Accepted Manuscript

Title: Structural insights into RNA synthesis by the influenza

virus transcription-replication machine

Authors: Alexander Pflug, Maria Lukarska, Patricia

Resa-Infante, Stefan Reich, Stephen Cusack

PII: S0168-1702(16)30782-1
DOI: http://dx.doi.org/doi:10.1016/j.virusres.2017.01.013
Reference: VIRUS 97056

To appear in: Virus Research

Received date: 28-11-2016

Revised date: 31-12-2016
Accepted date: 13-1-2017

Please cite this article as: Pflug, Alexander, Lukarska, Maria, Resa-Infante,
Patricia, Reich, Stefan, Cusack, Stephen, Structural insights into RNA synthesis
by the influenza virus transcription-replication machine.Virus Research
http://dx.doi.org/10.1016/j.virusres.2017.01.013

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
Structural insights into RNA synthesis by the influenza virus
transcription-replication machine

Alexander Pflug, Maria Lukarska, Patricia Resa-Infante, Stefan Reich and Stephen Cusack1#

1
European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, CS
90181, 38042 Grenoble Cedex 9, France

#Corresponding author: Stephen Cusack, European Molecular Biology Laboratory, Grenoble

Outstation, Grenoble Cedex 9, France. Tel. (33)476207238, Email: [email protected]

Review for Virus Research

Editors Luis Menéndez-Arias and Raul Andino
Submitted 27th November 2016.
Revised version 31st December 2017

1
Highlights
 Influenza polymerase is a dynamic machine with distinct functional configurations
 Crystal structures explain the mechanics of cap-dependent transcription
 Cap-snatching requires that influenza polymerase directly binds Pol II CTD
 A priming loop is required to initiate ‘unprimed’ vRNA to cRNA replication
 Influenza polymerase presents multiple targets for antiviral drug development

Abstract
Influenza virus is a segmented, negative strand RNA virus with each genome segment
being packaged in a distinct ribonucleoprotein particle (RNP). The RNP consists of the
heterotrimeric viral RNA-dependent RNA polymerase bound to the conserved 5' and 3' ends of
the genome segment (the viral promoter) with the rest of the viral RNA (vRNA) being covered
by multiple copies of nucleoprotein. This review focusses on the new insights that recent crystal
structures have given into the detailed molecular mechanisms by which the polymerase performs
both transcription and replication of the vRNA genome. Promoter binding, in particular that of 5'
end, is essential to allosterically activate all polymerase functions. Transcription is initiated by
the hijacking of nascent, capped host transcripts by the process of ‘cap-snatching’, for which the
viral polymerase makes an essential interaction with the C-terminal domain (CTD) of cellular
RNA polymerase II. The structures allow a coherent mechanistic model of the subsequent cap-
snatching, cap-dependent priming, elongation and self-polyadenylation steps of viral mRNA
synthesis. During replication, the vRNA is copied without modification into complementary
RNA (cRNA) which is packaged into cRNPs. A priming loop located in the polymerase active
site is required for the unprimed synthesis of cRNA from vRNA, but is not required for cRNA to
vRNA replication due to differences in the mode of initiation of RNA synthesis. Overall a
picture emerges of influenza polymerase being a highly complex, flexible and dynamic machine.
The challenge remains to understand in more detail how it functions within the RNP and how
interacting host factors modulate its activity in the cellular context. Finally, these detailed
insights have opened up new opportunities for structure-based antiviral drug design targeting
multiple aspects of polymerase function.

2
Abbreviations : cRNA, complementary RNA; CTD, C-terminal domain of Pol II; Pol II, cellular
RNA polymerase II; RNP, ribonucleoprotein particles; vRNA, viral genomic RNA;

Keywords: Influenza polymerase, RNA synthesis, replication, transcription, cap-snatching,

Pol II C-terminal domain, anti-influenza drug design

1. Introduction and scope of review

Influenza virus is a segmented negative-strand RNA virus of the family
Orthomyxoviridae. There are three officially recognised influenza genera denoted A, B and C
and potentially a fourth (D) recently discovered (Center for Disease Control, 2016). Influenza A
and B strains, which both have eight genome segments coding for at least 11 proteins, cause
widespread seasonal disease in humans. Influenza A is a zoonotic pathogen present in multiple,
antigenetically distinct strains in the aquatic bird reservoir, most of which are not transmissible
to humans. However it shows rapid and unpredictable evolution due to the possibility of segment
reassortment both within avian strains and between avian and mammalian strains. This can
generate novel human transmissible viruses with pandemic potential. Alternatively, acquisition
of point mutants in avian strains can allow them to more efficiently enter and replicate in
mammalian cells resulting in sporadic outbreaks particular in East Asia. Influenzas C and D both
have seven segments (the hemagglutinin and neuraminidase of A and B strains are replaced by
one dual function glycoprotein in C and D strains) and infect mainly humans and cattle
respectively (Swinehealth Information Center, 2015). Recently, genomic RNAs corresponding to
divergent influenza A like viruses have been isolated from various New World bat species where
they seem to have evolved independently from the avian/mammalian reservoir (Tong et al.,
2013).
The single-stranded RNA genome segments (vRNA) are packaged inside the virus in
distinct ribonucleoprotein particles (RNPs)(Eisfeld et al., 2015). Each RNP comprises the viral
RNA-dependent RNA polymerase bound to its promoter, which consists of the conserved and
quasi-complementary 5′ and 3′ extremities of the vRNA. Promoter binding to the polymerase
thus pseudo-circularises the vRNA, the remainder of the RNA being coated by multiple copies of
the viral nucleoprotein (NP) arranged in an anti-parallel, superhelical, flexible rod-shape (Figure
1A). In the infected cell nucleus, where viral RNA synthesis occurs, each RNP acts as an

3
autonomous transcription-replication unit (Figure 1BC). The same vRNA template can be either
transcribed into capped and poly-adenylated viral mRNA (the cap is obtained by a unique
process called ‘cap-snatching’, (Plotch et al., 1981), see below) or copied without modification
into complementary RNA (cRNA). Early in infection, transcription is the predominant process
allowing build-up of a pool of viral proteins. Subsequently, newly synthesised polymerase and
NP is used to assemble and stabilise first cRNPs and then progeny vRNPs, which are generated
through back replication of the anti-genomic cRNA intermediate. Finally vRNPs are selectively
exported from the nucleus, transported to the plasma membrane (Lakdawala et al., 2016) and,
together with the other major structural proteins (notably the M protein and surface glycoproteins
HA and NA), packaged into progeny virions which bud from the cell surface (Rossman and
Lamb, 2011). Specific RNA-RNA interactions between the different vRNPs are thought to
ensure packaging of a full complement of genomic vRNPs (Giese et al., 2016; Hutchinson et al.,
2010).
Over many years an enormous body of knowledge has built up concerning all aspects of
the influenza virus life cycle. Here we will focus on the role of the influenza RNA-dependent
RNA polymerase in viral RNA synthesis. The recent breakthroughs in the structure
determination of the complete promoter-bound polymerase has resolved long-standing
controversies and led to new insights into the functioning of the viral transcription/replication
machine as well as opening up new opportunities for structure based anti-viral drug design
(Stevaert and Naesens, 2016; Te Velthuis and Fodor, 2016).

2. The structure of influenza polymerase

2.1 The structure of individual polymerase domains
Influenza A polymerase is a ~270 kDa heterotrimeric complex composed of subunits PA
(polymerase acidic protein), PB1 (polymerase basic protein 1) and PB2 (polymerase basic
protein 2) of respectively 716 (726 in influenza B), 757 (752) and 759 (770) residues, which are
encoded by the three longest viral genome segments 1-3. During 2007-2008 the first structures of
independently folded fragments of the polymerase subunits emerged from crystallographic
analysis (Boivin et al.). First was the small, C-terminal, nuclear localisation signal (NLS)
containing domain of PB2 in complex with the nuclear import factor importin-α (Tarendeau et
al., 2007). Secondly was solved the PB2 627-domain and the 627-NLS double domain

4
(Tarendeau et al., 2008; Yamada et al., 2010). The 627-domian is so-called because it contains
the important host-determining residue, Lys627 in human strains and Glu627 in avian strains
(Long et al., 2016; Subbarao et al., 1993). The two stable inter-subunit interfaces were also
characterised structurally, that between the large C-terminal domain of PA and the N-terminal
extremity of PB1 (He et al., 2008; Obayashi et al., 2008) and a helical bundle composed of the
C-terminal region of PB1 and the N-terminal extremity of PB2 (Sugiyama et al., 2009),
confirming that the three subunits are linked head-to-tail in the order PA-PB1-PB2. Finally, the
locations of the two key domains for cap-snatching were identified and their structures solved,
thus resolving years of controversy about their nature and location. The cap-binding domain,
which binds the nascent-capped Pol II transcripts, is located in the central region of PB2 and has
a unique fold. The only similarity to known cap-binding proteins such as the cytoplasmic and
nuclear cap-binding proteins, eIF4E and CBC respectively, is that the methylated guanosine of
the cap (m7G) is specifically recognised by stacking between aromatic residues (Guilligay et al.,
2008). The endonuclease, responsible for cleaving the pirated host transcript 10-14 nucleotides
downstream from the 5′ cap, is located in the N-terminal domain of the PA subunit (Dias et al.,
2009; Yuan et al., 2009). It has a core fold characteristic of the PD-(D/E)xK superfamily of
nucleases (which includes several restriction enzymes and resolvases) and a two divalent cation
dependent active site (Crepin et al., 2010; Kowalinski et al., 2012). The isolated endonuclease
domain binds RNA weakly and there is still no structure revealing how the substrate is bound
although biochemical analysis suggests that there might be some sequence preference at the
cleavage site (Datta et al., 2013). Both the cap-binding domain and endonuclease are actively
being targeted for the development of new anti-influenza drugs (see below).

