Abstract
Parvoviral genome translocation from the plasma membrane into the nucleus is a coordinated multistep process mediated by capsid proteins. We used fast confocal microscopy line scan imaging combined with image correlation methods including auto-, pair- and cross-correlation, and number and brightness analysis, to study the parvovirus entry pathway at the single-particle level in living cells. Our results show that the endosome-associated movement of virus particles fluctuates from fast to slow. Fast transit of single cytoplasmic capsids to the nuclear envelope is followed by slow movement of capsids and fast diffusion of capsid fragments in the nucleoplasm. The unique combination of image analyses allowed us to follow the fate of intracellular single virus particles and their interactions with importin β revealing previously unknown dynamics of the entry pathway.
Highlights
Recent studies elucidating the dynamics of nuclear-replicating virus entry into host cells have shown that the capsid transport from the plasma membrane into the nucleus consists of mechanistically distinct dynamic stages[1,2,3,4,5,6]
To follow viral trafficking towards the nucleus, we analysed the cytoplasmic distribution of canine parvovirus (CPV) capsids as a prototype for other parvoviruses
We combined different advanced image analysis techniques and showed that the motility of capsids ranges from fast to slow in the cytoplasm, at the nuclear envelope (NE) and in the nucleoplasm
Summary
Recent studies elucidating the dynamics of nuclear-replicating virus entry into host cells have shown that the capsid transport from the plasma membrane into the nucleus consists of mechanistically distinct dynamic stages[1,2,3,4,5,6]. Number and brightness (N&B) analysis[15] and fluorescence correlation spectroscopy (FCS) -derived advanced image analyses along with confocal line scans were used to quantify the dynamics of capsid movement in living cells on their way towards the nucleus[16]. Cross-correlated image pCF of the capsids and importin β revealed their simultaneous transport across the NE independently of the brightness and total number of particles[16] Together, these techniques allowed a precise analysis and visualization of capsid transport across the NE thereby permitting a more complete description of single capsid dynamics. Nuclear admission of the viral capsids is followed by a so far unknown process of viral genome release into the nucleoplasm Due to their small size, the imaging of parvovirus entry by conventional microscopy techniques is challenging, in real time in living cells on the single capsid level. Our results describe the varied intracellular kinetics of capsid entry, and the discovery of capsid interaction with importin β during their nuclear import, increasing our comprehensive understanding of the parvoviral nuclear entry pathway
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