Tracking the Replication-Competent Zika Virus with Tetracysteine-Tagged Capsid Protein in Living Cells

Area of Science:

• Virology
• Cell Biology
• Molecular Biology

Background:

• Real-time imaging of virus-host interactions is crucial for understanding viral pathogenesis.
• Zika virus (ZIKV), a flavivirus causing microcephaly, presents challenges for genetic engineering due to its monocistronic genome.
• A fluorescently labeled, replication-competent ZIKV is needed for advanced imaging studies.

Purpose of the Study:

• To generate a replication-competent, fluorescently labeled ZIKV suitable for real-time imaging.
• To investigate ZIKV-host interactions at the molecular and single-particle levels.
• To establish a novel tool for studying ZIKV entry and intracellular dynamics.

Main Methods:

• Introduction of a biarsenical tetracysteine (TC) tag into the ZIKV capsid protein at six different sites.
• Rescue and characterization of genetically engineered ZIKV strains for replication and infectivity.
• Fluorescent labeling of the TC-tagged ZIKV with biarsenical reagents for live-cell imaging and single-particle tracking.

Main Results:

• Successful generation of a replication-competent ZIKV (TC27 ZIKV) by inserting the TC tag at amino acids 27/28 of the capsid protein.
• Visualization of dynamic nuclear import of the ZIKV capsid protein using fluorescently labeled TC27 ZIKV.
• Real-time imaging evidence of ZIKV movement along cellular filopodia and entry via endocytosis using single-particle tracking.

Conclusions:

• A feasible strategy for generating replication-competent, TC-tagged ZIKV for real-time imaging has been established.
• The TC27 ZIKV facilitates the study of ZIKV-host interactions at both protein and viral particle levels.
• This tool opens new avenues for investigating ZIKV pathogenesis and antiviral screening.