Quantitative microscopy of fluorescent adenovirus entry

Area of Science:

• Cell biology
• Virology
• Microscopy techniques

Background:

• Fluorescence imaging is crucial for studying cellular dynamics, including organelle, protein, and virus movement.
• Fluorescent adenoviruses serve as a model to investigate intracellular cargo transport from the cell surface to the nucleus.

Purpose of the Study:

• To develop and validate a quantitative method for measuring adenovirus-associated fluorescence within different subcellular compartments.
• To analyze the dynamics of wild-type (wt) adenovirus and a specific mutant (ts1) nuclear transport post-infection.

Main Methods:

• Utilized CCD camera-captured fluorescence imaging of entire cells.
• Applied fast Fourier transformation for image deblurring, followed by background subtraction and image merging.
• Verified deblurring accuracy using confocal laser scanning microscopy and quantified fluorescence intensity and particle homogeneity.

Main Results:

• Nuclear fluorescence of wt adenovirus peaked at 90 minutes postinfection (p.i.), decreased at 120 min, and increased again at 240 min p.i.
• The ts1 mutant, unable to escape endosomes, exhibited significantly reduced nuclear fluorescence compared to wt adenovirus.
• The initial nuclear transport phase of wt adenovirus correlated with cell peripheral region increase and cytoplasmic region decrease, suggesting signal-dependent cell contraction.

Conclusions:

• Adenovirus nuclear targeting appears to be a multiphasic and regulated process.
• Endosomal escape is critical for efficient adenovirus nuclear entry.
• Adenovirus infection can induce cell shape changes, potentially linked to its nuclear transport mechanism.