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The papain-like protease (PLpro) is a critical target due to its dual role in viral replication and immune evasion, particularly through the removal of ISG15 modifications from host proteins. However, nanobody-based strategies targeting PLpro for combined antiviral and anti-inflammatory purposes remain underdeveloped. This study reports the identification of two nanobodies, NbP1 and NbP2, that specifically disrupt the PLpro\/ISG15 interaction interface. We characterized their binding specificity and affinity using yeast surface display and competitive fluorescence polarization assays. To ensure intracellular efficacy, the nanobodies were conjugated with cell-penetrating peptides (CPPs), resulting in significant inhibition of viral replication and the attenuation of inflammatory cytokine responses in both human colonic cells and a murine colitis model. Structural epitope mapping revealed that both nanobodies bind to key conserved residues within the PLpro\/ISG15 and PLpro\/ubiquitin interfaces. Our findings demonstrate that nanobodies targeting the PLpro\/ISG15 interface can achieve synergistic antiviral and immunomodulatory effects, providing a proof-of-concept for a novel therapeutic approach to combat SARS-CoV-2 and potentially other emerging coronaviruses.<\/jats:p>\n                    <jats:sec>\n                      <jats:title>\n                        <jats:bold>IMPORTANCE<\/jats:bold>\n                      <\/jats:title>\n                      <jats:p>The COVID-19 pandemic caused by SARS-CoV-2 has resulted in millions of deaths worldwide. Despite the emergence of antiviral drugs and vaccines targeting 3CLpro and RNA-dependent RNA polymerase (Rdrp), the virus\u2019s continuous mutation underscores the need for novel therapeutic approaches that target highly conserved viral regions. PLpro is an attractive target due to its roles in viral replication and host immune regulation. However, research on nanobodies targeting PLpro remains in its infancy. This study provides significant insights into the antiviral and anti-inflammatory functions of two nanobodies, NbP1 and NbP2, which specifically disrupt the PLpro\/ISG15 interaction interface. Our findings have important implications for drug development targeting SARS-CoV-2, highlighting the potential of nanobody-based therapeutics to simultaneously inhibit viral replication and suppress pathological inflammation.<\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/jvi.00599-26","type":"journal-article","created":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T13:00:24Z","timestamp":1787922024000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Nanobodies targeting SARS-CoV-2 papain-like protease exert dual antiviral and anti-inflammatory effects"],"prefix":"10.1128","volume":"100","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-2704-3137","authenticated-orcid":false,"given":"Guolong","family":"Liu","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01vjw4z39","id-type":"ROR","asserted-by":"publisher"}],"name":"Guangdong 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