Functional L polymerase of La Crosse virus allows in vivo reconstitution of recombinant nucleocapsids
Gjon Blakqori,
Georg Kochs,
Otto Haller and
Friedemann Weber
Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, D-79008 Freiburg, Germany
Correspondence
Friedemann Weber
fweber{at}ukl.uni-freiburg.de
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ABSTRACT
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La Crosse virus (LACV), a member of the family Bunyaviridae, is the primary cause of paediatric encephalitis in the United States. In this study, a functional RNA polymerase (L) gene of LACV was cloned and a reverse genetics system established. A reporter minireplicon mimicking the viral genome was constructed by flanking the Renilla luciferase gene with the 3' and 5' noncoding regions of the genomic M segment. These noncoding regions serve as promoters for the viral polymerase. Both L and nucleocapsid (N) genes were expressed by means of T7 RNA polymerase, which was provided by the recombinant T7-expressing modified vaccinia virus Ankara. Renilla reporter activity in transfected cells reflected reconstitution of recombinant nucleocapsids by functional L and N gene products. Time-course experiments revealed a rapid increase in minireplicon activity from 10 to 18 h after the onset of L and N expression. Minireplicon activity was found to be dependent on the correct ratio of L to N plasmids, with too much of either construct resulting in downregulation. Furthermore, a specific inhibitory effect of LACV NSs protein on minireplicon activity was found. In passaging experiments using parental helper virions, it was demonstrated that the recombinant nucleocapsids are a useful model for transcription, replication and packaging of LACV.
The nucleotide sequence data of the LACV L gene reported in this paper has been deposited in EMBL, GenBank and DDBJ under accession no. AF525489.
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INTRODUCTION
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La Crosse encephalitis is the most frequently reported mosquito-borne disease in the United States (CDC, 2000
; Elliott, 1997
; Griot et al., 1993
; McJunkin et al., 2001
). It is caused by La Crosse virus (LACV), a member of the family Bunyaviridae, and mostly affects children and young adults. Usually, the disease is associated with fever, headache and neurological sequelae that resolve with time. Rarely, meningitis, convulsions, mental disorientation, coma and even death occur. Around 75100 cases resulting in hospitalization are reported annually but the majority of infections are subclinical and it has been estimated that there may be more than 1000 infections per recognized case (Calisher, 1994
; Gonzalez-Scarano & Nathanson, 1996
).
Bunyaviruses are a large group of viruses that are transmitted mainly by arthropods (Elliott, 1996
). Some members are human pathogens and can cause encephalitis, febrile illnesses or haemorrhagic fevers, among them are LACV, Oropouche virus, Hantaan virus, Rift Valley fever virus (RVFV) and Crimean-Congo haemorrhagic fever virus (Elliott, 1997
; Weber & Elliott, 2002
). All members of the Bunyaviridae are enveloped and have a tri-segmented, single-stranded RNA genome of negative or ambisense polarity, replicate in the cytoplasm and bud into the Golgi apparatus. They encode four common structural proteins: the viral polymerase (L) on the large (L) segment, two glycoproteins (G1 and G2) on the medium (M) segment and the viral nucleocapsid protein (N) on the smallest (S) segment. Viruses within some genera also encode nonstructural proteins, either on the M segment (termed NSm) or on the S segment (termed NSs). We have shown recently that the NSs proteins of Bunyamwera virus (BUNV) and RVFV act as virulence factors by suppressing the production of interferon
/
(Bouloy et al., 2001
; Weber et al., 2002
). In addition, NSs of BUNV was found to inhibit the viral polymerase (Weber et al., 2001
).
The general features of bunyavirus transcription and RNA replication are similar to those of other negative-stranded RNA viruses (Bishop, 1996
). Genomic RNA segments contain untranslated sequences on both the 5' and the 3' ends that serve as promoters for replication of the segment and transcription of the encoded reading frames. They are encapsidated by N protein and associate with L protein both intracellularly and in the virion, and only these nucleocapsids are functional templates for mRNA synthesis and RNA replication by the viral polymerase.
