Efficient production of Rift Valley fever virus-like particles - Core

Virology 385 (2009) 400–408

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Virology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y v i r o

Efficient production of Rift Valley fever virus-like particles: The antiviral protein MxA can inhibit primary transcription of bunyaviruses Matthias Habjan a,b, Nicola Penski a, Valentina Wagner a, Martin Spiegel a,1, Anna K. Överby a, Georg Kochs a, Juha T. Huiskonen c,d, Friedemann Weber a,b,⁎ a

Department of Virology, University of Freiburg, D-79008 Freiburg, Germany Centre for Biological Signalling Studies (bioss), Albert-Ludwigs-Universität Freiburg, Germany Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany d Institute of Biotechnology, University of Helsinki, P.O. Box 65, FI-00014, Helsinki, Finland b c

a r t i c l e

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Article history: Received 24 September 2008 Returned to author for revision 30 October 2008 Accepted 8 December 2008 Available online 19 January 2009 Keywords: Rift Valley fever virus Bunyavirus Phlebovirus Reverse genetics Minireplicon system Virus-like particles Primary transcription Interferon MxA protein Antiviral mechanism

a b s t r a c t Rift Valley fever virus (RVFV) is a highly pathogenic member of the family Bunyaviridae that needs to be handled under biosafety level (BSL) 3 conditions. Here, we describe reverse genetics systems to measure RVFV polymerase activity in mammalian cells and to generate virus-like particles (VLPs). Recombinant polymerase (L) and nucleocapsid protein (N), expressed together with a minireplicon RNA, formed transcriptionally active nucleocapsids. These could be packaged into VLPs by additional expression of viral glycoproteins. The VLPs resembled authentic virus particles and were able to infect new cells. After infection, VLP-associated nucleocapsids autonomously performed primary transcription, and co-expression of L and N in VLP-infected cells allowed subsequent replication and secondary transcription. Bunyaviruses are potently inhibited by a human interferon-induced protein, MxA. However, the affected step in the infection cycle is not entirely characterized. Using the VLP system, we demonstrate that MxA inhibits both primary and secondary transcriptions of RVFV. A set of infection assays distinguishing between virus attachment, entry, and subsequent RNA synthesis confirmed that MxA is able to target immediate early RNA synthesis of incoming RVFV particles. Thus, our reverse genetics systems are useful for dissecting individual steps of RVFV infection under non-BSL3 conditions. © 2008 Elsevier Inc. All rights reserved.

Introduction Rift Valley fever virus (RVFV) is a serious emerging pathogen affecting humans and livestock in sub-Saharan Africa, Egypt, Yemen, and Saudi Arabia. Since the first description of an outbreak in Kenya in 1931 (Daubney et al., 1931), there have been recurrent epidemics, killing thousands of animals, hundreds of humans, and causing significant economic losses (Balkhy and Memish, 2003). To animals, RVFV is mainly transmitted by mosquitoes, but humans are often infected by close contact with infected animal material (Morrill and McClain, 1996). A wide array of symptoms is connected with the human disease, ranging from an uncomplicated acute febrile illness to retinitis, hepatitis, meningoencephalitis, severe hemorrhagic disease, and death (Balkhy and Memish, 2003). The severity of RVFV zoonosis as well as the capability to cause major epidemics in livestock and

⁎ Corresponding author. Fax: +49 761 203 6634. E-mail address: [email protected] (F. Weber). 1 Present address: Institute for Virology, University Medicine Goettingen, D-37075 Goettingen, Germany. 0042-6822/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2008.12.011

humans has prompted authorities to list RVFV as a notifiable disease and a potential biological weapon (Borio et al., 2002). RVFV belongs to the genus Phlebovirus, family Bunyaviridae (Elliott, 1997). Bunyaviruses are enveloped and have a tri-segmented single-strand RNA genome of negative or ambisense polarity. They replicate in the cytoplasm and bud into the Golgi apparatus. RVFV encodes five structural proteins: the viral polymerase (L) on the large (L) segment, two glycoproteins (Gn and Gc) and the 78 kDa protein on the medium (M) segment, and the viral nucleocapsid protein (N) on the smallest (S) segment (Struthers et al., 1984). RVFV also expresses two nonstructural proteins, encoded on the M segment (termed NSm) and the S segment (termed NSs). These accessory proteins are dispensable for viral multiplication in cell culture (Gerrard et al., 2007; Vialat et al., 2000; Won et al., 2006), but play important roles for pathogenesis in vivo. NSm was found to suppress virus-induced apoptosis (Won et al., 2007) and to enhance intrahost viral spread (Bird et al., 2007a), whereas NSs is important to suppress the antiviral interferon system (Billecocq et al., 2004; Bouloy et al., 2001; Le May et al., 2004, 2008). The general features of RVFV transcription and replication are similar to those of other negative-stranded RNA viruses (Elliott, 1996).

