Influenza virus inhibits RNA polymerase II elongation.
ABSTRACT 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.
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ABSTRACT: Influenza virus RNA-dependent RNA polymerase (RdRp) is a heterotrimer composed of PA, PB1 and PB2 subunits. RdRp is required for both transcription and replication of influenza viral RNA taking place in the nucleus of infected cells. A 'cap-snatching' mechanism is employed to generate a 5'-capped primer for transcription, in which the cap-binding domain of PB2 (PB2cap) captures the 5' cap of the host pre-mRNA. Our statistical analysis of PB2 sequences showed that residue Lys339 located in the cap-binding pocket of H5N1 PB2cap was gradually replaced by Thr339 over the past decade. To understand the role of this amino acid polymorphism, we solved the crystal structures of PB2cap with or without a pre-mRNA cap analog m7GTP in the presence of Lys339 or Thr339. The structures showed that Lys339 contributes to binding the γ-phosphate group of m7GTP, and the replacement of Lys339 by Thr eliminates this interaction. Isothermal titration calorimetry analysis showed that Thr339 attenuated the PB2cap cap-binding activity in vitro compared to Lys339. Further functional studies confirmed that Thr339-PB2-containing RNP has a reduced influenza polymerase activity and RNA synthesis activity, and a reconstituted H5N1 virus containing the Thr339 substitution exhibited a lower virulence to mice while more active replication in MDCK cells. The K339T substitution in the cap-binding pocket of PB2 modulates the polymerase activity and virulence by regulating the cap-binding activity. It is informative to track variations in the cap-binding pocket of PB2 in surveillance of the evolution and spread of influenza virus.Journal of Biological Chemistry 02/2013; · 4.65 Impact Factor
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ABSTRACT: The segmented genome of an influenza virus is encapsidated into ribonucleoprotein complexes (RNPs). Unusually among RNA viruses, influenza viruses replicate in the nucleus of an infected cell, and their RNPs must therefore recruit host factors to ensure transport across a number of cellular compartments during the course of an infection. Recent studies have shed new light on many of these processes, including the regulation of nuclear export, genome packaging, mechanisms of virion assembly and viral entry and, in particular, the identification of Rab11 on recycling endosomes as a key mediator of RNP transport and genome assembly. This review uses these recent gains in understanding to describe in detail the journey of an influenza A virus RNP from its synthesis in the nucleus through to its entry into the nucleus of a new host cell.Viruses 01/2013; 5(10):2424-2446. · 2.51 Impact Factor
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ABSTRACT: We have examined the requirements for virus transcription and replication, and thus the roles of input and progeny genomes, in the generation of IFN-inducing PAMPs by influenza A viruses using inhibitors of these processes. Using IRF3 phosphorylation as a marker of activation of the IFN induction cascade that occurs upstream of the IFN-β promoter, we demonstrate strong activation of the IFN induction cascade in A549 cells infected with a range of influenza A viruses in the presence of cycloheximide or NP siRNA, which inhibit viral protein synthesis and thus cRNP and progeny vRNP synthesis. In contrast, activation of the IFN induction cascade by influenza viruses was very effectively abrogated by treatment with actinomycin D and other transcription inhibitors, which correlated with the inhibition of the synthesis of all viral RNA species. Furthermore, 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole, an inhibitor that prevents viral RNA export from the nucleus, was also a potent inhibitor of IRF3 activation; thus, both viral RNA synthesis and nuclear export are required for IFN induction by influenza A viruses. Whilst the exact nature of the viral PAMPs remains to be determined, our data suggests that in this experimental system the major influenza A virus PAMPs are distinct from incoming genomes or progeny vRNPs.IMPORTANCE The host interferon system exerts an extremely potent antiviral response that efficiently restricts virus replication and spread; the interferon response can thus dictate the outcome of a virus infection and it is therefore important to understand how viruses induce interferon. Both input and progeny genomes have been linked to interferon induction by influenza viruses. However, our experiments in tissue culture cells show that viral RNA synthesis and nuclear export are required to activate this response. Furthermore, the interferon induction cascade is activated under conditions in which the synthesis of progeny genomes is inhibited. Therefore, in tissue culture cells, input and progeny genomes are not the predominant inducers of interferon generated by influenza A viruses; the major viral interferon inducer/s still remain to be identified.Journal of Virology 01/2014; · 5.08 Impact Factor
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: email@example.com (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.
211 A.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).
212 A.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.
213 A.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