Groupe des Bunyaviridés1 and Unité décologie des systèmes vectoriels2, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France
Author for correspondence: Michèle Bouloy. Fax +33 1 40 61 31 51. e-mail mbouloy{at}pasteur.fr
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Abstract |
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Main text |
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Homologous interference has been described for several arboviruses: togaviruses (Karpf et al., 1997 ; Stollar & Shenk, 1973
), flaviviruses (Schmaljohn & Blair, 1977
), bunyaviruses (Beaty et al., 1983
; Elliott & Wilkie, 1986
) and RVFV (Turell et al., 1990
). More recently, B. J. Beatys group has developed recombinant Sindbis virus-based expression systems to produce intracellular immunity or pathogen-derived resistance against La Crosse virus or dengue virus in mosquitoes and in mosquito cells (Gaines et al., 1996
; Olson et al., 1996
; Powers et al., 1994
, 1996
). Using recombinant Semliki Forest virus (SFV) replicons as a vector, we investigated the possibility of producing mosquito cells resistant to RVFV and showed that homologous interference was induced by N but not NSs sequences and was RNA-mediated. This raises questions on the molecular mechanisms involved in virus resistance.
SFV replicons carrying partial or complete sequences of the RVFV MP12 strain S segment were prepared by inserting the viral sequences into pSFV-1 at the unique SmaI or BamHI site, transcribed in vitro from plasmids linearized at the SpeI site and electroporated into BHK-21 cells together with the Helper 2-RNA to produce suicide particles, as described by Liljeström & Garroff (1991 , 1995
).
To examine the effect of RVFV nucleoprotein sequences on replication of homologous virus we constructed pSFV-N, containing the sequence of the N ORF, and infected Aedes pseudoscutillaris (Ap61) cells with the recombinant SFV-N particles. Infection was monitored by immunofluorescence assay using the anti-SFV nsP3 or an anti-N monoclonal antibody and a fluorescein-labelled secondary antibody. In cells infected at an m.o.i. of 5 and examined at day 2 post-infection (p.i.), more than 80% of the cells exhibited a bright cytoplasmic fluorescence corresponding to the specific distribution of the RVFV N or SFV nsP3 proteins (Fig. 1A, B). Of the infected cells 50% were still positive on day 7 and 20% on day 12 p.i. (not shown), but staining was less intense, indicating that the replicons persisted in the cells but were diluted or became inefficient for protein expression as cells proliferated.
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Because the S segment encodes two proteins in opposite orientations, we investigated the possible effect of the NSs sequences and constructed replicons SFV-NSs and SFV-NSsanti containing the NSs sequences in the sense and antisense orientation, respectively. Fluorescent nuclear filaments, as observed in RVFV infection, were seen in Ap61 cells infected with SFV-NSs (Fig. 1C). However, although the amounts of recombinant genomic and subgenomic RNAs estimated by Northern blot analysis were equivalent to those present in SFV-N-infected cells (Fig. 1E
), expression of NSs sequences in Ap61 cells did not induce any homologous interference (Table 1
). To eliminate a possible antagonistic effect of NSs protein toward putative inhibition induced by the NSs nucleotide sequence, we mutated the AUG of the initiation codon of SFV-NSs, generating replicon SFV-NSsstop. Infection with this virus did not induce any protection to superinfection by MP12 (Table 1
), confirming that the NSs gene possesses no interfering potential.
Since the 3' and 5' halves of the S segment induced different effects on intracellular immunity, we tested the effect of complete S segment expression by generating SFV-Sg and SFV-Sag. Cells infected with SFV-Sag were resistant to MP12 infection to the same degree as cells infected with SFV-N but interference was significantly lower in cells expressing the S segment in the genomic orientation (Table 1). The degree of inhibition induced by SFV-Sg and SFV-Sag was not due to different amounts of RNAs synthesized by the recombinant SFVs, as revealed by Northern blot (Fig. 1E
); increasing the m.o.i. of SFV-Sg to 20 IU per cell had no effect.
To address the question of whether the observed interference due to the N sequence of RVFV was restricted to the homologous virus or could be extended to other viruses, we challenged SFV-N-infected Ap61 cells with the heterologous RVFV Clone 13, the related virus Toscana phlebovirus, and the unrelated Germiston bunyavirus and yellow fever flavivirus (17D strain). A similar experiment with SFV-Nanti led to similar results. Pre-infection with SFV-N inhibited Clone 13 replication although to a lesser extent than MP12 (Table 2). Interestingly, the sequences encoding the N protein of Clone 13 and MP12 share 97·8% identity [16 mutations over 736 nucleotides (Müller et al., 1995
)]. If intracellular immunity involves hybridization between the inducer and challenge RNAs, the fact that Clone 13 partially overcomes the resistance induced by the N sequences is likely to be due to an increased level of replication compared to MP12, a property repeatedly observed in cells infected with Clone 13. The sequences of RVFV N protein did not induce resistance toward Germiston or yellow fever viruses but resulted in a slightly reduced yield of Toscana virus (Table 2
), the progeny of which contained a considerable amount of a small plaque variant when compared to the progeny grown in control cells. This result indicates that the related Toscana virus, sharing 59% identity, was inhibited to some extent by the RVFV N sequences. A similar observation was reported by Powers et al. (1996)
who showed that La Crosse bunyavirus sequences, too, had an effect on the production of the related Trivittatus virus (62% identity).
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In their studies, B. J. Beaty and colleagues utilized recombinant Sindbis viruses to express La Crosse or dengue virus sequences and showed that mosquito cells as well as mosquitoes were resistant to homologous infection (Olson et al., 1996 ; Powers et al., 1996
). Here, for biosafety we used SFV suicide particles to orally infect Aedes aegypti mosquitoes but unfortunately, we were unable to detect replicon-driven expression probably because a very small number of cells had been infected. A replicative vector efficient for oral infection would be necessary to test the interfering effect in mosquitoes.
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Acknowledgments |
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References |
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Received 5 May 2000;
accepted 19 June 2000.