To investigate LACV replication on a molecular level and to elucidate virushost cell interactions, a system would be useful that allows targeted manipulation of the virus genome. In such a reverse genetics system, viral nucleocapsids are reconstituted from cloned L and N cDNAs that are expressed in cell culture. A cotransfected, genomic RNA-like minireplicon containing a reporter gene allows the activity of the recombinant nucleocapsids to be monitored. Genome fragments of most human pathogenic bunyaviruses have been cloned and sequenced previously (Elliott, 1996
). Reverse genetic systems, however, have so far been established only for the human pathogenic RVFV (Lopez et al., 1995
) and the less pathogenic BUNV (Dunn et al., 1995
), Uukuniemi virus (Flick & Pettersson, 2001
) and Toscana virus (Accardi et al., 2001
). In fact, BUNV was the first negative-stranded RNA virus with a segmented genome to be reconstituted entirely from cDNA plasmids (Bridgen & Elliott, 1996
). It is highly desirable to have a similar system at hand to manipulate the RNA segments of the medically important LACV. Here, we report the full-length sequence of an active L polymerase cDNA of LACV and the in vivo reconstitution of functional nucleocapsids from transfected L and N cDNAs. The minireplicon system was shown to be competent for transcribing, replicating and packaging a virus-like minireplicon RNA. Furthermore, the system allowed the establishment of conditions for rescuing recombinant nucleocapsids into virus particles.
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METHODS
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Cells and viruses.
Vero and BHK-21 cells were cultivated in Dulbecco's modified Eagle's medium supplemented with 10 % foetal calf serum. LACV was kindly provided by R. Raju, Meharry Medical College, Nashville, TN, USA. Recombinant vaccinia virus Ankara expressing T7 RNA polymerase (MVA-T7) was a gift from G. Sutter, GSF-Neuherberg, Germany (Sutter et al., 1995
).
Preparation of first-strand cDNA from virus sequences.
BHK-21 cells were infected with plaque-purified LACV at an m.o.i. of 0·02. The supernatant was harvested 48 h later. Virus particles in the supernatant were purified by ultracentrifugation (100 000 g for 2 h using a SW28 rotor) through a 30 % glycerol cushion and resuspended in PBS. RNA was isolated from the purified virus using Trifast reagent (PeqLab) according to the manufacturer's instructions. First-strand cDNA was synthesized by reverse transcription using random hexanucleotide primers. One reaction contained 1 µg viral RNA, 100 ng primers, 1 mM dNTPs, 20 mM DTT, 40 units RNasin (Promega) and 200 units SuperScript II reverse transcriptase (Gibco-BRL) in 1x first-strand buffer (Gibco-BRL). The reaction was carried out at 42 °C for 1 h and terminated by a 5 min incubation step at 95 °C.
Plasmid constructs.