2.2 The structure of the heterotrimeric influenza polymerase

The goal of determining the structure of the intact heterotrimer was for a long time
hampered by the difficulty of obtaining sufficient amounts of pure and active influenza A
polymerase. Eventually this problem was circumvented by focussing on bat influenza A/H17N10
(Pflug et al., 2014), influenza B (Reich et al., 2014; Thierry et al., 2016) and influenza C
(Hengrung et al., 2015) polymerases, which express well in insect cells. For unclear reasons,
human and avian influenza A polymerases remain difficult to express in large amounts as full-
length heterotrimers (Swale et al., 2016), although PB2-truncated variants express very well

5
(Swale et al., 2016) and allowed a cryo-EM structure of a dimeric form to be determined at good
resolution (Chang et al., 2015).
The original structures determined of bat influenza A and human influenza B with bound
promoter are very similar despite considerable divergence in amino acid sequence (overall, bat
and influenza B polymerases are respectively 75 and 35 % identical to human (or avian)
influenza A polymerase). The shapes of the three individual subunits and how they assemble to
form the promoter bound trimer are shown in Figure 2. The overall polymerase is U shaped with
the two upper protuberances being the endonuclease (PA-N) and the PB2 cap-binding domain
respectively (Figure 2A). The endonuclease is held in place by one of its helices extending the
helical bundle formed by the interface between the C-terminus of PB1 and the N-terminus of
PB2. The extended PA linker comprises three short helices that are packed on one side of the
PB1 subunit and connects to the large PA-C domain, which forms the bottom of the U. The PB1
subunit fills the interior of the U and has the characteristic fold of other viral RNA polymerases
with fingers, palm and thumb surrounding a central active site cavity where RNA synthesis
occurs (Figure 2B). The N-terminal third of PB2 (PB2-N) forms a series of modules that pack on
the opposite side of PB1 from the PA linker (Figure 2A). The structural stabilisation of PB1 by
large interfaces with the PA linker, PA-C and PB2-N is consistent with the fact that PB1 cannot
be expressed in soluble form on its own. The C-terminal two-thirds of PB2 (PB2-C) form one
side of the U and comprises an arc of independently folded domains including the cap-binding
domain, which is an insertion into the mid-link domain. These are followed by the PB2 627 and
NLS domains, which are juxtaposed in the same configuration as observed in crystal structures
of the 627-NLS double domain (Tarendeau et al., 2008).

2.3 Promoter binding

In the absence of polymerase, the promoter, composed of the conserved and quasi
complementary 3′ (nts 1-12) and 5′ (nts 1-13) ends of the vRNA (or cRNA), can form a non-
canonically base-paired duplex referred to as the panhandle (Hsu et al., 1987). However this is
not how the promoter binds to the polymerase as was originally shown biochemically (Flick et
al., 1996; Kim et al., 1997). In the promoter bound structure, nucleotides 1-10 of the 5′ end form
an intramolecular stem-loop or ‘hook’ with four base-pairs (two canonical base-pairs flanked
each side by an A-A base-pair) (Figure 3A). This compact RNA structure is bound with high

6
affinity in a pocket at the interface between PA and PB1 (Figure 3B). The loop of the 5′ hook
(nts 5-6) is close to the polymerase active site. Indeed binding of the promoter 5′ end
allosterically activates multiple polymerase functions (Li et al., 1998; Thierry et al., 2016) and
this corresponds structurally to ordering of the fingertips loop and other parts of the conserved
polymerase active site upon 5′ end binding (Gerlach et al., 2015). The distal part of the 5′ end of
the promoter makes at least three conserved canonical base-pairs with the 3′ end (specifically 5′
nts 11-13 and 3′ nts 10-12) whilst the proximal 3′ end nucleotides 1-9 are single-stranded and
must enter the polymerase active site through the template entrance tunnel (see below) to allow
initiation of RNA synthesis. In the first promoter bound polymerase structures, 3′ nucleotides 1-6
were not observed in the internal active site cavity being either not visible due to disorder or
bound on the polymerase surface (Pflug et al., 2014; Reich et al., 2014). However a new
influenza B polymerase structure co-crystallised with capped RNA primer shows that the 3′ end
can be relocated into the active site by threading through the narrow template entrance tunnel
with only minor perturbations in the structure (Figure 3B)(Reich et al., 2017).

2.4 Alternative configurations of the heterotrimer

Additional crystal structures of influenza A, B (Thierry et al., 2016) and C (Hengrung et
al., 2015) polymerases and biophysical characterization in solution have shown that the
heterotrimer has considerable conformational flexibility and likely exists in several distinct
functional states (Figure 4). Furthermore, the propensity to be in one or other state depends on
whether the full, partial or no promoter is bound (Hengrung et al., 2015; Thierry et al., 2016).
These studies show that there is an invariant core comprising PB1 stabilized by the PA linker,
PA-C and PB2-N, whereas the flexibly linked peripheral domains, the PA endonuclease (PA-N)
and the PB2-C mid-link, cap-binding, 627 and NLS domains can pack together in strikingly
different configurations (Figure 4). A direct comparison can be made between two radically
different conformations of influenza B polymerase, one with the full vRNA promoter bound
(Reich et al., 2014)(Figure 4B) and the other with just the c5′ end bound (this forms a very
similar stem-loop structure to the v5′ end)(Thierry et al., 2016)(Figure 4C). All the peripheral
domains are displaced by large amounts, the most striking example being the PB2 C-terminal
NLS domain, which in the c5′ structure has separated from 627-domain (see also (Delaforge et
al., 2015)) and translated 93 Å to interact with the endonuclease domain. The bipartite NLS

7
peptide (Tarendeau et al., 2007) forms an α-helix which packs on the endonuclease, without
apparently affecting intrinsic endonuclease function (Thierry et al., 2016). The same
juxtaposition of the endonuclease and the NLS domains is observed in the influenza C
polymerase structure with no bound promoter (Hengrung et al., 2015), which overall exhibts a
similar but not identical conformation to the influenza B c5′ structure (Figure 4D). The general
significance of this alternative conformation is further reinforced by the fact that the a multi-
domain PB2-C construct comprising the mid-link, cap-binding and 627 domains of influenza
A/H5N1 polymerase pack together in the same way as observed in the influenza B c5′ structure
(Thierry et al., 2016). The full promoter bound polymerase structure is consistent with a cap-
dependent transcriptionally active conformation (i.e. transcriptase, see below), whereas the apo-
influenza C and c5′ B structures are not, due to the unfavourable juxtaposition of the cap-binding
and endonuclease domains. This is consistent with the requirement of v5′ end or full promoter
binding for optimal cap-snatching activity (Thierry et al., 2016). However the exact functional
role of the apo-FluC and c5′ Influenza B conformation remains to be elucidated. It could
correspond to the newly synthesised apo-polymerase before assembly into a progeny RNP (for
vRNA to cRNA replication, such an apo-polymerase would bind first the nascent c5′ end that
emerges first from the replicating RNP and only at the end of replication would bind the c3′ end)
or it could be the replicase, that is the replication competent form of the polymerase.

2.5 Influenza RNP structure

At the next level of structural organisation, RNPs appear as flexible rods comprising two
anti-parallel superhelical strands of RNA bound NPs with a loop at one end and the polymerase
holding together the viral RNA extremities at the other end (Klumpp et al., 1997)(Figure 1A).
Significant progress is being made in the structure determination of RNPs by cryo-electron
microscopy (Arranz et al., 2012; Coloma et al., 2009; Moeller et al., 2012). However this is
hindered by the considerable flexibility of RNPs and the resolution is not yet high enough to
unambiguously define the interactions between the NPs, how the RNA binds to NP and its path
through the RNP and how the NPs bind to the polymerase. Recently a method has been
described allowing isolation of cRNPs from cells showing that at low resolution cRNPs resemble
vRNPs (York et al., 2013).

8
2.6 Structural similarities with other viral polymerases.
The other two major families of segmented negative-strand viruses, arena- (e.g. Lassa
virus) and bunya- (e.g. La Crosse, Rift-Valley and Crimean-Congo Haemorrhagic Fever) viruses,
with respectively two and three genome segments, possess a single chain polymerase (L protein)
(Ferron et al., 2017). Similar to influenza polymerase, arena- and bunyaviral polymerases bind to
both, highly complementary ends of each vRNA segment, pseudo-circularising the RNP, and
perform transcription by cap-snatching, but differ in that they replicate in the cytoplasm
(Reguera et al., 2014). Based on the finding that the N-terminal domain of the arena- and
bunyaviral L protein corresponded to the cap-snatching endonuclease (Morin et al., 2010;
Reguera et al., 2010) and the presence of the polymerase motifs in the central region, it was
proposed that the L protein would be architecturally equivalent to the head-to-tail concatenation
of the PA, PB1 and PB2 subunits of influenza polymerase (Reguera et al., 2010). The recent
structure determination of the C-terminally truncated La Crosse orthobunyavirus polymerase
confirmed this hypothesis, despite the high sequence divergence and idiosyncratic structural
differences (Gerlach et al., 2015). Furthermore, the allosteric 5′ end of the vRNA promoter was
bound in a similar hook conformation as in influenza polymerase, whereas, unlike influenza, the
3′ end is sequestered by highly specific interactions in a surface groove of the polymerase
(Gerlach et al., 2015). The first structure of a non-segmented negative-strand viral polymerase,
that of Vesicular stomatitis virus (VSV) (Liang et al., 2015), shows a more distant relationship
with influenza polymerase with conservation only of the polymerase core buttressed by a PA-C
like structure. The notable difference is that the domains relevant for cap-snatching in segmented
negative-strand polymerases are replaced by capping enzymes in non-segmented negative-strand
polymerases, reflecting the quite different mechanism for mRNA 5′ end formation in these two
viral families (Liang et al., 2015; Reguera et al., 2016; Whelan, 2017).

3. RNA synthesis by influenza polymerase

3.1 General features of RNA synthesis by influenza polymerase
Influenza RNPs can perform either replication or transcription of the same template and
an outstanding question is what determines which it does? The two processes are overall quite
different mechanistically even if the core catalytic activity of template-directed nucleotide
addition is the same. Replication corresponds to unprimed synthesis of a full-length, unmodified

9
copy of the template coupled with packaging of the nascent replicate into a progeny RNP of
reverse polarity. Transcription is primed by cap-snatching from nascent host Pol II transcripts
which yields a 10-14 nt capped primer of variable sequence that precedes the virally encoded
sequences in the resultant chimeric viral mRNA. In either case, RNA synthesis is catalysed by
the PB1 subunit within its internal active site cavity, which is formed by the highly conserved
polymerase motifs A-F (Jacome et al., 2015; Pflug et al., 2014). Motifs A and C contribute the
three conserved aspartates (Asp 305, Asp 445 and Asp 446) that coordinate two divalent cations
and promote nucleophilic attack of the terminal 3' OH from the growing transcript (or primer) on
the α-phosphate of the incoming NTP. Template and product RNA trafficking, as well as
substrate NTP accessibility, occurs via tunnels linking the exterior to the internal active site
chamber. Similar to other negative strand (Gerlach et al., 2015) (Liang et al., 2015) and double-
stranded RNA viral polymerases (Reguera et al., 2016; Tao et al., 2002), influenza has four
tunnels corresponding to template and NTP entry and template and product exit (Figure 5).

