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RNAi-Mediated Depletion of Poxvirus Proteins

Caroline Martin1, Samuel Kilcher2

  • 1MRC-Laboratory for Molecular Cell Biology, University College London, London, UK.

Methods in Molecular Biology (Clifton, N.J.)
|June 27, 2019
PubMed

Insights

RNA interference (RNAi) enables targeted protein depletion for studying viral infections. This chapter details RNAi methods for poxvirus gene discovery and high-throughput screening of viral infection levels.

Area of Science:

  • Molecular Biology
  • Virology
  • Genetics

Background:

  • RNA interference (RNAi) is a tool for transiently depleting proteins.
  • Previous RNAi screens identified host factors crucial for viral infections like HIV and Vaccinia virus (VACV).

Purpose of the Study:

  • To adapt RNAi for functional genomics of poxvirus genes.
  • To establish high-throughput methods for assessing viral infection.

Main Methods:

  • Utilizing a 96-well format for RNAi screening.
  • Employing flow cytometry and automated microscopy for infection assessment.
  • Using a VACV engineered with a fluorescent reporter under viral gene promoters.

Main Results:

  • The chapter outlines the adaptation of RNAi for poxvirus gene function studies.
  • Two high-throughput methods for quantifying viral infection are described.

Conclusions:

  • RNAi is a versatile tool for poxvirus research.
  • High-throughput screening methods facilitate efficient analysis of viral infection dynamics.
Keywords:
High-throughput screeningPoxvirus genesProtein depletionTransfectionsiRNA

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