Advertisement

Coronaviruses pp 231-240 | Cite as

Investigation of the Functional Roles of Host Cell Proteins Involved in Coronavirus Infection Using Highly Specific and Scalable RNA Interference (RNAi) Approach

  • Jean Kaoru Millet
  • Béatrice NalEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1282)

Abstract

Since its identification in the 1990s, the RNA interference (RNAi) pathway has proven extremely useful in elucidating the function of proteins in the context of cells and even whole organisms. In particular, this sequence-specific and powerful loss-of-function approach has greatly simplified the study of the role of host cell factors implicated in the life cycle of viruses. Here, we detail the RNAi method we have developed and used to specifically knock down the expression of ezrin, an actin binding protein that was identified by yeast two-hybrid screening to interact with the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) spike (S) protein. This method was used to study the role of ezrin, specifically during the entry stage of SARS-CoV infection.

Key words

RNA interference (RNAi) Small interfering RNA (siRNA) Ezrin Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Virus–host interactions 

References

  1. 1.
    Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2:279–289CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Fire A, Xu S, Montgomery MK et al (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811CrossRefPubMedGoogle Scholar
  3. 3.
    Sen GL, Blau HM (2006) A brief history of RNAi: the silence of the genes. FASEB J 20:1293–1299CrossRefPubMedGoogle Scholar
  4. 4.
    Ma H, Kien F, Manière M et al (2012) Human annexin A6 interacts with influenza A virus protein M2 and negatively modulates infection. J Virol 86:1789–1801CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Millet JK, Kien F, Cheung C-Y et al (2012) Ezrin interacts with the SARS coronavirus spike protein and restrains infection at the entry stage. PLoS One 7:e49566CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Wang PG, Kudelko M, Lo J et al (2009) Efficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteins. PLoS One 4:e8325CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Kudelko M, Brault JB, Kwok K et al (2012) Class II ADP-ribosylation factors are required for efficient secretion of dengue viruses. J Biol Chem 287:767–777CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Microbiology and ImmunologyCornell UniversityIthacaUSA
  2. 2.Department of Life Sciences, College of Health and Life SciencesBrunel UniversityLondonUK

Personalised recommendations