3.2 Structure based model for viral mRNA transcription

The recent high resolution structures of influenza polymerase provide evidence for a
highly plausible structure-based mechanism for cap-dependent transcription and
polyadenylation, consistent with many previous biochemical results (Figure 6). The critical
observation was that the PB2 cap-binding domain can rotate in situ, presumably stochastically,
over a large angular range of at least 70° (Reich et al., 2014). The four key steps in the structure
based model for synthesis of poly-adenylated viral mRNA are: (1) Cleavage by the PA
endonuclease of the donor host nascent Pol II transcript bound by its cap to the PB2 cap binding
domain (Figure 6A). (2) Rotation of the cap-binding domain directs the short capped primer
down the exit tunnel towards the catalytic centre of PB1 (Figure 6B). It is thought that those
primers with 3′ ends complimentary to the 3′ extremity of the template are preferentially selected
for transcription initiation (Geerts-Dimitriadou et al., 2011; Hagen et al., 1995)(Figure 6C). (3)
Progression into processive elongation with the translocating template and product being
extruded through separate tunnels (Figure 6D). (4) Formation of the poly(A) tail by reiterative
copying of the 5′ proximal oligo-U stretch of the template corresponding to nucleotides 17-22
from the 5′ end (Poon et al., 1999; Robertson et al., 1981)(Figure 6E, F). The growing poly(A)
tail is able to slip on the oligo-U stretch whilst the template is prevented from translocating by

10
the tight binding of the terminal 5′ hook to the polymerase. Not requiring the cellular machinery
for poly-adenylation, the virus actually inhibits this machinery using its NS1 protein (Nemeroff
et al., 1998), just one of the multiple mechanisms by which the virus limits host cell gene
expression (Bercovich-Kinori et al., 2016; Vreede and Fodor, 2010). Other mechanisms include
cap-snatching, degradation of Pol II (see below) and specific host mRNA degradation by the
recently discovered free-standing endonuclease PA-X derived from the PA gene by ribosomal
frame-shifting (Jagger et al., 2012; Khaperskyy et al., 2016). It is not known what causes the
eventual release of the mature transcript after the poly(A) tail reaches a length of 30-180
(Pritlove et al., 1998), nor how the template rebinds to the polymerase to initiate another round
of transcription. Finally, viral mRNAs are further processed by host machineries for splicing (for
the M and NS segments and for some viruses, the PB2 segment (Yamayoshi et al., 2015)),
nuclear export and translation similarly to host pre-mRNAs (Bier et al., 2011).
Further structural data are needed to confirm each step of this hypothetical transcription
model and clarify for instance how it applies in the RNP context, where the template has to be
disengaged from the proximal nucleoprotein prior to entering the polymerase active site and then
rebind to nucleoprotein after exiting. Towards this goal, a new influenza B polymerase structure
attempted to trap the transcription initiation state (Step 2), by co-crystallisation with a 13-mer
capped RNA primer ending in ApG. It shows for the first time the template 3′ end passing
through the entrance tunnel into the active site (Figure 3B) (unlike in the original influenza A or
B promoter bound structures) as well as the cap proximal part of the primer, which makes
multiple contacts to both the cap-binding domain and the mid domain (Reich et al., 2017). This
might explain the enhanced binding of capped RNA to the intact polymerase compared to the
isolated cap-binding domain (Cianci et al., 1997). However the expected base-pairing of the
primer terminal ApG-3′ with the template terminal 3′-UpCpG in the active site is not observed.
This could be because this base-pairing is only transiently stable, especially in the absence of the
next required incoming NTP (CTP). Alternatively it could be because stable formation of the
transcription initiation state requires breaking of the distal 3′-5′ base-pairs of the promoter to
allow the template to translocate freely, whereas in the crystal these base-pairs are still present.
These observations are consistent with a recent FRET study of the dynamics of the template
RNA during initiation of RNA synthesis (Robb et al., 2016).

11
3.3 Cap-snatching in the cellular context
Transcription by cap-snatching is a highly efficient process such that six hours post
infection around 50% of the mRNA in the infected cell is viral mRNA (Bercovich-Kinori et al.,
2016), but how does influenza polymerase get robust access to capped host-cell Pol II
transcripts? Furthermore it has been recently recognised that non-coding RNAs such as the very
abundant snRNAs are pirated as much as pre-mRNAs (Koppstein et al., 2015)(Gu et al., 2015),
raising the question as to whether there is preferential targeting of particular transcription
complexes. An interaction between influenza polymerase and the C-terminal domain (CTD) of
Pol II has been suggested to recruit the viral polymerase to the host transcription machinery
(Engelhardt et al., 2005; Martinez-Alonso et al., 2016). Mammalian Pol II CTD consists of 52
heptad repeats with consensus sequence Tyr1Ser2Pro3Thr4Ser5Pro6Ser7 (Palancade and Bensaude,
2003) and all of the serine, threonine and tyrosine residues can be reversibly phosphorylated
during transcription. The CTD serves as a scaffold to recruit RNA-processing factors and its
different phosphorylation states temporarily regulate this association. Capping of nascent
transcripts is performed whilst Pol II is paused soon after initiation due to the combined effect of
negative-elongation factors NELF and DSIF and when the CTD is predominantly Ser5-
phosphorylated. The 5′ triphosphate end of the nascent RNA emerging from the Pol II exit tunnel
is modified successively by RNG-TT (RNA guanylyltransferase and 5′ phosphatase), RNMT
(guanine-7-methyltransferase) and CMTR1 (cap-specific mRNA (nucleoside-2'-O-)-
methyltransferase 1), which are recruited to the Ser5-phosphorylated CTD. Once complete the
cap is rapidly sequestered by the nuclear cap-binding complex (CBC). Concomitantly Pol II
transitions to the elongation phase of transcription. This is induced by phosphorylation of DSIF
and NELF by the kinase pTEF-b and conversion of the CTD to a predominantly serine-2 (Ser2)
phosphorylated state (Lidschreiber et al., 2013). Thus influenza polymerase only has a short time
window to intervene and pirate capped transcripts. During infection influenza polymerase has
been shown to associate with initiating Pol II, when the CTD is Ser5-phosphorylated, but not to
the Ser2-phosphorylated form (Chan et al., 2006; Engelhardt et al., 2005; Loucaides et al., 2009).
Furthermore influenza polymerase inhibits transcription elongation and induces Pol II
degradation (Rodriguez et al., 2007; Vreede et al., 2010), which contributes to the inhibition of
cellular gene-expression (‘host shut-off’) (Vreede et al., 2010).

12
 A recent structural and biophysical analysis reveals that influenza A polymerase binds
directly to four consecutive heptad repeats of the Ser5-phosphorylated CTD (Lukarska et al.,
2016)(Figure 7A-D). Specifically there are two phosphoserine binding sites on the PA subunit
with a conserved lysine and arginine strongly interacting with the phosphate in each site, K635,
R638 for site 1 (Figure 7B) and K289, R454 for site 2 (Figure 7C). These residues are fully
conserved in all influenza A polymerases (including bat) but only site 1 is conserved in influenza
B. In vitro, mutation to alanine of both basic residues in either site reduces CTD peptide binding
affinity but has no effect on the intrinsic cap-dependent transcription activity of recombinant
polymerase (provided a capped RNA is supplied), consistent with the CTD binding sites being
remote from regions involved in cap-snatching and RNA synthesis. However in a minigenome
assay even single mutations in either site are highly deleterious to overall polymerase activity,
suggesting that any reduction in CTD binding prevents efficient access to capped RNA in the
cellular context. Similarly, rescued recombinant viruses with the single PA mutations K289A,
R454A, K635A or R638A are highly attenuated and genetically unstable. Indeed genome
segment sequencing revealed that only with additional, presumably compensatory mutations,
could the recombinant viruses be further propagated. Confirmation of these results comes from
previous work on the highly attenuated R638A mutant virus where a revertant with near wild-
type infectivity was obtained with the second site mutation C453R in PA (Fodor et al., 2003).
The crystal structure clearly shows that R453 could compensate for the loss of R638 by binding
the phosphoserine in CTD binding site 1. It is interesting that the R638A mutant virus promoted
the formation of defecting interfering RNAs in which genome segments acquire internal
deletions. This could be explained by the fact that the transcription impaired R638A mutant (due
to reduced CTD binding) does not produce enough viral protein (e.g. NP) to generate correctly
packaged cRNPs. These defective cRNPs may then be more susceptible to replication errors that
generate internal deletions in the progeny vRNPs. Furthermore some conservative substitutions
in CTD binding site 2 have been implicated in virulence and Pol II degradation (I550L,
(Llompart et al., 2014; Rolling et al., 2009)) or host specificity (T552S, (Mehle et al., 2012)).
Finally, these results suggest that to be able to perform transcription by cap-snatching, viral
RNPs must be bound to Pol II CTD, whereas RNPs not so associated can only replicate. Thus
saturation of Pol II sites may be an important factor in the transition from transcription, which is

13
prevalent early in infection, to replication, which predominates later (Heldt et al., 2013;
Kawakami et al., 2011).

3.4 The mechanism of viral RNA replication

Replication occurs through de novo synthesis, that is, with no externally supplied primer. This
requires that both ATP and GTP are simultaneously and correctly assembled in the polymerase
active site opposite the template 3′-UpC to catalyse formation of the first phosphodiester bond
yielding pppApG (Figure 8A). Not surprisingly this is the rate-limiting step in replication
initiation making replication much less efficient than transcription, which is primed by a capped-
oligomer ideally ending in ApG. Indeed a recent in vitro analysis of the initiation of RNA
synthesis by recombinant influenza B polymerase shows that the initial rate of RNA synthesis is
some 300 times slower for unprimed replication compared to cap-primed transcription and that
the rate limiting step in replication can be largely bypassed by supplying ApG as a ready formed
dinucleotide primer (Reich et al., 2017). However there are some interesting additional
considerations that need to be mentioned here. It has been known for some time that RNA
polymerases that perform unprimed RNA synthesis often have a ‘priming loop’ that stabilises
assembly of the initiation complex by aromatic stacking against the terminal base-pair (Butcher
et al., 2001; Lescar and Canard, 2009). A recent structure of hepatitis C polymerase replication
initiation complex well illustrates this (Appleby et al., 2015). The crystal structure of influenza
polymerase revealed that residues 641–657 of PB1 form a β-hairpin loop, very similarly located
to the priming loop of hepatitis C polymerase, with the loop tip containing the highly conserved
648-AHGP motif (Reich et al., 2014)(Figure 8B). Consistent with this, it has now been shown
that truncation of the PB1 priming loop (or simply making the mutation P651A) reduced by a
factor of ten the rate of initiation of vRNA to cRNA replication but had little effect on cRNA to
vRNA replication or on cap-dependent transcription (Te Velthuis et al., 2016). Superposition of
influenza polymerase with for instance polio virus RNA polymerase with a template-product
duplex in the active site shows that the priming loop has to be displaced once elongation starts
(Reich et al., 2014)(Figure 8D). The fact that the priming loop plays no significant role in cRNA
to vRNA replication is plausibly explained by the fact that this sense of replication has been
shown to occur by initiation at 4-UC-5, followed by back-tracking of the template so that
internally formed pppApG primer realigns to 1-UC-2 (Deng et al., 2006; Zhang et al.,