M. Habjan et al. / Virology 385 (2009) 400–408

The viral genomic RNA (vRNA) segments contain untranslated regions (UTRs) on both the 3′ and the 5′ ends that serve as promoters for replication of the segment and for transcription of the encoded reading frames. The vRNAs are encapsidated by N protein and associate with L protein to form ribonucleoprotein particles (RNPs). The RNPs are functional templates for mRNA synthesis and RNA replication by the viral polymerase. After glycoprotein-mediated entry into the cytoplasm, the negative-sense vRNAs of RVFV are transcribed into mRNAs by the RNPassociated L and N proteins (“primary transcription”). The products of these immediate early mRNAs then provide the machinery for replication of the genome and further mRNA synthesis (“secondary transcription”), and later drive the packaging of newly assembled RNPs into budding particles. A specific feature of bunyaviruses is that their mRNA synthesis is strictly dependent on host translation (Bellocq and Kolakofsky, 1987; Ikegami et al., 2005b; Vialat and Bouloy, 1992). Most likely, ongoing translation prevents premature termination of transcription by destroying inhibitory secondary structures which build up on the nascent mRNA (Barr, 2007). The infection cycle of bunyaviruses is strongly inhibited by the human interferon-induced protein MxA (Frese et al., 1996). MxA is a cytoplasmic, large dynamin-like GTPase with antiviral activity against a wide range of RNA viruses (Haller and Kochs, 2002; Haller et al., 2007). The antiviral power of MxA against bunyaviruses is best demonstrated by the protection of MxA transgenic mice from a lethal dose of La Crosse bunyavirus (LACV) (Hefti et al., 1999). A functional GTP binding activity is crucial for the antiviral activity of MxA, and biochemical analysis suggested a physical interaction of newly synthesized viral N protein with GTP-bound MxA (Kochs et al., 2002). This interaction resulted in the formation of large, oligomeric MxA/N copolymers that accumulate in virus-infected cells (Kochs et al., 2002). The current model is that depletion of free N by MxA cuts viral RNPs off the N supply, thus leading to a strong inhibition of bunyaviral RNA replication and secondary transcription (Reichelt et al., 2004). However, the question whether primary transcription of bunyaviruses is also affected, as was described for vesicular stomatitis virus and the Thogoto-Orthomyxovirus (Kochs and Haller, 1999; Pavlovic et al., 1992), is still unsolved. This lack of data is caused by the experimental hurdle that bunyavirus transcription is blocked by translation inhibitors, the traditional means to separate primary transcription from later steps. Reverse genetics systems to rescue infectious RVFV entirely from cloned cDNA plasmids have been developed (Billecocq et al., 2008; Gerrard et al., 2007; Habjan et al., 2008; Ikegami et al., 2006). These systems allow the targeted manipulation of the viral genome and proved to be extremely useful to demonstrate the contribution of NSs and the NSm proteins to viral pathogenesis (Bird et al., 2007a; Ikegami et al., 2006) and to identify transcriptional termination signals (Albarino et al., 2007; Ikegami et al., 2007). However, research with these recombinant RVFV systems is hampered and restricted since the pathogen can only be handled under biosafety level 3 (BSL3) conditions. Therefore, it would be desirable to have tools at hand which allow to study aspects of RVFV infection cycle and the host response under non-BSL3 conditions. Here, we report the establishment of a RVFV reverse genetics system which recapitulates individual steps of the replication cycle in a modular manner. A minireplicon system was developed to measure RVFV polymerase activity. Additional expression of viral glycoproteins allowed efficient and helper-free packaging of minireplicon RNPs into virus-like particles (VLPs) which were released into the medium. By incubating cells with VLPs, we were able to reconstitute the first steps of RVFV infection. Importantly, our system allowed us to demonstrate that the L polymerase complexed with incoming RNPs is highly active in primary transcription. Furthermore, if both L and the nucleoprotein N are additionally provided in trans, RNPs are active in replication as well. Using this modular approach we demonstrate that the human

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MxA protein is able to interfere with viral primary transcription, thus blocking RVFV early in infection. Results and discussion Establishment of a RVFV minireplicon system Minireplicon systems are useful tools to investigate viral polymerase activity and promoter sequences. Bunyavirus minireplicon systems usually consist of a reporter gene flanked by viral 5′ and 3′ UTRs, and recombinant L and N proteins (Accardi et al., 2001; Blakqori et al., 2003; Dunn et al., 1995; Gauliard et al., 2006; Ikegami et al., 2005a; Weber et al., 2001). The reporter construct is transfected into mammalian cells along with the L and N expression constructs and will be transcribed intracellularly into a minireplicon resembling a viral gene segment. The products of the expression constructs encapsidate, transcribe and replicate the minireplicon, thus resulting in reporter activity. For RVFV, minireplicon systems based on the coexpressed RNA polymerase of the bacteriophage T7 have been established earlier (Accardi et al., 2001; Gauliard et al., 2006; Ikegami et al., 2005a). We decided to generate a T7-independent version in which the expression constructs were under control of a promoter for the cellular RNA polymerase II (RNAP II), and the minireplicon was transcribed intracellularly by RNAP I. The L and N genes were expressed by the mammalian expression vector pI.18. A minireplicon consisting of the antisense gene for the Renilla Luciferase (Ren-Luc) flanked by RVFV M segment UTRs was cloned into the RNAP I vector pHH21 (Neumann et al., 1999). Human 293T cells were transfected with these plasmids, and minireplicon activity was assessed by measuring Ren-Luc activity of cell extracts after overnight incubation. Fig. 1 shows that coexpression of L, N and the minireplicon upregulated the Ren-Luc activity (black bars), whereas omission of the L construct resulted in low activity. As the minireplicon plasmid was designed to produce negative sense RNA, Ren-Luc activity clearly indicates successful packaging of RNA into RNPs and transcription by L and N. Moreover, the expression of an internal control, consisting of the RNAP II-driven gene for the firefly luciferase (FF-Luc), was independent of the full set of RVFV plasmids (Fig. 1, grey bars), as expected. Thus, our RVFV minireplicon system which is entirely based on the activity of cellular RNA polymerases was able to efficiently reconstitute recombinant RVFV RNPs.

