This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Överby, A. K.
Right arrow Articles by Pettersson, R. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Överby, A. K.
Right arrow Articles by Pettersson, R. F.

 Previous Article  |  Next Article 

Journal of Virology, November 2006, p. 10428-10435, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.01362-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Generation and Analysis of Infectious Virus-Like Particles of Uukuniemi Virus (Bunyaviridae): a Useful System for Studying Bunyaviral Packaging and Budding{triangledown}

Anna K. Överby,1 Vsevolod Popov,2 Etienne P. A. Neve,1 and Ralf F. Pettersson1*

Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institute, Box 240, SE-17177 Stockholm, Sweden,1 Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-06092

Received 28 June 2006/ Accepted 10 August 2006


arrow
ABSTRACT
 
In the present report we describe an infectious virus-like particle (VLP) system for the Uukuniemi (UUK) virus, a member of the Bunyaviridae family. It utilizes our recently developed reverse genetic system based on the RNA polymerase I minigenome system for UUK virus used to study replication, encapsidation, and transcription by monitoring reporter gene expression. Here, we have added the glycoprotein precursor expression plasmid together with the minigenome, nucleoprotein, and polymerase to generate VLPs, which incorporate the minigenome and are released into the supernatant. The particles are able to infect new cells, and reporter gene expression can be monitored if the trans-acting viral proteins (RNA polymerase and nucleoprotein) are also expressed in these cells. No minigenome transfer occurred in the absence of glycoproteins, demonstrating that the glycoproteins are absolutely required for the generation of infectious particles. Moreover, expression of glycoproteins alone was sufficient to produce and release VLPs. We show that the ribonucleoproteins (RNPs) are incorporated into VLPs but are not required for the generation of particles. Morphological analysis of the particles by electron microscopy revealed that VLPs, either with or without minigenomes, display a surface morphology indistinguishable from that of the authentic UUK virus and that they bud into Golgi vesicles in the same way as UUK virus does. This infectious VLP system will be very useful for studying the bunyaviral structural components required for budding and packaging of RNPs and receptor binding and may also be useful for the development of new vaccines for the human pathogens from this family.


arrow
INTRODUCTION
 
The Bunyaviridae family is divided into five different genera, Phlebovirus, Nairovirus, Hantavirus, Orthobunyavirus, and Tospovirus, and contains more than 300 members, most of them being arthropod-borne viruses. Many of them are serious human pathogens causing encephalitis or hemorrhagic fever, and no vaccines are currently available for any of them. Members of this family of viruses share many characteristics: they are enveloped and spherical (90 to 110 nm in diameter) with a trisegmented negative-stranded RNA genome with replication occurring in the cytoplasm and budding taking place in the Golgi compartment (9).

The Uukuniemi (UUK) virus, a member of the Phlebovirus genus, has been utilized as a model virus for studying this family for over 35 years. The UUK virus consists of three RNA segments L, M, and S encoding four structural proteins and one nonstructural protein. The L segment encodes the RNA-dependent RNA polymerase (L), and the M segment encodes the glycoprotein precursor (p110), which is cotranslationally cleaved into the two viral spike proteins GN and GC (13, 44). Both glycoproteins are type 1 transmembrane proteins, which are transported as heterodimers (40) from the endoplasmic reticulum to the Golgi where they are retained due to a Golgi retention signal located in the GN cytoplasmic domain (2). The S segment utilizes an ambisense strategy to express two proteins, the nucleoprotein (N) in antisense direction and the nonstructural protein (NSs) in the sense direction (46). The N protein is a cytoplasmic protein which binds viral RNA and cRNA, generating functional templates, such as ribonucleoproteins (RNPs) for polymerase transcription and replication. After replication, the RNPs accumulate around the Golgi region and are packaged into progeny particles through an interaction involving the glycoproteins, which are situated in the viral envelope (28, 29). The UUK NSs protein is a cytoplasmic protein that was shown to be associated with the 40S ribosomal subunit (47); however, its function is still unknown. All three genomic RNA segments have noncoding regions (NCRs) flanking the open reading frames, containing cis-acting signals important for viral transcription, replication, encapsidation, and packaging (15, 16).

Reverse genetic systems are often used to study different aspects of the viral life cycle. Three such rescue systems have so far been developed for the Bunyaviridae family. Bunyamwera virus was the first to be rescued in 1996 (8) and was later optimized for a more efficient rescue (35). The La Crosse virus was rescued in 2005 (7), and more recently, the rescue of Rift Valley fever virus was reported (23). In the absence of a reverse genetic system, viruses can be studied using minigenome systems, in which artificial RNA genome segments are generated containing a reporter gene (e.g., chloramphenicol acetyltransferase [CAT], green fluorescent protein [GFP], or luciferase) flanked by the NCR from the corresponding viral genomic segment. Many such systems have been developed for members in the Bunyaviridae family (6, 11, 14, 17-19, 34, 53) and were used to study transcription (4), replication (3, 22), and packaging (15, 26). However, these systems are limited, and studying structural components involved in particle assembly and maturation, RNP packaging, and receptor binding requires either a full rescue system or a virus-like particle (VLP) system (12, 20, 48, 55). VLP systems have been shown to be very useful for the analysis of other virus families, since their cellular uptake, intracellular trafficking, budding, and release are often similar to those of the wild-type (wt) virus. The structural components can be studied to analyze which protein or combination of proteins drives budding, RNP packaging, particle assembly, and receptor binding (1, 21, 31-33, 45). An additional advantage with the VLP system is that highly pathogenic viruses can be studied in a lower biosafety level (48, 54), thereby greatly facilitating its analysis.

Here, we describe the development of an infectious VLP system for UUK virus. By adding the glycoprotein expression plasmid to our recently developed functional polymerase I (PolI) minigenome system (15, 18), UUK minigenomes containing reporter genes are encapsidated, replicated, and packaged into VLPs. These VLPs bud into the Golgi and are transported to the plasma membrane where they are subsequently released into the supernatant. The supernatant containing these VLPs can be used to infect new cells, where they will replicate and generate new VLPs if the required trans-acting viral proteins (RNA polymerase, nucleoprotein, and glycoprotein) are also expressed. In the present study, we have optimized the generation of infectious UUK VLPs, biochemically and morphologically characterized them, and determined which proteins are required for their formation.


arrow
MATERIALS AND METHODS
 
Plasmids. pUUK-GN/GC, pUUK-L, and pUUK-N are plasmids expressing the Uukuniemi viral glycoprotein precursor (p110), RNA-dependent RNA polymerase, and nucleoprotein, respectively, under the control of a cytomegalovirus (CMV) promoter (18). M-CAT, L-CAT, and S-CAT are the PolI-driven UUK minigenome plasmids containing the NCRs from the M, L, and S segments, respectively, flanking the CAT reporter gene (15). The pUUK-GN and pUUK-GC plasmids were generated using the pUUK-GN/GC plasmid as a template using standard PCR cloning amplifying bp 18 to 1556 and 1473 to 3045, respectively. These regions were chosen to ensure correct processing of the proteins (37).

