Phosphorylation of SARS-CoV-2 Host-Interacting Proteins during Infection
The recently published SARS-CoV-2 virus-human protein-protein interaction map identified 332 human proteins interacting with 27 (26 wild-type and 1 mutant) viral proteins (Gordon et al., 2020). Here we found some of these host proteins (40 of 332) to be significantly differentially phosphorylated upon infection (Figure 3 ). Virus-host protein-protein interactions could drive changes in phosphorylation by affecting host protein subcellular localization or by sterically blocking kinase access. Furthermore, phosphorylation of these proteins upon infection may signify an additional mode of functional control over these potential dependency and restriction factors.
Figure 3 Phosphorylation on SARS-CoV-2 Virus-Human Interacting Proteins
The SARS-CoV-2 virus-host protein-protein interaction map (Gordon et al., 2020) found 332 human proteins interacting with 27 (26 wild-type and 1 mutant) viral proteins. Here we found 40 of 332 proteins significantly differentially phosphorylated across at least two time points (adjusted p < 0.05 and absolute value of log2 fold change [abs(log2FC)] > 1). Viral proteins are shown as red diamonds. Interacting human proteins are shown as gray circles. PHs emanate from human proteins, colored by their log2 fold change compared with uninfected control samples (red, increase; blue, decrease) at each time point (0, 2, 4, 8, 12, and 24 h after infection) in a clockwise fashion. An interactive version of phosphorylation data can be found at https://kroganlab.ucsf.edu/network-maps.
The SARS-CoV-2 N protein is known to interact with several RNA-processing proteins that are differentially phosphorylated during infection, including LARP1 and RRP9. Here LARP1 phosphorylation decreases on several sites, which is known to consequently increase LARP1 affinity for 3′ untranslated regions (UTRs) of mRNAs encoding ribosomal proteins, driving inhibition of protein synthesis (Hong et al., 2017). This mechanism may be utilized by SARS-CoV-2 to prioritize synthesis of viral proteins over host proteins. In addition, ORF6 interacts with the NUP98/RAE complex, and NUP98 phosphorylation was observed to increase at S888, a site within its peptidase domain. NUP98 autocatalytic cleavage is required for localization to the nuclear pore; thus, it is possible that NUP98 interaction with ORF6 and/or its virus-induced phosphorylation prevents host mRNA export through the nuclear pore (Krull et al., 2010; Hodel et al., 2002). A similar mechanism is employed by vesicular stomatitis virus (VSV) matrix protein to block host mRNA export by targeting the NUP98/RAE complex, leading to exclusive translation of cytoplasmic VSV mRNAs (Quan et al., 2014).
For Nsp12, the majority of its protein interactors displayed decreased phosphorylation during infection. Because Nsp12 is known to encode the RNA-dependent RNA polymerase, responsible for replicating the viral genome, and several of these interacting proteins are related to RNA processing (LARP4B and CRTC3), their regulation may possess functional implications for Nsp12 in viral RNA replication. In addition, Nsp8 interacts with several proteins whose phosphorylation increases (LARP7 and MPHOSPH10) and decreases (CCDC86) on several sites. Notably, LARP7 and MEPCE are important regulators of RNA polymerase II-mediated transcription elongation as part of the 7SK small nuclear ribonucleoprotein particle (snRNP) complex. Regulation of these phosphorylation sites may contribute to the regulation of positive transcription elongation factor b (P-TEFb [CDK9]) and transcriptional regulation of the virus, similar to how these proteins are regulated during HIV infection (Mbonye et al., 2015).