Fig. 1. Minireplicon system for RVFV. Human 293T cells were transfected with expression plasmids for the nucleocapsid protein (N) and the viral polymerase (L), together with a minireplicon construct containing the Ren-Luc reporter gene in antisense flanked by the untranslated regions of the M segment (vM-Ren). As a negative control the L expression plasmid was omitted. An FF-Luc plasmid was cotransfected to control transfection efficiency. Cell lysates were assayed 24 h post transfection for RenLuc and FF-Luc activity. Luciferase counts were normalized to the negative control, numbers on top of each column indicate fold induction. Absolute luciferase values were in the range of 105 and 106 relative light units for Ren-Luc and FF-Luc, respectively (data not shown). Results from a representative experiment are shown.

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Generation of VLPs for RVFV VLP systems consisting of minireplicon RNPs packaged by viral glycoproteins have been established for two bunyaviruses, Bunyamwera virus and Uukuniemi virus. These systems have proven useful for the analysis of particle assembly and RNP packaging (Overby et al., 2007a, 2006, 2007b; Shi et al., 2006). To generate VLPs of RVFV, we transfected an M expression construct (encoding Gn, Gc, NSm, and the 78 kDa protein) together with the minireplicon system plasmids into 293T cells (donor cells). Twenty-four hours later, supernatants were harvested and used to infect BSR-T7/5 cells expressing RVFV L and N (indicator cells). An outline of the experiment is provided in Fig. 2A. Two additional measures were taken to minimize unspecific reporter activities in indicator cells caused by carryover transfection. Firstly, any residual plasmids in the VLP supernatants were destroyed by extensive treatment with DNase. Secondly, the species-specificity of the human RNAP I-driven minireplicon construct excluded background expression in the non-human indicator cells in the possible case of unwanted transfection. Moreover, RNAP II-driven coexpression of FF-Luc in donor cells served as a specificity control, because the FFLuc RNA lacks any RVFV sequences and should therefore not be packaged into VLPs. Reporter assays demonstrated the specific minireplicon activity mediated by L and N (Fig. 2B, left panel). Coexpression of the M segment-encoded glycoproteins had no detrimental effects on reporter activity. Importantly, only the supernatant of cells that had been co-transfected with the M expression construct produced a strong Ren-Luc activity in indicator cells (Fig. 2B,

right panel). Transfer of FF-Luc activity, in contrast, was negligible. These data imply that coexpression of the M segment-encoded genes results in efficient and specific packaging of minireplicon RNPs into VLPs, which are subsequently released into the medium and are able to infect new cells. RVFV VLPs resemble authentic virions To further characterize the VLPs, we analyzed their immunological characteristics, protein composition and morphology. Firstly, VLPcontaining supernatants were incubated with different antisera derived from RVFV-infected mice or humans, and then used to infect indicator cells expressing RVFV L and N. A strong reduction in Ren-Luc activity occurred in indicator cells when RVFV-specific antisera were used (Fig. 3A). In contrast, a control antibody directed against the LACV Gc envelope protein as well as sera from an uninfected mouse or individual had no influence on the reporter activity. These results indicate that the VLPs were specifically neutralized by RVFV-specific antisera. Secondly, we determined the protein composition of VLPs. Supernatants from 293T cells transfected with different plasmid combinations were concentrated by ultracentrifugation and subjected to Western blot analysis. Supernatants from RVFV-infected cells served as positive control (Fig. 3B, lane 1). As expected, there was no release of viral proteins from minireplicon-expressing cells lacking glycoprotein expression (Fig. 3B, lane 3). However, when the full set of plasmids for the VLP system had been transfected, both the viral Gn

Fig. 2. Formation of RVFV VLPs. (A) Outline of the procedure used to generate VLPs. Donor cells were transfected with expression plasmids for N, L and the viral glycoproteins (M), together with the reporter minireplicon construct vM-Ren. Release of VLPs into the supernatant of donor cells was detected by transfer of supernatants onto indicator cells expressing both N and L. (B) Luciferase activities in donor and indicator cells. 293T cells (donor cells) were transfected with minireplicon system plasmids alone as described in Fig. 1 (left panel, columns 1 and 2), or together with an RNAP II-driven M expression plasmid (left panel, column 3). Supernatants (1 ml) were collected 24 h later and used to infect BSR-T7/5 indicator cells (right panel). FF-Luc and Ren-Luc activities in donor cells were measured after removal of supernatants, and indicator cells were analyzed 24 h post infection. Luciferase counts were normalized to the experiment where L was omitted, and numbers on top of each column display fold induction. Results from a representative experiment are shown.


