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. 2003 Aug;77(15):8541-7.
doi: 10.1128/jvi.77.15.8541-8547.2003.

Microtubule network facilitates nuclear targeting of human cytomegalovirus capsid

K Ogawa-Goto  1 , K Tanaka, W Gibson, E Moriishi, Y Miura, T Kurata, S Irie, T Sata
Affiliations

Microtubule network facilitates nuclear targeting of human cytomegalovirus capsid

K Ogawa-Goto et al. J Virol. 2003 Aug.

Abstract

We assessed the requirement of the host cytoskeleton for the intracytosolic transport of incoming human cytomegalovirus (HCMV) capsids. Treatments with microtubule (MT)-depolymerizing drugs nocodazole and colchicine led to a drastic decrease in levels of IE1 antigen, whereas cytochalasin B had no effect on the level of IE1 as determined by Western blot analyses. Sequential treatment including nocodazole washout and removal of cell surface virion revealed that HCMV entry into the cells occurred normally in the absence of the MT network. This finding was also supported by data obtained by monitoring pUL83 signals with an immunofluorescent assay (IFA). Furthermore, we demonstrated a close association of incoming HCMV capsids with MTs by IFA and ultrastructural analyses. In the absence of the MT network, the capsids which had entered the cytoplasm did not move to close proximity of the nucleus. These data suggest that HCMV capsids associate with the MT network to facilitate their own movement to the nucleus before the onset of immediate-early (IE) gene expression and that this association is required to start efficient IE gene expression.

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Figures

FIG. 1.
FIG. 1.
Efficient IE1 antigen expression requires an intact MT network. (A and B) HEL cells were infected with HCMV at an MOI of 3 PFU/cell in the presence of various drugs, and effects on expression levels of the IE1 antigen (72 kDa) were examined at 12 (A) and 18 (B) hpi. (A) Lane 1, 5 μM colchicine (Co); lane 2, 10 μM Taxol (Tx); lane 3, 0.5 μM cytochalasin B (CB); lane 4, no drug; lane 5, 10 μM nocodazole (Noc). (B) Lane 1, no drug; lane 2, 0.2 μM nocodazole; lane 3, 2 μM nocodazole; lane 4, 10 μM nocodazole. (C) K-1034 and Caco-2 cells were mock infected (lanes 1 and 4) or HCMV-infected (lanes 2, 3, 5, and 6) at an MOI of 3 PFU/cell. Effects of nocodazole on expression levels of the IE1 antigen (72 kDa) were examined at 24 hpi in the absence (lanes 2 and 5) or presence (lanes 3 and 6) of 10 μM nocodazole.
FIG. 2.
FIG. 2.
HCMV enters into the cytoplasm in the absence of the MT network. Effects of nocodazole removal on expression levels of IE1 were examined with HEL cells infected with HCMV at an MOI of 3 PFU/cell. (A) Schematic representation of sequential treatments with nocodazole, treatment with proteinase K (PK), and inoculation. C1 and C2, controls 1 and 2. (B) Lysates were taken at the times indicated in panel A and subjected to Western blotting analysis to compare IE1 antigens. Relative signal intensities of IE1 normalized by signals of beta-actin are shown. Values represent means ± standard deviations (n = 3 or 4).
FIG. 3.
FIG. 3.
pUL83 release in nocodazole-treated HCMV-infected cells. HEL cells were mock infected (a and b) or infected with HCMV at an MOI of 5 PFU/cell in the absence (c and d) or presence (e and f) of 10 μM nocodazole. At 2 hpi, the cells were fixed and stained for pUL83 (a, c, and e). Nuclei were visualized with TOPRO3 (b, d, and f). Bars, 50 μm.
FIG.4.
FIG.4.
IF analyses of incoming HCMV capsids labeled with anti-pUL32 antibody. (a to c) Untreated (a and b) or nocodazole-treated (c) HEL cells were mock infected (a) or HCMV-infected (b and c) in the presence of 0.5 mM cycloheximide at an MOI of 5 PFU/cell. At 4 hpi, the cells were fixed and costained with anti-pUL32 antibody. (Insets a1 to c1) The nucleus (N) was stained with MAb against the nuclear pore complex. (d to g) HEL cells were infected at an MOI of 5 PFU/cell in the presence of cycloheximide, fixed at 2 (g) or 4 (d to f) hpi, and double labeled with anti-pUL32 antibody (green) and MAb against α-tubulin (red). The laser-scanning microscopy images were sequentially obtained with 1-μm intervals. (d to g) Merged signals. (Insets d1 to g1) α-Tubulin. (Insets d2 to g2) pUL32. (h) Schematic representation of levels of sectioning. The sectioning levels are indicated by dotted lines marked with letters d to g, which correspond to the merged images shown in panels d to g. Colocalization of pUL32-labeled capsids with MTs was clearly demonstrated, especially beneath the nucleus and on the top surface near the MTOC. Bars, 10 μm.
FIG. 5.
FIG. 5.
The incoming HCMV capsids closely associated with MTs and an endoplasmic reticulum (ER)-like membrane structure. HEL cells were infected with HCMV in the presence of 0.5 mM cycloheximide at an MOI of 20 PFU/cell and processed for TEM at 4 hpi as described in the text. (a to d) The cytoplasmic capsids were surrounded by a fibrillar tegument layer. In the perinuclear region, MTs projected very close to the nuclear pores (small arrows) from the MTOC (M). The cytoplasmic capsids with core densities (large arrows) were associated with MTs in the vicinity of the nucleus. Three types of capsids, those with core densities, those without core densities, and broken capsids (large arrowheads), were observed. (e) Capsid located in MT bundles near the NE. (f to g) At the NE, capsids without core densities directly docked to the nuclear pore complexes. (g and h) Occasionally, capsids under releasing their contents were also observed. RER, rough endoplasmic reticulum; Mt, mitochondria; NPC, nuclear pore complexes. Bars, 1 μm (a); 0.5 μm (b to e); 0.2 μm (f); 0.5 μm (g and h).
FIG. 5.
FIG. 5.
The incoming HCMV capsids closely associated with MTs and an endoplasmic reticulum (ER)-like membrane structure. HEL cells were infected with HCMV in the presence of 0.5 mM cycloheximide at an MOI of 20 PFU/cell and processed for TEM at 4 hpi as described in the text. (a to d) The cytoplasmic capsids were surrounded by a fibrillar tegument layer. In the perinuclear region, MTs projected very close to the nuclear pores (small arrows) from the MTOC (M). The cytoplasmic capsids with core densities (large arrows) were associated with MTs in the vicinity of the nucleus. Three types of capsids, those with core densities, those without core densities, and broken capsids (large arrowheads), were observed. (e) Capsid located in MT bundles near the NE. (f to g) At the NE, capsids without core densities directly docked to the nuclear pore complexes. (g and h) Occasionally, capsids under releasing their contents were also observed. RER, rough endoplasmic reticulum; Mt, mitochondria; NPC, nuclear pore complexes. Bars, 1 μm (a); 0.5 μm (b to e); 0.2 μm (f); 0.5 μm (g and h).
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