14
2010)(Figure 8C). The biological utility of this ‘prime-and-realign’ mechanism may be that it
allows repair of an eventual deletion of the c3′ terminal nucleotide thus ensuring nevertheless
synthesis of a full-length genomic vRNA. Internal initiation is more favourable energetically
than terminal initiation and therefore does not require the priming loop (Te Velthuis et al., 2016).
These observations raise the question as to what determines the different behaviour of vPol
(polymerase bound to the vRNA promoter) compared to cPol (polymerase bound to the cRNA
promoter)? To allow internal initiation, the 3′ end of the cRNA template must project further into
the internal polymerase chamber so that 4-UC-5 is correctly positioned at the active site. This is
partly explained by the fact that the cRNA/vRNA sequence differences imply that the distal
promoter duplex region is predicted to involve 5′ nts 11-13 base-pairing with 3′ nts 12-14 for
cRNA compared to 3′ nts 10-12 for cRNA. However a structure with the cRNA promoter bound
is needed to confirm this hypothesis as well as to give insight into the mechanism of template
realignment. In any case, there is a further barrier to elongation which is the need to break the 3′-
5′ base-paired region of the vRNA or cRNA promoter. One role of this duplex region, as just
mentioned, is to allow correct initial positioning of the template 3′ end in the active site, but
elongation requires that the template can thereafter translocate freely. Recent studies propose that
breaking promoter base-pairs is mechanistically coupled to the making of new template-product
base-pairs in the active site (Reich et al., 2017; Robb et al., 2016). A structural feature of PB2,
denoted the helical lid (Reich et al., 2014), is likely involved in separating the two strands of the
template product duplex formed in the active site cavity and directing them into their respective
exit tunnels (Figure 8D).
As with transcription there are still many unclear details about the mechanism of
replication particularly within the RNP context. It remains controversial whether replication
requires the presence of more than one polymerase (Jorba et al., 2009), or whether this might
only be the case for cRNA to vRNA replication (York et al., 2013). Recently a method has been
described allowing isolation of cRNPs from cells (York et al., 2013). At low resolution cRNPs
resemble vRNPs, but unlike vRNPs, they are inactive in replication without the presence of a
trans-activating polymerase, which itself does not have to be catalytically active (York et al.,
2013). Certainly it is plausible that an incoming apo-polymerase is required to bind the emerging
5′ end of the replicate and this could initiate progeny RNP assembly. Another unknown, is why
during replication, the mechanism proposed for poly-adenylation is not active and instead the

15
template is faithfully copied to the 5′ end, although, surprisingly, poly-adenylation during
replication has been reported (Zheng et al., 1999)?

4. Influenza polymerase as a drug target

Currently the only clinically relevant anti-influenza drugs available for use are
neuraminidase inhibitors, although resistant strains have been detected in some years (McKimm-
Breschkin, 2013). It is generally thought that new effective anti-influenza drugs, preferably
active against all influenza A and B strains, are urgently needed for treatment of severe cases or
for prophylactic use, particular in the event of a pandemic where a vaccine may not be available.
The polymerase is an excellent target for anti-influenza drug design, since its inhibition will
directly and quickly reduce or eliminate viral replication. Furthermore it is highly conserved
amongst all strains. There are numerous potential target sites on the polymerase where small
molecule binding could disrupt its function. These include not only the cap-binding site (Cianci
et al., 1997; Clark et al., 2014; Pautus et al., 2013), the endonuclease (Carcelli et al., 2016;
Hastings et al., 1996; Jones et al., 2016; Kowalinski et al., 2012; Sagong et al., 2014; Song et al.,
2016) and the PB1 polymerase active site (Furuta et al., 2013) but also the PA-PB1 inter-subunit
interface (Massari et al., 2016), binding pockets that allosterically inhibit functional dynamics,
the vRNA promoter binding site and binding sites for interacting host factors (e.g. the Pol II
CTD binding site). A recent review gives a detailed compilation of the multitude of small
molecules proposed as polymerase inhibitors (Stevaert and Naesens, 2016). Here we give brief
details on three of the more promising candidate polymerase inhibitors, all of which are in
clinical trials.
An extremely potent and selective inhibitor, VX-787, targeting the cap-binding site and
active against all influenza A strains but not influenza B, was discovered by Vertex using a
phenotypic screening approach (Clark et al., 2014) (Figure 9A). The compound protects mice
from lethal influenza infection even when administered 48 hours after virus inoculation, unlike
neuraminidase inhibitors currently in clinical use (Clark et al., 2014). In vitro selection
experiments generated influenza virus variants with reduced susceptibility to VX-787, the
mutations being located near or in the PB2 cap-binding domain (Boyd et al., 2015). In particular,
mutation of Phe323, which makes key stacking interactions with the inhibitor, leads to resistance
and the natural resistance of influenza B strains is likely because this residue is in any case

16
substituted by a glutamine. It remains to be seen if this relative ease of resistance development is
an impediment to further development of this class of molecules, although it could perhaps be
circumvented by combination with other anti-influenza drugs.
Numerous potential endonuclease inhibitors have been described (Stevaert and Naesens,
2016). Most of them consist of a head-group which chelates the two divalent cations in the active
site linked to a tail group that interacts with other residues in the active site. The endonuclease
active site is quite large with several sub-pockets available but also flexible elements
(Kowalinski et al., 2012). Unlike for cap-binding inhibitors, it appears difficult to obtain
endonuclease inhibitor resistance mutants spontaneously in vitro (Song et al., 2016), which is
potentially a good sign. However the same study showed that after random mutagenesis of the
PA gene in vitro it was possible to rescue resistant strains carrying mutations in the endonuclease
active site which reduced inhibitor binding but maintained sufficient nuclease activity to support
viral replication (Song et al., 2016). One of a series of endonuclease inhibitors developed by
Shionogi has recently been described which is active against all tested influenza A and B strains
and shows considerable promise in a variety of in vitro assays (Jones et al., 2016)(Figure 9B).
Finally there is considerable interest in T705 (favipiravir), a compound developed by
Toyama Chemical that targets the RNA-dependent RNA polymerases of a broad spectrum of
RNA viruses, including influenza virus (Furuta et al., 2013) and ebola (Oestereich et al., 2014).
The compound is a pro-drug that is phosphoribosylated by cellular enzymes to become a
nucleotide triphosphate analogue (Figure 9C). It is thought that T705 can be incorporated instead
of guanosine and adenosine during viral RNA synthesis by influenza polymerase thus eventually
causing lethal mutagenesis of the viral RNA (Baranovich et al., 2013; Jin et al., 2013; Sangawa
et al., 2013). More generally, there is considerable scope to use the now available structural
information to develop or optimise influenza specific nucleotide analogues that might act as
chain terminators or mutagens by targeting the active site for RNA synthesis directly. Such
polymerase inhibitors are already in clinical use for treatment of hepatitis C (Appleby et al.,
2015) and under development for respiratory syncytial virus (RSV)(Fearns and Deval, 2016;
Wang et al., 2015) and Ebola (Reynard et al., 2015).

5. Perspectives

17
 Apart from Pol II CTD, it has been shown by various biochemical and proteomic
techniques that numerous other cellular factors potentially directly interact with influenza
polymerase (Bortz et al., 2011; Bradel-Tretheway et al., 2011; Eisfeld et al., 2015; Jorba et al.,
2008; Mayer et al., 2007; Watanabe et al., 2014; York et al., 2014). Some of these have been
further characterised and shown to be involved in different aspects of polymerase function e.g.
factors that promote polymerase subunit nuclear import and assembly, PB2 import factor
importin-α (Gabriel and Fodor, 2014; Pumroy et al., 2015; Tarendeau et al., 2007), PA-PB1
import factor RanBP5 (Hutchinson et al., 2011; Swale et al., 2016) and chaperone Hsp90 (Naito
et al., 2007); RNP intracellular transport, Rab11 (Amorim et al., 2011; Hutchinson and Fodor,
2013); viral replication, ANP32 (Long et al., 2016; Sugiyama et al., 2015), MCM (Kawaguchi et
al., 2011); viral transcription, Pol II (Engelhardt and Fodor, 2006), hCLE (Rodriguez et al.,
2011), SFPQ (Landeras-Bueno et al., 2011); and viral mRNA splicing, NS1-BP for the M
segment (Tsai et al., 2013), RED-SMU for the NS1 segment (Fournier et al., 2014). Other
polymerase interacting factors, notably from the innate immune system, are detrimental to viral
replication e.g. ZAPL (Liu et al., 2015), HAX-1(Hsu et al., 2013), TRIM32 (Fu et al., 2015). An
important aspect of these cellular factors is the potential connection with inter-species
transmission, host adaptation and pathogenicity. For instance it is well-known that polymerase
from avian strains replicates poorly in mammalian cells without some adaptive mutations, of
which E627K in PB2 is the most well-known but not only example (Gabriel et al., 2005;
Subbarao et al., 1993). Intriguingly, a recent study has linked the E627K adaptation to the
species differences in the host protein ANP32A, which has a 33 residue insertion in the avian
compared to the human variant (Long et al., 2016). It has been suggested that ANP32A promotes
cRNA to vRNA replication (Sugiyama et al., 2015) but the exact mechanism of action of
ANP32A and how it relates to residue 627 of PB2 remain to be elucidated. It is beyond the scope
of this review to discuss all these factors further, but a clear focus of future work will be to
understand the structural and mechanistic details of the modulation of polymerase function in
infected cells through its interaction network with not only host factors, but also other viral
proteins, for example, the viral nuclear export protein (NEP) (Bullido et al., 2001; Robb et al.,
2009).

Funding: This work was supported by ERC Advanced Grant V-RNA (322586). Thanks

18
to Edward Hutchinson for the schematic RNP model used in Figure 1.