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and N protein could be detected in the supernatants (Fig. 3B, lane 2). This indicates that the composition of VLPs is similar to that of RVFV particles. We noted, however, that the relative proportion of the 78 kDa protein (which is recognized by the monoclonal antibody against Gn) was substantially higher in the VLPs than in the authentic virus particles. This might be due to space restrictions inside the RVFV particles compared to the single-segmented VLPs, since the 78 kDa protein probably contains an additional cytoplasmic tail compared to Gn (Gerrard and Nichol, 2007). Another possibility is that the expression of the M segment-encoded proteins and their incorporation into particles differs slightly between plasmid-transfected and virus-infected cells. Notably, we were unable to detect release of glycoproteins in the absence of a co-transfected minireplicon system (data not shown). This is in contrast to results obtained with the VLP system of the related Phlebovirus Uukuniemi for which it was shown that overexpressed glycoproteins are sufficient to form VLPs that are released into the supernatant (Overby et al., 2007a; Overby et al., 2006). Apparently, RVFV requires additional structural components for assembly and release of VLPs. Finally, we compared the structure of VLPs and RVFV particles using negative stain transmission electron microscopy (TEM). Supernatants with VLPs or RVFV particles were concentrated by ultracentrifugation, fixed with glutaraldehyde at pH 7.4 and stained with uranyl acetate. The negative stain pictures shown in Fig. 3C demonstrate that VLPs resemble RVFV particles in size and morphology. Together, these data indicate that the VLPs which we generated by coexpression of the minireplicon system and the M-encoded glycoproteins can serve as a model for authentic RVFV particles. Optimization of VLP production

Fig. 3. VLPs resemble authentic RVFV particles. (A). Neutralization of VLPs by RVFVspecific antisera. VLP-containing supernatants (100 μl) were incubated with 5 μl of mouse or human antisera specific for RVFV for 1 h at 37 °C. As controls, 5 μl of serum from a non-seroconverted mouse and human (ctrl) or 5 μl of a monoclonal antibody against La Crosse bunyavirus glycoprotein Gc (anti-LACV MAb) were used. RVFV N and L-expressing indicator cells were then infected with antibody-treated or untreated VLP preparations, or the supernatant from minireplicon-expressing cells (SN MR). Ren-Luc activity was measured 24 h later and is shown as percent activity of untreated VLPs. Mean values and standard deviations were calculated from three independent experiments. (B) Western blot analysis of RVFV particles and VLPs. Concentrated supernatants from RVFV Clone 13-infected cells (lane 1) and 293T cells transfected with different plasmid combinations (lanes 2 to 4) were analyzed by Western blotting using a monoclonal antibody specific for RVFV Gn and 78 kDa protein (upper panel), and an antibody against the nucleoprotein N (lower panel). (C) Negative staining TEM of concentrated VLPs and RVFV particles (2 examples shown for each) fixed with glutaraldehyde and stained with uranyl acetate. Scale bar represents 50 nm.

We performed a time-course analysis to study the kinetics of VLP formation (Fig. 4). Donor cells were transfected with either the minireplicon alone or together with the M expression construct used to generate VLPs. Cell lysates and supernatants were harvested from parallel dishes after 24, 48 or 72 h. Reporter assays of donor cell lysates showed again that the addition of the M segment expression plasmid had no major effect on minireplicon activity (Fig. 4A, columns 2 and 3; see also Fig. 2B, left panel). Both the minireplicon and the VLP system were strongly active until 48 h post-transfection, and displayed a sharp decrease at the 72 h time point. This indicates robust and continual production of recombinant RVFV components and, most probably, exhaustion after longer expression times. When supernatants of the time course were used to infect indicator cells, reporter assays confirmed that the M segment expression plasmid was necessary to generate infectious VLPs (Fig. 4B). Moreover, VLP activities steadily increased in the supernatants harvested from 24 h to 72 h after the transfection of donor cells. The apparent discrepancy between donor cells (peak at 48 h time point) and indicator cells (steady increase until 72 h) may simply be due to the fact that VLPs are relatively stable and accumulate in the supernatants over the whole time course, whereas gene expression in donor cells fades out. Phlebovirus particles are indeed known to be remarkably stable under various conditions (Hardestam et al., 2007). To determine the stability of our RVFV VLPs, we incubated them for 3 days at 37 °C and compared their reporter activity to VLP preparations which were either kept at −80 °C or incubated for 10 min at 95 °C. The experiments showed that the RVFV VLPs lose on average only about 20% of their activity after 3 days at 37 °C (Fig. 4C). This indicates that, during the time course of VLP production, the high activity measured in the indicator cells at 72 h post-transfection (see Fig. 4B) is most likely due to an accumulation of environmentally stable VLPs. To estimate VLP yields in terms of infectious units, we constructed a GFP minireplicon plasmid and produced VLPs expressing GFP (GFP-

