Configuration of Viral Ribonucleoprotein Complexes within the Influenza A Virion
ABSTRACT
The influenza A virus possesses an eight-segmented, negative-sense, single-stranded RNA genome (vRNA). Each vRNA segment binds to multiple copies of viral nucleoproteins and a small number of heterotrimeric polymerase complexes to form a rod-like ribonucleoprotein complex (RNP), which is essential for the transcription and replication of the vRNAs. However, how the RNPs are organized within the progeny virion is not fully understood. Here, by focusing on polymerase complexes, we analyzed the fine structure of purified RNPs and their configuration within virions by using various electron microscopies (EM). We confirmed that the individual RNPs possess a single polymerase complex at one end of the rod-like structure and that, as determined using immune EM, some RNPs are incorporated into budding virions with their polymerase-binding ends at the budding tip, whereas others align with their polymerase-binding ends at the bottom of the virion. These data further our understanding of influenza virus virion morphogenesis.
INTRODUCTION
Influenza A virus is a member of the family Orthomyxoviridae, with an eight-segmented and single-stranded genomic RNA (vRNA) of negative polarity. The vRNA is associated with multiple copies of nucleoproteins (NPs) and a small number of heterotrimeric RNA-dependent RNA polymerase complexes that comprise PA, PB1, and PB2, which together form the ribonucleoprotein complex (RNP) (1–3). The RNP functions as a minimal unit responsible for the transcription and replication of the vRNA.
RNPs have twisted rod-like structures of approximately 13 nm in diameter; a string of NP beads folds back on itself, forming a loop structure at one end (4). These rod-like structures range in length from 30 to 110 nm, which is consistent with the lengths of the respective vRNA segments (5). The conserved and partially complementary sequences of the 5′ and 3′ ends of the vRNA form a promoter region, with which the viral polymerase complex is thought to associate (6–10). Immunoelectron microscopy (immuno-EM) of purified RNPs suggested that only one or a few polymerase complexes are located at the end of the rod-like RNP (1, 3) and that therefore the polymerase complexes are located at the end opposite to the RNP loop. In addition, recent single-particle analyses revealed the three-dimensional (3D) structure of native RNPs, in which the polymerase complex was located at the end (11, 12). However, the polymerase complex has still not been directly visualized without averaging techniques, because the 3D structure generated by single-particle analysis is the average image from many different RNPs; it cannot provide information about the structure of individual RNPs.
Recent studies have shown that the eight unique vRNA segments that form RNPs are selectively packaged into each virion through segment-specific packaging signals that are present at the 5′ and 3′ ends of the vRNA segments (13–17). It has also been demonstrated by EM that the eight RNPs within the virions are arranged in a characteristic pattern—seven RNPs surround a central RNP—and that this set of eight RNPs associates vertically with an inner viral envelope at the tip of the budding virion (14, 15). On the basis of these recent findings, the eight RNPs are thought to be coordinately arranged and incorporated into progeny virions. However, the configuration of the eight RNPs within budding virions is still not fully understood.
Here, we sought to determine the configuration of RNPs within the budding virion in more detail to better understand virion morphogenesis. To this end, we first conducted scanning transmission electron microscopy (STEM) tomography to clarify the number and exact location of the polymerase complexes in individual RNPs without using averaging techniques. Then, based on the information obtained from STEM tomography, we analyzed the configuration of the RNPs within the budding virion by using immuno-EM.
MATERIALS AND METHODS
Antibodies.
Mouse anti-NP (2S347/3), anti-PA (65/4), anti-PB1 (8/9), anti-PB2 (18/1), and anti-DYKDDDDK tag (anti-FLAG; Wako Pure Chemical Industries) monoclonal antibodies were used for immuno-EM. Goat anti-mouse IgG antibody conjugated to 10-nm gold particles was purchased from BBInternational.
Virus preparation.
A stock of the A/Puerto Rico/8/34 (PR8; H1N1) strain was prepared by growing the virus in the allantoic cavity of 10-day-old chicken eggs at 37°C for 2 days. The virions were purified via sucrose density gradient ultracentrifugation.
RNP purification.
Purified PR8 virions were lysed in a solution containing 50 mM Tris-HCl (pH 8.0), 100 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol (DTT), 2% lysolecithin, 2% Triton X-100, 5% glycerol, and 1 U/μl RNase inhibitor (Promega) for 1 h at 30°C. The sample was then directly ultracentrifuged through a 30% to 70% glycerol gradient at 245,000 × g for 3 h at 4°C.
