Architecture of ribonucleoprotein complexes in influenza A virus particles

Article (PDF Available)inNature 439(7075):490-2 · February 2006with43 Reads
DOI: 10.1038/nature04378 · Source: PubMed
Abstract
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
Takeshi Noda
1,2,3
, Hiroshi Sagara
4
, Albert Yen
5
, Ayato Takada
3,6
, Hiroshi Kida
2
, R. Holland Cheng
5,7
& Yoshihiro Kawaoka
1,3,6,8
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
1
. Inside each envelope is a viral genome
consisting of eight single-stranded negative-sense RNA segments
of 890 to 2,341 nucleotides each
1
. 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
2–4
. 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
5
,
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
6–12
. 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
13
, 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
2–4
. 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).
LETTERS
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).
1
Internal Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
2
Laboratory of
Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Kita-ku, Sapporo 060-0818, Japan.
3
Core Research for Evolutional
Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan.
4
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.
5
Department of Biosciences, Karolinska Institute,
141 57 Huddinge, Sweden.
6
Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
7
Molecular and Cellular Biology, University of California, Davis, California 95616, USA.
8
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
490
© 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,
are filamentous
1
, 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
2–4
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
RNPs.
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
resolution
14
. 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
5
or
selectively
6,7
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
8
, which
may then control intersegmental association. Our study contributes
Figure 2 | Rod-like structures in a developing virion. ac, 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
profile.
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
2–4
. 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
491
© 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.
METHODS
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
described
15
. 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
16,17
, 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
2
A
22
. The alignment of the projections was calculated by using IMOD
software
18
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
voxels.
Received 26 August; accepted 26 October 2005.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
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
492
    • "Recent studies in IAV indicate that for each segment, RNA packaging signals interact with other vRNA elements in coding and noncoding regions to facilitate segment specific co-incorporation [82][83][84][85][86][87][88][89]. Influenza virions, however, exhibit RNP densities indicative of organized RNP densities with seven RNPs surrounding a central eighth RNP [90,91]. In contrast, cryo-EM of model phleboviruses UUKV and RVFV give no such indication of distinctly separate RNP densities, but show highly ordered interactions between N and the glycoprotein tails [92]. "
    [Show abstract] [Hide abstract] ABSTRACT: The Bunyaviridae represents the largest family of segmented RNA viruses, which infect a staggering diversity of plants, animals, and insects. Within the family Bunyaviridae, the Phlebovirus genus includes several important human and animal pathogens, including Rift Valley fever virus (RVFV), severe fever with thrombocytopenia syndrome virus (SFTSV), Uukuniemi virus (UUKV), and the sandfly fever viruses. The phleboviruses have small tripartite RNA genomes that encode a repertoire of 5-7 proteins. These few proteins accomplish the daunting task of recognizing and specifically packaging a tri-segment complement of viral genomic RNA in the midst of an abundance of host components. The critical nucleation events that eventually lead to virion production begin early on in the host cytoplasm as the first strands of nascent viral RNA (vRNA) are synthesized. The interaction between the vRNA and the viral nucleocapsid (N) protein effectively protects and masks the RNA from the host, and also forms the ribonucleoprotein (RNP) architecture that mediates downstream interactions and drives virion formation. Although the mechanism by which all three genomic counterparts are selectively co-packaged is not completely understood, we are beginning to understand the hierarchy of interactions that begins with N-RNA packaging and culminates in RNP packaging into new virus particles. In this review we focus on recent progress that highlights the molecular basis of RNA genome packaging in the phleboviruses.
    Full-text · Article · Jul 2016
    • "The precise role of M1 during vRNP recruitment is currently unclear, however, due to its reported interactions with all transmembrane proteins and the vRNPs [64][65][66][80][81][82], M1 is believed to function as an adaptor, connecting the vRNPs to the viral membrane during 'genome packaging'. During 'genome packaging', the eight vRNPs arrange in a marked '(7 + 1) order' with seven peripheral vRNPs surrounding a central one [76,83]. Notably, all studies found the vRNPs to be visually interconnected, suggesting that interactions play a decisive role during the incorporation process. "
    [Show abstract] [Hide abstract] ABSTRACT: Influenza A viruses (IAVs) harbor a segmented RNA genome that is organized into eight distinct viral ribonucleoprotein (vRNP) complexes. Although a segmented genome may be a major advantage to adapt to new host environments, it comes at the cost of a highly sophisticated genome packaging mechanism. Newly synthesized vRNPs conquer the cellular endosomal recycling machinery to access the viral budding site at the plasma membrane. Genome packaging sequences unique to each RNA genome segment are thought to be key determinants ensuring the assembly and incorporation of eight distinct vRNPs into progeny viral particles. Recent studies using advanced fluorescence microscopy techniques suggest the formation of vRNP sub-bundles (comprising less than eight vRNPs) during their transport on recycling endosomes. The formation of such sub-bundles might be required for efficient packaging of a bundle of eight different genomes segments at the budding site, further highlighting the complexity of IAV genome packaging.
    Full-text · Article · Jun 2016
    • "A failure to package some segments is not, however, supported by recent fluorescence in situ hybridization data that show a high percentage of virus particles contain eight different viral RNAs [55]. Although not quantitative, electron microscopic analyses of RNPs within IAV virions also show the presence of eight segments arranged in an ordered fashion [56,57]. The possibility that SI particles carry eight fully functional vRNA molecules but fail to deliver one or more to the nucleus is feasible but weakened by FISH analysis of IAV genomes in infected cells, which suggests that the segments remain associated prior to nuclear import [58] . "
    [Show abstract] [Hide abstract] ABSTRACT: A high particle to infectivity ratio is a feature common to many RNA viruses, with ~90–99% of particles unable to initiate a productive infection under low multiplicity conditions. A recent publication by Brooke et al. revealed that, for influenza A virus (IAV), a proportion of these seemingly non-infectious particles are in fact semi-infectious. Semi-infectious (SI) particles deliver an incomplete set of viral genes to the cell, and therefore cannot support a full cycle of replication unless complemented through co-infection. In addition to SI particles, IAV populations often contain defective-interfering (DI) particles, which actively interfere with production of infectious progeny. With the aim of understanding the significance to viral evolution of these incomplete particles, we tested the hypothesis that SI and DI particles promote diversification through reassortment. Our approach combined computational simulations with experimental determination of infection, co-infection and reassortment levels following co-inoculation of cultured cells with two distinct influenza A/Panama/2007/99 (H3N2)-based viruses. Computational results predicted enhanced reassortment at a given % infection or multiplicity of infection with increasing semi-infectious particle content. Comparison of experimental data to the model indicated that the likelihood that a given segment is missing varies among the segments and that most particles fail to deliver !1 segment. To verify the prediction that SI particles augment reassortment, we performed co-infections using viruses exposed to low dose UV. As expected, the introduction of semi-infectious particles with UV-induced lesions enhanced reassortment. In contrast to SI particles, inclusion of DI particles in modeled virus populations could not account for observed reassortment outcomes. DI particles were furthermore found experimentally to suppress detectable reas-sortment, relative to that seen with standard virus stocks, most likely by interfering with production of infectious progeny from co-infected cells. These data indicate that semi-infectious particles increase the rate of reassortment and may therefore accelerate adaptive evolution of IAV.
    Full-text · Article · Nov 2015
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