to serine-5
phosphorylated CTD which is associated with initiating Pol II
(Engelhardt et al., 2005). In contrast, the viral RdRp exhibited
no detectable affinity for the serine-2 phosphorylated CTD
known to be associated with elongating Pol II.
To confirm these results, we performed a similar analysis on
another Pol II gene, the DHFR gene (Fig. 2B, also see
Supplementary Table 2). Essentially, we observed the same
results showing that there is a significant decrease in Pol II
densities in the coding region of the DHFR gene in the infected
cells compared to mock-infected cells. Also, the viral RdRp
Fig. 2. Statistical analysis of the distribution of Pol II and viral RdRp along Pol II genes. ChIP was performed with the N20 Pol II-specific antibody (left panels) or with
an antibody against the PA subunit of the viral RNA polymerase (right panels) using lysates from influenza virus-infected or mock-infected HeLa cells. ChIP without
specific antibody served as a negative control. Quantitation was performed by real-time PCR using primers specific for the promoter or downstream regions of the β-
actin (A) and DHFR (B) genes. A summary of data from five independent ChIP experiments with the N20 or PA-specific antibody is shown. In each experiment, ratios
of the concentrations of DNA immunoprecipitated from infected and uninfected cells were calculated for each gene region tested. The average and standard deviations
of the five ratios are shown. A two-tailed unpaired Student's t test was performed to assess whether the ratios in the downstream regions were significantly different
from the ratio in the promoter region. Schematic diagrams of the β-actin (A) and DHFR (B) genes are shown. DNA fragments for PCR amplification are shown as bars
under the genes and their approximate positions relative to the site of initiation are indicated in kilobases (kb).
212A.Y. Chan et al. / Virology 351 (2006) 210–217
preferentially accumulated in the promoter region as observed
for the β-actin gene (Fig. 2B, right panel).
Viral RdRp requires Pol II initiation but not elongation
The findings described above suggest that the viral RdRp
might have an inhibitory effect on Pol II elongation. However,
as viral mRNA production is dependent on Pol II-associated
activities (Lamb and Choppin, 1977; Mark et al., 1979), we
were interested to examine whether viral transcription depends
on Pol II elongation. For this, we used the kinase inhibitor DRB
(5,6-dichloro-1-β-D-ribofuranosylbenzimidazole) which inhi-
bits RNA Pol II elongation by preventing CTD hyperpho-
sphorylation but does not interfere with transcription initiation
and capping (Giardina and Lis, 1993; Medlin et al., 2003;
Yamaguchi et al., 1998). As shown in Fig. 3A (lane 4), viral
primary transcription was not affected by DRB treatment.
However, viral replication (cRNA and vRNA synthesis) and
secondary mRNA transcription appeared to be strongly
inhibited by DRB treatment, in agreement with previously
published work describing the inhibition of influenza virus
infection by DRB (Tamm et al., 1954; Tamm and Tyrrell, 1954).
This pattern of transcription is strongly reminiscent of that
obtained following treatment of infected cells with cyclohex-
imide (Vreede et al., 2004), an inhibitor of protein expression.
As viral replication and secondary transcription rely on the
expression of viral proteins (Barrett et al., 1979; Hay et al.,
1977), we speculated that the inhibition of these processes could
be caused by the inhibition of viral protein synthesis in DRB-
treated cells. In fact, it appears that DRB treatment inhibits viral
protein synthesis possibly by the inhibition of downstream
events after viral transcription, i.e., packaging of viral mRNAs
into translation-competent ribonucleoprotein complexes (P.
Digard, personal communication). In order to examine whether
this was the case, we pre-expressed the viral RdRp subunits (3P)
and the nucleoprotein (NP), the minimal set of viral proteins
required for viral RNA synthesis prior to viral infection. When
these transfected 293T cells were then infected as before (Fig.