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per ml. As a clearly detectable GFP signal in the indicator cells depends on their successful transfection with L and N plasmids, these numbers most likely represent an underestimate. RVFV infections typically yield titers between 106 and 108 infectious particles per ml (Billecocq et al., 2008; Gerrard et al., 2007; Habjan et al., 2008). Thus, although the VLPs are unable to undergo several cycles of infection, their plasmid-based production is only 10- to 100-fold less efficient compared to infectious RVFV particles. Dissection of RVFV primary and secondary transcriptions using the VLP system Conventionally, primary transcription of negative-strand RNA viruses is measured by using translational inhibitors, e.g. cycloheximide. Cutting off the supply of newly synthesized viral proteins allows to measure the activity of incoming polymerase complexes without the amplification loop established by genome replication. However, the fact that bunyavirus mRNA transcription is strictly dependent on concurrent translation (Ikegami et al., 2005b; Patterson and Kolakofsky, 1984; Vialat and Bouloy, 1992) precludes the dissection of primary and secondary transcriptions by pharmaceutical means. We investigated whether our VLP system allows the measurement of RVFV primary transcription. BSR-T7/5 cells were infected with VLPs and monitored by measuring Ren-Luc over a period of 48 h. To distinguish between primary and secondary transcriptions of the VLPs, the indicator cells were either left untransfected or were transfected with constructs for the polymerase complex proteins N and L prior to infection, respectively (Fig. 5). In parallel, supernatants

Fig. 4. Optimization of VLP production and VLP stability. VLPs were generated as described in Fig. 2, and VLP release from donor cells was followed over the course of three days. Supernatants from parallel dishes were harvested at the time points indicated and used to infect indicator cells expressing N and L. (A) Ren-Luc and FF-Luc activities in donor cells were measured upon removal of supernatants. (B) Analysis of luciferase activities in indicator cells was performed 24 h post-infection. Luciferase counts were normalized to the controls where L was omitted. Data from a representative experiment are shown, numbers on top of each column displaying fold induction. (C) Stability of RVFV VLPs at 37 °C. VLP-containing supernatants (100 μl) were incubated for 3 days at 37 °C or for 10 min at 95 °C and compared with a preparation of freshly thawed VLPs which had been stored at −80 °C. RVFV L- and Nexpressing indicator cells were infected with VLP preparations, or with the supernatant from minireplicon-expressing cells (SN MR). Ren-Luc activity was measured 24 h later and is shown as percent activity of the VLPs stored at −80 °C. Mean values and standard deviations were calculated from three independent experiments.

VLPs). Supernatants of donor cells harvested at 72 h after transfection were used to infect BSR-T7/5 cells which had been previously transfected with L and N constructs. These indicator cells were infected with ten-fold serial dilutions of the GFP-VLP-containing supernatants. After overnight incubation, GFP-positive indicator cells were counted by fluorescence microscopy and titers were calculated accordingly. Table 1 shows that 3 independent repetitions of this experiment yield titers of approximately 106 infectious particles

Table 1 Titers of RVFV VLPs expressing GFP Experimenta

VLP titer/mlb

#1 #2 #3

1.2 × 106 7.5 × 105 9.5 × 105

a BSR-T7/5 indicator cells expressing RVFV L and N were incubated overnight with 10-fold dilutions of supernatants containing GFP-VLPs. b VLP titers were calculated from the number of GFP-positive indicator cells and the respective dilution step.

Fig. 5. Primary and secondary transcriptions in VLP-infected cells. (A) BSR-T7/5 cells were either mock transfected (open squares), or transfected with N and L expression plasmids (filled squares) prior to infection with VLPs harbouring Ren-Luc RNPs. As a negative control, cells transfected with N and L plasmids were infected with supernatants from minireplicon-expressing donor cells in the absence of M expression plasmid (crosses). Ren-Luc activities in VLP-infected cells were then measured at different time points over a period of 48 h. (B) The same data is shown as in A, but with the y-axis adjusted to visualize activity of VLPs in mock transfected indicator cells. Mean values and standard deviations were calculated from three independent experiments.


from minireplicon-expressing cells were used to detect potential background reporter activity. As expected, we observed strong RenLuc activity in VLP-infected cells where polymerase complexes were present (Fig. 5A). This activity steadily increased until 48 h postinfection, the last time point measured, indicating continuous replication and transcription. Supernatants from minireplicon-only transfected cells, by contrast, did not elicit any detectable Ren-Luc activities as expected. Interestingly, a clear and reproducible Ren-Luc activity over background was also measured in VLP-infected indicator cells in the absence of additional N and L helper plasmids, albeit to a much lower extent than in the presence of these plasmids (Fig. 5B). Maximum activity was reached after 24 h and remained at an almost constant level for further 24 h. This indicates that the VLP-associated

