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Journal of Virology, September 2001, p. 7904-7912, Vol. 75, No. 17
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.17.7904-7912.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Posttranslational Processing of Infected Cell
Proteins 0 and 4 of Herpes Simplex Virus 1 Is Sequential and
Reflects the Subcellular Compartment in Which the Proteins
Localize
Sunil J.
Advani,1,2
Ryan
Hagglund,1
Ralph R.
Weichselbaum,2 and
Bernard
Roizman1,*
The Marjorie B. Kovler Viral Oncology
Laboratories1 and Department of
Radiation and Cellular Oncology,2 The
University of Chicago, Chicago, Illinois 60637
Received 21 March 2001/Accepted 18 May 2001
The herpes simplex virus 1 (HSV-1) infected cell proteins 0 and 4 (ICP0 and ICP4) are multifunctional proteins extensively posttranscriptionally processed by both cellular and viral enzymes. We
examined by two-dimensional separations the posttranslational forms of
ICP0 and ICP4 in HEp-2 cells and in human embryonic lung (HEL)
fibroblasts infected with wild-type virus, mutant R325, lacking the
sequences encoding the US1.5 protein and the overlapping carboxyl-terminal domain of ICP22, or R7914, in which the aspartic acid
199 of ICP0 was replaced by alanine. We report the following (i) Both
ICP0 and ICP4 were sequentially posttranslationally modified at least
until 12 h after infection. In HEL fibroblasts, the processing of
ICP0 shifted from A+B forms at 4 h to D+G forms at 8 h and finally to G, E, and F forms at 12 h. The ICP4 progression was from the A' form noted at 2 h to B' and C' forms noted at 4 h to the additional D' and E' forms noted at 12 h. The progression tended to be toward more highly charged forms of the proteins. (ii)
Although the overall patterns were similar, the mobility of proteins
made in HEp-2 cells differed from those made in HEL fibroblasts. (iii)
The processing of ICP0 forms E and F was blocked in HEL fibroblasts
infected with R325 or with wild-type virus and treated with
roscovitine, a specific inhibitor of cell cycle-dependent kinases cdc2,
cdk2, and cdk5. R325-infected HEp-2 cells lacked the D' form of ICP4,
and roscovitine blocked the appearance of the most highly charged E'
form of ICP4. (iv) A characteristic of ICP0 is that it is translocated
into the cytoplasm of HEL fibroblasts between 5 and 9 h after
infection. Addition of MG132 to the cultures late in infection resulted
in rapid relocation of cytoplasmic ICP0 back into the nucleus. Exposure
of HEL fibroblasts to MG132 late in infection resulted in the
disappearance of the highly charged ICP0 G isoform. The G form of ICP0
was also absent in cells infected with R7914 mutant. In cells infected
with this mutant, ICP0 is not translocated to the cytoplasm. (v) Last,
cdc2 was active in infected cells, and this activity was inhibited by
roscovitine. In contrast, the activity of cdk2 exhibited by immunoprecipitated protein was reduced and resistant to roscovitine and
may represent a contaminating kinase activity. We conclude from these
results that the ICP0 G isoform is the cytoplasmic form, that it may be
phosphorylated by cdc2, consistent with evidence published earlier
(S. J., Advani, R. R. Weichselbaum, and B. Roizman, Proc.
Natl. Acad. Sci. USA 96:10996-11001, 2000), and that the processing is reversed upon relocation of the G isoform from the cytoplasm into the nucleus. The processing of ICP4 is also affected by
R325 and roscovitine. The latter result suggests that ICP4 may also be
a substrate of cdc2 late in infection. Last, additional modifications
are superimposed by cell-type-specific enzymes.
*
Corresponding author. Mailing address: The Marjorie B. Kovler Viral Oncology Laboratories, The University of Chicago, 910 E. 58th St., Chicago, IL 60637. Phone: (773) 702-1898. Fax: (773) 702-1631. E-mail: bernard{at}cummings.uchicago.edu.
Journal of Virology, September 2001, p. 7904-7912, Vol. 75, No. 17
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.17.7904-7912.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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