Genes affecting viral entry were selectively identified with
enhanced resolution with the CRE/floxed reporter over the lentiviral
reporter.
We predicted that the known envelope receptors would be discovered by
the screen with signal strength specificity of the selection. In other
words, as the specificity of the molecular events is increased toward
viral entry and fewer genes associated with other viral activities
affect the selection, the frequency of sgRNAs targeting genes that
influence entry will be enriched as there will be fewer competing gene
sgRNA sequencing amplicons in the low-infection population. Thus, we
expected that that the normalized Log fold change, a normalized
frequency count and the primary statistical output of MaGeCK robust rank
aggregation (RRA) score will increase as the pathway specificity
increases, resulting in fewer genes affected (Li et al., 2014). Indeed,
ACE2 was identified for Spike-mNG entry but not for VSVG-mNG or VSVG-CRE
(Figure 3). Even though ACE2 expression in the ACE2-293T was driven from
a virally integrated cDNA, the sgRNAs in the Brunello library were able
to cut and disable the gene thereby preventing entry of Spike-mNG as
expected. 293T cells without the ACE2 transgene were highly resistant to
the reporter virus prior to expression of ACE2 (unpublished data, (Ou et
al., 2020). We attempted to conduct the comparative screen using
Spike-CRE, however we were unable to generate sufficient Spike-CRE
reporter virus. In the case of VSVG-CRE, our screen identified the
low-density lipoprotein receptor (LDLR, Figure 3C) which is a known
receptor for VSV-G (Finkelshtein et al., 2013; Nikolic et al., 2018).
The VSVG-mNG screen did not identify the LDLR (Figure 3B) with the LDLR
producing a modes LFC (0.83) and weakly significant RRA score (0.0023)
and rank of 205, which is trending toward importance for viral entry but
much weaker than in the VSVG-CRE which had a LFC (1.32) and a highly
significant RRA score (9.23*10-6) and is ranked 13th.
We interpret this improvement in the detection of the LDLR within the
VSVG-CRE reporter screen over the VSV-G-mNG screen due to the reduction
in overall noise arising from the additional genes influencing retro
transcription and viral integration present in the VSVG-mNG screen, but
not in the VSVG-CRE screen.
To compare the effect of the reporter with the same envelope, we plotted
the negative Log (RRA) of VSVG-mNG against negative Log (RRA) of
VSVG-CRE for the top 15 ranked genes for loss of viral reporter
expression in each screen (Figure 3D). Similarly, we plotted the Log
(RRA) for VSVG-mNG against Spike-mNG to allow for comparison of the same
reporter with different envelopes (Figure 3E). In these plots, genes
strongly affecting both screens will appear in the upper righthand
corner. In both plots, SLC35B2 appeared in the upper left indicating a
role for facilitating viral entry (Figure 3D and 3E). This Golgi
associated protein is a transporter for 3’-phosphoadenosine
5’-phosphosulfate and is required for sulfation of heparan sulfate
proteoglycans and has been shown to contribute to entry for SARS-CoV-2,
seasonal coronavirus, and a wide range of other viruses, likely by
heparan sulfate mediated recruitment of viral particles to the cell
surface (Clausen et al., 2020; Park et al., 2017; Schneider et al.,
2021; Tandon et al., 2021; Wang et al., 2021; Q. Zhang et al., 2020).
The ER chaperone Gp96 (HSP90B1) was identified in VSVG-mNG and VSVG-CRE
screens, but not in the Spike-mNG screen indicating that it is specific
to VSV-G mediated entry (Figure 3). Prior work has shown that Gp96
facilitates the correct folding of proteins and is essential for the
presence of functional LDLR at the cell surface and entry of VSV (Hastie
et al., 2013). Its absence in our screen against Spike-mNG is consistent
with it not being identified in other SARS-CoV-2 screens (Baggen et al.,
2021; Daniloski et al., 2021; Schneider et al., 2021; Wang et al., 2021;
Wei et al., 2021) and is consistent with HSP90B1 being a host factor
associated with the cell surface expression of the LDLR and VSV-G
mediated entry.