Abstract
Understanding the detailed nuclear import kinetics of adeno-associated virus (AAV) through the nuclear pore complex (NPC) is essential for the application of AAV capsids as a nuclear delivery instrument as well as a target for drug development. However, a comprehensive understanding of AAV transport through the sub-micrometer NPCs in live cells calls for new techniques that can conquer the limitations of conventional fluorescence microscopy and electron microscopy. With recent technical advances in single-molecule fluorescence microscopy, we are now able to image the entire nuclear import process of AAV particles and also quantify the transport dynamics of viral particles through the NPCs in live human cells. In this review, we initially evaluate the necessity of single-molecule live-cell microscopy in the study of nuclear import for AAV particles. Then, we detail the application of high-speed single-point edge-excitation sub-diffraction (SPEED) microscopy in tracking the entire process of nuclear import for AAV particles. Finally, we summarize the major findings for AAV nuclear import by using SPEED microscopy.
Highlights
New Features for the Nuclear Transport of associated virus (AAV) Particles Obtained by SPEED Microscopy
We found significant evidence for nuclear import of intact AAV2 capsids our study, we AAV2 foundparticles significant evidence formoving nuclearthrough importsingle of intact by In tracking individual across the nuclear envelope (NE) and
Compared to the higher transport efficiency of transport receptor Impβ1, ~50% [13], we concluded that the nuclear import of AAV2 may be another rate-limiting step for AAV2 transduction
Summary
Viruses are obligate intracellular pathogens that hijack the cellular components of an infected host cell to produce viral progeny. Influenza A will form the viral ribonucleoprotein (vRNP, ~10–15 nm in diameter [22]) complex, consisting of two NLSs that will be recognized by Impα and Impβ transport receptors [31,32] (Figure 1B) Some parvoviruses, such as adeno-associated virus 2 (AAV2, ~25 nm in diameter [25]) (Figure 1C), will hijack the facilitated transport mechanism for the translocation of the entire viral capsid into the nucleus [33]. They found the capsid to interact with nuclear basket Nups and that it was independent of the GTP-binding protein, Ran. Fluorescence resonance energy transfer (FRET) is a distance-dependent physical process for probing an inter- or intra-molecular distance of
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