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Journal of Virology, August 2000, p. 7600-7609, Vol. 74, No. 16
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification of an Export Control Sequence and a
Requirement for the KH Domains in ICP27 from Herpes Simplex Virus
Type 1
Tarik M.
Soliman and
Saul J.
Silverstein*
Department of Microbiology, College of
Physicians and Surgeons, Columbia University, New York, New York 10032
Received 22 February 2000/Accepted 12 May 2000
The herpes simplex virus type 1 (HSV-1) immediate-early protein
ICP27 is an RNA-binding protein that performs multiple functions required for the expression of HSV-1 genes during a productive infection. One essential function involves shuttling between the nucleus and the cytoplasm. Some of the domains identified in ICP27 include a leucine-rich nuclear export sequence (NES), a nuclear localization signal, three KH-like RNA-binding domains, and an RGG-box
type RNA-binding motif. To study the contribution of two of the
essential domains in ICP27 to HSV gene expression, we generated recombinant herpesviruses carrying deleterious mutations in the NES and
KH domains of ICP27. To accomplish this, we fused the green fluorescent
protein (GFP) to ICP27 and utilized fluorescence as a marker to isolate
recombinant herpesviruses. Fusion of GFP to wild-type ICP27 did not
disturb its localization or function or significantly reduce virus
yield. Analysis of HSV gene expression in cells infected with a
recombinant virus carrying a point mutation in the first KH-like
RNA-binding domain revealed that nuclear export of ICP27 was not
blocked, and the expression of only a subset of ICP27-dependent late
genes was affected. These findings suggest that individual KH-like
RNA-binding motifs in ICP27 may be involved in binding distinct RNAs.
Analysis of recombinant viruses carrying a lethal mutation in the NES
of ICP27 was not accomplished because this mutation results in a strong
dominant-negative phenotype. Finally, we demonstrate that shuttling by
ICP27 is regulated by an export control sequence adjacent to its NES
that functions like the inhibitory sequence element found adjacent to
the NES of NS1 from influenza virus.
*
Corresponding author. Mailing address: Department of
Microbiology, College of Physicians and Surgeons, Columbia University, 701 W. 168th St., New York, NY 10032. Phone: (212) 305-8149. Fax: (212)
305-5106. E-mail: sjs6{at}columbia.edu.
Journal of Virology, August 2000, p. 7600-7609, Vol. 74, No. 16
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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