Viral genes were amplified from the first-strand cDNA by PCR using specific primers (Table 1
) and cloned by standard molecular biology methods (Ausubel et al., 1992
). All expression plasmids were verified by sequencing. The protein expression plasmids pTM-LACV-L, pTM-LACV-S, pTM-LACV-N and pTM-LACV-NSs contained the appropriate coding sequences under the control of a T7 promoter and the encephalomyocarditis virus internal ribosome entry site (IRES) in the vector pTM1 (Moss et al., 1990
). The LACV L segment gene was amplified using the Expand Long PCR kit (Roche) with primers 5'Esp3I-LACV_L (containing an Esp3I site) and 3'XhoI-LACV_L (containing a XhoI site) and 3 µl LACV particle-derived cDNA as template. PCR was carried out in a GeneAmp PCR System 9600 (Perkin Elmer) and included 30 cycles of 10 s at 95 °C, 30 s at 60 °C and 10 min at 68 °C. After 10 cycles, the elongation time was extended for 20 s in every cycle. The PCR product was TA-cloned into the vector pCR4-TOPO (Invitrogen). The LACV L gene was recovered using Esp3I/XhoI digestion and ligated into NcoI/XhoI-digested pTM1. The LACV S segment and the NSs gene were cloned as described for the L segment gene except that Taq DNA polymerase (Roche) and the primer pairs 5'Esp3I-LACV_S/3'XhoI-LACV_S and 5'Esp3I-LACV_NSs/3'XhoI-LACV_NSs, respectively, were used. In order to abrogate NSs expression on the S gene plasmid pTM-LACV-S, the two ATG start codons (nt 2025 of the S segment-encoding region) were changed to ACG by primer mutagenesis. These alterations do not affect the N protein amino acid sequence. In brief, two PCR products that contain the primer-encoded alterations in overlapping regions were generated using the primer pairs 5'Esp3I-LACV_S/LACV-SdelNSs(-) and LACV-SdelNSs(+)/3'XhoI-LACV_S. Both fragments served as template in second-round PCR with primers 5'Esp3I-LACV_S and 3'Xho-LACV_S to give a full-length N-encoding DNA fragment, which was then cloned between the NcoI and the XhoI sites of pTM1.
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Table 1. DNA oligonucleotide primers
Restriction sites are indicated in capital letters. Nucleotides corresponding to LACV sequences are indicated in bold. Nucleotides are numbered according to the database entries U12396 (L segment), U18980 (M segment) and K00610 (S segment).
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Reporter plasmid pLACV-cREN contains the Renilla (sea pansy) luciferase gene (REN-Luc) in sense orientation, flanked by the consensus sequences of the 5'- and 3'-terminal noncoding sequences of the LACV M cRNA. The noncoding sequences (which serve as promoter for the LACV polymerase) were attached to the REN-Luc sequence by PCR. Due to the length of the noncoding sequences (61 nt at the 5' end and 139 nt at the 3' end), a two-step PCR protocol with overlapping primer sequences was used. Two additional bridging primers were involved in second-round PCR to cover the whole 3' noncoding region. In first-round PCR, the REN-Luc gene was amplified by PCR from the template plasmid pRL-SV40 (Promega) using primers 5'first_cLACV_M and 3'first_cLACV_M. The PCR product was gel purified and served as template for second-round PCR using four primers, 5'Esp3I-cLAC_M_huPolI, 3'Esp3I-cLAC_M, Sense_anneal and Antisense_anneal, in order to equip the REN-Luc gene with sense-oriented M segment untranslated sequences and Esp3I restriction sites. The final PCR product was cloned into the BsmBI site of plasmid pHH21 (kindly provided by G. Hobom, University of Giessen, Germany), resulting in plasmid pLACV-cREN. This plasmid contains the artificial LACV minigenome in sense orientation between the human RNA polymerase I promoter and terminator. Reporter plasmid pLACV-vREN contains REN-Luc in antisense orientation, flanked by the untranslated regions of the LACV M viral RNA. It was constructed by amplifying pLACV-cREN by PCR with primers 5'Esp3I-vLACV_M and 3'Esp3I-vLACV_M and cloning the PCR product as described for plasmid pLACV-cREN. A similar approach to that described for plasmid pLACV-cREN was used to generate the reporter plasmid pLACV-cGFP, which harbours the enhanced green fluorescent protein (EGFP). Primers 5'GFP-LACV_M and 3'GFP-LACV_M were used in first-round PCR to amplify the EGFP gene.
Plasmid pTM-FF-Luc harbours the Photinus (firefly) luciferase gene (FF-Luc) under the control of the T7 promoter and this served as an internal control for transfection efficiency (Weber et al., 2001
).
In vivo reconstitution of LACV nucleocapsids.