3A), we observed significant levels of all three viral RNA
species (lane 3). Although we do not fully understand every
aspect of the effects of DRB on the Pol II transcription cycle and
RNA processing, there is ample evidence demonstrating that
DRB blocks Pol II elongation by preventing serine-2 phos-
phorylation of the CTD (Giardina and Lis, 1993; Medlin et al.,
2003, 2005; Yamaguchi et al., 1998). Since we were able to
demonstrate synthesis of capped viral mRNAs in the presence
of DRB (Fig. 3, lane 3), our results clearly show that the viral
RdRp is able to utilize capped RNA primers obtained in the
absence of elongating Pol II.
In contrast, inhibition of Pol II by α-amanitin, which blocks
both Pol II initiation and elongation, abolished the accumulation
of all three types of viral RNA transcripts in infected cells (Fig.
3B, compare lanes 2 and 3). The only RNA detected under this
condition was the vRNA introduced into the cell via viral
infection (input vRNA). Pre-expression of the viral RdRp and
NP (3P/NP) prior to α-amanitin treatment and viral infection
resulted in the accumulation of cRNA and vRNA showing that
Pol II activity is essential only for viral mRNA transcription, but
not cRNA and vRNA synthesis (lane 5). The lack of cRNA and
vRNA accumulation in the absence of pre-expressed viral RdRp
and NP (lanes 3 and 4) is not due to an inhibition of replication.
Rather, the lack of viral protein synthesis, caused by the
inhibition of mRNA transcription, results in insufficient
quantities of viral RdRp and NP to prevent degradation of
and to replicate cRNA synthesized by the input viral RdRp
(Vreede et al., 2004). In order to exclude the possibility that α-
Fig. 3. The effect of DRB and α-amanitin on viral RdRp activity. 293Tcells in 35-mm dishes were transfected with 1 μg of each of pcDNA-PB1, -PB2, -PA, and -NP
(3P/NP),4μg ofpcDNA3A(Vectoralone)(Fodoret al.,2002),4 μgof constructsexpressingα-amanitinresistant PolII (rPol IIandΔ5 rPolII) (Gerber etal., 1995), or
mock transfected (−). At 22 h post-transfection, DRB (A) or α-amanitin (B) was added to a final concentration of 250 μM or 20 μg/ml, respectively. Cells were
incubatedfor a further 1 h (A) or 5 h (B) beforeinfection with influenzaA/WSN/33 virus at an MOI of 5. Cells were harvested 3 h post-infection and RNAwas isolated
using TRIzol (Invitrogen). Viral RNA species were analyzed by an NA gene-specific primer extension assay (Vreede et al., 2004). 5S rRNAwas used as an internal
control. Size markers in nucleotides are shown on the left.
213A.Y. Chan et al. / Virology 351 (2006) 210–217
amanitin directly interfered with transcriptional activity of the
viral RNA polymerase, we used an α-amanitin-resistant clone
of Pol II (Gerber et al., 1995) (lane 6). Under these conditions,
we observed significant levels of all three viral RNA products,
indicating that the inhibition of viral mRNA synthesis in the
presence of α-amanitin is due to the inhibition of Pol II
function. However, deletion of the CTD of the α-amanitin-
resistant Pol II clone, abolished this activity (lane 7). Taken
together, these results show that viral transcription is dependent
on functional Pol II transcription.
It has been known for decades that the influenza virus
RdRp depends on cellular functions associated with Pol II
(Lamb and Choppin, 1977; Mark et al., 1979). Viral mRNA
synthesis by the viral RdRp is initiated by capped RNA
primers which are derived from Pol II transcripts. Recently, it
was demonstrated that the viral RdRp interacts with the CTD
of initiating Pol II, and it was proposed that this interaction is
required for the viral RdRp to gain efficient access to 5′ cap
structures of nascent Pol II transcripts (Engelhardt et al.,
2005). In this paper, we provide evidence that the influenza
virus RdRp is associated with Pol II, but not Pol I or Pol III,
promoter DNA. We found that the viral RdRp is specifically
associated with the promoter region of Pol II genes, with
much lower levels being associated with the regions
downstream of the promoter. This distribution of the viral
RdRp reflects the higher densities of Pol II in the promoter
region compared to the downstream regions and the fact that
it preferentially binds to the serine-5 phosphorylated CTD of
Pol II that is known to be associated with the promoter
region. We also show that viral infection does not interfere
with promoter recognition by Pol II, but it interferes with
events downstream of Pol II promoters. More specifically, we
found decreased amounts of Pol II associated with the internal
gene regions in infected cells, suggesting that Pol II
elongation is inhibited by influenza virus.
