Respiratory virus infections are a major source of morbidity, mortality, and economic loss in both developed and under-developed countries. The emergence of SARS-CoV and the increased incidence of avian influenza highlight the potential importance of new vaccines and therapeutics to prevent disease and disease spread around the world. The rationale for this study is that SARS-CoV, like all other obligate intracellular pathogens, requires host proteins for its life cycle and that inhibiting the expression of critical host proteins should block viral replication. We have successfully used gene trap insertional mutagenesis to randomly inactivate cellular genes as a tool for rapidly identifying novel host genes required for influenza A, HIV-1, Marburg, Ebola, or reovirus replication, but not host cell survival. Viral infection normally kills the chosen host cell line unless a gene essential for the viral life cycle has been inactivated. We identified several pathways used by unrelated viruses suggesting the existence of conserved mechanisms for viral replication, and that inhibiting such pathways using small interfering RNA (siRNA) may allow broad spectrum therapeutic intervention. The objectives of the current application are to extend gene trap technology towards the study of novel host genetic requirements for SARS-CoV infection, and to use RNA interference as a strategy to independently confirm the requirement of these genes in viral replication.
Our Specific Aims are to 1) use Vero E6 cell gene trap libraries to select cells resistant to SARS- CoV infection and identify candidate genes whose disruption confer the resistant phenotype, and 2) to characterize the effects of silencing candidate genes identified in Specific Aim 1 on SARS-CoV replication and host cell viability and growth rates. This study is designed to identify specific siRNAs that inhibit SARS-CoV replication without causing detrimental effects to host cells. The benefit to public health is that the transient knock- down of host gene mRNAs to block infectious pathogens could confer broad spectrum therapeutic intervention that is difficult, if not impossible, to circumvent through mutation of the pathogen. The goal of this application is to determine novel host genetic requirements essential for SARS-CoV replication. Candidate host genes performing critical roles in SARS-CoV replication will be identified by randomly mutating host genes, and then identifying disrupted genes in surviving clonal SARS-CoV-resistant cell lines. The requirement of candidate genes in SARS-CoV replication will be confirmed in RNA interference (RNAi) studies. Following RNAi screening, cell lines expressing short hairpin (shRNA) against critical genes will be generated to determine the effects of long term target gene silencing on cellular growth rates and viability, and to prepare for prospective mechanistic studies beyond the term of this application. ? ? ?