Cell culture and transfection. BHK-21 cells (American Type Cell Culture) were grown in plastic dishes in minimum essential medium with Earle's salts supplemented with 5% fetal calf serum, 5% tryptose phosphate broth, 2 mM L-glutamine, 50 IU penicillin/ml, and 50 µg streptomycin/ml (Invitrogen).

For the VLP reporter gene system, BHK-21 cells were transfected with pUUK-L, pUUK-N, pUUK-GN/GC, and the PolI-driven minigenome (M-CAT, L-CAT, or S-CAT) using Lipofectamine 2000 reagent (Invitrogen). Transfections were performed as described previously (18) with a few modifications. Briefly, the transfection medium was removed 6 h posttransfection before fresh medium (minimum essential medium with 2% fetal calf serum and tryptose phosphate broth) was added. Cells were analyzed for reporter gene expression 24 h posttransfection, and the corresponding supernatants were used for VLP infection of new cells. Twenty-four hours prior to the supernatant transfer, BHK-21 cells were transfected with L and N expression plasmids to support minigenome replication and transcription to enable CAT detection. Fresh medium was added 3 h after VLP infection, and cells were incubated for 48 h before reporter gene analysis.

CAT assays. Cells were resuspended in 50 µl of 0.25 M Tris-HCl (pH 7.5) and lysed by two freeze-thawing cycles. Cells were centrifuged for 10 min at 16,000 x g, and CAT activity was determined using the commercially available Flash Cat kit (Molecular Probes) as described previously (18). The reaction products were visualized by UV illumination and documented by photography.

Purification of UUK VLPs. Supernatant from VLP-expressing cells was collected and clarified by centrifugation (4,000 x g, 10 min at 4°C). The particles were concentrated through a 20% (wt/vol) sucrose cushion and dissolved in TN buffer (0.05 M Tris-HCl, pH 7.4, and 0.1 M NaCl) by centrifugation at 100,000 x g for 1 h at 4°C. The pellet was dried for 10 min before resuspension in nonreducing Laemmli/sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer for subsequent Western blot analysis.

Western blot analysis. Precasted 10% polyacrylamide gels (Bio-Rad) were used according to the manufacturer's recommendations. Rabbit polyclonal antibodies recognizing both the UUK GN and GC proteins were used to detect the GN and GC proteins, and polyclonal antibodies recognizing the UUK N protein were used to detect the N protein (29).

TEM. VLPs and UUK virus used for negative staining in transmission electron microscopy (TEM) were fixed with glutaraldehyde in phosphate buffer to a final concentration of 0.5% at 4°C before concentration through the sucrose cushion, as described above. The pellet was resuspended in water, and Formvar carbon-coated copper grids were floated on the drops of the virus or VLP suspensions and after blotting were stained with 2% aqueous uranyl acetate (UA).

For immunogold electron microscopy, the samples were placed on Formvar carbon-coated nickel grids, treated with UUK virus immune serum diluted 1:500 in dilution buffer (1% bovine serum albumin in 0.05 M Tris-buffered saline) for 1 h at room temperature (RT), rinsed four times in washing buffer (0.1% bovine serum albumin and 0.01 M glycine in 0.05 M Tris-buffered saline), and incubated for 1 h at RT with secondary antibodies diluted 1:20 in dilution buffer. For secondary antibodies, we used goat anti-rabbit immunoglobulin G conjugated with 15-nm colloidal gold (AuroProbe EM GAR G15, RPN422V; Amersham Life Science). After incubation, the samples were rinsed in washing buffer and distilled H2O and negatively stained with 2% aqueous UA.

For ultrathin-section TEM, transfected BHK-21 cell monolayers were fixed 24 h posttransfection in a mixture of 2.5% formaldehyde, 0.1% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.2, to which 0.03% trinitrophenol and 0.03% CaCl2 were added (modified Ito's fixative [42]) for 1 h at RT, washed in 0.1 M cacodylate buffer, scraped off the plastic, and concentrated by centrifugation (10,000 x g, 10 min). The pellets were postfixed in 1% OsO4 in 0.1 M cacodylate buffer, stained en bloc with 2% aqueous UA, dehydrated in ethanol, followed by propylene oxide, and embedded in Poly/Bed 812 (Polysciences). Ultrathin sections were cut on a Reichert-Leica Ultracut S ultramicrotome and stained with lead citrate. Negative-stained samples and ultrathin sections were examined in a Philips 201 or Philips CM-100 electron microscope at 60 kV.


arrow
RESULTS AND DISCUSSION
 
Generation of infectious VLPs for UUK virus. In order to verify that infectious VLPs could be generated, we used our previously described PolI-driven minigenome system (15, 18) to explore whether UUK structural proteins can package the three minigenome segments (M-CAT, L-CAT, and S-CAT) into VLPs, which can be used for infection of new cells (Fig. 1A). The success of incorporation of the minigenome segments into the VLPs and transfer of these segments to new cells, previously transfected with pUUK-L and pUUK-N plasmids, can be monitored by measuring the CAT activity. CAT expression is then an indirect measure of VLP generation. Cells transfected with the minigenome constructs (M-CAT, L-CAT, or S-CAT) together with plasmids expressing only the L and N protein (Fig. 1B, top panels, lanes 1, 3, and 5) or with plasmids expressing the L, N, and GN/GC proteins (lanes 2, 4, and 6) all displayed CAT activity. Supernatants from these transfected cells were collected and used to infect new cells that were pretransfected with the expression plasmids pUUK-L and pUUK-N to support minigenome replication and transcription. VLP-infected cells were analyzed for reporter gene expression 48 h postinfection (Fig. 1B, bottom panels). No CAT activity could be transferred to new cells with any of the minigenomes tested when the glycoprotein expression plasmid was omitted (Fig. 1B, bottom panels, lanes 1, 3, and 5), illustrating that the transfected cells did not release free CAT enzyme or infectious particles. However, reporter gene activity was readily detected in VLP-infected cells for M-, L-, and S-segment-based minigenomes when the glycoprotein precursor was added to the VLP system (Fig. 1B, bottom panels, lanes 2, 4, and 6), demonstrating that the transfer of CAT activity to new cells is mediated by and dependent on the UUK viral glycoproteins. We observed that the reporter activity in cells infected with the VLPs containing the M-CAT minigenome appeared to be higher than the activity for the VLPs containing the L-CAT and S-CAT minigenomes. This may reflect the differences in promoter strength of the three segments, with M having the highest activity and S the lowest (15). In the following experiments we chose to show only the M-segment-based minigenome due to the strong and reproducible reporter gene activity. Except for the reduced CAT levels due to different promoter strengths, the use of the L- and S-segment-based minigenomes gave results identical to that of the M-segment-based minigenome (data not shown).


