Proceedings
The 12th International Conference of
THE ASSOCIATION OF INSTITUTIONS FOR
TROPICAL VETERINARY MEDICINE
Montpellier, France
Proceedings of the 12th International conference of the Association of Institutions of Tropical Veterinmy Medicine
CONTROL OF MORBILLIVIRUS REPLICATION BY RNAI
SERVAN DE ALMEIDA R.*, KEITA D., LIBEAU G., ALBINA E.
Cirad, Contrai of Emerging and Exotic Animal Diseases Research Unit
Campus international de Baillarguet, 34398 Montpellier Cedex 5, France
ABSTRACT
The Morbillivirus genus includes measles virus (MV), peste des petits ruminants virus
(PPRV) and rinderpest virus (RPV). Although preventive vaccines are available against
these three viruses, efficient therapeutics for virus control under emergency situations
are desirable. Inhibition of morbillivirus replication can be acbieved by post-transcriptional silencing of the nucleoprotein (N) gene by RNA interference (RNAi).
The viral N protein, a well conserved protein among the genus, plays a central role in
the replication of the virus. Using a comprehensive siRNA-based screening of the
conserved sequences of the N gene, we have identified, three cornmon positions on the
N gene, for the design of siRNA evenly effectives for PPRV, RPV and MV. siRNA
silencing resulted in more than 80% decrease of the viral replication in infected Vero
cell, as sbown by real-time quantitative PCR, flow cytometry and virus titration. In a
second step, a recombinant replication-defective buman type 5 adenovirus (Ad-5)
encoding one of the functional sequences directed against the N gene of PPRV was
constructed and shown to reduce the PPRV replication in vitro. These results illustrate
that adenovirus vector could be a prornising candidate for the development of siRNA
antiviral treatments against morbilliviruses.
INTRODUCTION
RNA interference (RNAi) is a natural process whereby introduction of double-stranded RNA (dsRNA) into cells results in degradation of homologous mRNA and, consequently, post-transcriptional gene
silencing. Specific inhibition of virus genes by RN Ai can be triggered by
the cellular introduction of synthetic 21- to 25-nucleotide duplexes of
RNA (siRNA) as was obtained to foot and mouth disease virus (1),
influenza virus (2), HIV (3) and human hepatitis virus (4). In this study, we report on the identification of three conserved loci in nucleoprotein
gene of PPRV and RPV which can be targeted for inhibition virus
replication by RNA interference. One of these loci was also effective on
*Contact au th or : E-mai 1 : Renata.servan _de_ almeida@cirad.fr
MV. As indicators of siRNA functionality, we have measured viral RNA and viral protein, the formed infectious particles as well as cytopathic effects induced in infected cell cultures.
MA TERIALS AND METHODS
Conserved regions were identified on multiple alignments of the N gene sequences of all morbilliviruses within which three conserved loci were selected. Nineteen base-long siRNAs covering these loci were synthesized (Ambion). Vero cells were transfected with 100, 50, 25 and 12.5nM of siRNAs and Lipofectamine 2000 (lnvitrogen) using usual methodology. Twenty-four hours after transfection, cells were infected by using a MOI of 0.1 of PPRV, RPV or MV and 4 days later, the siRNA silencing effect was evaluated. In a second step of this work, an adenovirus vector containing the siRNA sequence NPPRVl (Ad-shRNANl) was constructed to be tested in vivo. To this end, we have used the BLOCK-iT Adenoviral RNAi Expression System (Invitrogen ™) according to manufacturer's instructions. Firstly, the knockdown capacity of the Ad-shRNANl was verified in infected Vero cell cultures transduced with the Ad-shRNANl (MOI of 80) and infected by PPRV (MOI of 0.1) after 80 hours. A kinetic analysis of PPRV multiplication in these cell cultures has been carried out through virus titration of cell suspension samples collected after different hours post-infection.
RESULTS
The results demonstrate that the siRNA targeting the conserved locus 1 (position 480-498) of PPRV severely inhibited the virus multiplication. The reduction in N PPRV protein expression assessed by flow cytometry was over 90% (Fig. 1). The production of PPRV N transcripts and PPRV progeny was efficiently shut down 100- and 10,000- fold, respectively, as demonstrated by real time PCR and virus titration (Fig. 2A). Afterwards,
this locus efficiently silenced on PPRV was evaluated by flow cytometry on RPV and MV, using siRNAs homologous to the N gene of these viruses. The siRNA NRPVl and NMVl markedly inhibited the replication of the corresponding viruses as confirmed by the decrease in N protein expression of 90% and 70%, respectively (Figure 1 ).
Additionally, we have demonstrated that siRNAs NPPRV6, NPPRV7, NRPV6 and NRPV7 targeting two other conserved loci of N gene of
PPRV and RPV (loci 6 and 7, positions 741-759 and 899-917, respecti-vely) clearly inhibited the replication of the viruses as assessed by the marked decrease in viral protein, with a maximum of 87% (Figure 1 ), a reduction of 100- to 1.000-fold in PPRV and RPV titers and 10- to 100-fold in PPRV and RPV RNA copy number, respectively (Figures 2B and C).
Functional loci were then mapped by testing 19 base-long siRNAs overlapping by one or two nucleotides upstream or downstream. Results shown in Tab. 1 demonstrate that locus 1 was efficient for the three morbilliviruses tested, although maximum efficacy for RPV required a single base shift within the locus towards the 5' end. Additionally, loci 6 and 7 efficiently inhibited two out of three morbilliviruses tested (they have failed to inhibit MV replication; data not shown) and the efficacy of the loci 7 for RPV inhibition was also higher with a frame shift of one base downwards.