Fig. 4. Schematic model for Pol II transcription cycle in uninfected cells (A) and in cells infected with influenza virus (B). The capping, splicing, and 3′ end processing
machineries associated with CTD are indicated. See text for details.
214 A.Y. Chan et al. / Virology 351 (2006) 210–217
Model for Pol II transcription cycle in cells infected with
We propose a model for the Pol II transcription cycle in the
presence of influenza virus infection (Fig. 4). In the absence of
viral infection, RNA Pol II enters elongation after transcription
initiation and capping, producing mature capped, spliced, and
polyadenylated transcripts (Fig. 4A). However, in the presence
of viral infection, Pol II is prevented from entering the
elongation phase due to the viral RdRp binding to the CTD of
initiating Pol II (Fig. 4B). We speculate that this could be due to
the following reasons: (i) removal of the cap structure from the
5′ end of Pol II transcripts results in their degradation by the 5′-
3′ exonuclease Xrn2. As the exonuclease catches up with the
elongating Pol II, transcription is prematurely terminated,
releasing Pol II from the template. This situation would be
analogous to that recently described for termination of Pol II
transcripts, induced by cotranscriptional cleavage, followed by
degradation of the downstream cleavage product by Xrn2
(torpedo model) (West et al., 2004). Alternatively, (ii) the
presence of the viral RdRp on the CTD of the initiating Pol II
might physically prevent its normal functioning. The viral
RdRp could compete with host factors normally binding to the
CTD that are required for transcription elongation, and/or it
could prevent phosphorylation of CTD by steric hindrance.
Both mechanisms could result in Pol II pausing, terminating
prematurely and possibly releasing the template. Our results,
showing reduced Pol II densities in the coding, but not in the
promoter region of Pol II genes, are consistent with this model.
According to this model, influenza virus infection leads to
the inhibition of Pol II elongation but not initiation. Inhibition of
Pol II initiation would be detrimental to the virus considering its
requirement for capped RNA primers that are synthesized by
Pol II. Viral mRNA synthesis is inhibited by α-amanitin, which
blocks both Pol II initiation and elongation. However, viral
transcription can be observed in the presence of DRB, which
affects only Pol II elongation, but not initiation. The observation
that viral transcription can proceed in the presence of DRB
shows that nascent capped transcripts, which are possibly still
associated with the Pol II transcriptional machinery, are
substrates for the viral endonuclease. This suggests that the
viral RdRp is not dependent on mature, fully processed
mRNAs, and that the viral RdRp is able to access nascent
transcripts at the site of their synthesis. In fact, fully processed
mature mRNAs packaged in ribonucleoprotein complexes
destined for rapid export from the nucleus might not be
accessible for the viral RdRp. To overcome this potential
problem, the virus may have evolved a mechanism to target
nascent transcripts at the site of their synthesis when they are not
yet associated with host proteins that might protect them from
Specific inhibition of Pol II elongation in cells infected with
influenza virus could contribute towards the inhibition of the
synthesis of host proteins, a phenomenon known as cell shut-
off. During influenza virus infection, there is a progressive
decline in the synthesis of cellular proteins leading to the almost
exclusive synthesis of viral proteins (Beloso et al., 1992; Inglis,
1982; Zurcher et al., 2000). This could be explained, at least in
part, by inhibition of host mRNA synthesis and processing. For
example, it was proposed that cap snatching could result in the
degradation of newly synthesized Pol II transcripts in the
nucleus (Katze and Krug, 1984), or that the viral NS1 protein
could inhibit mRNA processing leading to a block in mRNA
export (Nemeroff et al., 1998; Shimizu et al., 1999). Our data
extend these previous studies by suggesting additional mechan-
isms that could lead to a decrease in host mRNA levels and
consequently to cell shut-off.
