Human Papillomavirus Type 16 Entry: Retrograde Cell Surface Transport along Actin-Rich Protrusions

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Abstract
The lateral mobility of individual, incoming human papillomavirus type 16 pseudoviruses (PsV) bound to live HeLa cells was studied by single particle tracking using fluorescence video microscopy. The trajectories were computationally analyzed in terms of diffusion rate and mode of motion as described by the moment scaling spectrum. Four distinct modes of mobility were seen: confined movement in small zones (30–60 nm in diameter), confined movement with a slow drift, fast random motion with transient confinement, and linear, directed movement for long distances. The directed movement was most prominent on actin-rich cell protrusions such as filopodia or retraction fibres, where the rate was similar to that measured for actin retrograde flow. It was, moreover, sensitive to perturbants of actin retrograde flow such as cytochalasin D, jasplakinolide, and blebbistatin. We found that transport along actin protrusions significantly enhanced HPV-16 infection in sparse tissue culture, cells suggesting a role for in vivo infection of basal keratinocytes during wound healing. To replicate, viruses have to enter into host cells. Since they have no means of locomotion, they rely entirely on cellular transport systems to access the cellular compartments where replication occurs. Following individual virus particles by video microscopy, we found that human papillomavirus type 16, the main causative agent of cervical cancer, bound to long finger-like protrusions of cells. There, they were transported from the periphery to the cell body. The transport was mediated by a process termed actin retrograde flow, where viruses bound to cell surface molecules hooked up to filamentuos actin and were dragged along with the actin-like items on a transport belt. Entry into the cell occured at the cell body. The results raised the interesting possibility that viruses use retrograde flow when they infect wounded epidermal keratinocytes, where finger-like protrusions of cells are abundant.