Subconfluent monolayers of Vero cells were transfected with 0·5 µg plasmid pLACV-vREN, 0·5 µg pTM-LACV-L, 0·5 µg pTM-LACV-S or pTM-LACV-N and 0·1 µg pTM-FF-Luc using 5 µl DAC-30 transfection reagent (Eurogentec) in 200 µl serum-free medium (OptiMEM, Gibco-BRL). In some experiments, specific expression plasmids were omitted from the transfection mixture and substituted by the empty parental vector pTM1. After transfection, cells were infected with MVA-T7 at an m.o.i. of 10. At the indicated times post-infection (p.i.), cells were lysed in 200 µl Dual Luciferase Passive Lysis buffer (Promega). An aliquot of 20 µl of the lysate was assayed for FF-Luc and REN-Luc activity, as described by the manufacturer (Promega).
Passaging of LACV nucleocapsids.
Subconfluent monolayers of Vero cells (donor cells) were transfected with 0·5 µg of the respective reporter plasmid (pLACV-cREN, pLACV-vREN or pLACV-cGFP), 0·5 µg pTM-LACV-L, 0·5 µg pTM-LACV-S or pTM-LACV-N, and 0·1 µg pTM-FF-Luc using 5 µl DAC-30 in 200 µl OptiMEM. At 5 h after transfection, cells were infected with MVA-T7 at an m.o.i. of 10 and after a further incubation period of 12 h, cells were superinfected with LACV at an m.o.i. of 5. After 24 h of incubation, the supernatant was removed, centrifuged for 1 min at 14 000 g to pellet residual cells and used to infect Vero cells (indicator cells). The infected cells were washed with medium and then incubated for 18 h. To measure luciferase activity, both donor and indicator cells were lysed in 200 µl Dual Luciferase Passive Lysis buffer and 20 µl of the lysate was assayed for FF-Luc and REN-Luc activity, as described by the manufacturer (Promega). EGFP expression in donor and indicator cells was assayed by fluorescence microscopy.
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RESULTS AND DISCUSSION
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Establishment of an in vivo reconstitution system for LACV
We assumed that successful in vivo reconstitution of recombinant LACV nucleocapsids would require (i) correct promoter ends for the minireplicon, (ii) a functional N nucleocapsid protein cDNA and (iii) a functional L polymerase cDNA. In a first step, we constructed a minireplicon that contained the REN-Luc gene in negative sense, flanked by the consensus sequences of the LACV M segment 3' and 5' promoter ends (Fig. 1A
). Intracellular transcription of the minireplicon RNA occurred by the cellular RNA polymerase I. To obtain the N gene, we generated a LACV S cDNA construct (encoding both N and NSs in overlapping reading frames) by RT-PCR using viral RNA from purified LACV particles as template. The L polymerase gene was cloned in a similar manner, except that an extended RT-PCR protocol was used (see Methods). Expression of the L and S cDNA gene products was under the control of a T7 promoter. An IRES placed upstream of the reading frames ensured cap-independent translation. The L reading frame is approximately 7000 nt long and is thus difficult to obtain by RT-PCR as an error-free product. Therefore, several independent L cDNA clones were tested for function in the minireplicon system. Cells were transfected with the L and S expression constructs together with the minireplicon plasmid and then infected with MVA-T7, a noncytopathic vaccinia virus that provides T7 polymerase (Sutter et al., 1995
). It should be noted that the minireplicon RNA was transcribed by the cellular RNA polymerase I in the negative-sense orientation. Therefore, positive-sense REN-Luc mRNA was generated exclusively by the recombinant LACV polymerase complex and the amount of reporter protein reflected the activity of the reconstituted viral ribonucleoproteins. Fig. 1
(B) shows that cotransfection of L, S and the minireplicon constructs resulted in high expression of the reporter gene, indicating transcription and replication of the minireplicon by the recombinant L and S gene products. In contrast, omitting either the L or the S expression plasmids resulted in only background reporter activity. Overall, we tested six different L cDNA clones and found two of them to be active (data not shown). Thus, our strategy to use RNA from purified virus particles for cloning the appropriate cDNAs by RT-PCR and to screen them for minireplicon reporter activity resulted in a relatively high yield of cDNA sequences encoding a functional L polymerase.

