CK2 and N Co-localize at Virus-Induced Filopodial Protrusions
The phosphoproteomics data indicated regulation of several kinases and effector proteins related to cytoskeleton organization upon SARS-CoV-2 infection. Kinases downstream of the Rho/Rac/Cdc42 GTPases (PAK1/2 and ROCK1/2) and several well-characterized phosphorylation site targets of PAK1/2 kinase in vimentin (VIM S39 and S56) and stathmin (STMN1 S16 and S25) were found to be downregulated during infection (Figures 5A and 5B). The interaction of Nsp7 with RHOA (Gordon et al., 2020) may contribute to this downregulation. In contrast, signaling via CK2 is strongly upregulated, as determined by the increase in phosphorylation of well-characterized target sites (Figures 5A and 5B). Among the many roles of this kinase, we noted increased phosphorylation of cytoskeleton protein targets such as ɑ-Catenin (CTNNA1 S641) and the heavy chain of the motor protein Myosin IIa (MYH9 S1943). In addition to these kinase-mediated effects, the Nsp2 protein of SARS-CoV-2 also interacts directly with strumpellin (WASHC5), a subunit of the actin assembly-inducing WASH complex (Gordon et al., 2020), further implicating cytoskeleton regulation during infection. To study the relevance of these observations in a human infection model, high-resolution immunofluorescence imaging of fixed Caco-2 human colon epithelial cells was performed 24 h after infection (STAR Methods).
Figure 5 Colocalization of CK2 and Viral Proteins at Actin Protrusions
(A) Pathway of regulated PHs and SARS-CoV-2 interaction partners involved in cytoskeletal reorganization. Dashed lines indicate downregulation of activity, while solid lines indicate upregulation of activity.
(B) Regulation of individual kinase activity or PHs depicted in (A).
(C) Caco-2 cells infected with SARS-CoV-2 at an MOI of 0.1 for 24 h prior to immunostaining for F-actin and M protein, as indicated. Shown is a confocal section revealing M protein localization along and to the tip of filopodia (left) and magnification of the dashed box (right).
(D) Dot plot quantification of the number and length of filopodia in untreated (mock) or infected Caco-2 cells for 24 h with SARS-CoV-2. Filopodium length was measured from the cortical actin to the tip of the filopodium. Error bars represent SD. Statistical testing by Mann-Whitney test.
(E) Caco-2 cells infected with SARS-CoV-2 at an MOI of 0.01 for 24 h prior to immunostaining for F-actin, N protein, and casein kinase II (CK2) as indicated (left). Shown is magnification of the dashed box as single channels (right).
(F) Magnification of the dashed box from (E) with quantification of colocalization between CK2 and N protein throughout infected Caco-2 cells. Displayed is the proportion of N protein-positive particles colocalizing with CK2. Error bars represent SD.
(G and H) Scanning electron microscopy (G) and transmission electron microscopy (H) images of SARS-CoV-2 budding from Vero E6 cell filopodia (black arrow in H).
See also Figure S3.
SARS-CoV-2 infected Caco-2 cells were imaged for filamentous actin and the SARS-CoV-2 M protein, revealing prominent M protein clusters, possibly marking assembled SARS-CoV-2 viral particles, localized along the shafts and at the tips of actin-rich filopodia (Figures 5B and S3 B). SARS-CoV-2 infection induced a dramatic increase in filopodial protrusions, which were significantly longer and more branched than in uninfected cells (Figure 5D). Uninfected cells also exhibited filopodial protrusions, but their frequency and shape were dramatically different (Figure S3A). Reorganization of the actin cytoskeleton is a common feature of many viral infections and is associated with different stages of the viral life cycle (Taylor et al., 2011).
Figure S3 Microscopy Images Showing Response to SARS-CoV-2 Infection, Related to Figure 5
(A) Non-infected Caco2 cells co-stained for F-actin, CK2 and nuclei (DAPI). Magnification of the indicated area is displayed as a single channel and merged images on the right panels. (B) Caco2 cells infected with SARS-CoV-2 at an MOI of 0.1 for 24 h prior to immunostaining for F-actin and M-protein, as indicated. See lower (1) and right (2) panel for magnification of regions indicated by dashed boxes. (C) Scanning electron microscopy and (D) transmission electron microscopy image of SARS-CoV-2 budding from Vero E6 cell filopodia. (E) N protein was found to physically interact with casein kinase II subunits (cartoon, left), CSNK2B and CSNK2A2 (Gordon et al., 2020). To test whether N protein could directly control CK2 activity, N protein was transduced via lentivirus in Vero E6 cells and stably induced via doxycycline for 48 hours followed by phosphoproteomics analysis. Kinase activities were calculated as before (STAR Methods) and top up- (> 1.5, red) and downregulated (< 1.5, blue) kinases are shown. See Table S1 for full phosphoproteomics data and Table S4 for full list of predicted kinase activities.
We hypothesize that induction of virus-containing filopodia could be important for SARS-CoV-2 egress and/or cell-to-cell spread within epithelial monolayers. Given that Rho/PAK/ROCK signaling is downregulated, we next asked whether CK2 could play a role in this process. At 24 h, infected cells showed CK2 expression along the thin filopodial protrusions (Figure 5E), partially co-localized with SARS-CoV-2 N protein (Figure 5F). Scanning and transmission electron microscopy were used (Figures 5G, 5H, S3C, and S3D) to image the cellular protrusions at higher resolution. Assembled viral particles are clearly visible along these filopodia (Figure 5G), with instances where the viral particles appear to be budding from the protrusions (Figure 5H). Finally, we performed a global phosphoproteomics analysis of Vero E6 cells overexpressing N protein and observed CK2 activity to be significantly upregulated (Figure S3E; Tables S1 and S4). Because CK2 activity can promote actin polymerization (D’Amore et al., 2019), we hypothesize that N protein may allosterically control CK2 activity and regulate cytoskeleton organization.