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viral polymerase is active in primary transcription. The same observation was also made in several other cell lines infected with VLPs (data not shown). The established system thus enables the study of RVFV primary transcription. Effect of the antiviral protein MxA on RVFV transcription The VLP system developed in this study enabled us to investigate the effect of the antivirally active protein MxA on primary and secondary transcriptions of RVFV. Indicator cells were transfected with an expression construct for MxA and infected with RVFV VLPs. The MxA mutant T103A served as a negative control. Fig. 6A (upper part) shows that MxA expressed by indicator cells was capable of

Fig. 6. Inhibition of RVFV primary and secondary transcriptions by human MxA. (A) The influence of human MxA on RVFV primary transcription was analyzed by transfecting BSR-T7/ 5 cells with 1.5 μg of an expression plasmid for wt MxA or the inactive mutant MxA(T103A), respectively. Twenty-four hours post transfection, cells were infected with VLPs harbouring Ren-Luc RNPs. Ren-Luc activities in cell lysates were measured 24 h later and the values of the MxA(T103A) control were set to 100%. Mean values and standard deviations were calculated from three independent experiments. Equal amounts of lysates from a representative experiment were used for Western blot analysis to confirm expression of both MxA and RVFV N. (B) To study the effect of MxA on RVFV replication and secondary transcription, 1.5 μg of plasmid for wt MxA or MxA(T103A) were cotransfected with 0.5 μg of each N and L expression plasmids prior to VLP treatment. Reporter assays and Western blot analysis were performed as indicated. (C) Stage of the virus replication cycle at which MxA exerts its antiviral effect on RVFV. For the attachment assay, cells stably transfected with an MxA-expression plasmid (MxA) or an empty vector (CTRL) were inoculated with RVFV (MOI 10) for 1 h on ice, washed with PBS, and then the total RNA was extracted. For the entry and the RNA synthesis assays, cells were also infected on ice for 1 h, but prewarmed medium was added after the virus inoculum was removed. The cells were incubated for another 2 h (entry) or 5 h (RNA synthesis), washed with PBS, and trypsinated for 10 min to remove any residual cell-attached virus before RNA extraction. RVFV RNA in the different samples was measured by real time RT-PCR and normalized to γ-actin mRNA. RNA from mock infected cells was used as control. Mean values and standard deviations from three independent experiments are shown (rel.u., relative units).

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reducing the reporter activity of incoming VLPs by at least 30%. This indicates a weak but clearly inhibitory effect on viral primary transcription. Moreover, if N and L support constructs were expressed in addition to MxA, a similar inhibitory effect of about 50% was observed (Fig. 6B, upper part). Western blot analyses for MxA and RVFV N were performed to control recombinant gene expression (Figs. 6A and B, lower parts). To further characterize the MxA-sensitive step in early RVFV infection, we established a set of assays which allows to differentiate between virus attachment, entry into the cytoplasm, and early RNA synthesis (Aizaki et al., 2008). For the attachment assay, control cells and cells stably expressing MxA (Frese et al., 1995) were infected with RVFV for 1 h on ice, washed, and the RNA extracted. In the entry and the RNA synthesis assays, cells were also infected for 1 h on ice and washed, but then pre-warmed cell culture medium was added and cells were incubated at 37 °C for another 2 h (entry) or 5 h (RNA synthesis). For these assays, any cell-associated virus particles which had not entered the cytoplasm were removed by trypsin treatment before the RNA was extracted. Subsequently, in all samples the presence of RVFV RNA was analyzed by real time RT-PCR (Bird et al., 2007b) and normalized to the cellular γ-actin mRNA. Fig. 6C shows that attachment of RVFV particles onto cells at the applied MOI of 10 results in an approximately 100,000-fold enrichment of viral RNA over γ-actin mRNA. This was independent of the presence of MxA. Similar observations were made for virus entry. However, if virus particles were allowed to initiate RNA synthesis, a 10-fold multiplication of viral RNA occurred only in the control cells, whereas in MxA-positive cells viral RNA levels did not increase after entry. Although this assay does not distinguish between transcription and replication, it shows that MxA efficiently inhibits early viral RNA synthesis. By contrast, receptor binding or entry into the cytoplasm are not affected. Together with the data obtained with the VLPs (see Figs. 6A and B), these results indicate that MxA is capable of interfering with primary transcription of bunyaviruses. Hence, MxA interacts not only with the soluble N protein to prevent RNA replication (Kochs et al., 2002), but also with the incoming, already assembled RNPs. This overall inhibition of RNP function eventually results in a strong reduction of RVFV replication (Frese et al., 1996). Our proposed mechanism is in line with the current model for the molecular action of MxA against RNA viruses (Haller et al., 2007). For Thogoto virus, a tick-transmitted Orthomyxovirus, it has been shown that MxA directly interacts with viral RNPs in the cytoplasm and prevents their translocation into the nucleus, the place of viral transcription and replication (Kochs and Haller, 1999; Weber et al., 2000). In accordance with our recent data on LACV (Reichelt et al., 2004), we speculate that this interaction occurs at the surface of smooth ER membranes where MxA accumulates. Membrane-bound MxA was shown to directly bind viral nucleoprotein and most likely RNPs, leading to copolymerization of these components into large aggregates and thereby inhibiting viral transcription. Accordingly, MxA might relocalize RNPs of RVFV to intracellular locations that are not favourable for viral transcription. Conclusions We have established reverse genetics tools which enable the study of RVFV RNP activity, virus assembly, cell infection, primary transcription and antiviral mechanisms under non-BSL3 conditions. The modular build-up of this system allows dissecting the viral replication cycle in a systematic manner. A system to produce VLPs via baculoviral expression of Gn, Gc, and N was recently published (Liu et al., 2008). These particles physically resemble RVFV virions and are able to induce cell–cell fusion after pH shift. Our VLPs, in contrast, are more similar to RVFV particles as they contain active RNPs, infect target cells, transcribe a gene of interest, and can be neutralized by