Figure 1
View larger version (38K):
[in this window]
[in a new window]
  		FIG. 1. VLP transfer of UUK minigenomes is dependent on UUK glycoproteins. A. Schematic representation of the experimental setup. BHK-21 cells were transfected with minigenome (M-CAT, L-CAT, or S-CAT) together with the individual expression plasmids pUUK-GN/GC, pUUK-L, and pUUK-N. Supernatant containing VLPs was harvested and inoculated into BHK-21 cells, which were pretransfected with pUUK-L and pUUK-N. Cells were analyzed for reporter gene expression 48 h after VLP infection. Open reading frames are indicated in red, CMV and Pol1 promoters in blue, and NCRs of the M, L, and S in green. B. Comparison of the three different UUK virus-derived minigenomes M-CAT (lanes 1 and 2), L-CAT (lanes 3 and 4), and S-CAT (lanes 5 and 6) with respect to VLP transfer and reporter gene expression (negative controls without pUUK-GN/GC [lanes 1, 3, and 5]). The cell lysates in the CAT reaction were compensated with regard to promoter strength (1x M-CAT, 2x L-CAT, and 4x S-CAT) in both transfected and VLP-infected cells. Transfected cells (top panel) and VLP-infected cells (bottom panel) are shown. Data shown are representative of three independent experiments. C. Transfer of GFP-containing minigenome (M-GFP) by UUK VLPs. BHK-21 cells were transfected with M-GFP and the L, N, and GN/GC expression plasmids (top panel) or infected with GFP-containing VLPs (bottom panel).

Additionally, these results were confirmed by using GFP as a reporter instead of CAT in the minigenome segment. Cells transfected with M-GFP (15) instead of M-CAT in the presence of pUUK-L, pUUK-N, and pUUK-GN/GC and analyzed by microscopy 48 h later displayed strong GFP staining (Fig. 1C, top panel). The supernatant was used to infect cells pretransfected with the L, N, and GN/GC expression plasmid, and 48 h after VLP infection, these cells also showed GFP expression (Fig. 1C, bottom panel).

Thus, we have created an infectious VLP system for UUK virus that can be utilized for packaging and transfer of L-, M-, and S-segment-based minigenomes to new cells. Together these results not only demonstrate that the VLP system can be utilized for the packaging and transfer of minigenomes to new cells but also demonstrate that expression of the glycoproteins (GN/GC) is essential for generation of these infectious VLPs.

Optimization of the VLP system. Because the generation of VLPs is absolutely dependent on GN/GC, we decided to further study the dependence of VLPs on the glycoprotein concentration. Different amounts of pUUK-GN/GC were transfected into cells, and examination of the expression levels of GN and GC revealed that the glycoprotein levels correlated well with the amount of plasmid transfected (data not shown). Next, cells transfected with increasing amounts of pUUK-GN/GC (0, 0.5, 1.0, 2.5, and 5 µg) together with the M-CAT, L, and N expression plasmids were analyzed for CAT reporter gene expression (Fig. 2A, top panel), and the supernatants collected from these cells were used to infect new cells (Fig. 2A, bottom panel). We observed a clear dose-dependent increase of transfer of CAT activity in VLP-infected cells with increasing amounts of glycoprotein (Fig. 2A, bottom panel, lanes 1 to 5); no activity was observed without the addition of glycoprotein (lane 1), and the highest activity was seen with 5 µg transfected pUUK-GN/GC (lane 5).


Figure 2
View larger version (30K):
[in this window]
[in a new window]
  		FIG. 2. Optimization of VLP formation. A. Different amounts of pUUK-GN/GC (0, 0.5, 1.0, 2.5, and 5.0 µg) were transfected together with the other plasmids (M-CAT, pUUK-L, and pUUK-N), and at 24 h posttransfection, cell lysates were analyzed for CAT activity (top panel; transfected) and supernatants were used to infect new cells previously transfected with pUUK-L and pUUK-N (bottom panel; VLP infected). VLP-infected cells were analyzed 48 h postinfection. B. Kinetics of VLP formation. Cells were transfected with minigenome and pUUK-L, pUUK-N, and pUUK-GN/GC for the indicated times, analyzed for minigenome activity (top panel), and supernatants containing VLPs were collected. The collected supernatants were passaged to new cells and analyzed for CAT activity 48 h after VLP infection (bottom panel). C. Serial passage of VLPs. Cells were transfected with the M-CAT and plasmids expressing L and N proteins (top panel, lane 1) or with L, N, and GN/GC proteins (bottom panel, lane 1), and supernatants were passaged three times to new cells 24 h before infection with the VLP supernatant from cells transfected with pUUK-L, pUUK-N, and pUUK-GN/GC. Before CAT activity was analyzed 48 h posttransfection, supernatant was transferred to new cells. P1, P2, and P3 indicate the first, second, and third passages, respectively. Panels A, B, and C show representative CAT pictures from three independent experiments. Background spots from BHK-21 cells are indicated by the black asterisks.

