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The replication organelles (ROs) of coronavirus associate with modified host endomembrane; however, the molecular mechanisms underlying the expansion and modification of these membranes remain poorly understood. Here, we show that viral protein orf3a collaborates with nsp3, nsp4, nsp6 to facilitate the formation of ROs in SARS-CoV-2. Importantly, orf3a targets LDs to ROs, establishing novel membrane contact sites and induces host cell microlipophagy, which supplies essential lipids for RO biogenesis. Subsequently, Following the formation of ROs, nsp3, with assistance from nsp12, indirectly recruits phosphatidylinositol 4-kinase beta (PI4KB) to ROs, to produce phosphatidylinositol 4-phosphate (PI4P). This action creates a PI4P-enriched microenvironment that enhances SARS-CoV-2 replication. Our findings elucidate the mechanism governing RO generation during SARS-CoV-2 infection and suggest that targeting microlipophagy pharmacologically may represent a promising strategy for the development of anti-coronaviruses therapies.<\/jats:p>","DOI":"10.1371\/journal.ppat.1013676","type":"journal-article","created":{"date-parts":[[2025,11,21]],"date-time":"2025-11-21T18:45:48Z","timestamp":1763750748000},"page":"e1013676","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":8,"title":["Reprogramming of lipid metabolism by ORF3a-induced microlipophagy enhances biogenesis of SARS-CoV-2 replication 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