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.