To further optimize the efficiency of the VLP system, a time course analysis was performed to study the kinetics of the VLP formation. Cells were transfected with minigenome, pUUK-L, pUUK-N, and 2 µg of pUUK-GN/GC. The supernatants were collected at different time points (4, 8, 12, 16, 20, 24, 48, and 72 h) posttransfection, and the corresponding cells were analyzed for CAT reporter gene expression (Fig. 2B, top panel). The supernatants were transferred to new BHK-21 cells pretransfected (24 h) with the pUUK-L and pUUK-N expression plasmids. The VLP-infected cells were harvested 48 h after VLP infection and analyzed for CAT activity (Fig. 2B, bottom panel). As expected when the glycoprotein expression plasmid was omitted, no transfer of CAT activity could be detected in VLP-infected cells at any time point (data not shown). In the presence of glycoprotein, detectable amounts of reporter activity were detected as early as 12 h posttransfection (Fig. 2B, top panel) and gradually increased until 48 h posttransfection after which no further increase was observed. The reporter gene transfer by VLPs was first detected 12 h postinfection (Fig. 2B, bottom panel, lane 3) and increased over time till a plateau was reached 20 to 24 h postinfection (lanes 5 and 6), after which no significant increase in activity was observed (lanes 6 to 8). This indicated that infectious VLPs were formed and secreted as early as 12 h posttransfection. We noticed that compared to the authentic UUK virus growth curve, the lag period before detectable CAT activity was observed in new cells was prolonged compared to the reported first release of authentic UUK virus (41). wt UUK virus exhibits a one-step growth curve that starts with a lag phase of 5 to 6 h postinfection, followed by a period where the titer rises exponentially to about 24 to 30 h postinfection after which another lag phase is initiated (41). The reason for this difference in kinetics between the UUK virus and the VLP system is not clear but might be explained by the different expression systems. In the VLP system, to detect CAT activity, the PolI-driven minigenome plasmid has to enter the nucleus, be transcribed by PolI, and be exported to the cytoplasm where replication and encapsidation occurs. In contrast, for the UUK virus, these processes all occur in the cytoplasm. To be able to detect the released VLPs by reporter gene expression system, the VLP has to infect a cell that was previously transfected with the N and L expression plasmids. Since we observed a transfection efficiency of 20%, not all the generated VLPs containing minigenomes could be detected in our system. This is in contrast to the UUK virus where all replication-competent viruses can infect cells and be detected.

To test the reproducibility of the VLP system, VLPs were generated and serially passaged several times to new cells. The experimental setup was basically as described above; however, VLP-infected cells were transfected with all three expression plasmids (pUUK-N, pUUK-L, and pUUK-GN/GC) to support production of a new generation of VLPs. No CAT activity was transferred to new cells when the glycoprotein expression plasmid was omitted (Fig. 2C, top panel, lanes 2 to 4), and strong reporter gene activity was observed in all three passages when the glycoprotein was present (Fig. 2C, bottom panel, lanes 2 to 4). This shows that the VLP system for UUK virus is reproducible and that VLPs can be repeatedly generated and released at least three times, with only a slight reduction in activity.

Neutralization of VLP infection. To verify that the VLPs initiate infection in the same manner as authentic UUK viruses, we attempted to neutralize them with UUK-specific antibodies raised against the entire UUK virus particle and specifically recognizing the GN and GC proteins (see the top panels of Fig. 4). Previously, these antibodies were shown to reduce UUK virus infectivity (data not shown). Two concentrations (100 and 250 µl) of supernatant collected from cells transfected with pUUK-L, pUUK-N, pUUK-GN/GC, and M-CAT were incubated for 1 h at 37°C with UUK-specific antibodies (10 and 20 µl) or with the corresponding preimmune serum (20 µl) before inoculation onto new cells. Untreated supernatant was also used as an additional control (Fig. 3, lanes 1 and 2). A significant neutralization of the VLPs was seen for the largest amounts of antibodies added (lanes 7 and 8), while the same amount of preimmune serum did not have any effect on the infectibility (lanes 3 and 4). Also, a smaller amount of antibodies significantly inhibited VLP infection (lanes 5 and 6). This result clearly indicates that the UUK antibodies can recognize the GN/GC spike proteins in the VLPs and thereby block the infection of new cells using a mechanism reminiscent of that of the authentic UUK virus.


Figure 4
View larger version (41K):
[in this window]
[in a new window]
  		FIG. 4. Western blot analysis of UUK virus and UUK VLPs released in the supernatant. Supernatants from UUK virus-infected cells (lane 1) and cells transfected with different combinations of UUK plasmids (lanes 2 to 8) were concentrated and analyzed by Western blotting using antibodies specifically recognizing UUK glycoproteins (GN/GC) and nucleoprotein (N).


Figure 3
View larger version (30K):
[in this window]
[in a new window]
  		FIG. 3. Inhibition of VLP infection by UUK virus-specific antibodies. Different amounts (100 and 250 µl) of supernatants with VLPs containing the M-CAT minigenome were incubated with 10 or 20 µl of UUK virus immune sera recognizing GN/GC (lanes 5 and 6 and lanes 7 and 8, respectively) or with 20 µl preimmune (pre) serum (lanes 3 and 4) before inoculation onto new cells. The inoculating material was removed 1 h later, and CAT activity was measured 48 h after VLP infection. Untreated supernatants are shown in lanes 1 and 2. The neutralizing effect was seen in three independent experiments.

