Revealing the Role of Actin Cytoskeleton in Herpes Simplex Virus Type 1–Endosome Membrane Fusion by Single-Virus Tracking

The actin cytoskeleton is critical for viral membrane fusion and subsequent release of internal components. However, the actin cytoskeleton's specific role in viral membrane fusion is poorly understood due to its involvement in multiple infection steps and methodological limitations. Here, we achieved dual quantum dot (QD) labeling of both the envelope and capsid of herpes simplex virus type 1 (HSV-1) by combining the streptavidin-Strep-tag II system with a biotinylated phospholipid-incorporated viral membrane labeling strategy. QD-based single-virus tracking revealed that HSV-1 enters MDCK cells via actin-dependent endocytosis, followed by microtubule-mediated transport to the perinuclear region. Critically, actin assembly was dispensable for virus-endosome membrane fusion. And myosin IIB on the actin cytoskeleton provided the mechanical force required for this process. These findings directly demonstrate the role of the actin cytoskeleton in HSV-1 endocytic uptake and subsequent fusion with endosomal membranes. This study provides important insights into the HSV-1 infection mechanism and intracellular membrane fusion processes.