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Fig. 1. Reconstitution of LACV nucleocapsids from cloned cDNAs. (A) Schematic representation of the vREN minireplicon containing the REN-Luc gene in negative sense, flanked by the noncoding sequences (UTR) of the LACV M vRNA segment. Initiation and termination of RNA synthesis is accomplished by recognition sites for the cellular RNA polymerase I (Ppol I and Tpol I, respectively). (B) REN-Luc activity of reconstituted nucleocapsids. Cells were transfected with expression constructs for the L and S segment of LACV and the reporter minireplicon vREN. Cells were then infected 5 h later with MVA-T7. At 18 h post-transfection, cells were assayed for REN-Luc activity. Luciferase counts were normalized with respect to the background REN-Luc activity of the experiment with no expression plasmids. Data from a representative experiment are shown.
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Sequence of the L reading frame
The sequences of the two active L cDNA clones reported here are identical. The sequence was compared to the single entry for LACV L in the sequence database (GenBank accession no. U12396; Roberts et al., 1995
). Overall, seven nucleotide differences were observed and four amino acids differed from the published sequence (Table 2
). However, it is presently unknown whether these four amino acid positions are critical for polymerase activity, since, to our knowledge, the L sequence published previously was not tested for function. Furthermore, none of the changes were situated within the conserved RNA-dependent RNA polymerase motifs identified previously (Jin & Elliott, 1992
; Poch et al., 1989
). The new LACV L sequence has been submitted to GenBank (accession no. AF525489). The cDNA sequence for the S segment, which was also derived from LACV particles, was identical to the database entry K00610 (Patterson et al., 1983
).
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Table 2. Comparison of LACV L sequences
Codons and amino acids for U12396 were taken from Roberts et al. (1995) , whereas those for pTM-LACV-L were derived from this study. Amino acids that differ are indicated in bold.
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Kinetics of minireplicon activity
We were interested to establish the time-course of nucleocapsid reconstitution. Cells were transfected with the plasmids expressing L, S and the minireplicon and infected with MVA-T7. Subsequently, samples were taken every 2 h for a time-period of 20 h. To control for transfection efficiency as well as cellular polymerase I-mediated gene expression, parallel dishes were transfected with a construct that contained the positive-sense REN-Luc gene (cREN) in the same vector as the minireplicon (vREN). Due to the capping activity of MVA-T7, cREN transcripts can be translated directly. Fig. 2
(cREN) shows that control activity increased in an approximately linear manner, with measurable activity as early as 6 h p.i. The increase in control activity proceeded until 16 h p.i. and then reached a plateau. In contrast, measurable activity of the LACV minireplicon began later, at 10 h p.i. (Fig. 2
, vREN/L/N). It then increased rapidly and reached a plateau phase at around 18 h p.i. Thus, gene expression mediated by polymerase I (which supplies the minireplicon) and by the LACV recombinant nucleocapsids appear to follow different kinetics.

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Fig. 2. Time course of minireplicon and control reporter activity. Cells in parallel dishes were cotransfected with expression constructs for the complete minireplicon system (designated vREN/L/N) or with a positive-sense polymerase I control plasmid (cREN). At 12 h after transfection, the cells were infected with MVA-T7 and single dishes were analysed every 2 h for REN-Luc activity, reflecting either minireplicon replication and transcription (dishes with vREN/L/N) or cellular RNA polymerase I-mediated transcription (dishes with cREN only). Luciferase counts were normalized with respect to the experiment with the highest activity. Mean±SD from five independent experiments are shown.