Gel electrophoresis and staining.
After ultracentrifugation through 30% to 70% glycerol gradients, the fractions were mixed with 2× Tris-glycine SDS sample buffer (Invitrogen) and subjected to SDS-PAGE, followed by Coomassie brilliant blue staining (CBB Stain One; Nacalai Tesque).
Negative-staining immuno-EM.
Purified RNPs were adsorbed to Formvar-coated nickel grids and prefixed with 1% glutaraldehyde (GLA). The grids were washed, treated with blocking solution (Blocking One; Nacalai Tesque), and then incubated with an anti-NP, anti-PA, anti-PB1, anti-PB2 antibody, or anti-FLAG antibody conjugated with 5-nm gold particles (BBInternational). After being washed, the samples were fixed with 1% GLA and negatively stained with 1% uranyl acetate solution (UA). The images were recorded with a Tecnai F20 transmission EM (TEM) (FEI Company) operated at 200 kV.
STEM tomography and image analysis.
Purified RNPs were adsorbed onto carbon-coated copper grids, negatively stained with 1% UA, and then air dried. The single- or dual-axis tomographic images of the negatively stained RNPs were obtained on a high-angle annular dark-field detector (Fischione) with a field emission STEM operated at 200 kV (Tecnai F20; FEI Company). The x and y axis tilted series of the RNP images were collected by 2 cos θ° each up to ±60° (75 images) at 0.18 to 0.26 nm per pixel. The raw images were reconstructed into a 3D volume by using the Inspect3D software program (FEI Company). For dual-axis STEM tomography, two volumes obtained from the x and y axis tilted series were combined by using Inspect3D. Digital slices (0.18 to 0.26 nm thick) were visualized with the Avizo 6.2 image processing package (Visualization Science Group).
Ultrathin-section immuno-EM.
The chemically fixed sample of PR8-infected allantoic membranes was prepared as described previously (18). Sections of the sample (80 nm thick) were etched on nickel grids with saturated sodium periodate solution, washed with 0.2 M glycine in phosphate-buffered saline (PBS), and treated with the blocking solution. The grids were then incubated with an anti-NP, anti-PB2, anti-PB1, anti-PA, or anti-FLAG monoclonal antibody. After being washed with PBS, they were incubated with a goat anti-mouse immunoglobulin conjugated to 10-nm gold particles. The samples were washed, fixed with 1% GLA, and stained with 2% UA and Reynolds lead. The digital images were recorded with a TEM operated at 200 kV.
Distribution analysis of NP and polymerases within virions.
Longitudinally sectioned virions containing rod-like RNPs were randomly selected in the images from the ultrathin-section immuno-EM. The distribution of gold signals in the virions was examined by measuring the distances from the budding tip of the virions to the gold signal by using the ImageJ software program (http://rsbweb.nih.gov/ij/).
Theoretical models for molecular distribution patterns on RNPs.
We constructed four theoretical distribution models in which the molecules are evenly distributed on the eight RNPs (NP model), located at the ends of the RNPs only at the budding tip (polymerase model 1), located at the bottom end (polymerase model 2), or located at both ends (polymerase model 3). The lengths of the RNPs were assumed to be proportional to the numbers of nucleotides in the respective vRNA segments (100, 100, 95, 76, 67, 60, 44, and 38 nm for segment 1 to 8, respectively). For the NP model, each molecule was considered to be located at intervals of 2.0 nm on the RNPs, on the basis of previous reports showing that each NP molecule binds to 24 nucleotides of viral RNA (11). The gold signals that reacted to each molecule were considered to be normally distributed. The data analysis and graph creation were done by using R packaging, version 2.15.1 (19).
RESULTS
STEM tomography of purified RNPs.