Acknowledgments

19
References
Amorim, M.J., Bruce, E.A., Read, E.K., Foeglein, A., Mahen, R., Stuart, A.D., Digard, P., 2011. A Rab11-
and microtubule-dependent mechanism for cytoplasmic transport of influenza A virus viral RNA.
Journal of virology 85, 4143-4156.
Appleby, T.C., Perry, J.K., Murakami, E., Barauskas, O., Feng, J., Cho, A., Fox, D., 3rd, Wetmore, D.R.,
McGrath, M.E., Ray, A.S., Sofia, M.J., Swaminathan, S., Edwards, T.E., 2015. Viral replication.
Structural basis for RNA replication by the hepatitis C virus polymerase. Science 347, 771-775.
Arranz, R., Coloma, R., Chichon, F.J., Conesa, J.J., Carrascosa, J.L., Valpuesta, J.M., Ortin, J., Martin-
Benito, J., 2012. The structure of native influenza virion ribonucleoproteins. Science 338, 1634-
1637.
Baranovich, T., Wong, S.S., Armstrong, J., Marjuki, H., Webby, R.J., Webster, R.G., Govorkova, E.A., 2013.
T-705 (favipiravir) induces lethal mutagenesis in influenza A H1N1 viruses in vitro. Journal of
virology 87, 3741-3751.
Bercovich-Kinori, A., Tai, J., Gelbart, I.A., Shitrit, A., Ben-Moshe, S., Drori, Y., Itzkovitz, S., Mandelboim,
M., Stern-Ginossar, N., 2016. A systematic view on influenza induced host shutoff. eLife 5.
Bier, K., York, A., Fodor, E., 2011. Cellular cap-binding proteins associate with influenza virus mRNAs. The
Journal of general virology 92, 1627-1634.
Boivin, S., Cusack, S., Ruigrok, R.W., Hart, D.J., 2010. Influenza A virus polymerase: structural insights
into replication and host adaptation mechanisms. The Journal of biological chemistry 285,
28411-28417.
Bortz, E., Westera, L., Maamary, J., Steel, J., Albrecht, R.A., Manicassamy, B., Chase, G., Martinez-
Sobrido, L., Schwemmle, M., Garcia-Sastre, A., 2011. Host- and strain-specific regulation of
influenza virus polymerase activity by interacting cellular proteins. mBio 2.
Boyd, M.J., Bandarage, U.K., Bennett, H., Byrn, R.R., Davies, I., Gu, W., Jacobs, M., Ledeboer, M.W.,
Ledford, B., Leeman, J.R., Perola, E., Wang, T., Bennani, Y., Clark, M.P., Charifson, P.S., 2015.
Isosteric replacements of the carboxylic acid of drug candidate VX-787: Effect of charge on
antiviral potency and kinase activity of azaindole-based influenza PB2 inhibitors. Bioorganic &
medicinal chemistry letters 25, 1990-1994.
Bradel-Tretheway, B.G., Mattiacio, J.L., Krasnoselsky, A., Stevenson, C., Purdy, D., Dewhurst, S., Katze,
M.G., 2011. Comprehensive proteomic analysis of influenza virus polymerase complex reveals a
novel association with mitochondrial proteins and RNA polymerase accessory factors. Journal of
virology 85, 8569-8581.
Bullido, R., Gomez-Puertas, P., Saiz, M.J., Portela, A., 2001. Influenza A virus NEP (NS2 protein)
downregulates RNA synthesis of model template RNAs. Journal of virology 75, 4912-4917.
Butcher, S.J., Grimes, J.M., Makeyev, E.V., Bamford, D.H., Stuart, D.I., 2001. A mechanism for initiating
RNA-dependent RNA polymerization. Nature 410, 235-240.
Carcelli, M., Rogolino, D., Gatti, A., De Luca, L., Sechi, M., Kumar, G., White, S.W., Stevaert, A., Naesens,
L., 2016. N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal
binding modes. Scientific reports 6, 31500.
Center for Disease Control, 2016. Types of Influenza Virus.
http://www.cdc.gov/flu/about/viruses/types.htm.
Chan, A.Y., Vreede, F.T., Smith, M., Engelhardt, O.G., Fodor, E., 2006. Influenza virus inhibits RNA
polymerase II elongation. Virology 351, 210-217.
Chang, S., Sun, D., Liang, H., Wang, J., Li, J., Guo, L., Wang, X., Guan, C., Boruah, B.M., Yuan, L., Feng, F.,
Yang, M., Wang, L., Wang, Y., Wojdyla, J., Li, L., Wang, J., Wang, M., Cheng, G., Wang, H.W., Liu,
Y., 2015. Cryo-EM structure of influenza virus RNA polymerase complex at 4.3 A resolution.
Molecular cell 57, 925-935.

20
Cianci, C., Colonno, R.J., Krystal, M., 1997. Differential effect of modified capped RNA substrates on
influenza virus transcription. Virus Res 50, 65-75.
Clark, M.P., Ledeboer, M.W., Davies, I., Byrn, R.A., Jones, S.M., Perola, E., Tsai, A., Jacobs, M., Nti-Addae,
K., Bandarage, U.K., Boyd, M.J., Bethiel, R.S., Court, J.J., Deng, H., Duffy, J.P., Dorsch, W.A.,
Farmer, L.J., Gao, H., Gu, W., Jackson, K., Jacobs, D.H., Kennedy, J.M., Ledford, B., Liang, J.,
Maltais, F., Murcko, M., Wang, T., Wannamaker, M.W., Bennett, H.B., Leeman, J.R., McNeil, C.,
Taylor, W.P., Memmott, C., Jiang, M., Rijnbrand, R., Bral, C., Germann, U., Nezami, A., Zhang, Y.,
Salituro, F.G., Bennani, Y.L., Charifson, P.S., 2014. Discovery of a novel, first-in-class, orally
bioavailable azaindole inhibitor (VX-787) of influenza PB2. Journal of medicinal chemistry 57,
6668-6678.
Coloma, R., Valpuesta, J.M., Arranz, R., Carrascosa, J.L., Ortin, J., Martin-Benito, J., 2009. The structure of
a biologically active influenza virus ribonucleoprotein complex. PLoS pathogens 5, e1000491.
Crepin, T., Dias, A., Palencia, A., Swale, C., Cusack, S., Ruigrok, R.W., 2010. Mutational and metal binding
analysis of the endonuclease domain of the influenza virus polymerase PA subunit. Journal of
virology 84, 9096-9104.
Datta, K., Wolkerstorfer, A., Szolar, O.H., Cusack, S., Klumpp, K., 2013. Characterization of PA-N terminal
domain of Influenza A polymerase reveals sequence specific RNA cleavage. Nucleic acids
research 41, 8289-8299.
Delaforge, E., Milles, S., Bouvignies, G., Bouvier, D., Boivin, S., Salvi, N., Maurin, D., Martel, A., Round, A.,
Lemke, E.A., Jensen, M.R., Hart, D.J., Blackledge, M., 2015. Large-Scale Conformational Dynamics
Control H5N1 Influenza Polymerase PB2 Binding to Importin alpha. Journal of the American
Chemical Society 137, 15122-15134.
Deng, T., Vreede, F.T., Brownlee, G.G., 2006. Different de novo initiation strategies are used by influenza
virus RNA polymerase on its cRNA and viral RNA promoters during viral RNA replication. Journal
of virology 80, 2337-2348.
Dias, A., Bouvier, D., Crepin, T., McCarthy, A.A., Hart, D.J., Baudin, F., Cusack, S., Ruigrok, R.W., 2009. The
cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Nature 458,
914-918.
Eisfeld, A.J., Neumann, G., Kawaoka, Y., 2015. At the centre: influenza A virus ribonucleoproteins. Nature
reviews. Microbiology 13, 28-41.
Engelhardt, O.G., Fodor, E., 2006. Functional association between viral and cellular transcription during
influenza virus infection. Reviews in Medical Virology 16, 329-345.
Engelhardt, O.G., Smith, M., Fodor, E., 2005. Association of the influenza a virus RNA-dependent RNA
polymerase with cellular RNA polymerase II. Journal of virology 79, 5812-5818.
Fearns, R., Deval, J., 2016. New antiviral approaches for respiratory syncytial virus and other
mononegaviruses: Inhibiting the RNA polymerase. Antiviral research 134, 63-76.
Ferron, F., Weber, F., de la Torre, J.C., Reguera, J., 2017. Transcription and replication mechanisms of
Bunyaviridae and Arenaviridae L-proteins. Virus Reserach. This issue.
Flick, R., Neumann, G., Hoffmann, E., Neumeier, E., Hobom, G., 1996. Promoter elements in the
influenza vRNA terminal structure. RNA 2, 1046-1057.
Fodor, E., Mingay, L.J., Crow, M., Deng, T., Brownlee, G.G., 2003. A single amino acid mutation in the PA
subunit of the influenza virus RNA polymerase promotes the generation of defective interfering
RNAs. Journal of virology 77, 5017-5020.
Fournier, G., Chiang, C., Munier, S., Tomoiu, A., Demeret, C., Vidalain, P.O., Jacob, Y., Naffakh, N., 2014.
Recruitment of RED-SMU1 complex by Influenza A Virus RNA polymerase to control Viral mRNA
splicing. PLoS pathogens 10, e1004164.
Fu, B., Wang, L., Ding, H., Schwamborn, J.C., Li, S., Dorf, M.E., 2015. TRIM32 Senses and Restricts
Influenza A Virus by Ubiquitination of PB1 Polymerase. PLoS pathogens 11, e1004960.

21
Furuta, Y., Gowen, B.B., Takahashi, K., Shiraki, K., Smee, D.F., Barnard, D.L., 2013. Favipiravir (T-705), a
novel viral RNA polymerase inhibitor. Antiviral research 100, 446-454.
Gabriel, G., Dauber, B., Wolff, T., Planz, O., Klenk, H.D., Stech, J., 2005. The viral polymerase mediates
adaptation of an avian influenza virus to a mammalian host. Proceedings of the National
Academy of Sciences of the United States of America 102, 18590-18595.
Gabriel, G., Fodor, E., 2014. Molecular determinants of pathogenicity in the polymerase complex.
Current topics in microbiology and immunology 385, 35-60.
Geerts-Dimitriadou, C., Zwart, M.P., Goldbach, R., Kormelink, R., 2011. Base-pairing promotes leader
selection to prime in vitro influenza genome transcription. Virology 409, 17-26.
Gerlach, P., Malet, H., Cusack, S., Reguera, J., 2015. Structural Insights into Bunyavirus Replication and
Its Regulation by the vRNA Promoter. Cell 161, 1267-1279.
Giese, S., Bolte, H., Schwemmle, M., 2016. The Feat of Packaging Eight Unique Genome Segments.
Viruses 8.
Gu, W., Gallagher, G.R., Dai, W., Liu, P., Li, R., Trombly, M.I., Gammon, D.B., Mello, C.C., Wang, J.P.,
Finberg, R.W., 2015. Influenza A virus preferentially snatches noncoding RNA caps. RNA 21,
2067-2075.
Guilligay, D., Tarendeau, F., Resa-Infante, P., Coloma, R., Crepin, T., Sehr, P., Lewis, J., Ruigrok, R.W.,
Ortin, J., Hart, D.J., Cusack, S., 2008. The structural basis for cap binding by influenza virus
polymerase subunit PB2. Nat Struct Mol Biol 15, 500-506.
Hagen, M., Tiley, L., Chung, T.D., Krystal, M., 1995. The role of template-primer interactions in cleavage
and initiation by the influenza virus polymerase. The Journal of general virology 76 ( Pt 3), 603-
611.
Hastings, J.C., Selnick, H., Wolanski, B., Tomassini, J.E., 1996. Anti-influenza virus activities of 4-
substituted 2,4-dioxobutanoic acid inhibitors. Antimicrobial agents and chemotherapy 40, 1304-
1307.
He, X., Zhou, J., Bartlam, M., Zhang, R., Ma, J., Lou, Z., Li, X., Li, J., Joachimiak, A., Zeng, Z., Ge, R., Rao, Z.,
Liu, Y., 2008. Crystal structure of the polymerase PA(C)-PB1(N) complex from an avian influenza
H5N1 virus. Nature 454, 1123-1126.
Heldt, F.S., Frensing, T., Pflugmacher, A., Gropler, R., Peschel, B., Reichl, U., 2013. Multiscale modeling of
influenza A virus infection supports the development of direct-acting antivirals. PLoS
computational biology 9, e1003372.
Hengrung, N., El Omari, K., Serna Martin, I., Vreede, F.T., Cusack, S., Rambo, R.P., Vonrhein, C., Bricogne,
G., Stuart, D.I., Grimes, J.M., Fodor, E., 2015. Crystal structure of the RNA-dependent RNA
polymerase from influenza C virus. Nature 527, 114-117.
Hsu, M.T., Parvin, J.D., Gupta, S., Krystal, M., Palese, P., 1987. Genomic RNAs of influenza viruses are
held in a circular conformation in virions and in infected cells by a terminal panhandle.
Proceedings of the National Academy of Sciences of the United States of America 84, 8140-
8144.
Hsu, W.B., Shih, J.L., Shih, J.R., Du, J.L., Teng, S.C., Huang, L.M., Wang, W.B., 2013. Cellular protein HAX1
interacts with the influenza A virus PA polymerase subunit and impedes its nuclear
translocation. Journal of virology 87, 110-123.
Hutchinson, E.C., Fodor, E., 2013. Transport of the influenza virus genome from nucleus to nucleus.
Viruses 5, 2424-2446.
Hutchinson, E.C., Orr, O.E., Man Liu, S., Engelhardt, O.G., Fodor, E., 2011. Characterization of the
interaction between the influenza A virus polymerase subunit PB1 and the host nuclear import
factor Ran-binding protein 5. The Journal of general virology 92, 1859-1869.
Hutchinson, E.C., von Kirchbach, J.C., Gog, J.R., Digard, P., 2010. Genome packaging in influenza A virus.
The Journal of general virology 91, 313-328.

