Whilst their structure has been well studied, there is little information on the replication biology of tetraviruses because of the lack of suitable tissue-culture cell lines that support virus replication. In this study, the potential site of stunt virus replication was investigated by transient expression of the replicase protein fused to enhanced green fluorescent protein (EGFP) in mammalian and insect cells. When EGFP was present at the C terminus of the protein, fluorescence was located in punctate cytoplasmic structures that were distinct from the peripheral Golgi, endoplasmic reticulum, early endosomes, lysosomes and mitochondria, but overlapped partially with late endosomes. In experiments where targeting to endosomal compartments was examined further by using Cascade Blue–dextran in live cells, no overlap between the replicase and active endocytic organelles was apparent. Analysis of the punctate structures using time-lapse imaging in live cells revealed that they undergo fusion, fission and ‘kiss-and-run’ events. Whilst the source of the membranes used to form the punctate structures remains unclear, we propose that the replicase sequesters membranes from the late endosomes and actively excludes host proteins, either by normal recycling processes or by a replicase-dependent mechanism that may result in the destabilization of the associated membranes and a release of luminal contents into the cytosol. This is the first study describing the localization of a tetravirus.


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vol. , part 6, pp. 1514–1523

Transport of EGFP-containing structures along similar paths at different time points. REP –EGFP-associated structures were tracked manually as described in Methods. The left panel shows the movement of the punctate structures within a region in the cytoplasm of a single HeLa cell. The centre panel contains the colour overlay generated after two particles were tracked as they were transported along a similar path within the cell at two different time points, and the panel on the right shows the other two panels merged. The first particle (green) was observed to travel a distance of almost 9 µm in 10 s along a pathway before commencing far slower movements in random directions for the following 90 s. A second particle (red) was then observed to move rapidly along a similar path for approximately half the distance, before commencing similar random movements at a corresponding point within the cell. Bar, 5 µm.

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