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Journal of Virology, February 2000, p. 1200-1208, Vol. 74, No. 3
Department of Microbiology and Immunology,
Louisiana State University Health Sciences Center, Shreveport,
Louisiana 71130-3932
Received 30 July 1999/Accepted 2 November 1999
The EICP0 protein of equine herpesvirus 1 (EHV-1) is an early,
viral regulatory protein that independently trans-activates EHV-1 immediate-early (IE), early,
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization of the trans-Activation
Properties of Equine Herpesvirus 1 EICP0 Protein
1 late, and
2 late promoters. To assess whether this powerful trans-activator functions
in conjunction with three other EHV-1 regulatory proteins to activate
expression of the various classes of viral promoters, transient
cotransfection assays were performed in which effector plasmids
expressing the EICP22, EICP27, and IE proteins were used either singly
or in combination with an EICP0 effector construct. These analyses
revealed that (i) independently, the EICP0 and IE proteins are powerful trans-activators but do not function synergistically, (ii)
the IE protein inhibits the ability of the EICP0 protein to
trans-activate the IE,
1 late, and
2 late promoters,
(iii) the EICP22 and EICP0 proteins do not function together to
significantly trans-activate any EHV-1 promoter, and (iv)
the EICP27 and EICP0 proteins function synergistically to
trans-activate the early and
1 late promoters. A panel
of EICP0 truncation and deletion mutant plasmids was generated and used
in experiments to define the domains of the 419-amino-acid (aa) EICP0
protein that are important for the trans-activation of each
class of EHV-1 promoters. These studies revealed that (i)
carboxy-terminal truncation mutants of the EICP0 protein exhibited a
progressive loss of trans-activating ability as increasing
portions of the carboxy terminus were removed, (ii) the amino terminus of the EICP0 protein containing the RING finger (aa 8 to 46) and the
acidic region (aa 71 to 84) was necessary but not sufficient for
activation of all classes of EHV-1 promoters, (iii) the RING finger was
absolutely essential for activation of EHV-1 promoters, since deletion
of the entire RING finger motif (aa 8 to 46) or a portion of it (aa 19 to 30) completely abrogated the ability of these mutants to activate
any promoter tested, (iv) the acidic region contributed to the ability
of the EICP0 protein to activate the early and
1 late promoters, and
deletion of the acidic region enhanced the ability of this mutant to
activate the IE promoter, (v) the carboxy terminus (aa 325 to 419),
which is rich in glutamine residues, was dispensable for the EICP0
trans-activation function, (vi) a motif resembling a
nuclear localization signal (aa 289 to 293) was unnecessary for the
EICP0 protein to trans-activate promoters of any temporal
class, and (vii) the EICP0 protein was phosphorylated during infection,
and deletion of the serine-rich region (aa 210 to 217), a potential
site for phosphorylation, reduced by more than 70% the ability of the
EICP0 protein to activate the
2 late class of promoters.
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, Louisiana State University Health Sciences Center, 1501 Kings Highway, P.O. Box 33932, Shreveport, LA 71130-3932. Phone: (318) 675-5750. Fax: (318) 675-5764. E-mail:
DOCALL{at}LSUMC.EDU.
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