22
Jacome, R., Becerra, A., Ponce de Leon, S., Lazcano, A., 2015. Structural Analysis of Monomeric RNA-
Dependent Polymerases: Evolutionary and Therapeutic Implications. PloS one 10, e0139001.
Jagger, B.W., Wise, H.M., Kash, J.C., Walters, K.A., Wills, N.M., Xiao, Y.L., Dunfee, R.L., Schwartzman,
L.M., Ozinsky, A., Bell, G.L., Dalton, R.M., Lo, A., Efstathiou, S., Atkins, J.F., Firth, A.E.,
Taubenberger, J.K., Digard, P., 2012. An overlapping protein-coding region in influenza A virus
segment 3 modulates the host response. Science 337, 199-204.
Jin, Z., Smith, L.K., Rajwanshi, V.K., Kim, B., Deval, J., 2013. The ambiguous base-pairing and high
substrate efficiency of T-705 (Favipiravir) Ribofuranosyl 5'-triphosphate towards influenza A
virus polymerase. PloS one 8, e68347.
Jones, J.C., Marathe, B.M., Lerner, C., Kreis, L., Gasser, R., Pascua, P.N., Najera, I., Govorkova, E.A., 2016.
A Novel Endonuclease Inhibitor Exhibits Broad-Spectrum Anti-Influenza Virus Activity In Vitro.
Antimicrobial agents and chemotherapy 60, 5504-5514.
Jorba, N., Coloma, R., Ortin, J., 2009. Genetic trans-complementation establishes a new model for
influenza virus RNA transcription and replication. PLoS pathogens 5, e1000462.
Jorba, N., Juarez, S., Torreira, E., Gastaminza, P., Zamarreno, N., Albar, J.P., Ortin, J., 2008. Analysis of the
interaction of influenza virus polymerase complex with human cell factors. Proteomics 8, 2077-
2088.
Kawaguchi, A., Momose, F., Nagata, K., 2011. Replication-coupled and host factor-mediated
encapsidation of the influenza virus genome by viral nucleoprotein. Journal of virology 85, 6197-
6204.
Kawakami, E., Watanabe, T., Fujii, K., Goto, H., Watanabe, S., Noda, T., Kawaoka, Y., 2011. Strand-
specific real-time RT-PCR for distinguishing influenza vRNA, cRNA, and mRNA. Journal of
virological methods 173, 1-6.
Khaperskyy, D.A., Schmaling, S., Larkins-Ford, J., McCormick, C., Gaglia, M.M., 2016. Selective
Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus PA-X Host Shutoff
Protein. PLoS pathogens 12, e1005427.
Kim, H.J., Fodor, E., Brownlee, G.G., Seong, B.L., 1997. Mutational analysis of the RNA-fork model of the
influenza A virus vRNA promoter in vivo. The Journal of general virology 78 ( Pt 2), 353-357.
Klumpp, K., Ruigrok, R.W., Baudin, F., 1997. Roles of the influenza virus polymerase and nucleoprotein in
forming a functional RNP structure. Embo J 16, 1248-1257.
Koppstein, D., Ashour, J., Bartel, D.P., 2015. Sequencing the cap-snatching repertoire of H1N1 influenza
provides insight into the mechanism of viral transcription initiation. Nucleic acids research 43,
5052-5064.
Kowalinski, E., Zubieta, C., Wolkerstorfer, A., Szolar, O.H., Ruigrok, R.W., Cusack, S., 2012. Structural
Analysis of Specific Metal Chelating Inhibitor Binding to the Endonuclease Domain of Influenza
pH1N1 (2009) Polymerase. PLoS pathogens 8, e1002831.
Lakdawala, S.S., Fodor, E., Subbarao, K., 2016. Moving On Out: Transport and Packaging of Influenza
Viral RNA into Virions. Annual review of virology 3, 411-427.
Landeras-Bueno, S., Jorba, N., Perez-Cidoncha, M., Ortin, J., 2011. The splicing factor proline-glutamine
rich (SFPQ/PSF) is involved in influenza virus transcription. PLoS pathogens 7, e1002397.
Lescar, J., Canard, B., 2009. RNA-dependent RNA polymerases from flaviviruses and Picornaviridae.
Current opinion in structural biology 19, 759-767.
Li, M.L., Ramirez, B.C., Krug, R.M., 1998. RNA-dependent activation of primer RNA production by
influenza virus polymerase: different regions of the same protein subunit constitute the two
required RNA-binding sites. Embo J 17, 5844-5852.
Liang, B., Li, Z., Jenni, S., Rahmeh, A.A., Morin, B.M., Grant, T., Grigorieff, N., Harrison, S.C., Whelan, S.P.,
2015. Structure of the L Protein of Vesicular Stomatitis Virus from Electron Cryomicroscopy. Cell
162, 314-327.

23
Lidschreiber, M., Leike, K., Cramer, P., 2013. Cap completion and C-terminal repeat domain kinase
recruitment underlie the initiation-elongation transition of RNA polymerase II. Mol Cell Biol 33,
3805-3816.
Liu, C.H., Zhou, L., Chen, G., Krug, R.M., 2015. Battle between influenza A virus and a newly identified
antiviral activity of the PARP-containing ZAPL protein. Proceedings of the National Academy of
Sciences of the United States of America 112, 14048-14053.
Llompart, C.M., Nieto, A., Rodriguez-Frandsen, A., 2014. Specific residues of PB2 and PA influenza virus
polymerase subunits confer the ability for RNA polymerase II degradation and virus
pathogenicity in mice. Journal of virology 88, 3455-3463.
Long, J.S., Giotis, E.S., Moncorge, O., Frise, R., Mistry, B., James, J., Morisson, M., Iqbal, M., Vignal, A.,
Skinner, M.A., Barclay, W.S., 2016. Species difference in ANP32A underlies influenza A virus
polymerase host restriction. Nature 529, 101-104.
Loucaides, E.M., von Kirchbach, J.C., Foeglein, A., Sharps, J., Fodor, E., Digard, P., 2009. Nuclear dynamics
of influenza A virus ribonucleoproteins revealed by live-cell imaging studies. Virology 394, 154-
163.
Lukarska, M., Fournier, G., Pflug, A., Resa-Infante, P., Reich, S., Naffakh, N., Cusack, S., 2016. Structural
basis of an essential interaction between influenza polymerase and Pol II CTD. Nature.
Martinez-Alonso, M., Hengrung, N., Fodor, E., 2016. RNA-Free and Ribonucleoprotein-Associated
Influenza Virus Polymerases Directly Bind the Serine-5-Phosphorylated Carboxyl-Terminal
Domain of Host RNA Polymerase II. Journal of virology 90, 6014-6021.
Massari, S., Goracci, L., Desantis, J., Tabarrini, O., 2016. Polymerase Acidic Protein-Basic Protein 1 (PA-
PB1) Protein-Protein Interaction as a Target for Next-Generation Anti-influenza Therapeutics.
Journal of medicinal chemistry 59, 7699-7718.
Mayer, D., Molawi, K., Martinez-Sobrido, L., Ghanem, A., Thomas, S., Baginsky, S., Grossmann, J., Garcia-
Sastre, A., Schwemmle, M., 2007. Identification of cellular interaction partners of the influenza
virus ribonucleoprotein complex and polymerase complex using proteomic-based approaches. J
Proteome Res 6, 672-682.
McKimm-Breschkin, J.L., 2013. Influenza neuraminidase inhibitors: antiviral action and mechanisms of
resistance. Influenza and other respiratory viruses 7 Suppl 1, 25-36.
Mehle, A., Dugan, V.G., Taubenberger, J.K., Doudna, J.A., 2012. Reassortment and mutation of the avian
influenza virus polymerase PA subunit overcome species barriers. Journal of virology 86, 1750-
1757.
Moeller, A., Kirchdoerfer, R.N., Potter, C.S., Carragher, B., Wilson, I.A., 2012. Organization of the
influenza virus replication machinery. Science 338, 1631-1634.
Morin, B., Coutard, B., Lelke, M., Ferron, F., Kerber, R., Jamal, S., Frangeul, A., Baronti, C., Charrel, R., de
Lamballerie, X., Vonrhein, C., Lescar, J., Bricogne, G., Gunther, S., Canard, B., 2010. The N-
terminal domain of the arenavirus L protein is an RNA endonuclease essential in mRNA
transcription. PLoS pathogens 6, e1001038.
Naito, T., Momose, F., Kawaguchi, A., Nagata, K., 2007. Involvement of Hsp90 in assembly and nuclear
import of influenza virus RNA polymerase subunits. Journal of virology 81, 1339-1349.
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.
Molecular cell 1, 991-1000.
Obayashi, E., Yoshida, H., Kawai, F., Shibayama, N., Kawaguchi, A., Nagata, K., Tame, J.R., Park, S.Y.,
2008. The structural basis for an essential subunit interaction in influenza virus RNA polymerase.
Nature 454, 1127-1131.

