Specific inhibition of orthopoxvirus replication by a small interfering RNA targeting the D5R gene.
BACKGROUND
Concerns about the potential use of smallpox in bioterrorism have stimulated interest in the development of novel antiviral treatments. Currently, there are no effective therapies against smallpox and new treatment strategies are greatly needed.


METHODS
In this study, specifically designed small interfering RNAs (siRNAs), targeting five proteins essential for orthopoxvirus replication, were investigated for their ability to inhibit vaccinia virus strain Western Reserve (VACVWR) replication.


RESULTS
Among these siRNAs, 100 nM siD5R-2, an siRNA targeting the D5 protein, decreased VACVWR replication up to 90% when used either prophylactically or therapeutically in human lung carcinoma A549 cells. This siRNA induced a striking concentration-dependent inhibition of VACVWR replication and a prolonged prophylactic antiviral effect that lasted for 72 h, at a concentration of 100 nM. Confocal microscopy of Alexa-siD5R-2-treated VACVWR-infected cells confirmed a decrease in viral replication. Furthermore, siD5R-2 was shown to specifically reduce the D5R mRNA and protein expression using real-time reverse transcriptase-PCR and western blotting analysis, without inducing interferon-13 in A549 cells. We also demonstrated the antiviral potency of siD5R-2 against different pathogenic orthopoxviruses, such as cowpox and monkeypox viruses, which were inhibited up to 70% at the lowest concentration (1 nM) tested. Finally, siD5R-2 showed antiviral effects in VACVWR-infected human keratinocyte and fibroblast cell cultures.


CONCLUSIONS
These results suggest that siD5R-2 could be a potential candidate to treat poxvirus infections.
Virology Laboratory, Centre de Recherches du Service de Santé des Armées (CRSSA) Emile Pardé, Grenoble, France.
Solenne
S
Vigne
Raphaële
R
Germi
Sophie
S
Duraffour
Sylvie
S
Larrat
Graciela
G
Andrei
Robert
R
Snoeck
Daniel
D
Garin
Jean-Marc
JM
Crance
D015153:Blotting, Western; D045744:Cell Line, Tumor; D015608:Cowpox virus / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation; D005347:Fibroblasts / Q000378:metabolism / Q000821:virology; D005455:Fluorescent Antibody Technique; D015967:Gene Expression Regulation, Viral; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D016899:Interferon-beta / Q000378:metabolism; D015603:Keratinocytes / Q000378:metabolism / Q000821:virology; D008993:Monkeypox virus / Q000235:genetics / Q000378:metabolism; D018155:Orthopoxvirus / Q000235:genetics* / Q000378:metabolism; D011213:Poxviridae Infections / Q000378:metabolism / Q000628:therapy* / Q000821:virology; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics*
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014764:Viral Proteins; D016899:Interferon-beta