RNPs are not required for assembly and release of UUK VLPs. Next we analyzed the protein composition of the VLPs and also which proteins are required for assembly and release of UUK VLPs. Different combinations of plasmids were transfected into BHK-21 cells, and supernatants were analyzed by Western blotting 48 h posttransfection. Supernatants from UUK virus-infected cells served as a positive control (Fig. 4, lane 1), and both glycoproteins, GN and GC, and the N nucleoprotein were detected in the UUK viral particles. Cells transfected with the complete VLP system also showed an efficient release of both GN and GC proteins and N protein (Fig. 4, lane 2), indicating that the glycoprotein and nucleoprotein composition of the VLP released are similar to those of the UUK virus. When the minigenome was omitted, almost no nucleoprotein was incorporated into VLPs, showing that the minigenome is important for the incorporation of the nucleoprotein (Fig. 4, lane 3). One unexpected finding was that the expression of the UUK glycoprotein precursor alone was sufficient to produce and release VLPs in the supernatant, demonstrating that the only components required for efficient production and release of VLPs are the glycoproteins (Fig. 4, lane 4). Generation of VLPs for the bunyavirus Hantaan virus requires coexpression of both the N protein and the viral glycoproteins (5). The differences observed between these two VLP systems might be explained by the different expression systems used and the different viruses examined. In the Hantaan VLP system, two different expression systems (baculovirus-insect cells and recombinant vaccinia virus) were used, and VLPs were observed in the supernatant in the vaccinia virus system. Vaccinia virus is a lytic virus which expresses quite a lot of proteins that could interfere with VLP generation, compared to our plasmid-driven system. The presence of nucleocapsids to facilitate VLP generation has been shown to be important for some viruses (e.g., Semliki Forest virus [49], Hantaan virus [5], paramyxovirus simian virus 5 [45], murine leukemia virus [38], and Ebola virus [33]) but not for others (e.g., coronavirus [51], Marburg virus [50], influenza virus [20], vesicular stomatitis virus [25], and human parainfluenza virus type 1 [10]). As expected, no nucleoprotein was released into the supernatant when nucleoprotein was expressed alone (lane 5). Cells transfected with plasmids expressing only one of the glycoproteins (GN or GC) released no detectable amounts of proteins in the supernatant, suggesting that no particles were produced when the glycoproteins were expressed separately or together (Fig. 4, lanes 6 to 8). This was quite surprising since the two proteins accumulate in the Golgi and are processed the same way as the glycoprotein precursor when coexpressed from two plasmids (37). Although undetectable by Western blotting, immunofluorescence analysis revealed a small number of cells expressing these proteins, implying that expression of the GN and GC proteins separately under control of the CMV promoter was very inefficient (data not shown). Therefore, the choice of expression system appears to be important, since similar problems have been reported previously when the glycoproteins were expressed in a simian virus 40-based system (43). However, these problems were not observed in the T7 RNA polymerase-driven vaccinia virus expression system (37).

VLPs released from transfected cells are morphologically similar to UUK virus. UUK virus grown in cell cultures forms particles, which are spherical and measure about 95 nm in diameter. The lipid envelope contains glycoproteins that form characteristic projections that can be visualized efficiently by electron microscopy. The projections are clustered to form hollow cylindrical units arranged in a icosahedral surface lattice (hexons and pentons) with a T12 symmetry (52). Supernatants from UUK virus-infected cells and cells transfected with only the pUUK-GN/GC plasmid or with pUUK-GN/GC together with M-CAT, pUUK-L, and pUUK-N were collected, fixed with glutaraldehyde (0.7%) to preserve the surface morphology, concentrated by ultracentrifugation, and subjected to negative staining and electron microscopy (Fig. 5A, C, and E). UUK virus displayed the same morphological surface structure (Fig. 5E) as described before (52). The VLPs produced by transfection showed a particle morphology that was identical to that of the UUK virus particles (Fig. 5, compare panels E with panels A and C). VLPs generated with the complete system of plasmids (Fig. 5A) and empty VLPs generated with only the glycoproteins (Fig. 5C) showed no significant difference in size or morphology compared to that of the UUK virus. Some of the hollow cylindrical units formed by the glycoproteins were preserved during fixation and could be visualized. We also wanted to detect the glycoproteins immunologically with the immune gold technique using the UUK glycoprotein-specific antibodies. This was performed on unfixed particles due to the disruptive effect that glutaraldehyde has on surface antigens. Both the VLPs (Fig. 5B and D) and UUK virus (Fig. 5F) were labeled with UUK glycoprotein-specific antibodies, demonstrating that glycoproteins were present at the VLP surface.


Figure 5
View larger version (151K):
[in this window]
[in a new window]
  		FIG. 5. Morphology of the VLPs and UUK virus by transmission electron microscopy. Supernatant from cells transfected with pUUK-GN/GC, pUUK-L, pUUK-N, and M-CAT (A and B) or with only pUUK-GN/GC (C and D) or infected with UUK virus (E and F) were harvested and concentrated through a sucrose cushion. Particle morphology (A, C, and E) was analyzed by negative staining on glutaraldehyde-fixed particles. UUK glycoproteins were visualized by immunogold staining (B, D, and F) on unfixed particles using polyclonal antibodies recognizing GN/GC detected with secondary antibodies coupled to 15-nm gold particles. Bars, 100 nm.

Together these data show that the morphology and size of VLPs are similar to those of the UUK virus particles and confirm the presence of glycoproteins at the VLP surface. It further suggests that the VLP system is very useful for studying viral morphology and receptor binding.

VLPs are budding into the Golgi complex in the same way as UUK virus. The UUK virus structural proteins (N, GN, and GC) accumulate in the Golgi region after virus infection, and virus particles can be seen to bud into and mature in the Golgi complex (Fig. 6E and F) (29). Large vesicles containing the viral particles are then transported to the plasma membrane where the virus is released (29). In order to determine whether the site of budding for VLPs is the same as for the wt UUK virus, cells were transfected with the complete system of plasmids (Fig. 6A and B) or with the glycoproteins alone (Fig. 6C and D) or infected with UUK virus (Fig. 6E and F), fixed, and embedded for thin-section TEM. Several smooth vesicles could be detected containing particles in both virus-infected cells and in plasmid-transfected cells, showing that the VLPs bud in the same way as the UUK virus. In cells transfected with GN and GC, both glycoproteins were shown to colocalize with the Golgi marker GM130 by immunofluorescence microscopy (data not shown); this together with the TEM observations corroborates that the VLPs are budding into the Golgi. Again, VLPs generated by transfection of glycoproteins alone were identical to those produced by the complete system. This suggests that the glycoproteins alone have an intrinsic ability to organize themselves into particles in the same way as authentic UUK virus does and that the concentration of glycoproteins in the Golgi region might be one of the determining factors for initiation of budding.


Figure 6
View larger version (146K):
[in this window]
[in a new window]
  		FIG. 6. Morphology of VLP-producing BHK-21 cells by thin-section transmission electron microscopy. Cells transfected with pUUK-GN/GC, pUUK-L, pUUK-N, and M-CAT (A and B) or with only pUUK-GN/GC (C and D) or infected with UUK virus (E and F) at 24 h posttransfection or infection were fixed and embedded for TEM. Bars, 100 nm.

