Tripartite motif (TRIM)5 alpha has recently been identified as a host restriction factor that has the ability to block infection by certain retroviruses in a species-dependent manner. One interesting feature of this protein is that it is localized in distinct cytoplasmic clusters designated as cytoplasmic bodies. The potential role of these cytoplasmic bodies in TRIM5 alpha function remains to be defined. By using fluorescent fusion proteins and live cell microscopy, we studied the localization and dynamics of TRIM5 alpha cytoplasmic bodies. This analysis reveals that cytoplasmic bodies are highly mobile, exhibiting both short saltatory movements and unidirectional long-distance movements along the microtubule network. The morphology of the cytoplasmic bodies is also dynamic. Finally, photobleaching and photoactivation analysis reveals that the TRIM5 alpha protein present in the cytoplasmic bodies is very dynamic, rapidly exchanging between cytoplasmic bodies and a more diffuse cytoplasmic population. Therefore, TRIM5 alpha cytoplasmic bodies are dynamic structures more consistent with a role in function or regulation rather than protein aggregates or inclusion bodies that represent dead-end static structures.

Visualization of huTRIM5 Cytoplasmic Bodies. (A) HeLa cells were transiently transfected with YFP-huTRIM5, and the cells then fixed, stained for cellular DNA (blue), and TRIM5 localization (green) was examined. An enlargement highlighting the hollow body structures formed by YFP-hu-TRIM5 is shown in the top inset. (B) Electron microscopy confirms that YFP-huTRIM5 forms hollow structures, with a magnification of the structures observed shown in the bottom inset. The P-body markers mRFP-Dcp1a (C) and RFP-rck/p54 were expressed in cells stably expressing YFP-rhTRIM5. Cells were fixed and analyzed by confocal microscopy. The merged images demonstrates the lack of TRIM5 (green) and Dcp1a (red) (C) or RFP-rck/p54 (red) (D) colocalization.