Materials and methods
Chromatin immunoprecipitation (ChIP) assay
HeLa cells grown in 10-cm cell culture dishes (∼70%
confluency) were either infected with influenza A/WSN/33
virus at a multiplicity of infection (MOI) of 5 or mock infected
for 1 h at room temperature. Three hours post-infection,
formaldehyde was added to the cell culture medium to a final
concentration of 1%. Cross-linking reactions were stopped by
the addition of glycine to a final concentration of 125 mM. Cells
pooled from 3 dishes were resuspended in 1 ml cell lysis buffer
[50 mM Tris–HCl, pH 8.0, 10 mM NaCl, 1 mM EDTA, 1 mM
DTT, 0.5% Igepal CA-630 (Sigma), and 1 Complete Mini,
EDTA-free protease inhibitor cocktail tablet (Roche)/10 ml] and
incubated for 10 min on ice. Nuclei were lysed in 1 ml of cell
lysis buffer containing 200 mM NaCl and sonicated in a
Bioruptor (Diagenode) for 15 min. 300 μl clarified cell lysates
were incubated with various antibodies as indicated for 2 h at 4
°C, followed by the addition of protein A-Sepharose CL-4B
beads (Sigma) and a further 2-h incubation at 4 °C. The beads
were washed in 10 mM Tris–HCl, 1 mM EDTA and 0.1%
Igepal CA-630 containing 150 mM NaCl (1 wash), 1 M NaCl (3
washes), or 0.5 M LiCl (3 washes). Complexes were eluted and
cross-links reversed by the addition of 300 μl of elution buffer
(50 mM Tris–HCl, pH 6.8, 200 mM NaCl, 1 mM EDTA, 1%
SDS) and incubation at 65 °C for 12 h. 30 μl of cell extracts
mixed with 300 μl elution buffer was also incubated at 65 °C for
12 h to obtain an “input” DNA sample. DNA was isolated by
proteinase K treatment, extraction with phenol/chloroform and
precipitation with ethanol.
Quantitative PCR analysis
PCR was performed by using the QuantiTect SYBR
Green PCR Kit (Qiagen) and a Corbett Rotor-Gene RG-3000
cycler. Reactions were set up in triplicate, and data were
analyzed by using the Rotor-Gene 6 software. A standard
curve was obtained for each set of PCR primers by
analyzing serial dilutions of the input DNA. The following
sets of primers were used: DHFR-specific primers, promoter
≈6.0 kb (AACAGAATCTGGTGATTATGGG, TACT-
GATCTCCACTATGAGA), ≈17.0 kb (GTTCTATAGT-
TAAG), ≈27.0 kb (GAGTATGTTTCTGTCTTAGATTGG,
215A.Y. Chan et al. / Virology 351 (2006) 210–217
ATGAGAACCTGCTCGCTGAC) (Cheng and Sharp, 2003);
β-actin-specific primers, promoter (CCAATCAGCGTG-
≈1.5 kb (GACCTGAGTCTCCTTTGGAAC, TAATACA-
CACTCCAAGGCCGC), ≈3.0 kb (ACAATGTGGCCGAG-
GACTTTGA, ACACGAAAGCAATGCTATCAC), ≈4.5 kb
TTTCTTCTTGCGAG); GAPDH-specific primers, promoter
CCGGGTTTCTCT) (Medlin et al., 2005); 45S rRNA-specific
primers, promoter (GCGTTTTTGGGGACAGGTGTC,
CGCGCATCCGGAGGCCCAACC); 7SK RNA-specific pri-
mers, promoter (GCCCCACCCATCTGCAAGGCATTC,
RNA isolation and analysis of vRNA, mRNA, and cRNA by
primer extension assay
RNA isolation and analysis were performed as described
(Fodor et al., 2002; Vreede et al., 2004). To analyze NA-specific
viral RNAs, the following primers were used: TGGAC-
TAGTGGGAGCATCAT to detect NA vRNA and TCCAG-
TATGGTTTTGATTTCCG to detect NA mRNA and cRNA.
The expected size of the primer extension products in
nucleotides is 129 for the vRNA, 160 for the cRNA, and 169
to 177 (depending on the length of the capped primer) for the
TCCCAGGCGGTCTCCCATCC (K. Hara, personal com-
munication) was used as a primer to detect 5S rRNA.
