Architecture of ribonucleoprotein complexes in influenza A virus particles
In viruses, as in eukaryotes, elaborate mechanisms have evolved to protect the genome and to ensure its timely replication and reliable transmission to progeny. Influenza A viruses are enveloped, spherical or filamentous structures, ranging from 80 to 120 nm in diameter. Inside each envelope is a viral genome consisting of eight single-stranded negative-sense RNA segments of 890 to 2,341 nucleotides each. These segments are associated with nucleoprotein and three polymerase subunits, designated PA, PB1 and PB2; the resultant ribonucleoprotein complexes (RNPs) resemble a twisted rod (10-15 nm in width and 30-120 nm in length) that is folded back and coiled on itself. Late in viral infection, newly synthesized RNPs are transported from the nucleus to the plasma membrane, where they are incorporated into progeny virions capable of infecting other cells. Here we show, by transmission electron microscopy of serially sectioned virions, that the RNPs of influenza A virus are organized in a distinct pattern (seven segments of different lengths surrounding a central segment). The individual RNPs are suspended from the interior of the viral envelope at the distal end of the budding virion and are oriented perpendicular to the budding tip. This finding argues against random incorporation of RNPs into virions, supporting instead a model in which each segment contains specific incorporation signals that enable the RNPs to be recruited and packaged as a complete set. A selective mechanism of RNP incorporation into virions and the unique organization of the eight RNP segments may be crucial to maintaining the integrity of the viral genome during repeated cycles of replication.
© 2006 Nature Publishing Group
Architecture of ribonucleoprotein complexes in
inﬂuenza A virus particles
, Hiroshi Sagara
, Albert Yen
, Ayato Takada
†, Hiroshi Kida
, R. Holland Cheng
& Yoshihiro Kawaoka
In viruses, as in eukaryotes, elaborate mechanisms have evolved
to protect the genome and to ensure its timely replication
and reliable transmission to progeny. Inﬂuenza A v iruses are
enveloped, spherical or ﬁlamentous structures, ranging from 80
to 120 nm in diameter
. Inside each envelope is a viral genome
consisting of eight single-stranded negative-sense RNA segments
of 890 to 2,341 nucleotides each
. These segments are associated
with nucleoprotein and three polymerase subunits, designated PA,
PB1 and PB2; the resultant ribonucleoprotein complexes (RNPs)
resemble a twisted rod (10–15 nm in width and 30–120 nm in
length) that is folded back and coiled on itself
. Late in viral
infection, newly s ynthesized RNPs are transpor ted from the
nucleus to the plasma membrane, where they are incorporated
into progeny virions capable of infecting other cells. Here we
show, by transmission electron microscopy of serially sectioned
virions, that the RNPs of inﬂuenza A virus are organized in a
distinct pattern (seven segments of different lengths surrounding
a central segment). The individual RNPs are suspended from the
interior of the viral envelope at the distal end of the budding virion
and are oriented perpendicular to the budding tip. This ﬁnding
argues against random incorporation of RNPs into vir ions
supporting instead a model in which each segment contains
speciﬁc incorporation signals that enable the RNPs to be recruited
and packaged as a complete set
. A selective mechanism of RNP
incorporation into virions and the unique organization of the
eight RNP segments may be crucial to maintaining the integrity of
the viral genome during repeated cycles of replication.
To elucidate the architecture of the virion interior, we longitudi-
nally and transversely sectioned A/WSN/33 (H1N1) virions budding
from Madin–Darby canine kidney (MDCK) cells at 10 h after
infection. Although A/WSN/33 virions released into culture medium
are spherical in shape
, the budding virions in longitudinal sections
were elongated and contained rod-like structures that were associ-
ated with host-derived lipid bilayer envelopes and oriented perpen-
dicular to the budding tip of the virion (Fig. 1a and Supplementary
Fig. 1). They were ,12 nm in width and up to 130 nm in length,
consistent with the sizes of puriﬁed RNPs
. In transversely sec-
tioned budding virions, electron-dense dots, representing the rod-
like structures seen in longitudinal sections, were apparent inside
each virion (Fig. 1b). Notably, many of the virions contained eight
dots, arranged as seven in a circle surrounding one at the centre
(Fig. 1c); not more than eight dots were observed in a virion. To
determine the lengths of the rod-like structures, we compared
serial transverse sections of whole budding virions (Fig. 1d and
Supplementary Fig. 2). In all virions examined, the number of dots
decreased progressively with increasing distance from the budding
tip of the virion. Together, these results indicate that inﬂuenza A
virions contain a highly organized set of eight rod-like structures of
different lengths (Fig. 2).