Many enveloped negative-stranded viruses have a matrix protein, which is a structural protein linking the viral envelope with the viral core. These matrix proteins play an important role in determining the size and shape of the virus particles and in initiating the budding process (10, 12, 20, 24, 30, 36, 39, 56). Bunyaviruses do not have matrix protein, suggesting that the characteristic functions of such a protein have to be performed by another protein. Here, we show that UUK VLPs can be generated with only the two glycoproteins, indicating that the nucleoprotein does not have these "matrix" functions. Instead, it is suggested that the cytoplasmic tails of both glycoproteins might act as a link between the viral envelope and the RNPs, and indeed coimmunoprecipitation experiments showed that GN/GC interacts with the N protein (27). The ectodomain alone or in combination with the cytoplasmic tails might initiate budding and determine the size and shape of the virus particles. In conclusion, we have described an efficient infectious VLP system for a bunyavirus that can be used in future studies to analyze the packaging of RNPs and budding of particles. Especially for studying viruses classified as high containment agents, the development of VLP systems is of great practical use because subsequent work can be performed under lower biosafety conditions, thereby significantly facilitating its analysis (54). In addition, VLPs might also be useful agents for developing vaccines against other members of the Bunyaviridae family.


arrow
ACKNOWLEDGMENTS
 
Thanks to Anita Bergström for excellent assistance with cell cultures and to Violet C. Han and Julie W. Wen for expert assistance in electron microscopy and Thomas Bednarek for his excellent help with photographing/imaging procedures. We are grateful to Rene Rijnbrand and Ulf Eriksson for help with critically revising the manuscript.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Ludwig Institute for Cancer Research, Stockholm Branch, Karolinska Institute, Box 240, SE-17177 Stockholm, Sweden. Phone: 46 (0)8 524 871 01. Fax: 46 (0)8 33 28 12. E-mail: rpet{at}licr.ki.se. Back