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These data are in agreement with the illegitimate assembly model for negative-strand virus minireplicons proposed by Conzelmann (1998)
. In this model, recombinant nucleocapsid components need to accumulate to a critical level before they can promote encapsidation of the naked minireplicon RNA. This inefficient process would explain the time lag of 4 h when no measurable minireplicon activity was present. Then, the first-generation seeding nucleocapsids generate progeny nucleocapsids that assemble in a manner similar to the situation in infected cells, i.e. with direct encapsidation of nascent RNAs generated by the viral polymerase. This results in a much higher efficiency of minireplicon transcription and replication, reflected by the rapid increase of reporter gene expression from 10 h p.i. on. The final plateau phase reached by both the minireplicon and the T7-driven control most probably indicates exhaustion of the system, since even after prolonged incubation no further enhancement of reporter gene expression could be observed (data not shown).
Concentration-dependent effects of L, N and NSs proteins
The level of protein expression in a minireplicon system is directly dependent on the amount of plasmid transfected (Atreya et al., 1998
; Weber et al., 2001
). We varied LACV expression plasmid concentrations systematically and monitored minireplicon activity in order to find optimum levels. To control transfection efficiency and MVA-mediated gene expression, an additional construct was transfected that contained the firefly luciferase gene (FF-Luc) in the same vector as the LACV expression plasmids, i.e. under the control of a T7 promoter and an IRES. When LACV S plasmid concentration was kept constant, increasing amounts of LACV L plasmid resulted in a bell-shaped response curve of minireplicon activity (Fig. 3
A, REN-Luc). Optimal conditions for minireplicon activity were obtained with an L plasmid concentration of 0·5 µg per well, whereas lower or higher amounts of plasmid resulted in decreased activity. Significantly, the FF-Luc expression control remained largely unaffected, indicating a specific effect (Fig. 3A
, FF-Luc). Similarly, varying the amount of LACV S plasmid resulted in a bell-shaped dose-response curve of minireplicon activity with a maximum at 1 µg (Fig. 3B
, REN-Luc) but not of the expression control (Fig. 3B
, FF-Luc). Thus, the performance of the LACV polymerase appears to be highly dependent on the molar ratio of L to N protein, with too much of either protein resulting in downregulation of polymerase activity. This is similar to the situation with the related BUNV (Dunn et al., 1995
) and suggests that bunyavirus L and N have regulatory functions. In contrast, an approximately linear dose-dependency of minireplicon activity has been described for Uukuniemi phlebovirus (Flick & Pettersson, 2001
), suggesting a less tight regulation of polymerase activity.

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Fig. 3. Monitoring minireplicon activity. Cells were transfected with varying amounts of pTM-LACV-L (A) or pTM-LACV-S (B), as indicated, and standard concentrations of either LACV S (A) or LACV L (B) and vREN and FF-Luc expression constructs. Cells were infected with MVA-T7 5 h later and REN-Luc and FF-Luc activities were measured at 18 h post-transfection. Luciferase counts were normalized with respect to the experiment with the lowest activity. Data from a representative experiment are shown.
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In virus-infected cells and in recombinant expression systems, translation of the bunyavirus S mRNA results in two different proteins, the nucleocapsid protein N and the nonstructural protein NSs (Bishop, 1996
). In all previous experiments (Figs 1
3
), a LACV S plasmid was used that truthfully reflected this situation. For BUNV, we have shown previously that NSs negatively regulates minireplicon activity (Weber et al., 2001
). To investigate this for LACV, we deleted the NSs gene from the LACV S plasmid and tested minireplicon performance. Indeed, using the mutated plasmid coding only for LACV N resulted in an approximately twofold higher activity than the original LACV S plasmid expressing both N and NSs (Fig. 4
A, REN-Luc); the T7 expression control was not altered (Fig. 4A
, FF-Luc). As observed with the S cDNA (Fig. 3B
), varying the amount of N plasmid resulted in a bell-shaped dose-response curve of minireplicon activity (data not shown). To test the inhibitory potential of LACV NSs on minireplicon activity directly, we transfected LACV NSs on a separate plasmid together with optimum amounts of LACV N and LACV L plasmids. Indeed, coexpression of NSs reduced minireplicon activity in a specific manner (Fig. 4B
, REN-Luc). In contrast, the FF-Luc expression control was not affected (Fig. 4B
, FF-Luc). Thus, similar to BUNV NSs (Weber et al., 2001
), LACV NSs has the ability to downregulate minireplicon activity.

