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Comparative Study
. 2001 Apr 30;153(3):449-55.
doi: 10.1083/jcb.153.3.449.

Aggresomes resemble sites specialized for virus assembly

C M Heath  1 , M Windsor, T Wileman
Affiliations
Comparative Study

Aggresomes resemble sites specialized for virus assembly

C M Heath et al. J Cell Biol. .

Abstract

The large cytoplasmic DNA viruses such as poxviruses, iridoviruses, and African swine fever virus (ASFV) assemble in discrete perinuclear foci called viral factories. Factories exclude host proteins, suggesting that they are novel subcellular structures induced by viruses. Novel perinuclear structures, called aggresomes are also formed by cells in response to misfolded protein (Johnston, J.A., C.L. Ward, and R.R. Kopito. 1998. J. Cell Biol. 143:1883--1898; García-Mata, R., Z. Bebök, E.J. Sorscher, and E.S. Sztul. 1999. J. Cell Biol. 146:1239--1254). In this study, we have investigated whether aggresomes and viral factories are related structures. Aggresomes were compared with viral factories produced by ASFV. Aggresomes and viral factories were located close to the microtubule organizing center and required an intact microtubular network for assembly. Both structures caused rearrangement of intermediate filaments and the collapse of vimentin into characteristic cages, and both recruited mitochondria and cellular chaperones. Given that ASFV factories resemble aggresomes, it is possible that a cellular response originally designed to reduce the toxicity of misfolded proteins is exploited by cytoplasmic DNA viruses to concentrate structural proteins at virus assembly sites.

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Figures

Figure 1
Figure 1
Aggresomes and ASFV assembly sites cause redistribution of the intermediate filament protein vimentin. Vero cells were transfected with the GFP-250 plasmid and grown at 37°C for 48 h (A) or infected for 12 h with ASFV (B). Fixed and permeabilized cells were incubated with an antibody specific for vimentin, and antibodies specific for p34 (B). Protein distribution was visualized by using appropriate secondary antibodies conjugated to Alexa Fluor 488 (green) or 594 (red), and the DNA was labeled with DAPI (blue). In the lower images the GFP-250/p34, vimentin and cellular DNA images were digitally merged.
Figure 2
Figure 2
Aggresomes and viral factories locate close to the MTOC and recruit mitochondria. Vero cells were transfected with the GFP-250 plasmid (A and C) and grown at 37°C for 48 h, or infected for 12 h with ASFV (B and D–F). The green GFP signal indicates the aggresome in A and C, while virions in viral factories are identified in B, D, and E using antibodies specific for p73 and a secondary antibody coupled to Alexa Fluor 488 (green). Cellular and viral DNA was labeled with DAPI (blue). (A and B) Merged images where antibodies specific for γ-tubulin identify centrioles (red). (C–E) Merged images where Mito-Tracker red CMXRos identifies mitochondria (red). (F) Phase contrast image of cells in E.
Figure 3
Figure 3
Recruitment of chaperones into aggresomes and viral factories. Cells containing aggresomes were generated using GFP-250 (A), and distribution of Hsp70 in the same cell is shown in B. Vero cells infected with ASFV for 12 h were fixed and permeabilized and labeled with antibody specific for viral protein p34 (C and E) and Hsp70; antibodies specific for TCP1 identify the ring chaperonin (E and F).
Figure 4
Figure 4
Viral factories require an intact microtubule network. (A–C) Microtubules are required for the integrity of preassembled factories. Vero cells were infected with ASFV for 14 h, and then incubated at 37°C in the absence (A) or presence (B and C) of 10 μg/ml nocodazole for another 2 h, and then fixed and permeabilized. The viral factories were labeled with antibody raised against p34 (top) or antibody specific for p73 (C, middle). Microtubules were labeled with antibody specific for α-tubulin (A and B, middle). The proteins were visualized with the Alexa Fluor 488 or 594 conjugated to appropriate secondary antibodies. (D and E) Late gene expression requires microtubules. (D) Vero cells were transfected with a plasmid expressing p50/dynamitin; 2 h later, cells were infected with ASFV and incubated at 37°C for another 12 h before processing for immunofluorescence microscopy. Cells were triple stained using antibodies specific for the myc-tag in dynamitin, a biotinylated antibody specific for p30, and rabbit polyclonal antibody recognizing p73. The proteins were visualized using Marina blue, Alexa Fluor 488, or 594 conjugated to appropriate secondary antibodies. (E) Vero cells were infected with ASFV for 2 h, and then incubated at 37°C in the absence (−) or presence (+) of 10 μg/ml nocodazole for another 10 h. Cells were pulse labeled for 30 min, lysed, and immunoprecipitated using antibodies specific for p30 or p73. Proteins were resolved by SDS/PAGE and visualized by autoradiography.
Figure 5
Figure 5
Model for the recruitment of ASFV into aggresomes. ASF virions enter the cell by receptor-mediated endocytosis (1 and 2). The virus is uncoated and the virion cores enter the cytoplasm (3). The cores mimic large protein aggregates, initiate aggresome formation, and are transported to the newly established aggresomes on microtubules (4). DNA replication begins leading to late protein synthesis (5). Later, during infection, viral proteins synthesized on cytoplasmic ribosomes are carried by hypothetical viral carrier proteins along microtubules (6) to the aggresomes to facilitate further replication and assembly.
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