RVFV-specific antisera. Furthermore, as we describe in an accompanying vaccination paper (Näslund et al., 2009), the VLPs confer a high level of protection from lethal RVFV infection. It is hoped that the RVFV minireplicon and VLP systems might be instrumental for future research on this important pathogen. Materials and methods Chemicals, cells and viruses Geneticin G418 (Biochrom AG) was dissolved in H2O to 100 mg/ml and used at a concentration of 1 mg/ml cell culture medium. BSR-T7/5 cells (Buchholz et al., 1999), Vero cells and 293T cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS. BSR-T7/5 cells were additionally provided with G418 on every second passage. MxA expressing cell clones VA9 and the control cell line VN5 (Frese et al., 1995) were grown in DMEM with 5% serum supplemented with G418. RVFV strain Clone 13 was propagated in Vero cells. Antibodies The mouse monoclonal anti-LACV antibody #807-31 directed against the viral Gc protein was kindly provided by Samantha Soldan and Daniel Gonzales-Scarano, University of Pennsylvania (GonzalezScarano et al., 1982). The mouse control antiserum with specificity for influenza A virus strain PR/8/34 was kindly provided by Otto Haller. The human control serum was kindly provided by Ursula Meyer-König, Dept of Virology, Freiburg, and the human anti-RVFV sera #1, #2, and #3 were kindly obtained from Michele Bouloy (Institute Pasteur, Paris, France) and Patricia A. Nuttall (NERC Institute of Virology, Oxford, UK), respectively. The mouse monoclonal antibody 4D4 directed against the RVFV Gn and 78 kDa proteins was kindly provided by Jonathan F. Smith, (USAMRIID, Fort Detrick, USA). The mouse monoclonal antibody M143 for detection of human MxA was described previously (Flohr et al., 1999). Polyclonal mouse antisera recognizing RVFV N protein or RVFV particles were produced by inoculating mice with either recombinant N protein or RVFV strain Clone 13, respectively (A. Pichlmair and F. Weber, unpublished). Plasmids All plasmids were generated using standard molecular cloning techniques and confirmed by DNA sequencing. PCR was carried out with AccuPrime Pfx DNA Polymerase (Invitrogen; for cloning purposes) or Taq DNA Polymerase (Eppendorf; for diagnostic purposes). The constructs pI.18_RVFV_L, pI.18-RVFV_N and pI.18-RVFV_M containing the L, N and M segment coding sequences subcloned into the eukaryotic expression plasmid pI.18 have been described previously (Habjan et al., 2008). The reporter plasmid pHH21_RVFV_vMRen contains the Renilla luciferase gene (Ren-Luc) in antisense orientation, flanked by the 3′ and 5′ genomic untranslated regions (UTRs) of the RVFV M segment. This reporter plasmid was generated by cloning the Ren-Luc gene, amplified from plasmid pRL-SV40 (Promega) using specific oligonucleotides (sequences available on request), into pHH21_RVFV_vMPro (Habjan et al., 2008) via SapI restriction sites. Reporter plasmid pHH21_RVFV_vMGFP encoding the enhanced green fluorescent protein (EGFP) instead of Ren-Luc was constructed in a similar manner. Plasmid pGL3-control (Promega) expresses firefly luciferase (FF-Luc) under control of a mammalian RNA polymerase II promoter. Plasmid constructs encoding human MxA and the GTPase-deficient mutant T103A have been described previously (Ponten et al.,1997). For improved expression in mammalian cells we cloned the corresponding coding sequences into pI.18 via BamHI/EcoRI restriction sites, giving rise to plasmids pI.18-MxA and pI.18-HA-MxA(T103A).


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Subconfluent monolayers of 293T cells in 12-well plates were transfected with 0.4 μg each of pI.18_RVFV_N and pI.18_RVFV_L, 0.25 μg of pHH21_RVFV_vMRen, and 0.1 μg of pGL3-control using 3.5 μl Fugene HD transfection reagent (Roche) in 100 μl serum free medium (OptiMEM; Invitrogen). In some experiments, specific expression plasmids were omitted from the transfection mix. After transfection, cells were incubated for the indicated times and lysed in 100 μl Dual Luciferase Passive Lysis Buffer (Promega). An aliquot of 20 μl of the lysate was assayed for FF-Luc and Ren-Luc activities as described by the manufacturer (Promega).