{triangledown} Published ahead of print on 23 August 2006. Back


arrow
REFERENCES
 
    1
  1. Ako-Adjei, D., M. C. Johnson, and V. M. Vogt. 2005. The retroviral capsid domain dictates virion size, morphology, and coassembly of Gag into virus-like particles. J. Virol. 79:13463-13472.[Abstract/Free Full Text]
    2. 2
  2. Andersson, A. M., L. Melin, A. Bean, and R. F. Pettersson. 1997. A retention signal necessary and sufficient for Golgi localization maps to the cytoplasmic tail of a Bunyaviridae (Uukuniemi virus) membrane glycoprotein. J. Virol. 71:4717-4727.[Abstract]
    3. 3
  3. Barr, J. N., R. M. Elliott, E. F. Dunn, and G. W. Wertz. 2003. Segment-specific terminal sequences of Bunyamwera bunyavirus regulate genome replication. Virology 311:326-338.[CrossRef][Medline]
    4. 4
  4. Barr, J. N., J. W. Rodgers, and G. W. Wertz. 2006. Identification of the Bunyamwera bunyavirus transcription termination signal. J Gen. Virol. 87:189-198.[Abstract/Free Full Text]
    5. 5
  5. Betenbaugh, M., M. Yu, K. Kuehl, J. White, D. Pennock, K. Spik, and C. Schmaljohn. 1995. Nucleocapsid- and virus-like particles assemble in cells infected with recombinant baculoviruses or vaccinia viruses expressing the M and the S segments of Hantaan virus. Virus Res. 38:111-124.[CrossRef][Medline]
    6. 6
  6. Blakqori, G., G. Kochs, O. Haller, and F. Weber. 2003. Functional L polymerase of La Crosse virus allows in vivo reconstitution of recombinant nucleocapsids. J. Gen. Virol. 84:1207-1214.[Abstract/Free Full Text]
    7. 7
  7. Blakqori, G., and F. Weber. 2005. Efficient cDNA-based rescue of La Crosse bunyaviruses expressing or lacking the nonstructural protein NSs. J. Virol. 79:10420-10428.[Abstract/Free Full Text]
    8. 8
  8. Bridgen, A., and R. M. Elliott. 1996. Rescue of a segmented negative-strand RNA virus entirely from cloned complementary DNAs. Proc. Natl. Acad. Sci. USA 93:15400-15404.[Abstract/Free Full Text]
    9. 9
  9. Calisher, C. H. 1996. History, classification, and taxonomy of viruses in the family Bunyaviridae, p. 1-17. In R. M. Elliott (ed.), The Bunyaviridae. Plenum Press, New York, N.Y.
    10. 10
  10. Coronel, E. C., K. G. Murti, T. Takimoto, and A. Portner. 1999. Human parainfluenza virus type 1 matrix and nucleoprotein genes transiently expressed in mammalian cells induce the release of virus-like particles containing nucleocapsid-like structures. J. Virol. 73:7035-7038.[Abstract/Free Full Text]
    11. 11
  11. Dunn, E. F., D. C. Pritlove, H. Jin, and R. M. Elliott. 1995. Transcription of a recombinant bunyavirus RNA template by transiently expressed bunyavirus proteins. Virology 211:133-143.[CrossRef][Medline]
    12. 12
  12. Eichler, R., T. Strecker, L. Kolesnikova, J. ter Meulen, W. Weissenhorn, S. Becker, H. D. Klenk, W. Garten, and O. Lenz. 2004. Characterization of the Lassa virus matrix protein Z: electron microscopic study of virus-like particles and interaction with the nucleoprotein (NP). Virus Res. 100:249-255.[CrossRef][Medline]
    13. 13
  13. Elliott, R. M., E. Dunn, J. F. Simons, and R. F. Pettersson. 1992. Nucleotide sequence and coding strategy of the Uukuniemi virus L RNA segment. J Gen. Virol. 73:1745-1752.[Abstract/Free Full Text]
    14. 14
  14. Flick, K., J. W. Hooper, C. S. Schmaljohn, R. F. Pettersson, H. Feldmann, and R. Flick. 2003. Rescue of Hantaan virus minigenomes. Virology 306:219-224.[CrossRef][Medline]
    15. 15
  15. Flick, K., A. Katz, A. Overby, H. Feldmann, R. F. Pettersson, and R. Flick. 2004. Functional analysis of the noncoding regions of the Uukuniemi virus (Bunyaviridae) RNA segments. J. Virol. 78:11726-11738.[Abstract/Free Full Text]
    16. 16
  16. Flick, R., F. Elgh, and R. F. Pettersson. 2002. Mutational analysis of the Uukuniemi virus (Bunyaviridae family) promoter reveals two elements of functional importance. J. Virol. 76:10849-10860.[Abstract/Free Full Text]
    17. 17
  17. Flick, R., K. Flick, H. Feldmann, and F. Elgh. 2003. Reverse genetics for Crimean-Congo hemorrhagic fever virus. J. Virol. 77:5997-6006.[Abstract/Free Full Text]
    18. 18
  18. Flick, R., and R. F. Pettersson. 2001. Reverse genetics system for Uukuniemi virus (Bunyaviridae): RNA polymerase I-catalyzed expression of chimeric viral RNAs. J. Virol. 75:1643-1655.[Abstract/Free Full Text]
    19. 19
  19. Gauliard, N., A. Billecocq, R. Flick, and M. Bouloy. 2006. Rift Valley fever virus noncoding regions of L, M and S segments regulate RNA synthesis. Virology 351:170-179.[Medline]
    20. 20
  20. Gomez-Puertas, P., C. Albo, E. Perez-Pastrana, A. Vivo, and A. Portela. 2000. Influenza virus matrix protein is the major driving force in virus budding. J. Virol. 74:11538-11547.[Abstract/Free Full Text]
    21. 21
  21. Hsieh, P. K., S. C. Chang, C. C. Huang, T. T. Lee, C. W. Hsiao, Y. H. Kou, I. Y. Chen, C. K. Chang, T. H. Huang, and M. F. Chang. 2005. Assembly of severe acute respiratory syndrome coronavirus RNA packaging signal into virus-like particles is nucleocapsid dependent. J. Virol. 79:13848-13855.[Abstract/Free Full Text]
    22. 22
  22. Ikegami, T., C. J. Peters, and S. Makino. 2005. Rift Valley fever virus nonstructural protein NSs promotes viral RNA replication and transcription in a minigenome system. J. Virol. 79:5606-5615.[Abstract/Free Full Text]
    23. 23
  23. Ikegami, T., S. Won, C. J. Peters, and S. Makino. 2006. Rescue of infectious Rift Valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene. J. Virol. 80:2933-2940.[Abstract/Free Full Text]
    24. 24
  24. Jasenosky, L. D., G. Neumann, I. Lukashevich, and Y. Kawaoka. 2001. Ebola virus VP40-induced particle formation and association with the lipid bilayer. J. Virol. 75:5205-5214.[Abstract/Free Full Text]
    25. 25
  25. Justice, P. A., W. Sun, Y. Li, Z. Ye, P. R. Grigera, and R. R. Wagner. 1995. Membrane vesiculation function and exocytosis of wild-type and mutant matrix proteins of vesicular stomatitis virus. J. Virol. 69:3156-3160.[Abstract]
    26. 26
  26. Kohl, A., A. C. Lowen, V. H. Leonard, and R. M. Elliott. 2006. Genetic elements regulating packaging of the Bunyamwera orthobunyavirus genome. J. Gen. Virol. 87:177-187.[Abstract/Free Full Text]
    27. 27
  27. Kuismanen, E. 1984. Posttranslational processing of Uukuniemi virus glycoproteins G1 and G2. J. Virol. 51:806-812.[Abstract/Free Full Text]
    28. 28
  28. Kuismanen, E., B. Bang, M. Hurme, and R. F. Pettersson. 1984. Uukuniemi virus maturation: immunofluorescence microscopy with monoclonal glycoprotein-specific antibodies. J. Virol. 51:137-146.[Medline]
    29. 29
  29. Kuismanen, E., K. Hedman, J. Saraste, and R. F. Pettersson. 1982. Uukuniemi virus maturation: accumulation of virus particles and viral antigens in the Golgi complex. Mol. Cell. Biol. 2:1444-1458.[Abstract/Free Full Text]
    30. 30
  30. Latham, T., and J. M. Galarza. 2001. Formation of wild-type and chimeric influenza virus-like particles following simultaneous expression of only four structural proteins. J. Virol. 75:6154-6165.[Abstract/Free Full Text]
    31. 31
  31. Lee, K. J., M. Perez, D. D. Pinschewer, and J. C. de la Torre. 2002. Identification of the lymphocytic choriomeningitis virus (LCMV) proteins required to rescue LCMV RNA analogs into LCMV-like particles. J. Virol. 76:6393-6397.[Abstract/Free Full Text]
    32. 32
  32. Li, P. P., A. Naknanishi, M. A. Tran, K. Ishizu, M. Kawano, M. Phillips, H. Handa, R. C. Liddington, and H. Kasamatsu. 2003. Importance of Vp1 calcium-binding residues in assembly, cell entry, and nuclear entry of simian virus 40. J. Virol. 77:7527-7538.[Abstract/Free Full Text]
    33. 33
  33. Licata, J. M., R. F. Johnson, Z. Han, and R. N. Harty. 2004. Contribution of Ebola virus glycoprotein, nucleoprotein, and VP24 to budding of VP40 virus-like particles. J. Virol. 78:7344-7351.[Abstract/Free Full Text]
    34. 34
  34. Lopez, N., R. Muller, C. Prehaud, and M. Bouloy. 1995. The L protein of Rift Valley fever virus can rescue viral ribonucleoproteins and transcribe synthetic genome-like RNA molecules. J. Virol. 69:3972-3979.[Abstract]
    35. 35
  35. Lowen, A. C., C. Noonan, A. McLees, and R. M. Elliott. 2004. Efficient bunyavirus rescue from cloned cDNA. Virology 330:493-500.[CrossRef][Medline]
    36. 36
  36. Mebatsion, T., M. Konig, and K. K. Conzelmann. 1996. Budding of rabies virus particles in the absence of the spike glycoprotein. Cell 84:941-951.[CrossRef][Medline]
    37. 37
  37. Melin, L., R. Persson, A. Andersson, A. Bergstrom, R. Ronnholm, and R. F. Pettersson. 1995. The membrane glycoprotein G1 of Uukuniemi virus contains a signal for localization to the Golgi complex. Virus Res. 36:49-66.[CrossRef][Medline]
    38. 38
  38. Muriaux, D., S. Costes, K. Nagashima, J. Mirro, E. Cho, S. Lockett, and A. Rein. 2004. Role of murine leukemia virus nucleocapsid protein in virus assembly. J. Virol. 78:12378-12385.[Abstract/Free Full Text]
    39. 39
  39. Noda, T., H. Sagara, E. Suzuki, A. Takada, H. Kida, and Y. Kawaoka. 2002. Ebola virus VP40 drives the formation of virus-like filamentous particles along with GP. J. Virol. 76:4855-4865.[Abstract/Free Full Text]
    40. 40
  40. Persson, R., and R. F. Pettersson. 1991. Formation and intracellular transport of a heterodimeric viral spike protein complex. J. Cell Biol. 112:257-266.[Abstract/Free Full Text]
    41. 41
  41. Pettersson, R. F. 1974. Effect of Uukuniemi virus infection on host cell macromolecule synthesis. Med. Biol. 52:90-97.[Medline]
    42. 42
  42. Popov, V., X. Yu, and D. Walker. 2000. The 120 kDa outer membrane protein of Ehrlichia chaffeensis: preferential expression on dense-core cells and gene expression in Escherichia coli associated with attachment and entry. Microb. Pathog. 28:71-80.[CrossRef][Medline]
    43. 43
  43. Ronnholm, R. 1992. Localization to the Golgi complex of Uukuniemi virus glycoproteins G1 and G2 expressed from cloned cDNAs. J. Virol. 66:4525-4531.[Abstract/Free Full Text]
    44. 44
  44. Ronnholm, R., and R. F. Pettersson. 1987. Complete nucleotide sequence of the M RNA segment of Uukuniemi virus encoding the membrane glycoproteins G1 and G2. Virology 160:191-202.[CrossRef][Medline]
    45. 45
  45. Schmitt, A. P., G. P. Leser, D. L. Waning, and R. A. Lamb. 2002. Requirements for budding of paramyxovirus simian virus 5 virus-like particles. J. Virol. 76:3952-3964.[Abstract/Free Full Text]
    46. 46
  46. Simons, J. F., U. Hellman, and R. F. Pettersson. 1990. Uukuniemi virus S RNA segment: ambisense coding strategy, packaging of complementary strands into virions, and homology to members of the genus Phlebovirus. J. Virol. 64:247-255.[Abstract/Free Full Text]
    47. 47
  47. Simons, J. F., R. Persson, and R. F. Pettersson. 1992. Association of the nonstructural protein NSs of Uukuniemi virus with the 40S ribosomal subunit. J. Virol. 66:4233-4241.[Abstract/Free Full Text]
    48. 48
  48. Strecker, T., R. Eichler, J. Meulen, W. Weissenhorn, H. Dieter Klenk, W. Garten, and O. Lenz. 2003. Lassa virus Z protein is a matrix protein and sufficient for the release of virus-like particles. J. Virol. 77:10700-10705. (Erratum, 77:12927.)[Abstract/Free Full Text]
    49. 49
  49. Suomalainen, M., P. Liljestrom, and H. Garoff. 1992. Spike protein-nucleocapsid interactions drive the budding of alphaviruses. J. Virol. 66:4737-4747.[Abstract/Free Full Text]
    50. 50
  50. Swenson, D. L., K. L. Warfield, K. Kuehl, T. Larsen, M. C. Hevey, A. Schmaljohn, S. Bavari, and M. J. Aman. 2004. Generation of Marburg virus-like particles by co-expression of glycoprotein and matrix protein. FEMS Immunol. Med. Microbiol. 40:27-31.[CrossRef][Medline]
    51. 51
  51. Vennema, H., G. J. Godeke, J. W. Rossen, W. F. Voorhout, M. C. Horzinek, D. J. Opstelten, and P. J. Rottier. 1996. Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes. EMBO J. 15:2020-2028.[Medline]
    52. 52
  52. von Bonsdorff, C. H., and R. Pettersson. 1975. Surface structure of Uukuniemi virus. J. Virol. 16:1296-1307.[Abstract/Free Full Text]
    53. 53
  53. Walpita, P., and R. Flick. 2005. Reverse genetics of negative-stranded RNA viruses: a global perspective. FEMS Microbiol. Lett. 244:9-18.[CrossRef][Medline]
    54. 54
  54. Watanabe, S., T. Watanabe, T. Noda, A. Takada, H. Feldmann, L. D. Jasenosky, and Y. Kawaoka. 2004. Production of novel Ebola virus-like particles from cDNAs: an alternative to Ebola virus generation by reverse genetics. J. Virol. 78:999-1005.[Abstract/Free Full Text]
    55. 55
  55. Watanabe, T., S. Watanabe, G. Neumann, H. Kida, and Y. Kawaoka. 2002. Immunogenicity and protective efficacy of replication-incompetent influenza virus-like particles. J. Virol. 76:767-773.[Abstract/Free Full Text]
    56. 56
  56. Ye, Z., T. Liu, D. P. Offringa, J. McInnis, and R. A. Levandowski. 1999. Association of influenza virus matrix protein with ribonucleoproteins. J. Virol. 73:7467-7473.[Abstract/Free Full Text]

