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Journal of Virology, November 2001, p. 10856-10869, Vol. 75, No. 22
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.22.10856-10869.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Insertions in the gG Gene of Pseudorabies Virus Reduce Expression
of the Upstream Us3 Protein and Inhibit Cell-to-Cell Spread of
Virus Infection
Gretchen L.
Demmin,1
Amanda C.
Clase,1
Jessica A.
Randall,1
L. W.
Enquist,2 and
Bruce W.
Banfield1,*
Department of Microbiology, University of
Colorado Health Sciences Center, Denver, Colorado
80262,1 and Department of Molecular
Biology, Princeton University, Princeton, New Jersey
085442
Received 22 May 2001/Accepted 15 August 2001
The alphaherpesvirus Us4 gene encodes glycoprotein G (gG), which is
conserved in most viruses of the alphaherpesvirus subfamily. In the
swine pathogen pseudorabies virus (PRV), mutant viruses with internal
deletions and insertions in the gG gene have shown no discernible
phenotypes. We report that insertions in the gG locus of the attenuated
PRV strain Bartha show reduced virulence in vivo and are defective in
their ability to spread from cell to cell in a cell-type-specific
manner. Similar insertions in the gG locus of the wild-type PRV strain
Becker had no effect on the ability of virus infection to spread
between cells. Insertions in the gG locus of the virulent NIA-3 strain
gave results similar to those found with the Bartha strain. To examine
the role of gG in cell-to-cell spread, a nonsense mutation in the gG
signal sequence was constructed and crossed into the Bartha strain.
This mutant, PRV157, failed to express gG yet had cell-to-cell spread properties indistinguishable from those of the parental Bartha strain.
These data indicated that, while insertions in the gG locus result in
decreased cell-to-cell spread, the phenotype was not due to loss of gG
expression as first predicted. Analysis of gene expression upstream and
downstream of gG revealed that expression of the upstream Us3 protein
is reduced by insertion of lacZ or egfp
at the gG locus. By contrast, expression of the gene immediately
downstream of gG, Us6, which encodes glycoprotein gD, was not affected
by insertions in gG. These data indicate that DNA insertions in gG have
polar effects and suggest that the serine/threonine kinase encoded by
the Us3 gene, and not gG, functions in the spread of viral infection
between cells.
*
Corresponding author. Mailing address: Department of
Microbiology, University of Colorado Health Sciences Center, Campus Box B175, 4200 E. Ninth Ave., Denver, CO 80262. Phone: (303) 315-5285. Fax:
(303) 315-6785. E-mail: Bruce.Banfield{at}uchsc.edu.
Journal of Virology, November 2001, p. 10856-10869, Vol. 75, No. 22
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.22.10856-10869.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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