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Fig. 4. Effect of NSs on minireplicon activity. (A) LACV nucleocapsids were reconstituted from plasmid constructs for vREN, LACV L and either LACV S (expressing both N and NSs) or LACV N alone. The control construct pTM-FF-Luc was included in the transfection mixture. Luciferase counts were normalized with respect to the experiment with the lowest activity. To indicate the differences between the LACV S and LACV N expression constructs, the respective translation products are depicted below the graph. (B) Cells were transfected with expression plasmids encoding vREN, LACV L, LACV N and the control construct pTM-FF-Luc. In addition, empty vector pTM1 or 0·125 µg pTM-LACV-NSs was added to the plasmid mixture. Data from a representative experiment are shown.
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Packaging of minireplicons into virus particles
We analysed whether the recombinant LACV nucleocapsids could be packaged into virus particles. To this aim, donor cells expressing the minireplicon system and the FF-Luc control construct were superinfected with LACV and supernatants containing newly produced virus particles were taken to infect indicator cells. In general, superinfecting LACV reduced the performance of the minireplicon system by approximately 50 % (data not shown). To investigate a possible influence of NSs, either S (expressing N and NSs) or N (expressing N) plasmids were used for nucleocapsid reconstitution. Furthermore, vRNA-sense (as used in all previous experiments) and cRNA-sense minireplicons were compared. First, minireplicon activity was analysed in superinfected donor cells (Fig. 5
A). As expected (see Fig. 4
), LACV-infected cells transfected with the S expression plasmid (Fig. 5A
, columns 1 and 3, REN-Luc) displayed a somewhat lower minireplicon activity than cells expressing the N expression plasmid (Fig. 5A
, columns 2 and 4, REN-Luc). This difference, however, was less pronounced than that observed previously (see Fig. 4
), most probably because the superinfecting LACV contributes NSs also in the N-transfected dishes. Furthermore, under optimal conditions, the cRNA-sense minireplicon (Fig. 5A
, column 4, REN-Luc) appeared to have a higher activity than the vRNA-sense minireplicon (Fig. 5A
, column 2, REN-Luc). As shown before (see Fig. 2
), a high transcription rate of the positive-sense cRNA template was also observed when L expression was omitted, due to the capping activity of MVA-T7 (Fig. 5A
, columns 5, REN-Luc). In contrast, very little minireplicon activity was present in LACV-infected donor cells expressing only minireplicon vRNA, or the minireplicon vRNA together with the S or the N gene but not the L gene (data not shown). Apparently, LACV is not able to use naked or nonphysiologically packaged minireplicon RNA as template for its polymerase. This suggests that, during infection, viral N and L are not produced in sufficient amounts to encapsidate the minireplicon, or they localize to a different cellular compartment.

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Fig. 5. Passaging of recombinant nucleocapsids expressing REN-Luc. (A) Donor cells. Nucleocapsids expressing the reporter minireplicon were reconstituted from transfected plasmid constructs for LACV L and either LACV S (columns 1 and 3) or LACV N (columns 2 and 4) and infected with LACV. The reporter minireplicon was supplied as either a genome-sense vREN construct (columns 1 and 2) or an antigenome-sense cREN construct (columns 3 to 5). As a control, L expression was omitted (columns 5). All plasmid mixtures contained the control construct pTM-FF-Luc. Luciferase counts were normalized with respect to the experiment with LACV S and vREN transfection (columns 1). (B) Indicator cells. Cells were exposed to supernatants from the donor cells described in (A). After incubation for 18 h, cells were harvested and analysed for luciferase activities. Both REN-Luc and FF-Luc activities were normalized to the donor cells transfected with LACV S and vREN (A, columns 1). Data from a representative experiment are shown.