Figure 1 | Budding virions show a speciﬁc arrangement of eight rod-like
structures of different lengths.
a, Rod-like structures, 12 nm in width, are
associated with the viral envelope at the distal end of the budding virion.
b, c, Electron-dense dots (N # 8), representing transversely sectioned rods,
were observed in each virion in a characteristic conﬁguration (several
peripheral dots surrounding a core dot). d, Serial sections of a vi rion cut
from the distal end. As the distance from the end increased the number of
dots decreased, suggesting that the eight rod-like structures differed in
length. e, Lower magniﬁcation views of the serial ultrathin sections in d,
demonstrating that the serial section shown in d represents the same virion
(circled in red). Other virions are indicated by arrowheads. Scale bars, 50 nm
(a, c, d); 200 nm (b, e).
Internal Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Kita-ku, Sapporo 060-0818, Japan.
Core Research for Evolutional
Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan.
Fine Morphology Laboratory, Department of Basic Medical Science, and
Division of Virology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Department of Biosciences, Karolinska Institute,
141 57 Huddinge, Sweden.
Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Molecular and Cellular Biology, University of California, Davis, California 95616, USA.
Department of Pathological Science, School of Veterinary Medicine, University of
Wisconsin-Madison, Madison, Wisconsin 53706, USA. †Present address: Research Center for Zoonosis Control, Hokkaido University, Kita-ku, Sapporo 060-0818, Japan.
Vol 439|26 January 2006|doi:10.1038/nature04378
© 2006 Nature Publishing Group
To determine whether or not the arrangement of the virion
interior in spherical A/WSN/33 virus is unique to that strain, we
examined MDCK cells infected with other strains of spherical
inﬂuenza A viruses isolated from humans (A/Aichi/2/68 (H3N2)
and A/Puerto Rico/8/34 (H1N1)), ducks (A/duck/Hong Kong/836/
80 (H3N1) and A/mallard/New York/6750/78 (H2N2)), and swine
(A/swine/Chiba/1/91 (H1N2); see Supplementary Fig. 3). In all
strains tested, the virions showed the same organization of rod-like
structures, in which a central dot was surrounded by seven dots,
duplicating the pattern seen in A/WSN/33 virions.
Because some inﬂuenza A viruses, especially newly isolated strains,
, we next examined the architecture of the ﬁlamen-
tous A/Udorn/307/72 (H3N2) virus. Although most of the transverse
sections of ﬁlamentous virions lacked electron-dense material (Sup-
plementary Fig. 4a), some showed the typical conﬁguration of seven
dots with a single core dot (Supplementary Fig. 4b). In longitudinally
sectioned ﬁlamentous virions, the rod-like structures were conﬁned
to the distal end of each ﬁlamentous particle (Supplementary Fig. 4c);
the remainder of the virion was empty, consistent with the apparent
lack of internal structures in transverse sections of ﬁlamentous
virions. Thus, both spherical and ﬁlamentous inﬂuenza virions
budding from cells possess an organized set of eight rod-like
structures that are associated with the envelope at the budding end
of the virion.
