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. 2001 Jan;75(2):1039-43.
doi: 10.1128/JVI.75.2.1039-1043.2001.

A single amino acid substitution in the ICP27 protein of herpes simplex virus type 1 is responsible for its resistance to leptomycin B

T Murata  1 , F Goshima, T Koshizuka, H Takakuwa, Y Nishiyama
Affiliations

A single amino acid substitution in the ICP27 protein of herpes simplex virus type 1 is responsible for its resistance to leptomycin B

T Murata et al. J Virol. 2001 Jan.

Abstract

Leptomycin B (LMB) is a specific inhibitor of Crm1-dependent nuclear export of proteins. The replication of herpes simplex virus (HSV) is normally highly sensitive to LMB; a resistant HSV variant, however, was isolated by serial passages of the virus. Analysis of marker transfer and viral DNA sequences revealed that a single amino acid substitution within the ICP27 gene is responsible for conferring this resistance.

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Figures

FIG. 1
FIG. 1
Inhibition of HSV-1 growth by LMB and the generation of a resistant virus. (A) Vero cells were infected with HSV-1 KOS at a multiplicity of infection of 0.1 in the presence or absence of LMB. At the indicated times postinfection, cells and the culture medium on dishes were kept at −80°C. The quantity of virus in each was determined by virus titration on Vero cells. (B) Concentrations of LMB and PAA required to inhibit HSV replication. Vero cells were infected with HSV-1 KOS in the presence of various concentrations of either LMB or PAA. After 1.5 days, the numbers of plaques were counted. (C) Isolation of an LMB-resistant mutant. The HSV-1 WT strain, KOS, was passaged 11 times in increasing concentrations of either LMB or PAA. In the series of passages, a portion of each sample was titrated with or without the inhibitors. The drug-resistant plaque numbers per total plaque numbers were plotted against the passage number. (D) Dose dependence of the virus resistant to LMB. The plaque numbers of WT KOS and the LMB-resistant mutant following 11 passages (p11) were counted. (E and F) Multistep growth curve of WT (KOS) and resistant (Cl1) HSV-1 incubated with or without LMB (10 ng/ml). Intracellular (E) and extracellular (F) infectious particles were quantified.
FIG. 2
FIG. 2
(A) Schematic arrangement of the DNA sequence of HSV-1 and the ICP27 gene. (Top) EcoRI restriction fragments of HSV-1 DNA were designated alphabetically according to their sizes. (Middle) HSV-1 EcoRI restriction fragment B containing genes from UL52 to ICP4. (Bottom) Schematic representation of the ICP27 coding region displaying the putative NES, the essential acidic domain, and other motifs. (B) Result of marker transfer experiments. After cotransfection of DNA fragment plasmids in conjunction with the WT KOS infectious DNA and a subsequent 2-day incubation, samples were frozen at −80°C, thawed, and analyzed by plaque formation assay in the presence or absence of LMB. Alphabetical letters represent the EcoRI restriction fragments, and I-V, P-V, and V-I represent the 8.5-kb EcoRI-EcoRV, 5.9-kb PstI-EcoRV, and 12.9-kb EcoRV-EcoRI fragments contained within the B fragment derived from the resistant mutant, Cl1, respectively. The DNA designated ACT contains the same sequence as the WT, 5.9-kb PstI-EcoRV fragment with a mutation (ATG [Met] to ACT [Thr]) at the 50th residue of ICP27.
FIG. 3
FIG. 3
Subcellular localization of ICP27. (A to F) Fluorescent images of ICP27 (fluorescein isothiocyanate) and the nucleus (red). Vero cells infected with WT KOS (A, B, and E) or resistant Cl1 (C and D) or mock infected (F) with 10 ng of LMB per ml (B and D) or 10 μg of actinomycin D per ml (E) were fixed. The nonspecific binding of antibodies was blocked by preincubation with human serum (22). Cells were treated with RNase in order not to permit the binding of propidium iodide to RNA. After treatment of cells with a primary anti-ICP27 antibody (H1113; Goodwin Institute, Plantation, Fla.), cells were washed extensively and then incubated with fluorescein isothiocyanate-conjugated, anti-mouse immunoglobulin G antibodies. Propidium iodide was added at a concentration of 10 mg/ml to the staining mixture to identify the nucleus. Cells were washed again and examined with a Bio-Rad MRC-1024 confocal microscope. (G) Vero cells infected with either WT KOS or resistant Cl1 were incubated with or without LMB. At 4 h postinfection, cells were lysed, separated into soluble (C) and insoluble (N) fractions, and subjected to Western blotting analysis. As a loading control, part of the blot was subjected to amido black protein staining.
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