24
Oestereich, L., Ludtke, A., Wurr, S., Rieger, T., Munoz-Fontela, C., Gunther, S., 2014. Successful
treatment of advanced Ebola virus infection with T-705 (favipiravir) in a small animal model.
Antiviral research 105, 17-21.
Palancade, B., Bensaude, O., 2003. Investigating RNA polymerase II carboxyl-terminal domain (CTD)
phosphorylation. Eur J Biochem 270, 3859-3870.
Pautus, S., Sehr, P., Lewis, J., Fortune, A., Wolkerstorfer, A., Szolar, O., Guilligay, D., Lunardi, T., Decout,
J.L., Cusack, S., 2013. New 7-methylguanine derivatives targeting the influenza polymerase PB2
cap-binding domain. Journal of medicinal chemistry 56, 8915-8930.
Pflug, A., Guilligay, D., Reich, S., Cusack, S., 2014. Structure of influenza A polymerase bound to the viral
RNA promoter. Nature 516, 355-360.
Plotch, S.J., Bouloy, M., Ulmanen, I., Krug, R.M., 1981. A unique cap(m7GpppXm)-dependent influenza
virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA
transcription. Cell 23, 847-858.
Poon, L.L., Pritlove, D.C., Fodor, E., Brownlee, G.G., 1999. Direct evidence that the poly(A) tail of
influenza A virus mRNA is synthesized by reiterative copying of a U track in the virion RNA
template. Journal of virology 73, 3473-3476.
Pritlove, D.C., Poon, L.L., Fodor, E., Sharps, J., Brownlee, G.G., 1998. Polyadenylation of influenza virus
mRNA transcribed in vitro from model virion RNA templates: requirement for 5' conserved
sequences. Journal of virology 72, 1280-1286.
Pumroy, R.A., Ke, S., Hart, D.J., Zachariae, U., Cingolani, G., 2015. Molecular determinants for nuclear
import of influenza A PB2 by importin alpha isoforms 3 and 7. Structure 23, 374-384.
Reguera, J., Cusack, S., Kolakofsky, D., 2014. Segmented negative strand RNA virus nucleoprotein
structure. Curr Opin Virol 5, 7-15.
Reguera, J., Gerlach, P., Cusack, S., 2016. Towards a structural understanding of RNA synthesis by
negative strand RNA viral polymerases. Current opinion in structural biology 36, 75-84.
Reguera, J., Weber, F., Cusack, S., 2010. Bunyaviridae RNA polymerases (L-protein) have an N-terminal,
influenza-like endonuclease domain, essential for viral cap-dependent transcription. PLoS
pathogens 6, e1001101.
Reich, S., Guilligay, D., Cusack, S., 2017. An in vitro fluorescence based study of initiation of RNA
synthesis by influenza B polymerase. Submitted.
Reich, S., Guilligay, D., Pflug, A., Malet, H., Berger, I., Crepin, T., Hart, D., Lunardi, T., Nanao, M., Ruigrok,
R.W., Cusack, S., 2014. Structural insight into cap-snatching and RNA synthesis by influenza
polymerase. Nature 516, 361-366.
Reynard, O., Nguyen, X.N., Alazard-Dany, N., Barateau, V., Cimarelli, A., Volchkov, V.E., 2015.
Identification of a New Ribonucleoside Inhibitor of Ebola Virus Replication. Viruses 7, 6233-6240.
Robb, N.C., Smith, M., Vreede, F.T., Fodor, E., 2009. NS2/NEP protein regulates transcription and
replication of the influenza virus RNA genome. The Journal of general virology 90, 1398-1407.
Robb, N.C., Te Velthuis, A.J., Wieneke, R., Tampe, R., Cordes, T., Fodor, E., Kapanidis, A.N., 2016. Single-
molecule FRET reveals the pre-initiation and initiation conformations of influenza virus
promoter RNA. Nucleic acids research 44, 10304-10315.
Robertson, J.S., Schubert, M., Lazzarini, R.A., 1981. Polyadenylation sites for influenza virus mRNA.
Journal of virology 38, 157-163.
Rodriguez, A., Perez-Gonzalez, A., Nieto, A., 2007. Influenza virus infection causes specific degradation of
the largest subunit of cellular RNA polymerase II. Journal of virology 81, 5315-5324.
Rodriguez, A., Perez-Gonzalez, A., Nieto, A., 2011. Cellular human CLE/C14orf166 protein interacts with
influenza virus polymerase and is required for viral replication. Journal of virology 85, 12062-
12066.

25
Rolling, T., Koerner, I., Zimmermann, P., Holz, K., Haller, O., Staeheli, P., Kochs, G., 2009. Adaptive
mutations resulting in enhanced polymerase activity contribute to high virulence of influenza A
virus in mice. Journal of virology 83, 6673-6680.
Rossman, J.S., Lamb, R.A., 2011. Influenza virus assembly and budding. Virology 411, 229-236.
Sagong, H.Y., Bauman, J.D., Patel, D., Das, K., Arnold, E., LaVoie, E.J., 2014. Phenyl substituted 4-
hydroxypyridazin-3(2H)-ones and 5-hydroxypyrimidin-4(3H)-ones: inhibitors of influenza A
endonuclease. Journal of medicinal chemistry 57, 8086-8098.
Sangawa, H., Komeno, T., Nishikawa, H., Yoshida, A., Takahashi, K., Nomura, N., Furuta, Y., 2013.
Mechanism of action of T-705 ribosyl triphosphate against influenza virus RNA polymerase.
Antimicrobial agents and chemotherapy 57, 5202-5208.
Song, M.S., Kumar, G., Shadrick, W.R., Zhou, W., Jeevan, T., Li, Z., Slavish, P.J., Fabrizio, T.P., Yoon, S.W.,
Webb, T.R., Webby, R.J., White, S.W., 2016. Identification and characterization of influenza
variants resistant to a viral endonuclease inhibitor. Proceedings of the National Academy of
Sciences of the United States of America 113, 3669-3674.
Stevaert, A., Naesens, L., 2016. The Influenza Virus Polymerase Complex: An Update on Its Structure,
Functions, and Significance for Antiviral Drug Design. Medicinal research reviews 36, 1127-1173.
Subbarao, E.K., London, W., Murphy, B.R., 1993. A SINGLE AMINO-ACID IN THE PB2-GENE OF
INFLUENZA-A VIRUS IS A DETERMINANT OF HOST RANGE. Journal of virology 67, 1761-1764.
Sugiyama, K., Kawaguchi, A., Okuwaki, M., Nagata, K., 2015. pp32 and APRIL are host cell-derived
regulators of influenza virus RNA synthesis from cRNA. eLife 4.
Sugiyama, K., Obayashi, E., Kawaguchi, A., Suzuki, Y., Tame, J.R., Nagata, K., Park, S.Y., 2009. Structural
insight into the essential PB1-PB2 subunit contact of the influenza virus RNA polymerase. Embo J
28, 1803-1811.
Swale, C., Monod, A., Tengo, L., Labaronne, A., Garzoni, F., Bourhis, J.M., Cusack, S., Schoehn, G., Berger,
I., Ruigrok, R.W., Crepin, T., 2016. Structural characterization of recombinant IAV polymerase
reveals a stable complex between viral PA-PB1 heterodimer and host RanBP5. Scientific reports
6, 24727.
Swinehealth Information Center, 2015. Influenza C and influenza D viruses.
http://www.swinehealth.org/wp-content/uploads/2016/03/Influenza-virus-C-IVC-and-influenza-
virus-D-IVD.pdf.
Tao, Y., Farsetta, D.L., Nibert, M.L., Harrison, S.C., 2002. RNA synthesis in a cage--structural studies of
reovirus polymerase lambda3. Cell 111, 733-745.
Tarendeau, F., Boudet, J., Guilligay, D., Mas, P.J., Bougault, C.M., Boulo, S., Baudin, F., Ruigrok, R.W.,
Daigle, N., Ellenberg, J., Cusack, S., Simorre, J.P., Hart, D.J., 2007. Structure and nuclear import
function of the C-terminal domain of influenza virus polymerase PB2 subunit. Nat Struct Mol Biol
14, 229-233.
Tarendeau, F., Crepin, T., Guilligay, D., Ruigrok, R.W., Cusack, S., Hart, D.J., 2008. Host determinant
residue lysine 627 lies on the surface of a discrete, folded domain of influenza virus polymerase
PB2 subunit. PLoS pathogens 4, e1000136.
Te Velthuis, A.J., Fodor, E., 2016. Influenza virus RNA polymerase: insights into the mechanisms of viral
RNA synthesis. Nature reviews. Microbiology 14, 479-493.
Te Velthuis, A.J., Robb, N.C., Kapanidis, A.N., Fodor, E., 2016. The role of the priming loop in influenza A
virus RNA synthesis. Nature microbiology 1, 16029.
Thierry, E., Guilligay, D., Kosinski, J., Bock, T., Gaudon, S., Round, A., Pflug, A., Hengrung, N., El Omari, K.,
Baudin, F., Hart, D.J., Beck, M., Cusack, S., 2016. Influenza Polymerase Can Adopt an Alternative
Configuration Involving a Radical Repacking of PB2 Domains. Molecular cell 61, 125-137.
Tong, S., Zhu, X., Li, Y., Shi, M., Zhang, J., Bourgeois, M., Yang, H., Chen, X., Recuenco, S., Gomez, J.,
Chen, L.M., Johnson, A., Tao, Y., Dreyfus, C., Yu, W., McBride, R., Carney, P.J., Gilbert, A.T.,

