title: RNA interference-mediated inhibition of Semliki Forest virus replication in mammalian cells.
abstract: RNA interference (RNAi) has recently shown promise as a mode of inhibition of slowly replicating viruses causing chronic diseases such as hepatitis C. To investigate whether RNAi is also feasible for rapidly growing RNA viruses such as alphaviruses, we tested the ability of expressed short hairpin RNAs (shRNAs) to inhibit the Semliki Forest virus (SFV), a rapidly replicating positive-strand RNA virus. Plasmids expressing shRNAs targeting SFV target sequences under the control of a human U6 promoter were introduced into BHK-21 cells. The targets included sequences encoding nonstructural (nsP1, 2, and 4) and structural (capsid) proteins as well as nonviral sequences serving as control targets. Twenty-four to 48 hours following transfection with shRNA plasmids, the cells were infected with replication-competent or replication-deficient recombinant SFV expressing green fluorescent protein (GFP) at a multiplicity of infection (MOI) of approximately 5. Viral replication was monitored by fluorescence microscopy and flow cytometry. Specific and marked reduction of viral replication was observed with shRNAs targeting nsP1 and nsP4. The degree of inhibition of the replication-deficient SFV was >or=70% over a 5-day period, a level similar to the transfection efficiency, suggesting complete inhibition of nonreplicating virus in the transfected cell population. However, only nsP1 shRNA was inhibitory against replication-competent SFV (approximately 30%-50% reduction), and this effect was transient. No inhibition was observed with control shRNAs. In contrast to the recent success of RNAi approaches for slowly growing viruses, these results illustrate the challenge of inhibiting very rapidly replicating RNA viruses by RNAi. However, the addition of RNAi approaches to other antiviral modalities might improve the response to acute infections.
authors: SomaGenics, Inc., Santa Cruz, CA 95060, USA.
authors: Attila A
authors: AA
authors: Seyhan
authors: Babak N
authors: BN
authors: Alizadeh
authors: Kenneth
authors: K
authors: Lundstrom
authors: Brian H
authors: BH
authors: Johnston
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D005822:Genetic Vectors; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D012672:Semliki forest virus / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D014779:Virus Replication
chemical_list: D034741:RNA, Small Interfering