Uncovering the F-Actin-Based Nuclear Egress Mechanism of Newly Synthesized Influenza A Virus Ribonucleoprotein Complexes by Single-Particle Tracking.

Nuclear trafficking of viral genome is an essential cellular process in the life cycles of viruses. Despite substantial progress in uncovering a wide variety of complicated mechanisms of virus entry, intracellular transport of viral components, virus assembly, and egress, the temporal and spatial dynamics of viral genes trafficking within the nucleus remains poorly understood. Herein, using single-particle tracking, we explored the real-time dynamic nuclear trafficking of influenza A virus (IAV) genes packaged as the viral ribonucleoprotein complexes (vRNPs) by combining a four-plasmid DNA transfection system for the reconstruction of green fluorescent protein (GFP)-labeled vRNPs and a spinning disk super-resolution fluorescence microscope. We found that IAV infection significantly induced the formation of actin microfilaments (F-actin) in the nucleus. In combination with the fluorescent protein-tagged nuclear F-actin probe, we visualized the directed movement of GFP-labeled vRNPs foci along the nuclear F-actin with a speed of 0.18 μm/s, which is similar to the microfilaments-dependent slow directed motion of IAVs in the cytoplasm. The disruption of nuclear F-actin after treatment with microfilament inhibitors caused a considerable decrease in vRNPs motility and suppressed the nuclear export of newly produced vRNPs, indicating that the slow, directed movement plays a crucial role in facilitating the nuclear egress of vRNPs. Our findings identified a nuclear F-actin-dependent pathway for IAV vRNPs transporting from the nucleus into the cytoplasm, which may in turn uncover a novel target for antiviral treatment.