26
 Chang, J., Guo, Z., Davis, C.T., Paulson, J.C., Stevens, J., Rupprecht, C.E., Holmes, E.C., Wilson,
I.A., Donis, R.O., 2013. New world bats harbor diverse influenza A viruses. PLoS pathogens 9,
e1003657.
Tsai, P.L., Chiou, N.T., Kuss, S., Garcia-Sastre, A., Lynch, K.W., Fontoura, B.M., 2013. Cellular RNA binding
proteins NS1-BP and hnRNP K regulate influenza A virus RNA splicing. PLoS pathogens 9,
e1003460.
Vreede, F.T., Chan, A.Y., Sharps, J., Fodor, E., 2010. Mechanisms and functional implications of the
degradation of host RNA polymerase II in influenza virus infected cells. Virology 396, 125-134.
Vreede, F.T., Fodor, E., 2010. The role of the influenza virus RNA polymerase in host shut-off. Virulence
1, 436-439.
Wang, G., Deval, J., Hong, J., Dyatkina, N., Prhavc, M., Taylor, J., Fung, A., Jin, Z., Stevens, S.K.,
Serebryany, V., Liu, J., Zhang, Q., Tam, Y., Chanda, S.M., Smith, D.B., Symons, J.A., Blatt, L.M.,
Beigelman, L., 2015. Discovery of 4'-chloromethyl-2'-deoxy-3',5'-di-O-isobutyryl-2'-fluorocytidine
(ALS-8176), a first-in-class RSV polymerase inhibitor for treatment of human respiratory
syncytial virus infection. Journal of medicinal chemistry 58, 1862-1878.
Watanabe, T., Kawakami, E., Shoemaker, J.E., Lopes, T.J., Matsuoka, Y., Tomita, Y., Kozuka-Hata, H.,
Gorai, T., Kuwahara, T., Takeda, E., Nagata, A., Takano, R., Kiso, M., Yamashita, M., Sakai-
Tagawa, Y., Katsura, H., Nonaka, N., Fujii, H., Fujii, K., Sugita, Y., Noda, T., Goto, H., Fukuyama, S.,
Watanabe, S., Neumann, G., Oyama, M., Kitano, H., Kawaoka, Y., 2014. Influenza virus-host
interactome screen as a platform for antiviral drug development. Cell host & microbe 16, 795-
805.
Whelan, S.P., 2017. To be determined. Virus Research.
Yamada, S., Hatta, M., Staker, B.L., Watanabe, S., Imai, M., Shinya, K., Sakai-Tagawa, Y., Ito, M., Ozawa,
M., Watanabe, T., Sakabe, S., Li, C., Kim, J.H., Myler, P.J., Phan, I., Raymond, A., Smith, E., Stacy,
R., Nidom, C.A., Lank, S.M., Wiseman, R.W., Bimber, B.N., O'Connor, D.H., Neumann, G.,
Stewart, L.J., Kawaoka, Y., 2010. Biological and structural characterization of a host-adapting
amino acid in influenza virus. PLoS pathogens 6, e1001034.
Yamayoshi, S., Watanabe, M., Goto, H., Kawaoka, Y., 2015. Identification of a Novel Viral Protein
Expressed from the PB2 Segment of Influenza A Virus. Journal of virology 90, 444-456.
York, A., Hengrung, N., Vreede, F.T., Huiskonen, J.T., Fodor, E., 2013. Isolation and characterization of
the positive-sense replicative intermediate of a negative-strand RNA virus. Proceedings of the
National Academy of Sciences of the United States of America 110, E4238-4245.
York, A., Hutchinson, E.C., Fodor, E., 2014. Interactome analysis of the influenza A virus
transcription/replication machinery identifies protein phosphatase 6 as a cellular factor required
for efficient virus replication. Journal of virology 88, 13284-13299.
Yuan, P., Bartlam, M., Lou, Z., Chen, S., Zhou, J., He, X., Lv, Z., Ge, R., Li, X., Deng, T., Fodor, E., Rao, Z.,
Liu, Y., 2009. Crystal structure of an avian influenza polymerase PA(N) reveals an endonuclease
active site. Nature 458, 909-913.
Zhang, S., Wang, J., Wang, Q., Toyoda, T., 2010. Internal initiation of influenza virus replication of viral
RNA and complementary RNA in vitro. The Journal of biological chemistry 285, 41194-41201.
Zheng, H., Lee, H.A., Palese, P., Garcia-Sastre, A., 1999. Influenza A virus RNA polymerase has the ability
to stutter at the polyadenylation site of a viral RNA template during RNA replication. Journal of
virology 73, 5240-5243.

27
Figure Legends
Figure 1. Influenza viral RNA metabolism.
(a) Negative stain image of influenza RNPs purified from virions.
(b) Schematic of RNA synthesis performed by influenza polymerase
(c) Simplified life-cycle of influenza showing successive steps in the transcription and
replication of RNPs.
Figure 2. Structure of influenza polymerase subunits and heterotrimer.
(a) Left and middle: surface diagrams of each subunit and their assembly into the
heterotrimer. Right: the position of each domain within the subunit sequence together
with the colour code.
(b) Left: ribbon diagram of the PB1 subunit showing the fingers, palm and thumb domains
typical of RNA polymerases and the priming loop and fingertips in the active site cavity.
Middle: additional structural features of PB1, the β-ribbon (carries the PA-PB1 nuclear
localisation signal), β-hairpin (involved in promoter binding) and C-extension (subunit
interface with PB2). Right: the position of each PB1 element within the sequence
together with the colour code. Drawn using bat influenza polymerase PDB:WSB.
Figure 3. vRNA promoter binding to influenza polymerase.
(a) Sequence and secondary structure of influenza B vRNA promoter showing the 5′ stem-
loop (‘hook’) and the 3′-5′ duplex region.
(b) Ribbon diagram of vRNA promoter bound to the PB1 subunit showing the 3′ end
(yellow) entering the active site (Reich et al., 2017).
Figure 4. Alternative configurations of influenza polymerase.
(a) Schematic diagram showing the polymerase core and flexibly-linked peripheral domains.
(b) Two crystallographic structures of influenza B polymerase showing radically different
packing arrangements of the peripheral domains onto the core. The left structure with full
bound promoter (PDB:4WSA) is presumed to be configured for cap-snatching and
transcription initiation. The right structure has only the c5′ bound (PDB:5EPI).
(c) The structure of RNA-free influenza C polymerase (PDB:5D98) is similar to the
transcriptionally inactive form of influenza B polymerase.

28
Figure 5. Influenza polymerase internal tunnels.
Transparent surface representation of promoter bound bat influenza A polymerase
highlighting the internal tunnels (green tubes) converging on the catalytic chamber. The template
entrance and exit, NTP entrance and product exit are indicated.

Figure 6. Four key steps in cap-dependent transcription by influenza virus polymerase.

(a) Cap-snatching: binding and cleavage of capped, nascent Pol II transcript (red). The
polymerase core is represented as a grey cut-away surface, with the endonuclease (green),
cap-binding domain (orange) and 3′ (yellow) and 5′ (violet) ends of the vRNA.
(b) Rotation of the cap-binding domain directs the capped primer into the active site. Those
primers which can base-pair with the end of the template will be preferentially selected.
(c) Schematic arrangement of RNAs during transcription initiation with CTP being the next
nucleotide to be incorporated.
(d) In the elongation phase, the template processively translocates through the active site to
be copied. The resultant mRNA is chimeric with host sequences (red) capping the virally
encoded sequences (cyan).
(e) Self polyadenylation: the polymerase stutters on the oligo-U sequence proximal to the 5′
end thus generating the poly(A) tail.
(f) Schematic arrangement of the poly(A) signal at the 5′ end of the genomic vRNA.

Figure 7. Influenza polymerase binds the serine 5 phosphorylated C-terminal domain

(CTD) of cellular RNA polymerase II.

(a) Structure of bat influenza A polymerase in complex with four heptad repeats of a serine 5

phosphorylated CTD peptide (blue) showing two separated binding sites on the PA

subunit (green). Drawn from PDB:5M3H.

(b) Details of phosphoserine binding site 1 with key interacting basic residues K630 and

R633 (bat influenza A numbering). These correspond to K635 and R638 in human and

avian influenza polymerases.

29
 (c) Details of phosphoserine binding site 2 with key interacting basic residues K289 and

R449 (bat influenza A numbering). These correspond to K289 and R454 in human and

avian influenza polymerases.

(d) Linear representation of the polymerase bound CTD peptide with the unobserved linker

dotted or in italics.

Figure 8. Replication by influenza polymerase.

(a) Schematic diagram of model for terminal initiation of replication at the vRNA promoter.
Correct assembly of ATP and GTP in the active site is facilitated by a priming loop
allowing catalysis of pppApG formation.
(b) Three-dimensional arrangement of elements corresponding to (A) showing the tip of the
priming loop stabilising the nascent duplex (purple) and the three active site catalytic
aspartates (D305, D445, D446, red sidechains) together with the two divalent cations
(green spheres). Modelled using a superposition with the Norwalk virus polymerase
primer-template-CTP complex (PDB 3BSO).
(c) Schematic diagram of model for internal initiation of replication at the cRNA promoter.
The alternative 3′-5′ base-pairing of the cRNA allows the template to enter deeper into
the active site. The energetically more favourable internal assembly of ATP and GTP
does not require the priming loop. The self-made pppApG dinucleotide primer then
reanneals (arrow) to the end of the template.
(d) Structural model for the early stage of elongation showing next incoming NTP (black
sticks) and active site loop (motif C, red). The duplex formed by the orange product
strand (orange) and extended template strand (green) are derived by superposition with
the Polio virus polymerase elongation complex (PDB 3OL7). To enter into the elongation
phase, the 3′-5′ duplex must break to allow the template to translocate and the priming
loop must displace to accommodate the initial product-template duplex. Note that the
outgoing template strand would clash with the PB2 helical lid (residues 153-212, red
cartoon) whereas the product strand does not. Thus the two stands are forced to separate
and are directed out through their respective exit tunnels (Figure 5)

30
Figure 9. Inhibitors of influenza polymerase.
(a) Chemical structure and binding site in the cap-binding domain (orange surface) of
compound VX787 (green sticks) developed by Vertex.
(b) Chemical structure and binding site in the endonuclease domain (green surface with
divalent cations as magenta spheres) of compound developed by Shionogi (yellow
sticks).
(c) Chemical structure of the pro-drug T705 (favipiravir) which is phosphoribosylated into
the active triphosphate form by cellular enzymes.

31
Figure 1.
A Influenza RNPs
B
REPLICATION TRANSCRIPTION
(+) cRNA (-) vRNA mRNA
Anti-genome Genome

(2) Viral mRNA (3) Viral proteins

C (8) Assembly and

budding of
(6) Secondary progeny virions
(1) Primary transcription transcription

(7) Nuclear Export

Incoming vRNP Progeny vRNP
(5) cRNA to vRNA replication
and vRNP assembly
(genome amplification)
(4) vRNA to cRNA replication
and cRNP assembly
Nucleus Cytoplasm

cRNP
Figure 2.

Endo
PB2-C

B
Figure 3.

PB1 β-ribbon

3′ vRNA
A B 3'-5' duplex

5′ vRNA
5' stem-loop
(‘hook’)
G A
PB1 β-hairpin
A A
U A 5′

G C
5′ppp-A A 15
1 AGGGU 3′

Template strand
3′OH- UCGUUUUCGUCCCA Priming
loop
1 3'-5′ 14
duplex PB1 active site
hairpin loop
(motif C)
Figure 4.

B C D
Figure 5.

Endonuclease
Cap-binding

Product exit
- mRNA (transcription)
Template - cRNA or vRNA (replication)
exit

5′ vRNA

3′ vRNA Catalytic chamber

for RNA synthesis
Template
entrance NTP entrance
tunnel
5′ hook
Figure 6.
A B

D E F
Figure 7.

A Cap-binding Endonuclease

Promoter C

R633 K289
site 1 R449
pS5a
K630 site 2
pS5c

D
Y1a S2a P3a T4a S5a P6a (S7a-Y1bS2bP3bT4bS5b) P6b S7b-Y1c S2c P3c T4c S5c P6c S7c-Y1d
I545
PO4 PO4 PO4
T547
site 1 site 2
Figure 8.
A 1 6
C 1 6
U-C-G-U-C- U U-C-A-U-C- A-U-U
U G
Priming | | C 10 | | | | U 12
U-C-U-C-C 3′ cRNA
G- U-C-U-C 3′ vRNA pppApG

{ GTP
ATP
{ GTP
loop
ATP

|||| ||| |
A-G-A-G 5′ vRNA G-A-G-G 5′ cRNA
pppApG A-A-C-A10 pppApG A-G-C-A10
U| || | A| |||

-
-

G-A-U-G-Appp G-A-C-G-Appp
1 1

3'-5' duplex 3'-5' duplex

PB2 helical must break
3′ vRNA 5′ vRNA lid
B D
Priming loop with
conserved tip
648-AHGP

U C
P651

A G Priming loop
H649 must displace
D445 Incoming
Mg NTP
D446
D305
Figure 9.

A B