The particles were concentrated by ultracentrifugation at 100,000 ×g and 4 °C for 2 h, and the pellet was dried for 5 min before resuspension in phosphate-buffered saline (PBS). Proteins were separated by SDSPAGE and transferred to a PVDF membrane (Amersham), followed by incubation in saturation buffer (PBS containing 10% non-fat dry milk and 0.05% Tween). The membrane was then incubated for one hour with primary antibodies, washed three times with 0.05% PBS-Tween, and incubated with a horseradish-peroxidase (HRP)-conjugated secondary antibody. After three additional washing steps, detection was performed using the SuperSignal West Femto chemiluminescence kit (Pierce). Primary antibody concentrations used were 1:1000 for mouse-α-RVFV N, 1:2000 for mAb M143 and 1:5000 for mAb 4D4.

Preparation of VLPs

Attachment, entry, and RNA synthesis assays

Subconfluent monolayers of 293T cells in 6-well plates (donor cells) were transfected with 0.5 μg of plasmids pI.18_RVFV_N, pI.18_RVFV_L, pI.18_RVFV_M, pHH21_RVFV_vMRen, and with 0.1 μg of pGL3-control using 6.3 μl Fugene HD transfection reagent. At the indicated time points, supernatants were harvested and donor cells were lysed with 200 μl Passive Lysis Buffer to measure luciferase activities as described above. The supernatants were treated with 1 μl/ml Benzonase (Novagen) at 37 °C for 3 h and centrifuged at 12,000 ×g for 5 min to remove cellular debris. Aliquots of the treated supernatants were then used to infect new cells (indicator cells). In some experiments, replication and transcription of RNPs was supported by transfecting indicator cells with N and L expression plasmids pI.18_RVFV_N and pI.18_RVFV_L (0.5 μg each) 16 h prior to the transfer of supernatant.

Vero cells stably transfected with an MxA expression plasmid (VA9) or an empty plasmid vector (VN5) (Frese et al., 1995) were seeded in six-well dishes at 80% confluency. Cells were infected with RVFV strain Clone 13 at an MOI of 10 for 1 h on ice to allow attachment but impede virus entry. After three washes with PBS, RNA was extracted to assay the amount of cell-bound virus. To assay the subsequent infection steps, virus inocula were removed after 1 h of binding on ice, cells were washed, pre-warmed medium was added, and the cells were incubated for another 2 h (entry) or 5 h (RNA synthesis) at 37 °C, respectively. Before RNA extraction, cells were once washed with PBS, trypsinated for 10 min, and again washed three times with PBS to remove any cell-associated virus which had not entered the cytoplasm.

Titration of VLPs

Real time RT-PCR

GFP-expressing VLPs were generated in 6-well plates as described above, using the minireplicon construct containing the GFP gene (pHH21_RVFV_vMGFP) in place of the Renilla luciferase gene. Supernatants containing VLPs were titrated on N and L expressing cells as follows. First, BSR-T7/5 cells grown in 12-well plates were transfected with 0.4 μg of each pI.18-RVFV-L and pI.18-RVFV-N using Nanofectin transfection reagent (PAA Laboratories), and incubated overnight. Then, cells were infected with ten-fold serial dilutions of GFP-VLPcontaining supernatants. Twenty-four hours post infection the number of GFP-positive cells was determined by fluorescence microscopy and titers were calculated accordingly. Western blot analysis confirmed that the amounts of GFP-VLPs and the Ren-VLPs used throughout the manuscript were comparable.

All real-time RT-PCR reactions were performed with a LightCycler 1.0 (Roche). One microgram of RNA was used to synthesize cDNA with the Quantitect reverse transcription kit (Qiagen) according to the manufacturer's recommendations. In the subsequent PCR reactions, the γ-actin mRNA was detected with QuantiTect primers QT00996415 and the QuantiTect SYBR Green RT-PCR Kit (Qiagen). RVFV RNA was detected as described by Bird et al. (2007b) using the QuantiFast Probe PCR Kit (Qiagen), and normalized to the γ-actin mRNA signal.

RVFV minireplicon system

Transmission electron microscopy (TEM) Supernatants containing Ren-VLPs or RVFV Clone 13 were collected and clarified by centrifugation (6000 ×g, 10 min, 4 °C), and pH 7.4 was set by adding phosphate buffer (final concentration of 50 μM). The particles were fixed with glutaraldehyde (Sigma; final concentration of 0.5%) and centrifuged through a 20% (wt/vol) sucrose cushion in TN buffer (50 mM Tris–HCl pH 7.4, and 100 mM NaCl) at 100,000 ×g for 2 h at 4 °C. The pellet was resuspended in TN buffer. For TEM, aliquots of VLP or virus suspension were pipetted on EM grids with a thin continuous carbon film, washed once with TN buffer, and stained for 1 min with 2% aqueous uranyl acetate. Images were recorded at a calibrated magnification of 68,000× using a Tecnai F20 transmission electron microscope equipped with an Eagle CCDcamera (FEI, Eindhoven, The Netherlands). Western blot analysis For detection of proteins in the supernatant of VLP-expressing or RVFV Clone 13-infected cells, supernatants were collected and clarified from cell debris by centrifugation (6000 ×g, 10 min at 4 °C).

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