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To investigate whether the recombinant nucleocapsids could be passaged by LACV particles, indicator cells were incubated with supernatants of the donor cells and analysed for reporter activities (Fig. 5B
). Interestingly, indicator cells treated with supernatants from N- and L-transfected donor cells (cRNA minireplicon) displayed a higher REN-Luc activity than S- and L-transfected donor cells (Fig. 5B
, columns 3 and 4, REN-Luc). Furthermore, a better transfer rate was achieved when a cRNA instead of a vRNA minireplicon was used in the donor cells (Fig. 5B
, columns 1/2 and 3/4, REN-Luc). In contrast, no passaging of the minireplicon was achieved when L expression was omitted in the donor cells (Fig. 5B
, columns 5, REN-Luc). In all indicator cells, FF-Luc control activity was absent (Fig. 5B
, FF-Luc), demonstrating that the measured REN-Luc activity was indeed due to specific transfer by virus particles. Omitting LACV infection of donor cells did not result in detectable activity in indicator cells (data not shown). Thus, the recombinant nucleocapsids were competent to be packaged into LACV particles and to be transferred into indicator cells.
To investigate further the packaging of artificial genome segments by LACV, we expressed a GFP-encoding minireplicon under optimal conditions (i.e. in cRNA-sense and with L and N expression plasmids) and directly visualized positive donor and indicator cells by fluorescence microscopy. Fig. 6
(left panels) shows that a high amount of the donor cells expressed GFP, independent of whether they were superinfected with LACV or not. As expected, GFP expression was only detected in indicator cells when supernatants from infected donor cells were used (Fig. 6
, right panels). Similar to the situation with REN-Luc minireplicon packaging (see Fig. 5B
), approximately 1 % of indicator cells were positive for GFP expression. Further passaging of the recombinant nucleocapsids, however, was not possible.

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Fig. 6. Passaging of recombinant nucleocapsids expressing GFP. Nucleocapsids expressing the GFP minireplicon in cRNA-sense were reconstituted from transfected plasmids encoding LACV L and LACV N expression plasmids. Donor cells (left panels) were transfected and either infected with LACV (upper panels) or left uninfected (lower panels), as described in Fig. 5 . Indicator cells (right panels) were incubated with supernatants of donor cells. Data from a representative experiment are shown.
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In both systems, using either REN-Luc or GFP as a reporter, efficient passaging of recombinant nucleocapsids was achieved when NSs expression was omitted. It is, therefore, possible that NSs is not required for packaging of virus particles but may have a negative effect. Furthermore, our data show that a positive-sense, i.e. cRNA-like minireplicon RNA is a more efficient template for passaging than a negative-sense one. Interestingly, the first rescues of negative-sense RNA viruses were all accomplished when cRNA-like plasmid constructs were used as genomic constructs instead of vRNA-like plasmids (reviewed by Conzelmann, 1998
). Thus, cRNA templates appear to be intrinsically better suited for rescuing recombinant nucleocapsids, possibly because the naked vRNA templates produced initially could hybridize with viral mRNAs and be degraded.
Our data demonstrate that the recombinant nucleocapsids derived from our cloned cDNAs are competent for transcription and replication and for being packaged into virus particles. Both L and N but not NSs are necessary for these activities, and the molar ratio of L, N and NSs determines polymerase efficiency, whereby NSs has an inhibiting effect.
Taken together, the sequence of an active L polymerase clone and the in vivo reconstitution of LACV nucleocapsids represent an important step towards the genetic manipulation of this important human pathogen.
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ACKNOWLEDGEMENTS
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We would like to thank Ramaswamy Raju, Gerd Sutter and Gerd Hobom for providing LACV, MVA-T7 and pHH21, respectively. Furthermore, we thank Martin Spiegel and Peter Staeheli for critically reading the manuscript and Simone Gruber for excellent technical assistance. This work was supported by Deutsche Forschungsgemeinschaft grants We 2616/1-1 and 1-2 to F. W.
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Received 4 October 2002;
accepted 6 January 2003.