Isolation of Zika Virus Replication Complex Reveals a Proviral Nuclear Factor

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Abstract Zika virus (ZIKV) is an arthropod-borne flavivirus of international public health impact. ZIKV has a positive-sense, single-stranded RNA genome and remodels intracellular membranes to form replication complexes (RCs). The objective of this study was to isolate and characterize the RCs from ZIKV-infected cells and to identify host-cell components recruited to participate in viral replication. Here, we isolated the RCs from ZIKV-infected Vero cells by detergent treatment and flotation centrifugation. Fractional flotation analysis demonstrated that ZIKV proteins NS2B, NS3, and NS5, and ZIKV RNA were present in the detergent-resistant membranous fraction. In contrast, the ER-resident protein calnexin and a mitochondrial protein were present in the detergent-soluble fractions. The isolated RCs were functional for ZIKV RNA synthesis, as shown by quantitative PCR. To determine the components of the RCs, we conducted mass spectrometry analysis and identified numerous cellular proteins. Among them is the replication factor C subunit 2 (RFC2), an accessory protein of DNA polymerase. RFC2 is involved in ATP binding and hydrolysis and may promote cell survival. ZIKV infection increased the RFC2 protein level and induced its relocation to the cytoplasm. RNAi-mediated silencing of RFC2 reduced ZIKV replication. Together, our results provide insights into ZIKV replication and virus-cell interaction. IMPORTANCE By isolating and characterizing the ZIKV replication complexes (RCs) from infected Vero cells, this research provides valuable insights into the virus’s strategy for hijacking host intracellular membranes and proteins. The identification of ZIKV proteins (NS2B, NS3, and NS5) and RNA in detergent-resistant, lipid-rich membranous structures and the confirmation of functional RCs in RNA synthesis demonstrate successful RC purification. Mass spectrometry and proteomic analysis of the RCs identified numerous cellular proteins that may contribute to ZIKV RC formation and RNA synthesis. Notably, among them is the replication factor C subunit 2 (RFC2), an accessory protein of DNA polymerase in the nucleus. Our results demonstrate that ZIKV increases the level of RFC2 and relocates it to the cytoplasm and that RFC2 plays a role in ZIKV replication, as its depletion reduces viral yield. These results provide insights into ZIKV-cell interactions and potentially facilitate future development of novel antiviral strategies.

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