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Arf1 directly recruits the Pik1-Frq1 PI4K complex to regulate the final stages of Golgi maturation.

ABSTRACT: Proper Golgi complex function depends on the activity of Arf1, a GTPase whose effectors assemble and transport outgoing vesicles. Phosphatidylinositol 4-phosphate (PI4P) generated at the Golgi by the conserved PI 4-kinase Pik1 (PI4KIIIβ) is also essential for Golgi function, although its precise roles in vesicle formation are less clear. Arf1 has been reported to regulate PI4P production, but whether Pik1 is a direct Arf1 effector is not established. Using a combination of live-cell time-lapse imaging analyses, acute PI4P depletion experiments, and in vitro protein-protein interaction assays on Golgi-mimetic membranes, we present evidence for a model in which Arf1 initiates the final stages of Golgi maturation by tightly controlling PI4P production through direct recruitment of the Pik1-Frq1 PI4-kinase complex. This PI4P serves as a critical signal for AP-1 and secretory vesicle formation, the final events at maturing Golgi compartments. This work therefore establishes the regulatory and temporal context surrounding Golgi PI4P production and its precise roles in Golgi maturation.

SUBMITTER: Highland CM

PROVIDER: S-EPMC8101487 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Hazara nairovirus (HAZV) is an enveloped trisegmented negative-strand RNA virus classified within the Nairoviridae family of the Bunyavirales order and a member of the same subtype as Crimean-Congo hemorrhagic fever virus, responsible for fatal human disease. Nairoviral subversion of cellular trafficking pathways to permit viral entry, gene expression, assembly, and egress is poorly understood. Here, we generated a recombinant HAZV expressing enhanced green fluorescent protein and used live-cell fluorescent imaging to screen an siRNA library targeting genes involved in cellular trafficking networks, the first such screen for a nairovirus. The screen revealed prominent roles for subunits of the coat protein 1 (COPI)-vesicle coatomer, which regulates retrograde trafficking of cargo between the Golgi apparatus and the endoplasmic reticulum, as well as intra-Golgi transport. We show the requirement of COPI-coatomer subunits impacted at least two stages of the HAZV replication cycle: an early stage prior to and including gene expression and also a later stage during assembly and egress of infectious virus, with COPI-knockdown reducing titers by approximately 1,000-fold. Treatment of HAZV-infected cells with brefeldin A (BFA), an inhibitor of Arf1 activation required for COPI coatomer formation, revealed that this late COPI-dependent stage was Arf1 dependent, consistent with the established role of Arf1 in COPI vesicle formation. In contrast, the early COPI-dependent stage was Arf1 independent, with neither BFA treatment nor siRNA-mediated ARF1 knockdown affecting HAZV gene expression. HAZV exploitation of COPI components in a noncanonical Arf1-independent process suggests that COPI coatomer components may perform roles unrelated to vesicle formation, adding further complexity to our understanding of cargo-mediated transport.IMPORTANCE Nairoviruses are tick-borne enveloped RNA viruses that include several pathogens responsible for fatal disease in humans and animals. Here, we analyzed host genes involved in trafficking networks to examine their involvement in nairovirus replication. We revealed important roles for genes that express multiple components of the COPI complex, which regulates transport of Golgi apparatus-resident cargos. COPI components influenced at least two stages of the nairovirus replication cycle: an early stage prior to and including gene expression and also a later stage during assembly of infectious virus, with COPI knockdown reducing titers by approximately 1,000-fold. Importantly, while the late stage was Arf1 dependent, as expected for canonical COPI vesicle formation, the early stage was found to be Arf1 independent, suggestive of a previously unreported function of COPI unrelated to vesicle formation. Collectively, these data improve our understanding of nairovirus host-pathogen interactions and suggest a new Arf1-independent role for components of the COPI coatomer complex.

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Arf1 directly recruits the Pik1-Frq1 PI4K complex to regulate the final stages of Golgi maturation.

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