Differential polymerase activity in avian and mammalian cells determines host range of influenza virus. J. Virol., 81: 9601-9604.

Abstract

As recently shown, mutations in the polymerase genes causing increased polymerase activity in mammalian cells are responsible
for the adaptation of the highly pathogenic avian influenza virus SC35 (H7N7) to mice (G. Gabriel et al., Proc. Natl. Acad.
Sci. USA 102:18590-18595, 2005). We have now compared mRNA, cRNA, and viral RNA levels of SC35 and its mouse-adapted variant
SC35M in avian and mammalian cells. The increase in levels of transcription and replication of SC35M in mammalian cells was
linked to a decrease in avian cells. Thus, the efficiency of the viral polymerase is a determinant of both host specificity
and pathogenicity.

Full text

JOURNAL OF VIROLOGY, Sept. 2007, p. 9601–9604 Vol. 81, No. 17
0022-538X/07/$08.00⫹0 doi:10.1128/JVI.00666-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Differential Polymerase Activity in Avian and Mammalian Cells
Determines Host Range of Influenza Virus
䌤
G. Gabriel, M. Abram, B. Keiner, R. Wagner,† H.-D. Klenk,* and J. Stech‡
Institut fuer Virologie, Philipps-Universita¨t Marburg, Hans-Meerwein-Str. 2, 35043 Marburg, Germany
Received 28 March 2007/Accepted 6 June 2007
As recently shown, mutations in the polymerase genes causing increased polymerase activity in mam-
malian cells are responsible for the adaptation of the highly pathogenic avian influenza virus SC35
(H7N7) to mice (G. Gabriel et al., Proc. Natl. Acad. Sci. USA 102:18590–18595, 2005). We have now
compared mRNA, cRNA, and viral RNA levels of SC35 and its mouse-adapted variant SC35M in avian and
mammalian cells. The increase in levels of transcription and replication of SC35M in mammalian cells
was linked to a decrease in avian cells. Thus, the efficiency of the viral polymerase is a determinant of both
host specificity and pathogenicity.
Influenza A viruses have wild aquatic birds as natural
hosts, in which they occur with a large variety of different
strains defined by 16 HA and 9 NA subtypes (15). Since the
host barrier is not an insurmountable obstacle for these
viruses, they can occasionally be transmitted from their nat-
ural reservoir to terrestrial birds and mammals, including
humans. Most of these transmissions are transient. On rare
occasions, the viruses adapt to the new species and give rise
to a new lineage. Adaptation requires multiple mutations
and may involve gene reassortment after coinfection with
another virus. By these mechanisms, the human H1N1,
H2N2, and H3N2 viruses, which caused the pandemics in
1918, 1957, and 1968, respectively, were generated (24, 26).
More recently, avian influenza viruses of subtypes H5N1,
H7N7, and H9N2 were transmitted from chickens directly to
humans, posing a severe pandemic threat (2, 6, 16, 22).
Species specificity has long been known to be a multifacto-
rial trait depending on most viral genes and many host
factors (9, 10, 18, 20). However, there is increasing evidence
that some viral proteins are particularly important for host
adaptation, among them the viral polymerase, which cata-
lyzes both the transcription of viral genomic RNA (vRNA)
to mRNA and the replication of vRNA with cRNA as an
intermediate. The polymerase, a heterotrimeric complex
consisting of subunits PB2, PB1, and PA (1), is active in the
nucleus. Nuclear and cytoplasmic host proteins serve as
cofactors of the polymerase (3, 4). Several important mark-
ers of host range and pathogenicity have been identified in
the polymerase genes. Numerous studies have shown that
adaptation of an avian virus to a mammalian host was linked
to the PB2 mutation E627K (11, 14, 21). However, other
mutations in the polymerase proteins or the associated NP
protein may also be involved. Thus, when comparing the
avian SC35 strain (H7N7) with its mouse-adapted variant
SC35M, we previously identified six mutations in SC35M
responsible for the increased virulence in mice (L13P and
S678N in PB1, D701N and S714R in PB2, K615N in PA, and
N319K in NP). By showing that the increase in virulence
correlated with enhanced polymerase activity in mammalian
cells, we also provided an explanation for the mechanism
underlying the adaptation process (7). It was not clear, how-
ever, from this study whether the increased polymerase ac-
tivity of SC35M was host dependent and whether replication
or transcription, or both, were affected. Here, therefore, we
have analyzed mRNA, cRNA, and vRNA synthesis in avian
and mammalian cells. Furthermore, we have compared the
pathogenicity of SC35 and SC35M in chicken embryos.
Transmission and replication of SC35 and SC35M in avian
and mammalian cells. To compare the transcription and rep-
lication properties of SC35 and SC35M, we performed primer
extension assays as described previously (5, 25). In primary
chicken embryo fibroblasts, mRNA, cRNA, and vRNA levels
of SC35M were only 40%, 30%, and 70% of SC35 levels,
respectively (100%) (Fig. 1A). In quail fibrosarcoma (QT6)
cells, SC35M activities were also reduced (Fig. 1B). In human
embryo kidney (293T) cells, however, SC35M showed signifi-
cantly higher transcription and replication activities than SC35
(Fig. 1C). Results obtained for monkey kidney (Vero) cells
were similar to those for 293T cells (Fig. 1D). These data
confirm our previous observations showing that SC35M has a
replicative advantage over SC35 in mammalian cells (7). They
also demonstrate that, vice versa, in avian cells SC35 replicates
more efficiently than SC35M.
Effects of individual mutations on transcription and repli-
cation. To find out how individual mutations acquired during
the adaptation process contributed to the functional differ-
ences in avian and mammalian cells, we studied a series of
recombinant SC35 mutants comprising, on the one hand, sin-
gle-gene reassortants (SGR viruses) of SC35 in which one
complete polymerase gene was replaced with the correspond-
ing SC35M gene and, on the other hand, SC35 recombinants
with SC35M-specific single point mutations (SPM viruses) (7).
* Corresponding author. Mailing address: Institut fuer Virologie,
Philipps-Universita¨t Marburg, Hans-Meerwein-Str. 2, 35043 Marburg,
Germany. Phone: 49 6421 2866253. Fax: 49 6421 2868962. E-mail:
klenk@staff.uni-marburg.de.
† Present address: Paul Ehrlich Institut, Paul Ehrlich Str. 51-59,
63225 Langen, Germany.
‡ Present address: Friedrich-Lo¨ffler-Institut, Bundesforschungsinsti-
tut fu¨r Tiergesundheit, Institut fu¨r Molekularbiologie, Boddenblick 5a,
17493 Greifswald, Insel Riems, Germany.
䌤
Published ahead of print on 13 June 2007.
9601