Our serendipitous observation that some inﬂuenza A virions are
partially disrupted when freeze-dried enabled us to study the nature
of the rod-like structures inside virions. The widths of the structures
released by freeze-dried, negatively stained A/Puerto Rico/8/34
virions were ,12 nm on average (Fig. 3a), similar to the widths of
structures inside sectioned virions (Fig. 1). These released structures
were morphologically indistinguishable from the RNPs puriﬁed from
virions in previous studies
and reacted with anti-nucleoprotein
monoclonal antibodies conjugated to colloidal gold (Fig. 3b). To
determine further the nature of the rod-like structures observed
in virions, we performed ultrathin-section immunoelectron
microscopy using mouse monoclonal antibody against nucleo-
protein. The electron-dense dots in transversely sectioned virions
reacted with anti-nucleoprotein monoclonal antibodies conjugated
to gold particles (Fig. 3c), indicating that they were indeed viral
To analyse the interior architecture of virions in greater detail, we
used electron tomography, which constructs a three-dimensional
density map of molecules, the ‘tomogram’, at a near-molecular
. Taking computational thin slices from the map, Fig. 4
shows the results of electron tomography applied to virions budding
from MDCK cells. Within a virion, outlined in Fig. 4 by a bilayered
membrane with protruding viral glycoproteins, were eight RNPs
with a distorted round or square shape. Although some of the RNPs
were clearly isolated from each other, there were instances in which
peripheral RNPs, as well as central and peripheral RNPs, appeared to
be in close contact (Fig. 4, arrows). Whether such contact represents
nonspeciﬁc association of the RNPs inside the virion or speciﬁc
interactions with functional signiﬁcance remains to be determined.
Our ﬁndings address a long-standing controversy in inﬂuenza
virus research: are viral RNA segments incorporated randomly
into virions? Together with studies showing that all viral
RNPs possess segment-speciﬁc packaging signals (refs 8–12; and
Y. Muramoto and Y.K., and M. Ozawa and Y.K., unpublished data),
our morphological ﬁndings clearly favour a model of selective
incorporation. Still unclear is how the eight RNPs are organized
into a characteristic arrangement within virions. One possibility is
that coding regions of the viral RNAs possess signal sequences that
promote recruitment of the segments during virion assembly
may then control intersegmental association. Our study contributes
Figure 2 | Rod-like structures in a developing virion. a–c, Rod-like
structures align vertically with the plasma membrane. d, The fully developed
budding virion contains a set of eight rod-like structures. Virions in the
upper row are viewed from the top; those in the lower row are shown in side
Figure 3 | Identiﬁcation of rod-like structures in virions as viral RNPs.
a, Virions ruptured by freeze-drying release twisted rods, 12 nm in width,
which are indistinguishable from described puriﬁed RNPs
. b, Twisted rods
speciﬁcally immunolabelled with anti-nucleoprotein monoclonal antibodies
conjugated to 5-nm gold particles. c, Immunogold labelling of electron-
dense dots within transversely sectioned vir ions with anti-NP monoclonal
antibodies conjugated to 10-nm gold particles. Scale bars, 50 nm.
NATURE|Vol 439|26 January 2006 LETTERS
© 2006 Nature Publishing Group
fundamental knowledge to attempts aimed at elucidating the mecha-
nisms of genome incorporation into virions. Deﬁning the interior
architecture of inﬂuenza virions may speed up the development of
both antiviral compounds and more efﬁcient methods of gene
delivery and expression.
Cells and viruses. A stock of A/WSN/33 (H1N1) was prepared by growing the
virus in MDCK cells. Stocks of A/Aichi/2/68 (H3N2), A/Puerto Rico/8/34
(H1N1), A/duck/Hong Kong/836/80 (H3N1), A/mallard/New York/6750/78
(H2N2), A/swine/Chiba/1/91 (H1N2) and A/Udorn/307/72 (H3N2) were
prepared in embryonated chicken eggs aged 10–11 d.
Ultrathin section electron microscopy. Electron microscopy was done as
. In brief, MDCK cells were infected with virus at a multiplicity of
infection of more than 10 and were then preﬁxed with 2.5% glutaraldehyde in
0.1 M cacodylate buffer (pH 7.4) on ice for 1 h. After being washed with the same
buffer, the cells were post-ﬁxed with 2% osmium tetraoxide on ice for 1 h,
stained with uranyl aqueous solution en bloc, dehydrated with a series of ethanol
gradients followed by propylene oxide treatment, and embedded in Epon 812
Resin mixture. Ultrathin sections (,60-nm thick) were stained with 2% uranyl
acetate in 70% ethanol for 3 min at room temperature and in Raynold’s lead for
3 min at room temperature, and then examined with a JEM-1200EX electron
microscope (Jeol) operated at 80 kV. For ultrathin section-immunoelectron
microscopy, the sections were prepared on nickel grids as described above and
incubated with saturated sodium periodate solution
, followed by 0.2 M
glycine in PBS buffer. After being washed with PBS, the sections were incubated
with 1% bovine serum albumin (BSA) in PBS, and then with anti-inﬂuenza A
virus nucleoprotein mouse monoclonal antibodies. They were then washed
with PBS and incubated with a goat anti-mouse immunoglobulin conjugated to
10-nm gold particles (1:100 dilution, BBInternational).
