Protocol

Virus-Host Interactions

Volume 1064 of the series Methods in Molecular Biology pp 43-70

Date:

Discovery of Host–Viral Protein Complexes During Infection

  • Daniell L. RowlesAffiliated withDepartment of Molecular Biology, Princeton University
  • , Scott S. TerhuneAffiliated withDepartment of Microbiology and Molecular Genetics, Biotechnology and Bioengineering Center, Medical College of Wisconsin
  • , Ileana M. CristeaAffiliated withDepartment of Molecular Biology, Princeton University

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Abstract

Viruses have co-evolved with their hosts, developing effective approaches for hijacking and manipulating host cellular processes. Therefore, for their efficient replication and spread, viruses depend on dynamic and temporally regulated interactions with host proteins. The rapid identification of host proteins targeted by viral proteins during infection provides significant insights into mechanisms of viral protein function. The resulting discoveries often lead to unique and innovative hypotheses on viral protein function. Here, we describe a robust method for identifying virus–host protein interactions and protein complexes, which we have successfully utilized to characterize spatial–temporal protein interactions during infections with either DNA or RNA viruses, including human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-1), pseudorabies virus (PRV), human immunodeficiency virus (HIV-1), Sindbis, and West Nile virus (WNV). This approach involves cryogenic cell lysis, rapid immunoaffinity purification targeting a virus or host protein, followed by identification of associated proteins using mass spectrometry. Like most proteomic approaches, this methodology has evolved over the past few years and continues to evolve. We are presenting here the updated approaches for each step, and discuss alternative strategies allowing for the protocol to be optimized for different biological systems.

Key words

Virus–host interactions Infection Epitope tag Immunoaffinity purification Cryogenic cell lysis Proteomics Protein complex Protein–protein interactions Mass spectrometry