Endocytic and actin genes were most frequently detected by the VSVG-CRE screen.
With the goal of uncovering the host that contributes to viral entry, we compared the three screens for their ability to identify genes that contribute to viral entry from the cell surface. We observed that the ARP2/3 complex was prominent in the VSVG-Cre and Spike-mNG screens, with ACTR2 (both), ARPC4 (both) and ARPC3 (VSVG-Cre only), appearing in the top 15 genes (Figure 3D and 3E). The ARP2/3 complex consists of 7 proteins and is well-known for nucleating and branching actin (Goley & Welch, 2006; Mullins et al., 1998; Pollard, 2007; Robinson et al., 2001; Rouiller et al., 2008), which can aid in endosome formation contribute to SARS-CoV-2 infection (Daniloski et al., 2021; Schmidt et al., 2021; Zhu et al., 2021). The ARP2/3 complex also is important for entry of VSV and a wide range of other viruses (Paluck et al., 2021; Zhu et al., 2021). Given this finding, we expected that the VSVG-CRE screen would be superior at identifying genes contributing to viral entry. Indeed, plotting RRA for VSVG-CRE against VSVG-mNG showed that the VSVG-CRE screen placed multiple subunits of the Arp2/3 at the top, VSVG-mNG was more likely to identify genes associated with protein synthesis and gene expression (such as SEC61B, SEC63 and ARF1) (Figure 3D).
Additionally, the VSVG-CRE screen identified multiple subunits (ATP6V1C1, ATP61G1 and ATP6V0B) of the proton-transporting v-type ATPase complex: ATP6V0D1, ATP6AP2, ATP6V1C1, ATP6V1G1, CCDC115, ATP6V0B, APT6AP1 which are required for endosome acidification and support VSV-G membrane fusion (Breton & Brown, 2013; Icho et al., 2022; Lafourcade et al., 2008). However, we have not identified this pathway in other two screens even though as it has been shown that this complex is important SARS-CoV-2 entry (Icho et al., 2022). In addition, the VSVG-CRE screen identified MESDC2 (Figure 3D) an ER chaperone protein for LDLR folding for proper localization to the plasma membrane (Culi et al., 2004; Hsieh et al., 2003). Taken together, these findings indicate that bypassing the expression of a lentiviral reporter increased the frequency of identifying genes directly contributing to viral endocytosis and entry.
Our VSVG-mNG and Spike-mNG screens identified SEC63 and SEC61B, representing two of the three components of the translocon pore (Linxweiler et al., 2017). The SEC complexes are located in the plasma membrane of the ER and play an important role in the translocation of newly synthesized polypeptide into the ER. The VSVG-mNG screen, also identifies secretory related proteins ARF1, ARFRP1 and KIF13A (Figure 3D) which contribute to trafficking of proteins from the trans-Golgi network to the plasma membrane (Donaldson & Jackson, 2011). Together, these hits may reflect a requirement of an intact secretory pathway transporting ACE2 or LDLR to the plasma membrane.