Preparation of serial ultrathin sections. Samples for serial ultrathin sections
were prepared as described above. Sections (,20-nm thick) were continuously
cut with an Ultracut S ultramicrotome (Leica). The resultant series of ultrathin
sections were placed on a single-slot copper grid and examined with an H-7500
electron microscope (Hitachi) operated at 60 kV.
Negative staining of freeze-dried samples. Puriﬁed virions were adsorbed to
Formvar-carbon-coated copper grids and negatively stained with 2% phospho-
tungstic acid solution (PTA). Grids were immediately placed in liquid nitrogen
and transferred to a JEE-4X vacuum evaporator (Jeol) for freeze-drying.
Immunoelectron microscopy was done with freeze-dried samples on nickel
grids treated as described above, except that the 2% PTA staining step was
omitted. The grids were then treated with 1% BSA in PBS and mouse
monoclonal antibodies against inﬂuenza A virus nucleoprotein, washed with
PBS, and incubated with a goat anti-mouse immunoglobulin conjugated to
5-nm gold particles (1:50 dilution, BBInternational). After being washed, the
samples were ﬁxed with 2% glutaraldehyde and negatively stained with 2% PTA.
Tomographic reconstruction. Sections (,50-nm thick) were prepared as
described above and afﬁxed to 10-nm colloidal gold particles on the upper
surface, which served as ﬁducial markers for subsequent image alignment. They
were then placed in a CompStage specimen holder (FEI) and imaged in a Tecnai
G2 Shera (FEI) operating at 200 kV. Images were taken at a magniﬁcation of
58,000 every 28 (from 2658 to þ708) and recorded by a Gatan CCD camera
(1,024 £ 1,024 pixels with 0.43 nm per pixel) with an accumulative electron dose
of 400 e
. The alignment of the projections was calculated by using IMOD
and the 10-nm gold beads, and the three-dimensional reconstruction
was based on an R-weighted back projection. Galleries of slice tomograms were
displayed with Amira software (Template Graphics) at intervals of 2.15-nm
Received 26 August; accepted 26 October 2005.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements We thank J. Gilbert for editing the manuscript; M. Imai,
Y. Muramoto and K. Fujii for discussion; Y. Hirata, K. Aoyama and K. Inoke for
technical assistance with electron microscopic tomography; and Y. Kawaoka for
illustrations. This work was supported by CREST (Japan Science and Technology
Agency), by Grants-in-Aid by the Ministry of Education, Culture, Sports, Science
and Technology, by the Ministry of Health, Labor and Welfare, Japan, and by a
National Institute of Allergy and Infectious Disease Public Health Service
research grant (to Y.K.); and by Swedish Research Council grants and the STINT
Foundation (to R.H.C.). T.N. was the recipient of a fellowship from the
incorporated foundation SYOUSHISYA and a research fellowship from the Japan
Society for the Promotion of Science for Young Scientists.
Author Information Reprints and permissions information is available at
npg.nature.com/reprintsandpermissions. The authors declare no competing
ﬁnancial interests. Correspondence and requests for materials should be
addressed to Y.K. (Kawaoka@ims.u-tokyo.ac.jp).
Figure 4 | Electron tomography of RNP complexes in a budding virion. The
virion was reconstructed in three dimensions by electron tomography, and
three tomograms of its upper portion (at 2.15-nm intervals) containing
eight RNPs are shown. Note the close proximities (possible associations) of
the RNPs (arrows). Scale bar, 50 nm.
LETTERS NATURE|Vol 439|26 January 2006