Analysis of phosphomotifs coupled to phosphoproteome and interactome unveils potential human kinase substrate proteins in SARS-CoV-2

Vineetha Shaji^1,2Rajesh Raju^1*Ahmad Rafi¹Dr. Mukhtar Ahmed³Athira Perunelly Gopalakrishnan^1,2Sowmya Soman¹Amjesh Revikumar¹Ganesh Prasad⁴Abhithaj Jayanandan¹

• ¹Centre for Integrative Omics Data Science, Yenepoya (Deemed to be University), Mangalore, India
• ²Center for Systems Biology and Molecular Medicine, Yenepoya University, Mangalore, Karnataka, India
• ³Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia
• ⁴Department of Biochemistry, Yenepoya Medical College, Yenepoya (Deemed to be University), Mangalore, India., Mangalore, India

Employing the protein kinase substrate sequence-preference motifs derived by Poll B G. et. al., 2024, we performed kinase-substrate phosphomotif pattern analysis on the SARS-CoV-2 proteome. We identified major host kinases by analyzing SARS-CoV-2 perturbed phosphoproteomes from various studies and cell systems. These kinases were subjected to interactome analysis and literature-based validation, while conservation of viral phosphosites across SARS-CoV-2 variants was assessed; Subsequently, an in-silico protein-protein docking approach was employed to validate MAP2K1 (Mitogen-activated protein kinase kinase 1) as one of the kinases with several known and novel viral protein phosphosites.The human kinome-substrate phosphomotif analysis predicted 49 kinases capable of phosphorylating 639 phosphosites across 33 SARS-CoV-2 proteins. From these, 24 kinases were also perturbed in SARS-CoV-2-infected phosphoproteomes. Literature review identified seven kinases, including MAP2K1, whose inhibition may reduce viral replication. MAP2K1 was found to target key viral phosphosites, including N protein (S206, T198) and ORF9b (S50), conserved across SARS-CoV-2 variants. Docking analysis showed MAP2K1 forms stronger, closer interactions with N protein compared to SRPK1, highlighting MAP2K1 as a potential host kinase for therapeutic targeting in SARS-CoV-2 infection.This study presents a framework for predicting SARS-CoV-2 phosphosites by integrating kinase specificity, virus-host interactions, and post-translational modifications. MAP2K1 was identified as a key host kinase, showing stronger interactions than SRPK1, and is proposed as an antiviral drug target for repurposing in SARS-CoV-2 and other viral infections.