First, we examined the SGR viruses for transcription and
replication properties in QT6 cells. SC35-PB2
SC35M
, SC35-
PA
SC35M
, and SC35-NP
SC35M
showed mRNA, cRNA, and
vRNA levels similar to those of SC35. However, SC35-
PB1
SC35M
had lost the high transcription and replication prop
-
erties of SC35 (Fig. 2A). The reductions in transcription and
replication levels observed with SC35-PB1
SC35M
were similar
to the reductions obtained with SC35M, indicating that PB1 of
SC35 is a crucial factor for transcription and replication in
avian cells. We then analyzed SPM viruses with PB1 point
mutations in QT6 cells. SC35-PB1
13P
showed mRNA and
vRNA levels similar to those of SC35, whereas cRNA levels
were reduced. However, the mRNA and vRNA levels of SC35-
PB1
678N
were higher than those of SC35, while cRNA levels
were unchanged (Fig. 2A).
To determine which of the SC35M-specific mutations con-
tribute to the increased transcription and replication levels in
mammalian cells, we then analyzed RNA levels in Vero cells.
With SC35-PB1
SC35M
and SC35-NP
SC35M
, mRNA, cRNA and
vRNA levels were increased, while SC35-PA
SC35M
showed
only vRNA enhancement (Fig. 2B). Therefore, the PB1 or NP
gene segment of SC35M, when introduced into the SC35 back-
ground, significantly enhanced both transcription and replica-
tion activities in mammalian cells. The PB2 and PA gene seg-
ments of SC35M enhanced replication slightly, whereas
mRNA levels remained unchanged. Finally, to throw light on
the effects of the individual mutations in PB1 and PB2, we
analyzed the respective PB1 and PB2 SPM viruses. SC35-
PB1
13P
had increased mRNA and cRNA levels, while all RNA
species were strongly enhanced with SC35-PB1
678N
. SC35-
PB2
701N
and SC35-PB2
714R
also showed increased mRNA and
cRNA levels, but there was no significant effect on vRNA
(Fig. 2B).
Taken together, the data shown in Fig. 2A and B demon-
strate that, in Vero cells, all mutations elevated transcription
and replication efficiently; none of them affected transcription
or replication exclusively. With some mutations, including the
PB1 mutation S678N, the PB1 double mutation L13P S678N,
and the PB2 double mutation D701N S714R, mRNA, cRNA,
and vRNA levels were elevated to similar extents. With other
mutants, enhancement was disproportionate. PA mutation
K615N and NP mutation N319K stimulate vRNA synthesis
more than mRNA and cRNA synthesis. The PB2 mutations
D701N and S714R do not enhance vRNA levels as much as
mRNA and cRNA levels. However, except for PB1 mutation
L13P, all mutations caused similar mRNA and cRNA shifts,
suggesting that mRNA and cRNA synthesis are closely linked.
This observation is interesting because it is compatible with the
proposal that mRNA and cRNA are synthesized simulta-
neously early in infection (25), but it does not rule out the
classical model implicating a switch from early transcription to
subsequent replication (8, 13, 19). PB2 mutations D701N and
S714R and NP mutation N319K, which are convergent with
H5N1 strains (7), also enhance the polymerase activity of re-
constituted RNP complexes in mammalian cells when trans-
ferred into the PB2 and NP genes of an unrelated avian virus
(Fig. 3). This result demonstrates that these mutations act in
mammalian cells independently of the context of the virus
genome. Whereas each individual mutation alters polymerase
activities quite significantly in mammalian cells, the effects are
FIG. 1. Transcription and replication activities of SC35 and SC35M in avian and mammalian cells. mRNA, cRNA, and vRNA levels in SC35-
and SC35M-infected chicken embryo fibroblasts (CEF) (A), quail fibrosarcoma (QT6) cells (B), human embryo kidney (293T) cells (C), and
African green monkey kidney (Vero) cells (D) were determined by primer extension. Primer extension analysis was performed 14 h after
inoculation at a multiplicity of infection of 0.1. Transcription products of three independent experiments were quantified using TINA 2.0 software.
The results shown are derived from three independent experiments.
9602 NOTES J. V
IROL.