We thank J. L. Corden, T. Deng, K. Hara, and S. Murphy for
reagents; G. G. Brownlee and N. Proudfoot for the helpful
discussions. This work was supported by the MRC (senior non-
clinical research fellowship G117/457 to E. F. and cooperative
grant G9826944). F. T. V. was supported by MRC programme
grant G9523972 to G. G. Brownlee and A. Y. C. was supported
by a studentship from the Croucher Foundation.
Appendix A. Supplementary data
Supplementary data associated with this article can be found
in the online version at doi:10.1016/j.virol.2006.03.005.
Barrett, T., Wolstenholme, A.J., Mahy, B.W., 1979. Transcription and
replication of influenza virus RNA. Virology 98 (1), 211–225.
Beloso, A., Martinez, C., Valcarcel, J., Santaren, J.F., Ortin, J., 1992.
Degradation of cellular mRNA during influenza virus infection: its possible
role in protein synthesis shutoff. J. Gen. Virol. 73 (Pt. 3), 575–581.
Bentley, D.L., 2005. Rules of engagement: co-transcriptional recruitment of pre-
mRNA processing factors. Curr. Opin. Cell Biol. 17 (3), 251–256.
Cheng, C., Sharp, P.A., 2003. RNA polymerase II accumulation in the
promoter–proximal region of the dihydrofolate reductase and gamma-actin
genes. Mol. Cell. Biol. 23 (6), 1961–1967.
Engelhardt, O.G., Smith, M., Fodor, E., 2005. Association of the influenza A
virus RNA-dependent RNA polymerase with cellular RNA polymerase II.
J. Virol. 79 (9), 5812–5818.
Fodor, E., Brownlee, G.G., 2002. Influenza virus replication. In: Potter, C.W.
(Ed.), Influenza. Elsevier, Amsterdam, pp. 1–29.
Fodor, E., Mikulasova, A., Mingay, L.J., Poon, L.L., Brownlee, G.G.,
2000. Messenger RNAs that are not synthesized by RNA polymerase
II can be 3′ end cleaved and polyadenylated. EMBO Rep. 1 (6),
Fodor, E., Crow, M., Mingay, L.J., Deng, T., Sharps, J., Fechter, P., Brownlee,
G.G., 2002. A single amino acid mutation in the PA subunit of the
influenza virus RNA polymerase inhibits endonucleolytic cleavage of
capped RNAs. J. Virol. 76 (18), 8989–9001.
Gerber, H.P., Hagmann, M., Seipel, K., Georgiev, O., West, M.A., Litingtung,
Y., Schaffner, W., Corden, J.L., 1995. RNA polymerase II C-terminal
domain required for enhancer-driven transcription. Nature 374 (6523),
Giardina, C., Lis, J.T., 1993. Polymerase processivity and termination on
Drosophila heat shock genes. J. Biol. Chem. 268 (32), 23806–23811.
Hay, A.J., Lomniczi, B., Bellamy, A.R., Skehel, J.J., 1977. Transcription of the
influenza virus genome. Virology 83 (2), 337–355.
Inglis, S.C., 1982. Inhibition of host protein synthesis and degradation of
cellular mRNAs during infection by influenza and herpes simplex virus.
Mol. Cell. Biol. 2 (12), 1644–1648.
Jove, R., Manley, J.L., 1984. In vitro transcription from the adenovirus 2 major
late promoter utilizing templates truncated at promoter–proximal sites.
J. Biol. Chem. 259 (13), 8513–8521.
Katze, M.G., Krug, R.M., 1984. Metabolism and expression of RNA
polymerase II transcripts in influenza virus-infected cells. Mol. Cell. Biol.
4 (10), 2198–2206.
Lamb, R.A., Choppin, P.W., 1977. Synthesis of influenza virus polypeptides in
cells resistant to alpha-amanitin: evidence for the involvement of cellular
RNA polymerase II in virus replication. J. Virol. 23 (3), 816–819.
Lamb, R.A., Horvath, C.M., 1991. Diversity of coding strategies in influenza
viruses. Trends Genet. 7 (8), 261–266.
