SARS-CoV-2 infection remodels the host protein thermal stability landscape.

Bernhard Küster ,Frank Stein,Mandy Rettel,Athanasios Typas,Carlos G.p. Voogdt ,Nils Kurzawa,Iñigo Barrio-Hernandez ,Ralf Bartenschlager,André Mateus,Joel Selkrig,Karin Mitosch,Clement M Potel ,Philipp Walch,Megan L. Stanifer ,Anna Jarząb ,Heeyoung Kim,Pedro Beltrão ,Mikhail M. Savitski ,Steeve Boulant,Carmon Kee

doi:10.15252/msb.202010188

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is a global threat to human health and has compromised economic stability. In addition to the development of an effective vaccine, it is imperative to understand how SARS‐CoV‐2 hijacks host cellular machineries on a system‐wide scale so that potential host‐directed therapies can be developed. In situ proteome‐wide abundance and thermal stability measurements using thermal proteome profiling (TPP) can inform on global changes in protein activity. Here we adapted TPP to high biosafety conditions amenable to SARS‐CoV‐2 handling. We discovered pronounced temporal alterations in host protein thermostability during infection, which converged on cellular processes including cell cycle, microtubule and RNA splicing regulation. Pharmacological inhibition of host proteins displaying altered thermal stability or abundance during infection suppressed SARS‐CoV‐2 replication. Overall, this work serves as a framework for expanding TPP workflows to globally important human pathogens that require high biosafety containment and provides deeper resolution into the molecular changes induced by SARS‐CoV‐2 infection.

Highlights

SARS-CoV-2, the novel severe acute respiratory syndrome coronavirus has led to a worldwide pandemic that is upending economic stability and poses a tremendous burden on healthcare systems
To ensure compatibility with a biosafety level 3 (BSL3) working environment, we first adapted our standard thermal proteome profiling (TPP) protocol 18 by replacing centrifugation steps - which can otherwise lead to the generation of aerosols containing airborne pathogens - with a filtration step aided by vacuum for protein aggregate removal that can be performed inside a HEPA filtered laminar flow cabinet (Figure 1a; see Methods for details)
Caco-2 cells, which are permissive to SARS-CoV-2 infection[1,19], were infected in triplicate with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.5 for 1 hour, after which unbound viral particles were removed and samples were harvested at [1, 2, 4, 7, 12, 24] and 48 hours post infection (Figure 1a)

Summary

Introduction

SARS-CoV-2, the novel severe acute respiratory syndrome coronavirus has led to a worldwide pandemic that is upending economic stability and poses a tremendous burden on healthcare systems. SARS-CoV-2 hijacks host protein cell processes for its replication, packaging, and release, leading to alterations in cell signaling and protein expression [1,2,3,4]. Genetic approaches are shedding light onto the functional relevance of such large-scale proteomic information [5,6,7], complementary approaches are needed to expand our understanding of the biophysical changes that proteins can undergo within a live infection context. Many physiological changes to the functional state of a protein are reflected in altered protein thermostability [8,9]. Applying TPP to viral pathogens can provide a comprehensive and orthogonal view on various protein state changes during infection, and highlight key proteins and cellular processes required for viral replication. No study to date has applied TPP to the infection context under biosafety level 3 (BSL3) conditions

Methods

Results

Conclusion