The herpes simplex virus US11 protein effectively compensates for the gamma1(34.5) gene if present before activation of protein kinase R by precluding its phosphorylation and that of the alpha subunit of eukaryotic translation initiation factor 2 - PubMed (original) (raw)

The herpes simplex virus US11 protein effectively compensates for the gamma1(34.5) gene if present before activation of protein kinase R by precluding its phosphorylation and that of the alpha subunit of eukaryotic translation initiation factor 2

K A Cassady et al. J Virol. 1998 Nov.

Abstract

In herpes simplex virus-infected cells, viral gamma134.5 protein blocks the shutoff of protein synthesis by activated protein kinase R (PKR) by directing the protein phosphatase 1alpha to dephosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). The amino acid sequence of the gamma134.5 protein which interacts with the phosphatase has high homology to a domain of the eukaryotic protein GADD34. A class of compensatory mutants characterized by a deletion which results in the juxtaposition of the alpha47 promoter next to US11, a gamma2 (late) gene in wild-type virus-infected cells, has been described. In cells infected with these mutants, protein synthesis continues even in the absence of the gamma134.5 gene. In these cells, PKR is activated but eIF-2alpha is not phosphorylated, and the phosphatase is not redirected to dephosphorylate eIF-2alpha. We report the following: (i) in cells infected with these mutants, US11 protein was made early in infection; (ii) US11 protein bound PKR and was phosphorylated; (iii) in in vitro assays, US11 blocked the phosphorylation of eIF-2alpha by PKR activated by poly(I-C); and (iv) US11 was more effective if present in the reaction mixture during the activation of PKR than if added after PKR had been activated by poly(I-C). We conclude the following: (i) in cells infected with the compensatory mutants, US11 made early in infection binds to PKR and precludes the phosphorylation of eIF-2alpha, whereas US11 driven by its natural promoter and expressed late in infection is ineffective; and (ii) activation of PKR by double-stranded RNA is a common impediment countered by most viruses by different mechanisms. The gamma134.5 gene is not highly conserved among herpesviruses. A likely scenario is that acquisition by a progenitor of herpes simplex virus of a portion of the cellular GADD34 gene resulted in a more potent and reliable means of curbing the effects of activated PKR. US11 was retained as a gamma2 gene because, like many viral proteins, it has multiple functions.

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Figures

FIG. 1

Schematic representation of the HSV-1 genome and of genome domains relevant to this report. Line 1, sequence arrangement of the domains of the genes US10, US11, and α47 (US12) located at the terminus of unique short (US) sequence shown in the prototype (P) orientation. The coding sequences are shown as rectangular boxes; thin lines and arrows represent the transcriptional unit and polarity of the genes. Line 2, sequence arrangement of HSV-1 genome. The rectangular boxes represent the inverted repeat sequences ab and _b′a_′ flanking the unique long (UL) sequence and inverted repeats c′a′ and ca flanking the US sequence. Line 3, map location of the Δ134.5 gene. In wild-type virus, the Δ134.5 gene is present in both copies of the inverted repeats. Line 4, representation of the structure of R3616 DNA in which both copies of the Δ134.5 had been deleted. Line 5, structure of the single ab sequence of the R5103 genome in which the Δ134.5 gene had been replaced with the E. coli lacZ gene. Line 6, sequence arrangement of the R5103 genome. In both the R5103 and parent R7023 genomes (29), the genes US8 to -12 as well as most of the internal inverted repeats had been deleted. The US sequence is in an inverted (IS) arrangement. Line 7, sequence arrangement of DNA fragment inserted into the _Bgl_II site of the _Bam_HI Q fragment, resulting in construction of the R5104 recombinant virus. The thick line represents the _Bam_HI Q fragment disrupted by the insertion of the fragment consisting of the α47 gene fused to the coding domain of the US11 gene. Abbreviations: B, _Bam_HI; Bg, _Bgl_II; Bs, _Bst_EII; E, _Eco_RI; N, _Nco_I; Nr, _Nru_I; S, _Sal_I; Sc, _Sac_II; St, _Stu_I.

FIG. 2

Photograph of an immunoblot of lysates of wild-type and mutant viruses probed with US11 and ICP0 antibodies. Mock-infected SK-N-SH cells or SK-N-SH cells infected with HSV-1(F), R3616, R5103, or R5104 were harvested at 3, 6, 9, or 12 h after infection as described in Materials and Methods, lysed in disruption buffer, subjected to electrophoresis on an SDS–12.5% polyacrylamide gel, electrically transferred to a nitrocellulose sheet, and reacted with the anti-US11 or ICP0 monoclonal antibody as described in Materials and Methods. Positions of the US11 (labeled B) and ICP0 (labeled A) protein bands are indicated.