Lamb, R.A., Krug, R.M., 2001. Orthomyxoviridae: the viruses and their
replication, In: Knipe, D.M., Howley, P.M., Griffin, D.E., Lamb, R.A.,
Martin, M.A., Roizman, B., Straus, S.E. (Eds.), Fields Virology, 4th ed.
Lippincott Williams and Wilkins, Philadelphia, PA, pp. 1487–1530.
Mark, G.E., Taylor, J.M., Broni, B., Krug, R.M., 1979. Nuclear accumulation of
influenza viral RNA transcripts and the effects of cycloheximide,
actinomycin D, and alpha-amanitin. J. Virol. 29 (2), 744–752.
McCracken, S., Fong, N., Rosonina, E., Yankulov, K., Brothers, G., Siderovski,
D., Hessel, A., Foster, S., Shuman, S., Bentley, D.L., 1997. 5′-Capping
enzymes are targeted to pre-mRNA by binding to the phosphorylated
carboxy-terminal domain of RNA polymerase II. Genes Dev. 11 (24),
Medlin, J.E., Uguen, P., Taylor, A., Bentley, D.L., Murphy, S., 2003. The C-
terminal domain of pol II and a DRB-sensitive kinase are required for 3′
processing of U2 snRNA. EMBO J. 22 (4), 925–934.
Medlin, J., Scurry, A., Taylor, A., Zhang, F., Peterlin, B.M., Murphy, S., 2005.
P-TEFb is not an essential elongation factor for the intronless human U2
snRNA and histone H2b genes. EMBO J. 24 (23), 4154–4165.
Nemeroff, M.E., Barabino, S.M., Li, Y., Keller, W., Krug, R.M., 1998.
Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of
CPSF and inhibits 3′end formation of cellular pre-mRNAs. Mol. Cell 1
Palancade, B., Bensaude, O., 2003. Investigating RNA polymerase II carboxyl-
terminal domain (CTD) phosphorylation. Eur. J. Biochem. 270 (19),
Proudfoot,N.J., Furger, A.,Dye, M.J.,2002.Integrating mRNA processingwith
transcription. Cell 108 (4), 501–512.
Rasmussen, E.B., Lis, J.T., 1993. In vivo transcriptional pausing and cap
formation on three Drosophila heat shock genes. Proc. Natl. Acad. Sci.
U.S.A. 90 (17), 7923–7927.
Shimizu, K., Iguchi, A., Gomyou, R., Ono, Y., 1999. Influenza virus inhibits
cleavage of the HSP70 pre-mRNAs at the polyadenylation site. Virology
254 (2), 213–219.
Tamm, I., Tyrrell, D.A., 1954. Influenza virus multiplication in the
216A.Y. Chan et al. / Virology 351 (2006) 210–217
chorioallantoic membrane in vitro: kinetic aspects of inhibition by 5,6-
dichloro-1-beta-D-ribofuranosylbenzimidazole. J. Exp. Med. 100 (6),
Tamm, I., Folkers, K., Shunk, C.H., Horsfall Jr., F.L., 1954. Inhibition of
influenza virus multiplication by N-glycosides of benzimidazoles-N. J. Exp.
Med. 99 (3), 227–250.
Vreede, F.T., Jung, T.E., Brownlee, G.G., 2004. Model suggesting that
replication of influenza virus is regulated by stabilization of replicative
intermediates. J. Virol. 78 (17), 9568–9572.
West, S., Gromak, N., Proudfoot, N.J., 2004. Human 5′→3′ exonuclease Xrn2
promotes transcription termination at co-transcriptional cleavage sites.
Nature 432 (7016), 522–525.
Yamaguchi, Y., Wada, T., Handa, H., 1998. Interplay between positive and
negative elongation factors: drawing a new view of DRB. Genes Cells 3 (1),
Zurcher, T., Marion, R.M., Ortin, J., 2000. Protein synthesis shut-off induced by
influenza virus infection is independent of PKR activity. J. Virol. 74 (18),
217 A.Y. Chan et al. / Virology 351 (2006) 210–217