Functional interrogation of a SARS-CoV-2 host protein interactome identifies unique and shared coronavirus host factors

Abstract

SUMMARYThe ongoing SARS-CoV-2 pandemic has devastated the global economy and claimed nearly one million lives, presenting an urgent global health crisis. To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen four related coronaviruses (HCoV-229E, HCoV-NL63, HCoV-OC43 and SARS-CoV-2) at two physiologically relevant temperatures (33 °C and 37 °C), allowing us to probe this interactome at a much higher resolution relative to genome scale studies. This approach yielded several new insights, including unexpected virus and temperature specific differences in Rab GTPase requirements and GPI anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating COVID-19, and help prepare for future coronavirus outbreaks.HIGHLIGHTSFocused CRISPR screens targeting host factors in the SARS-CoV-2 interactome were performed for SARS-CoV-2, HCoV-229E, HCoV-NL63, and HCoV-OC43 coronaviruses.Focused interactome CRISPR screens achieve higher resolution compared to genome-wide screens, leading to the identification of critical factors missed by the latter.Parallel CRISPR screens against multiple coronaviruses uncover host factors and pathways with pan-coronavirus and virus-specific functional roles.The number of host proteins that interact with a viral bait protein is not proportional to the number of functional interactors.Novel SARS-CoV-2 host factors are expressed in relevant cell types in the human airway.

Functional genomic screens identify human host factors for SARS-CoV-2 and common cold coronaviruses

“…In particular, genes involved in cholesterol homeostasis were enriched in all screens and in the network propagation. Consistent with our data, two SARS-CoV-2 interactomes revealed binding of viral proteins to SCAP, and a recent CRISPR screen focused on the interactome components also identified SCAP as a host factor critical for SARS-CoV-2 replication 16,17,21 ; given the essentiality of SCAP for replication, viral proteins are likely to positively regulate SCAP activity and cholesterol levels. Cellular cholesterol homeostasis has previously been linked to viral entry and membrane fusion in the context of bunya-and hantavirus infections, suggesting a pro-viral function across different viral families [60][61][62] .…”

“…Several groups have already successfully applied this approach, yet with certain limitations, e.g. the use of Vero green monkey cells instead of a human cell line 20 , the use of a small CRISPR library only based on the SARS-CoV-2 protein interactome 21 , or the use of a SARS-CoV-2 harboring a deletion in the spike S1/S2 site due to cell culture adaptation 22 .…”

Genome-Scale Identification of SARS-CoV-2 and Pan-coronavirus Host Factor Networks

“…We screened Cas9-expressing Huh-7.5 hepatoma cells (Huh-7.5-Cas9), which endogenously express the SARS-CoV-2 cellular receptor, angiotensin-converting enzyme 2 ( ACE2 ), as well as transmembrane serine protease 2 ( TMPRSS2 ), a key mediator of SARS-CoV-2 entry ( Hoffmann et al., 2020 ). We recently showed that Huh-7.5-Cas9 cells are permissive to infection by SARS-CoV-2, HCoV-OC43, HCoV-NL63, and HCoV-229E and that they are a robust system for CRISPR-based genetic screening ( Hoffmann et al., 2020b ).

Figure 1 Genome-wide CRISPR Screens Identify Host Factors Required for SARS-CoV-2 Infection (A) Genome-wide CRISPR screening workflow.

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“…The HCoV-229E screen, although successful, was particularly stringent, resulting in a lower area under the curve (AUC) relative to the other screens in this study. As described in our recent study ( Hoffmann et al., 2020b ), we performed a Z score analysis and computed a gene essentiality (beta) score using a published maximum likelihood estimation (MLE) algorithm ( Li et al., 2014 ). The gene essentiality analysis allowed us to stratify candidate host factor targets based on their effects on cellular fitness under mock-infected conditions followed by identification of high-confidence gene hits in virus-infected cells.…”

Sex differences in viral entry protein expression and host transcript responses to SARS-CoV-2

“…In addition to reported immune response differences, we found vast differences in non-immune cells such as mitochondria functions, phagocytosis, and cholesterol biosynthesis, suggesting men and women have unique response trajectories after infection and latency. Both large-scale genome-wide CRISPR screenings confirmed that cholesterol synthesis is a key host response factor and targeting cholesterol synthesis using small molecules like 27-hydroxycholesterol could reduce SARS-CoV-2 infection in vitro [32][33][34] . Of note, independent of sex, younger people (<=60) have much more differentially expressed genes than older people (>60); independent of age, women tend to have more differentially expressed genes than men.…”