The polymerase complex is located at the end of the RNP, whose 3D structure was recently visualized by use of single-particle analysis (11, 12). This analysis, however, provides structural information by averaging data for many different molecules. Therefore, the polymerase complex has still not been directly visualized without averaging techniques. To determine the exact locations and numbers of polymerase complexes on individual RNPs, we extracted RNPs from purified virions. Gel electrophoresis and negative-staining EM of the purified RNPs showed that the specimen was highly purified, with few disrupted RNPs or debris from the other viral components (Fig. 1). We then conducted negative-staining immuno-EM with monoclonal antibodies against the respective RNP components. We found that the polymerase subunits were detected exclusively at one end of the rod-like RNP, whereas NP molecules were detected along the entire length of the rod-like structure of the RNP (Fig. 2). These results are in good agreement with findings of a previous study (1, 3). Next, we attempted to visualize the polymerase complex binding to the end of the RNP by using STEM tomography. Electron tomography is a technique used to construct detailed 3D structures of macromolecules; STEM tomography provides a better contrast and signal-to-noise ratio than does conventional electron tomography, producing high-resolution images (20, 21). Upon reconstruction of images by using STEM tomography, we were able to visualize the constituent molecules of the RNP (Fig. 3A). At one end of the RNP, a loop structure was formed by several molecules that were uniform in shape and about 4 by 6 nm (Fig. 3, arrowheads); this is consistent with a previous report of NP molecules of 3.5 by 6.2 nm forming a small rod-like structure (22). In 50 out of the 323 RNPs (15.5%), we observed a single molecule of about 10 nm in diameter only at the blunt end opposite the loop structure. The morphology of this molecule, which sometimes showed a holey or grooved structure (Fig. 3A, arrows in the left panels), is consistent with the polymerase complex visualized by single-particle analyses (11, 12, 23, 24). Serial slices of the RNPs showed that the molecule has an electron density different from that of NP molecules (Fig. 3A, arrows in the right small panels), suggesting that the molecule observed at the end of the RNP is most likely a single polymerase complex. We never observed two or more polymerase complexes on the RNPs. Taken together, our observations strongly suggest that only a single polymerase complex is present at the opposite end of the loop structure of a rod-like RNP.
Fig 1
Fig 2
Fig 3
Immuno-EM of thin-sectioned virions.
Within a budding virion, eight RNPs are associated vertically with the envelope at the tip (14, 15). However, it remains unclear whether the polymerase-binding ends of the eight RNPs are present at the tip or at the middle or bottom portions of the budding virion. To clarify this point and determine the configuration of the eight RNPs within the budding virion in more detail, we analyzed the locations of the polymerase subunits, which bind to the blunt end of the rod-like RNP (Fig. 3), within budding virions by using immuno-EM. After immunogold labeling with the respective monoclonal antibodies, we randomly chose longitudinally sectioned budding virions, in which the whole rod-like RNP complexes were visible (Fig. 4A to G). We saw no significant signal when immuno-EM was conducted with an anti-FLAG antibody or without a primary antibody (Fig. 4E and F). Then, the distances between the immunogold signal and the budding tip of the virions were measured (Fig. 4H). We found that the NP signals were distributed along the length of the rod-like RNPs in the virions with a broad peak at 30 to 90 nm from the virion budding tip (Fig. 4I, left); there were also a few signals with anti-FLAG. The reduction in NP signals at around 100 to 150 nm from the tip of the virion is consistent with reports that the eight RNPs within the virion are associated with the tip of the budding virion and are different in length (14, 15). On the other hand, the signals of the various polymerase subunits were distributed with a peak of around 10 to 40 nm from the virion budding tip; however, a considerable number of the signals were also found at around 60 to 120 nm from the virion budding tip (Fig. 4I, the three graphs on the right). These results indicate that most of the polymerases are present underneath the budding tip of the virion, but some are also present in the middle and at the bottom portion of the virions. Importantly, in some virions, the polymerase signals were found at both the top and the middle or the top and the bottom portions of the budding virion (Fig. 4G). Thus, our results indicate that some RNPs are incorporated with their polymerase-binding ends toward the budding tip, whereas others are incorporated with their polymerase-binding ends toward the bottom of a virion (see Fig. 6B).
Fig 4
Distribution modeling of the NP and polymerase.
To confirm our observations that polymerases are located at both the upper and lower portions of the virion, we constructed models for various distribution patterns on RNPs within the virion. The NP distribution model showed a single peak at around the middle part of the virion, which agreed with the distribution of the anti-NP signals (Fig. 4I and 5C, left). Polymerase model 1 showed a sharp peak at the budding tip of the virion. Because RNPs differ in length, the distribution of polymerase model 2 showed a broad peak(s) at the bottom part of the virion. The data obtained by immuno-EM of antipolymerase antibodies was fitted to polymerase model 3, in which the molecules are located at both the tip and the bottom of the virion (Fig. 4I and 5C, right). This distribution model is characterized by a major sharp peak at the budding tip and a minor broad peak(s) at the middle/bottom area of the virion. We analyzed the theoretical distributions by using three different standard deviations (10, 15, and 20 nm) of the normal distributions for the distribution of each molecule, because the size of the primary and secondary antibody complex ranges from 10 to 20 nm (Fig. 5C). We found that the distribution pattern for polymerase model 3 was similar to the histograms obtained from immuno-EM by using anti-PA, -PB1, or -PB2 antibodies (Fig. 4I and 5C). Therefore, the slight differences in the distribution patterns of the PA, PB1, and PB2 signals likely stem from differences among the respective normal distributions of the conjugated antibodies.