less distinct in avian cells. Only the PB1 double mutation L13P
S678N reduced transcription and replication activities to the
levels observed with SC35M. Interestingly, the PB1 mutation
S678N alone raised these activities in avian as well as mam-
malian cells, suggesting that the enhancing effect of this mu-
tation is host independent. Most mutations, however, did not
alter RNA synthesis in avian cells. These observations support
the notion that the virus, when crossing the species barrier,
goes through a phase that allows gradual acquisition of adap-
tive mutations without losing fitness for the old host. Such
conditions should favor the development of the constellation
of mutations necessary for optimal growth in mammalian cells.
Pathogenicity of SC35 and SC35M in chicken embryos. It
has been reported previously that both SC35 and SC35M are
pathogenic for chickens (12). However, that study was per-
formed with adult chickens and at a high inoculation dose of
10
6
PFU that did not allow quantitative assessment of the
virulence of these viruses. Therefore, we determined the 50%
egg lethal dose (ELD
50
) and the mean time to death (MDT)
for SC35 and SC35M in 11-day-old embryonated chicken eggs
as described previously (17, 24). Eggs were inoculated with
10-fold virus dilutions ranging from 10
6
to 10
⫺2
PFU. SC35M
showed lower virulence (ELD
50
, 11.5 log
10
PFU) and mortality
(MDT, 96 h) in chicken embryos than SC35 (ELD
50
, 0.2 log
10
FIG. 2. Transcription and replication activities of SGR and SPM viruses in avian and mammalian cells. Quail fibrosarcoma (QT6) cells (A) and
African green monkey kidney (Vero) cells (B) were infected with SC35, SGR viruses (SC35-PB1
SC35M
, SC35-PB2
SC35M
, SC35-PA
SC35M
, and
SC35-NP
SC35M
), and SPM viruses (SC35-PB2
701N
, SC35-PB2
714R
, SC35-PB1
13P
, and SC35-PB1
678N
) at a multiplicity of infection of 0.1. At 14 h
postinfection, mRNA, cRNA, and vRNA levels were determined by primer extension. The results shown are derived from three independent
experiments.
V
OL. 81, 2007 NOTES 9603

PFU; MDT, 24 h). These data demonstrate that the reduced
transcription and replication activity of SC35M in avian cells
corresponds to diminished virulence, while the high transcrip-
tion and replication properties of SC35 in avian cells corre-
spond to increased virulence in chicken embryos. Replication
efficiency is therefore a determinant not only of host range but
also of pathogenicity.
Our results suggest that the mutations observed with SC35M
mediate the adaptation of the viral polymerase complex to host
factors. This concept is supported by a recent study in which the
structure of the C-terminal domain of PB2 was elucidated. It
revealed that the mutation sites 701 and 714 are exposed at the
surface of the molecule, where they interact with a bipartite nu-
clear localization signal that promotes transport into the nucleus
by binding to importin ␣5 (23). Structural analysis of the NP
protein has shown that the mutation site 319 is also located at the
surface of the molecule, and it has been proposed that it may
mediate binding to other subunits of the polymerase (27). Thus, it
appears that the mutations responsible for adaptation to a new
host alter interaction with host factors by affecting the contact site
either directly, or indirectly by inducing structural changes in
other subunits of the polymerase.
We are indebted to the late R. Rott and the late M. Orlich, who
established the SC35–SC35M system. We thank G. G. Brownlee, E.
Fodor, T. Jung, and K. Hara for providing protocols on the primer
extension method and for helpful discussions.
This work was supported by the Deutsche Forschungsgemeinschaft,
grants SFB 593-TPB1 and DFG-Kl238/9-1; by the European Commis-
sion, SPSB-CT-2006-044263 (FLUPOL); and by the Fonds der Che-
mischen Industrie.
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FIG. 3. Effects of PB2 mutations D701N and S714R and NP mu-
tation N319K on RNP activities of A/Fowl plague virus (FPV)/Ros-
tock/34 H7N1 in mammalian cells. 293T cells were transfected as
indicated with plasmids containing FPV genes PB2, PB1, PA, and NP
and FPV mutant genes PB2 D701N, PB2 S714R, and NP N319K.
Polymerase activity was determined 24 h after transfection by measur-
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9604 NOTES J. VIROL.