FIG. 3

Autoradiogram and photograph of proteins from infected cell lysates that were bound to GST or GST-PKR, subjected to electrophoretic separation and autoradiography, and reacted with the anti-US11 antibody. Equivalent quantities of GST or GST-PKR bound to beads were reacted overnight with the radiolabeled, RNase-treated, and sonicated lysates from 2 × 106 HeLa cells infected with HSV-1(F) as described in Materials and Methods. The beads were then washed seven times with PBS containing 1% NP-40 and 1% DOC, denatured in disruption buffer, boiled, subjected to electrophoresis in a denaturing 12.5% polyacrylamide gel, electrically transferred to a nitrocellulose sheet, reacted with the anti-US11 antibody, and subjected to autoradiography. The position of the US11 protein band is indicated. (A) Autoradiographic image of the electrophoretically separated proteins bound to beads; (B) photograph of the immunoblot.

FIG. 4

Autoradiographic images of electrophoretically separated proteins from reaction mixtures containing lysates of cells infected with R3616 (γ134.5−) as a source of activated PKR, eIF-2, and either GST or GST-US11. Phosphorylation reactions contained the equivalent of 0.19 μl of lysates of R3616-infected cells (lanes 1 to 9) or R5104-infected cell lysate (lane 10), 5 pmol of eIF-2 (lanes 2 to 10), and GST-US11 (lanes 3, 5, and 7) or GST (lanes 4, 6, and 8) in concentrations shown in a final volume of 10 μl of TKM buffer. The mixtures were preincubated at 34°C for 30 s; the reaction was started by the addition of [γ-32P]ATP (final concentration, 0.04 mM) and terminated by the addition of an SDS denaturing solution after 1 min of further incubation at 34°C. The mixtures were subjected to electrophoresis in denaturing polyacrylamide gels and autoradiography. The positions of eIF-2α, eIF-2β, the _M_r-39,000 phosphoprotein, GST-US11, and GST are shown.

FIG. 5

Autoradiographic images of electrophoretically separated proteins from reaction mixtures containing activated or nonactivated purified PKR, eIF-2, and either GST or GST-US11. PKR, partially purified from rabbit reticulocyte ribosomes as indicated in Material and Methods, was preincubated with 0.10 mM ATP and 2.5 mM MgCl2, either without (nonactivated PKR) or with poly(I-C) (0.1 μg/ml), for 20 min at 34°C. The contents of the reaction mixtures are given above the autoradiogram. The additions were 5 pmol of eIF-2, 22 pmol of GST-US11 or GST, and a final concentration of 0.09 mM [γ-32P]ATP in a final volume of 10.5 μl of TKM buffer. The reaction was begun by addition of activated (lanes 1 to 8) or nonactivated (lanes 9 to 14) PKR and incubation at 34°C. Aliquots (5.0 μl) were removed from each reaction mixture and placed into disruption buffer after 6 and 15 min, boiled, and subjected to electrophoresis in denaturing gels and autoradiography. Positions of the US11 protein, _M_r-39,000 phosphoprotein, and eIF-2α bands are shown. The position of GST is indicated, although it was not labeled.

FIG. 6

Autoradiographic images of electrophoretically separated proteins from reaction mixtures in which PKR was activated before or after addition of the GST-US11 chimeric protein. The order of addition of the reactants in mixtures 1 through 14 is shown at the top. Phosphorylation reaction mixtures contained of eIF-2 (2.5 pmol) and PKR; poly(I-C) (0.1 μg/ml) and 0.09 mM [γ-32P]ATP were added after (lanes 1 to 7) or before (lanes 8 to 14) addition of GST, GST-US11, or GST-UL10 in the concentrations shown. All components were in TKM buffer, and the final volume was 5.25 μl. The reaction mixtures were incubated at 34°C for 15 min. The reaction was stopped by the addition of disruption buffer, and the proteins were separated on denaturing gels and subjected to autoradiography. Positions of the reacting proteins are shown.

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The herpes simplex virus US11 protein effectively compensates for the gamma1(34.5) gene if present before activation of protein kinase R by precluding its phosphorylation and that of the alpha subunit of eukaryotic translation initiation factor 2 - PubMed (original) (raw)