Results of the silencing capacity of the Ad-shRNANl in infected cell culture dernonstrated that it clearly in11ibited the replication of PPRV. This inhibitory effect was characterized by a remarkable decrease of CPE (data not shown). Additionally, the virus titration showed a decrease in PPRV titres higher than 2 logs by the Ad-shRNANl in cell suspensions collected after 96 hours post-infection (Figure 3). This Ad-shRNANl will be tested soon in goats superinfected with PPRV.
CONCLUSIONS
We were able to identify loci in the N gene of MV, PPRV and RPV which showed very strong in vitro antiviral effect when inhibited by siRNA. Ali these siRNAs may be used alone or in association for targeting multiple viral regions to prevent the emergence of escape mutants. Currently, we are studying delivery methods of siRNAs to be applied in vivo.
Acknowledgemen t
This work was partially supported by EU Pan-African prograrn for the Control of Epizooties, International Atornic Energy Agency, Marie Curie International Fellowship of Community 's 6th Frarnework Prograrn and the French Ministry of Foreign Affairs.
NR PVI 5'-AGUCUUACUGGUUUGAGAAn -3' 3'-nUCAGAAUGACCAAACUCUU -5 ' NPPRV6 5 ' -GGCGGUUCAUGGUAUCUCUtt -3 ' ~ 3 ' -ttCCGCCAAGUACCAUAGAGA -5 ' ·;;; N PPRV 7 5'-GCAUUAGGCCUUCACGAGUtt -3 ' 3 ' -nCGUAAUCCGGAAGUGCUCA -5 ' GAP DH 5 ' -AAGGUCAUCCAUGACAACUn -3 ' 3'-n UUCCAGUAGGUACUGUUGA -5' 1 1 1 1 0 1 l l 1 l i 1 1 1 0 , 1 0 ,2 Fi!!ure 1. siRNA functionalitv measured bv flow cvtometrv. 1 l 1 1 1 1 1 1 i 1 1 0 , 3 0 , 4 0 , 5 0 , 6 0 , 7 0 ,8 0 ,9 N viral prot e in expression
;z. ... 0 ... ~ " r-.0 " E~ :::> X "'-' >. > c. ~ 8 "-<!'. "-~ 1 2 Il 10 9 8 7 6 5 4 3 2 1 0 11 , 69
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0 0 , 0715 10000000000 > ;z. 6 c:: """" 100000000 "-0 5 .... ,.._, "-" 'D E ..c " 4 1000000 8 :2 ~ ~ :::> >< 3 10000 c:l '-' >, > 2 V) g. i:i::: 100 0 ü (,) 11.. 1 < 11.. f-~ Vero PPRV NPPRVI D RNA cop y nurnber e Virus tit e r-
--
---
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~ 6 1 s . • 2 0 ~ 5 > ~ 4 z '- ~ 3 1 .0 ~ 2 c: o. "'!
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1 1 0,5244 0 ,---, 0,072 1 35 Vero RPV NRPV6 NRPV7 D RNA copy nu m be r • Vir us t it er 10000000 5 ,~ 17 10000001
100000 Â Â 10000 1000 100 0 , 22653 IO 0 0 0!'.lO'.'lfi V e ro PPRV NPPRV6 NPPRV 7 RNA copy number • Virus liter ---1 0000000 1000000 10000 0 > ~ 10000 Ë 1 000 ~ 0 V) Cl 1 00 ü f-1 0 Figure 2. Functionality of the locus 1 (A) , loci 6 (B) and 7 (C) assessed by virus titration and rea l-tim e PCR.Table 1. Mapping of loci. Results are expressed as percentages of inhibition
(PI) of nucleoprotein expression as measured by flow cytometry.
Positions 477-495 478-496 479-497 480-498 481-499 482-500 NPPRVl NPPRVI- NPPRVI- NPPRVl-1 NPPRVl NPPRVl+l NPPRV1+2 3 2 Pl value 7% 15% 78% 90% 24% 28% NPRPVl nt* NRPVl-2 NRPVl-1 NRPVl NRPVJ+l nt PI value nt 35% 97% 70% 45% nt NMVl nt MVl-2 MVl-1 MVl MVl+l nt Pl value nt 35% 85% 90% 28% nt Positions 738-756 739-757 740-758 741-759 742-760 743-761 NPPRV6 nt NPPRV6-2 NPPRV6-l NPPRV6 NPPRV6+1 NPPRV6+2 Pl value nt 12% 35% 84% 55% 41% NRPV6 nt NRPV6-2 NRPV6-l NRPV6 NRPV6+1 NRPV6+2 Pl value nt 11% 0% 85% 36% 40% Positions 896-914 897-915 898-916 899-917 900-918 901-919 NPPRV7 nt NPPRV7-2 NPPRV7-l NPPRV7 NPPRV7+1 NPPRV7+2 Pl value nt 35% 44% 87% 62% 40% NRPV7 nt NRPV7-2 NRPV7-I NRPV7 NRPV7+1 NRPV7+2 Pl value nt 18% 36% 81% 86% 49% *nt = not tested 10000000 1000000 > ~ 100000 "-E 10000 -~ 1000 0 .,., 0 ü 100 1--IO 12h 24h 36h 48h 60h 72h 84h 96h Hours post-challenge DshRNA MOI 80 + PPRV 0 PPRV
Figure 3. Kinetic of PPRV multiplication in cell cultures transduced and
non-transduced with the Ad-shRNANl.
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