This study investigates the role of the papain-like protease (PLpro) in SARS-CoV-2 infection and innate immunity. PLpro is essential for viral replication as it processes viral polyproteins to form a functional replicase complex. It also cleaves host proteins, potentially evading immune responses. The study compares SARS-CoV-2 PLpro (SCoV2-PLpro) with SARS-CoV PLpro (SCoV-PLpro), revealing differences in their regulation of host interferon and NF-κB pathways. SCoV2-PLpro preferentially cleaves ISG15, while SCoV-PLpro targets ubiquitin chains. Structural analysis shows SCoV2-PLpro has a higher affinity for ISG15, contributing to its role in viral spread and immune evasion. Inhibiting SCoV2-PLpro with GRL-0617 reduces viral replication and enhances antiviral immunity. The study highlights the potential of targeting SCoV2-PLpro as a therapeutic strategy against SARS-CoV-2. The findings suggest that SCoV2-PLpro plays a critical role in modulating host immune responses, and its inhibition could help in developing dual therapeutic approaches to combat the virus. The research also underscores the importance of understanding the molecular mechanisms underlying the differences in pathogenicity between SARS-CoV and SARS-CoV-2.This study investigates the role of the papain-like protease (PLpro) in SARS-CoV-2 infection and innate immunity. PLpro is essential for viral replication as it processes viral polyproteins to form a functional replicase complex. It also cleaves host proteins, potentially evading immune responses. The study compares SARS-CoV-2 PLpro (SCoV2-PLpro) with SARS-CoV PLpro (SCoV-PLpro), revealing differences in their regulation of host interferon and NF-κB pathways. SCoV2-PLpro preferentially cleaves ISG15, while SCoV-PLpro targets ubiquitin chains. Structural analysis shows SCoV2-PLpro has a higher affinity for ISG15, contributing to its role in viral spread and immune evasion. Inhibiting SCoV2-PLpro with GRL-0617 reduces viral replication and enhances antiviral immunity. The study highlights the potential of targeting SCoV2-PLpro as a therapeutic strategy against SARS-CoV-2. The findings suggest that SCoV2-PLpro plays a critical role in modulating host immune responses, and its inhibition could help in developing dual therapeutic approaches to combat the virus. The research also underscores the importance of understanding the molecular mechanisms underlying the differences in pathogenicity between SARS-CoV and SARS-CoV-2.