Meta-analysis of genome-wide CRISPR screenings reveals host dependency and restriction factors of the SARS-CoV-2 replication cycle

SARS-CoV-2, the etiologic agent that causes COVID-19, remains a global threat to public health. Understanding the complex interplay between SARS-CoV-2 and its host cell is fundamental to identify novel targets for antiviral or anti-inflammatory therapy. Being provided with two genome-scale CRISPR/Cas9 loss-of-function screenings that were performed in the lung epithelial cell lines A549-ACE2 and Calu-3, I set out to identify analogies between the loss-of-function screenings conducted in our laboratory and previously published screenings that were performed in other cellular models of infection. The rationale behind this approach was to determine which host cell factors have conserved functions during SARS-CoV-2 infection in a variety of cell lines and might therefore be physiological contributors to SARS-CoV-2 propagation and pathogenesis. I employed thresholding to identify factors that exhibited proviral function in all of the six analyzed loss-of-function screenings. This approach yielded 24 potential host cell factors of which angiotensin converting enzyme 2 (ACE2), the primary receptor of SARS-CoV-2, showed the highest average enrichment, validating this approach. The remaining 23 factors have not been described in the context of SARS-CoV-2 infection so far. Due to its interesting location in the late endosome and its prominent involvement in membrane trafficking and endosome biogenesis, I selected RAB9A for detailed validation and mechanistic investigation. In addition, TIMM17B, an integral component of the TIM23 complex that imports presequence containing proteins into the mitochondrial matrix, was validated. RAB9A supported SARS-CoV-2 infection in a variety of cell lines and using a variety of virus isolates. As evidenced by a reduction of SARS-CoV-2 infection of cells depleted of the RAB9A guanine nucleotide exchange factor DENND2D, RAB9A’s proviral activity seems to require the conversion of its inactive GDP-bound form to active GTP-bound RAB9A. Furthermore, and in line with previous reports, RAB9A depletion resulted in morphological changes of the cellular lysosome population, confirming an involvement of RAB9A in endo-lysosome biogenesis. Interestingly, ectopic expression of the host protease TMPRSS2 that is required for SARS-CoV-2 entry at the plasma membrane mitigated the observed reduction in SARS-CoV-2 infection in RAB9A-depleted cells, indicating a specific involvement of RAB9A during endocytic entry, which can proceed in the absence of TMPRSS2 and instead requires the endosomal activity of cathepsin proteases. This assumption was confirmed by means of propagation-deficient vesicular stomatitis virus pseudotypes. Consistently, RAB9A depletion increased SARS-CoV-2 uptake, followed by a retention of virions in the late endosomal and lysosomal compartment, likely indicating deficits in the initiation of fusion during entry. The relevance of RAB9A for the fusion of SARS-CoV-2 with membranes of the late endosome was confirmed employing lentiviral pseudotypes. Interestingly, the second host cell factor identified in the meta-analysis, TIMM17B, does not support but significantly suppress SARS-CoV-2 infection in A549-ACE2 and Calu-3 lung epithelial cells, arguing that it is a restriction factor. It remains unclear how TIMM17B-targeting sgRNAs became enriched in all six analyzed loss-of-function screenings. Mechanistic follow-up studies show that TIMM17B is most likely involved in the early stages of the viral life cycle. However, the enhancement of entry in the absence of TIMM17B is very moderate, suggesting that TIMM17B might restrict other steps of SARS-CoV-2 replication. Further investigation will be necessary to address this question. In summary, by integrating the results of various CRISPR/Cas9 screenings I was able to identify RAB9A as a SARS-CoV-2 dependency factor that supports fusion during entry into various cellular models. In addition, TIMM17B was identified to be a SARS-CoV-2 restriction factor affecting, at least in part, the early stages of the viral life cycle. The meta-analysis approach used here will aid to identify relevant host cell factors and reduce the number of false-positive hits caused by technical or cell line specific artifacts.