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. 2002 Oct;76(19):9962-71.
doi: 10.1128/jvi.76.19.9962-9971.2002.

Intact microtubules support adenovirus and herpes simplex virus infections

Hélène Mabit  1 , Michel Y Nakano, Ute Prank, Bianca Saam, Katinka Döhner, Beate Sodeik, Urs F Greber
Affiliations

Intact microtubules support adenovirus and herpes simplex virus infections

Hélène Mabit et al. J Virol. 2002 Oct.

Abstract

Capsids and the enclosed DNA of adenoviruses, including the species C viruses adenovirus type 2 (Ad2) and Ad5, and herpesviruses, such as herpes simplex virus type 1 (HSV-1), are targeted to the nuclei of epithelial, endothelial, fibroblastic, and neuronal cells. Cytoplasmic transport of fluorophore-tagged Ad2 and immunologically detected HSV-1 capsids required intact microtubules and the microtubule-dependent minus-end-directed motor complex dynein-dynactin. A recent study with epithelial cells suggested that Ad5 was transported to the nucleus and expressed its genes independently of a microtubule network. To clarify the mechanisms by which Ad2 and, as an independent control, HSV-1 were targeted to the nucleus, we treated epithelial cells with nocodazole (NOC) to depolymerize microtubules and measured viral gene expression at different times and multiplicities of infections. Our results indicate that in NOC-treated cells, viral transgene expression was significantly reduced at up to 48 h postinfection (p.i.). A quantitative analysis of subcellular capsid localization indicated that NOC blocked the nuclear targeting of Ad2 and also HSV-1 by more than 90% at up to 7 h p.i. About 10% of the incoming Texas Red-coupled Ad2 (Ad2-TR) was enriched at the nucleus in microtubule-depleted cells at 5 h p.i. This result is consistent with earlier observations that Ad2-TR capsids move randomly in NOC-treated cells at less than 0.1 micro m/s and over distances of less than 5 micro m, characteristic of Brownian motion. We conclude that fluorophore-tagged Ad2 and HSV-1 particles are infectious and that microtubules play a prominent role in efficient nuclear targeting during entry and gene expression of species C Ads and HSV-1.

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Figures

FIG. 1.
FIG. 1.
Ad5-mediated gene expression requires intact MTs. (A to D) A549 cells were not treated or treated with the indicated concentrations of NOC for 1 h and then infected with β-Gal-Ad5 (A to C) or Luc-Ad5 (D) for the indicated times at 37°C. Gene expression was normalized to that of drug-free infected cells (A and B) or is reported as enzymatic units per milligram of protein per minute (C) or relative light units (D) at room temperature. The reversibility of the effects of NOC was tested by washing out the drug at 180 min, and the postentry effects of NOC were assessed by adding the drug at 180 min p.i. (C). Error bars indicate standard errors of the means. (E) Toxicity of NOC for β-Gal-Ad5-infected cells (a to c) and noninfected cells (d to f) at 7 h p.i. with ethidium bromide (a, d, and g) and calcein (b, e, and h) staining and with Nomarksi (Nom) differential interference contrast imaging (c, f, and i). Note that dead cells were EthD-1 positive and calcein negative; see, e.g., methanol (MetOH, 70% for 30 min)-treated cells (g to i). NOC-treated or infected cells were all alive. Bar, 40 μm.
FIG. 2.
FIG. 2.
NOC blocks nuclear targeting of Ad2-TR in A549 cells. Ad2-TR was cold bound to A549 cells not pretreated or pretreated with different concentrations of NOC. Cells were warmed for the indicated times at 37°C in the presence or in the absence of NOC and then fixed with PHEMO-fix. (A) Entire set of projected confocal laser scanning microscopy sections of representative cells depicting Ad2-TR (red), MTs (green), and the nucleus (4′,6′-diamidino-2-phenylindole; dark blue). Note the disappearance of the MTs with increasing concentrations of NOC and the lack of nuclear accumulation of Ad2-TR. Bar, 10 μm. (B) Quantification of the subcellular localization of Ad2-TR in the periphery, the cytoplasm, and the nuclear and perinuclear region of cells treated with different concentrations of NOC (n, number of cells analyzed). (C) Quantification of the subcellular localization of Ad2-TR in A549 control cells over time. Note that the nuclear transport of Ad2-TR peaks at 80 to 90 min p.i., with corresponding reductions in peripheral and cytoplasmic particles. (D) Quantification of the subcellular localization of Ad2-TR in the absence of MTs (NOC treatment). Note the lack of nuclear transport at times before 200 min p.i. and the slight increase in nuclear accumulation at 300 min p.i. Error bars indicate standard errors of the means.
FIG. 3.
FIG. 3.
Intact MTs are required for HSV-1 mediated gene expression. (A) PtK2 cells were treated with DMSO (control) or NOC for 1 h at 37°C and then incubated with the β-Gal-expressing HSV-1 strain [KOS]tk12 at 4°C for 2 h (MOI, 10). The unbound viruses were removed, and the cells were shifted to 37°C in the presence or in the absence of NOC to initiate infection. Cells were lysed at 4 h p.i., and the amount of β-Gal was quantitated (squares). Cells on parallel dishes were quantified by using the binding of crystal violet (circles). The means and standard deviations for duplicate samples are shown. Note that with increasing NOC concentrations, the amount of immediate-early viral gene expression was inhibited. (B) Time course of HSV-1 gene expression. Cells were infected in the absence (filled symbols) or in the presence (open symbols) of MTs with HSV-1 [KOS]tk12 (MOI of 2 [triangles] or MOI of 10 [squares]), lysed at different times p.i., and analyzed for β-Gal expression (absorbance at 420 nm). At both MOIs and at all times tested, NOC clearly reduced immediate-early viral gene expression. The crystal violet assay showed no significant loss of cells under these conditions (<5%) (data not shown).
FIG. 4.
FIG. 4.
NOC inhibits the nuclear targeting of HSV-1 in Vero and PtK2 cells. (A) Vero cells were infected in the absence or in the presence of different concentrations of NOC with 50 PFU of HSV-1 per cell for 2 h and then fixed in methanol after preextraction. Samples were stained with anti-HC (green) and antitubulin (red) and analyzed by confocal laser scanning microscopy. Note the disappearance of the MTs with increasing concentrations of NOC and the lack of nuclear HSV-1 capsids. (B to D) Quantification of subcellular localization of HSV-1 in PtK2 cells infected with 80 PFU per cell in the absence of drug (B), in the presence of 50 μM NOC (C), or in the presence of 10 μM paclitaxel (D). Cells were fixed at 30, 120, or 180 min p.i. and labeled with anti-LC. n, number of cells. Error bars indicate standard errors of the means. MTs in Vero cells were completely depolymerized at lower concentrations of NOC than were those in PtK2 cells (data not shown).
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