Journal of Virology, November 2006, p. 10428-10435, Vol. 80, No. 21
0022-538X/06/$08.00+0     doi:10.1128/JVI.01362-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Huiskonen, J. T., Overby, A. K., Weber, F., Grunewald, K. (2009). Electron Cryo-Microscopy and Single-Particle Averaging of Rift Valley Fever Virus: Evidence for GN-GC Glycoprotein Heterodimers. J. Virol. 83: 3762-3769 [Abstract] [Full Text]  
  • Overby, A. K., Pettersson, R. F., Grunewald, K., Huiskonen, J. T. (2008). Insights into bunyavirus architecture from electron cryotomography of Uukuniemi virus. Proc. Natl. Acad. Sci. USA 105: 2375-2379 [Abstract] [Full Text]  
  • Ogawa, Y., Sugiura, K., Kato, K., Tohya, Y., Akashi, H. (2007). Rescue of Akabane virus (family Bunyaviridae) entirely from cloned cDNAs by using RNA polymerase I. J. Gen. Virol. 88: 3385-3390 [Abstract] [Full Text]  
  • Overby, A. K., Popov, V. L., Pettersson, R. F., Neve, E. P. A. (2007). The Cytoplasmic Tails of Uukuniemi Virus (Bunyaviridae) GN and GC Glycoproteins Are Important for Intracellular Targeting and the Budding of Virus-Like Particles. J. Virol. 81: 11381-11391 [Abstract] [Full Text]  
  • Overby, A. K., Pettersson, R. F., Neve, E. P. A. (2007). The Glycoprotein Cytoplasmic Tail of Uukuniemi Virus (Bunyaviridae) Interacts with Ribonucleoproteins and Is Critical for Genome Packaging. J. Virol. 81: 3198-3205 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Överby, A. K.
Right arrow Articles by Pettersson, R. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Överby, A. K.
Right arrow Articles by Pettersson, R. F.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»