Viral highway to nucleus exposed by image correlation analyses

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.

Protoparvovirus Knocking at the Nuclear Door

“…Cargo-dynein interactions are based on various binding partners including the dynein–dynactin complex [ 58 ]. Dynein-mediated transport is also used by protoparvovirus capsids to reach the NE [ 43 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 ], which is consistent with the velocity of 3.5 and ~2.0 μm/s observed for AAV2 and AAV9 [ 41 , 69 , 70 ].…”

“…After the stepwise endosomal uptake, CPV and other parvoviruses, including adeno-associated virus 2 (AAV2), a member of the genus dependoparvovirus, are present in small vesicles scattered around the cytoplasm, and in large vesicles accumulated in the perinuclear area [ 30 , 39 , 40 , 41 , 42 , 43 ]. This is in line with earlier observations of size-distribution of cargo-filled endosomes and their perinuclear accumulation: small endosomes with little cargo are found in the cell periphery, whereas large endosomes rich in cargo are located close to the nucleus [ 44 , 45 , 46 ].…”

“…The fast movement could correspond to capsids located in small endosomes actively moving along microtubules (MTs) [ 43 ]. The slow movement corresponds to capsids accumulated in endosomes at the perinuclear area, where they move very slowly or are nearly immobile (velocity < 1 µm/s) [ 43 , 45 , 46 , 47 , 48 , 49 , 50 , 51 ]. In parvovirus entry, the endosomal transportation is followed by capsid release into cytoplasm, which appears to be inefficient.…”

“…The observation of the conserved NLS combined with the attachment of at least importin β to cytosolic CPV capsids suggest that the capsids follow the classical nuclear import pathway. In fact, capsids have been observed on the nuclear side of the NPC [ 43 ]. Observations using the AAV2 and the protoparvovirus PV H-1 showed that their attachment to NE causes its local degradation [ 107 ].…”

“…Although the molecular mechanisms of capsid disassembly are yet unknown, recent studies of intranuclear CPV capsids dynamics have indicated that slow moving nucleoplasmic CPV capsids are accompanied by the rapidly moving capsid-derived components, either remnants thereof or dissociated constituent proteins. This shows that capsids are disassembled after their entry into the nucleoplasm [ 43 ]. Within the nucleus, once the genome becomes released, transcription and replication are initiated via nuclear host proteins.…”