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Journal of Virology, September 1998, p. 7005-7011, Vol. 72, No. 9
The Marjorie B. Kovler Viral Oncology
Laboratories1 and
Department of
Pathology,2 The University of Chicago, Chicago,
Illinois 60637
Received 23 March 1998/Accepted 21 May 1998
In cells infected with the herpes simplex virus 1 (HSV-1)
recombinant R3616 lacking both copies of the
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Second-Site Mutation in the Herpes Simplex Virus Recombinants
Lacking the
134.5 Genes Precludes Shutoff of Protein
Synthesis by Blocking the Phosphorylation of eIF-2
134.5 gene,
the double-stranded protein kinase R (PKR) is activated, eIF-2
is
phosphorylated, and protein synthesis is shut off. Although PKR is also
activated in cells infected with the wild-type virus, the product of
the
134.5 gene, infected-cell protein 34.5 (ICP34.5),
binds protein phosphatase 1
and redirects it to dephosphorylate
eIF-2
, thus enabling sustained protein synthesis. Serial passage in
human cells of a mutant lacking the
134.5 gene yields
second-site, compensatory mutants lacking various domains of the
47
gene situated next to the US11 gene (I. Mohr and Y. Gluzman, EMBO J. 15:4759-4766, 1996). We report the construction of
two recombinant viruses: R5103, lacking the
134.5,
US8, -9, -10, and -11, and
47 (US12) genes;
and R5104, derived from R5103 and carrying a chimeric DNA fragment
containing the US10 gene and the promoter of the
47 gene
fused to the coding domain of the US11 gene. R5104
exhibited a protein synthesis profile similar to that of wild-type
virus, whereas protein synthesis was shut off in cells infected with R5103 virus. Studies on the wild-type parent and mutant viruses showed
the following: (i) PKR was activated in cells infected with parent or
mutant virus but not in mock-infected cells, consistent with earlier
studies; (ii) lysates of R3616, R5103, and R5104 virus-infected cells
lacked the phosphatase activity specific for eIF-2
characteristic of
wild-type virus-infected cells; and (iii) lysates of R3616 and R5103,
which lacked the second-site compensatory mutation, contained an
activity which phosphorylated eIF-2
in vitro, whereas lysates of
mock-infected cells or cells infected with HSV-1(F) or R5104 did not
phosphorylate eIF-2
. We conclude that in contrast to wild-type
virus-infected cells, which preclude the shutoff of protein synthesis
by causing rapid dephosphorylation of eIF-2
, in cells infected with
134.5
virus carrying the compensatory
mutation, eIF-2
is not phosphorylated. The activity made apparent by
the second-site mutation may represent a more ancient mechanism evolved
to preclude the shutoff of protein synthesis.
*
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.
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