Fig 5
DISCUSSION
We have not fully understood how the eight RNPs within the influenza virion are arranged, even though such information is important to our understanding of virion morphogenesis. Here, by combining STEM tomography and immuno-EM, we have confirmed that a single polymerase complex is associated with a rod-like RNP at the blunt end of the budding virion and have shown that the orientation of the polymerase-binding ends of the eight RNPs is not uniform within the budding virion.
A previous report estimated that there are more than nine polymerase complexes within an influenza virion (25). However, the accuracy of this estimation remains unclear, because our recent study (18) showed that influenza virus virions could be fused during virion purification that involves ultracentrifugation. In fact, immuno-EM and biochemical analyses indicate that a single polymerase complex is associated with one end of each of the eight RNPs (1, 3, 7–12). In this study, by using STEM tomography, we visualized the polymerase complex on a native RNP purified from virions (Fig. 3) without using averaging techniques, such as single-particle analysis (23, 24). We confirmed that each RNP likely possesses only a polymerase complex at one end of the RNP, although polymerase complexes could not be clearly observed in some RNPs (Fig. 3B and C). This was likely because some polymerase complexes may have been too close to neighboring NPs to be distinguished by STEM tomography or because they were inadvertently removed during the experimental manipulation. Since polymerase complexes can be dissociated during RNP purification that involves ultracentrifugation (1, 3, 7, 26), it remains unclear whether some RNPs lack the polymerase complex to begin with.
For decades, two conflicting genome packaging models have been considered: selective packaging and random packaging. Recent studies finally concluded that a set of eight vRNA segments (i.e., the eight RNPs) are selectively incorporated into every progeny virion (13–15, 17, 27). Reverse genetics studies further demonstrated that all eight vRNA segments possess segment-specific packaging signal sequences at both the 5′ and 3′ ends of the vRNAs (28, 29). We and others showed that mutations in the packaging signal of a vRNA segment affect the efficiency of packaging of the other vRNA segments into virions (17, 30–32). These findings imply that the eight RNPs within the virion interact with each other, possibly via the packaging signals (16, 17), which are close to the polymerase-binding promoter region (Fig. 6A). If all eight of the RNPs have their polymerase-binding ends at the tip or the bottom of the budding virion, then interactions among the eight RNPs may be restricted at the tip or at the middle and bottom portions of that virion, respectively (Fig. 6C and D) (16, 17). In our present study, however, the eight RNPs are differently oriented within virions (Fig. 6B). Therefore, interactions among RNPs do not appear to be restricted at a single side within the budding virion, which is consistent with our previous report that string-like intermediates exist between the RNPs throughout the virion (15). On the other hand, there may be as-yet-unidentified regions in the middle of the vRNA segments that are involved in efficient genome packaging. It remains unclear whether the orientation of the respective RNPs is the same within all budding virions. Further studies are needed to fully understand the selective packaging mechanism.
Fig 6
In conclusion, here we suggest that the eight RNPs are differently oriented within a virion. Our study provides a novel model of the influenza virus virion structure and important insights for elucidating the mechanism of influenza virus genome packaging.
ACKNOWLEDGMENTS
We thank Susan Watson for editing the manuscript, Sumiho Nakatsu for helpful discussions, and Eiryo Kawakami for model analysis.
This work was supported by Exploratory Research for Advanced Technology (Japan Science and Technology Agency), by a grant-in-aid for Specially Promoted Research from the Ministries of Education, Culture, Sport, Science, and Technology (MEXT), by a grant-in-aid from Health, Labor, and Welfare of Japan, by National Institute of Allergy and Infectious Disease Public Health Service research grants, and in part by the Global COE Program Center of Education and Research for Advanced Genome-Based Medicine for Personalized Medicine and the Control of Worldwide Infectious Diseases from MEXT. Y.S. is supported by a Grant-in-Aid from the Japan Society for the Promotion of Science. T.N. was supported by a Grant-in-Aid for Young Scientists from the Japan Society for the Promotion of Science, by the Uehara Memorial Foundation, and by the Takeda Science Foundation.
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© 2013 American Society for Microbiology.
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Received: 31 July 2013
Accepted: 16 September 2013
Published online: 1 December 2013
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