234    

Background:  TRIM5a has recently been identified as a host restriction factor that is directed against determinants located in the viral capsid. TRIM5a from different species has the ability to restrict infection by certain retroviruses. Although various models have been proposed for the mechanism of TRIM5a restriction, little is known about the cell biology of this class of proteins. By using fluorescent fusion proteins, live-cell deconvolution microcopy, and FRAP, we have investigated the cellular distribution, formation, and highly dynamic properties of huTrim5a and rhTrim5a cytoplasmic bodies.

Methods:  To study the cellular localization of TRIM5a, fluorescence protein-labeled fusion proteins were constructed. The movement of TRIM5a cytoplasmic bodies was monitored by time-lapse deconvolution microscopy. Fluorescence-recovery-after-photobleaching (FRAP) is used to study the dynamics of TRIM5a cytoplasmic bodies. This analysis was complemented using photo-activatable GFP that allows an alternative approach to study cytoplasmic body dynamics in living cells.

Results:  Both human and rhesus TRIM5a fluorescent fusion proteins form cytoplasmic bodies like their HA-tagged counterparts. TRIM5a cytoplasmic bodies were found to be heterogeneous in size and shape, typically showing a spherical morphology, although bodies with elongated wormy structures were also observed. The larger spherical cytoplasmic bodies were clearly hollow. No obvious association between cytoplasmic bodies and cellular markers of actin, microtubules, early endosomes, the trans-Golgi network, or lysosomes was observed. Time-lapse analysis of TRIM5a cytoplamic bodies revealed that the smaller bodies were highly mobile and that the merging of smaller bodies forms larger cytoplasmic bodies. Further, the TRIM5a creating the cytoplasmic bodies is highly dynamic. Photo-bleaching studies revealed that TRIM5a readily swapped between individual cytoplasmic bodies.

Conclusion:  TRIM5a continuously exchanges between the cytoplasmic bodies and a soluble form in the cytoplasm. This demonstrates that cytoplasmic bodies are dynamic in nature rather than protein aggregates or inclusion bodies that represent dead-end static structures. The unique structure and highly dynamic property of cytoplasmic bodies may provide valuable information to illuminate the mechanism of TRIM5a function.

Keywords: Trim5; restriction factor; cell biology