The second-site mutation in the herpes simplex virus recombinants lacking the gamma134.5 genes precludes shutoff of protein synthesis by blocking the phosphorylation of eIF-2alpha

Abstract

In cells infected with the herpes simplex virus 1 (HSV-1) recombinant R3616 lacking both copies of the gamma134.5 gene, the double-stranded protein kinase R (PKR) is activated, eIF-2alpha 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 gamma134.5 gene, infected-cell protein 34.5 (ICP34.5), binds protein phosphatase 1alpha and redirects it to dephosphorylate eIF-2alpha, thus enabling sustained protein synthesis. Serial passage in human cells of a mutant lacking the gamma134.5 gene yields second-site, compensatory mutants lacking various domains of the alpha47 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 gamma134. 5, US8, -9, -10, and -11, and alpha47 (US12) genes; and R5104, derived from R5103 and carrying a chimeric DNA fragment containing the US10 gene and the promoter of the alpha47 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-2alpha 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-2alpha in vitro, whereas lysates of mock-infected cells or cells infected with HSV-1(F) or R5104 did not phosphorylate eIF-2alpha. We conclude that in contrast to wild-type virus-infected cells, which preclude the shutoff of protein synthesis by causing rapid dephosphorylation of eIF-2alpha, in cells infected with gamma134.5(-) virus carrying the compensatory mutation, eIF-2alpha 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.

FIG. 1

Schematic representation of the DNA sequence arrangements of the wild-type and recombinant viruses used in this study. Line 1, schematic representation of the wild-type HSV-1 genome. The genome consists of two covalently linked components, L and S, each consisting of unique sequences (U_L and U_S) flanked by inverted repeats. The arrangement shown is the I_S isoform in which the S component is inverted relative to the prototypic orientation of the L component. The inverted repeat sequences designated ab and b′a′ flanking the U_L sequence are 9 kb in size, whereas the repeat sequences ac and c′a′ flanking the U_S sequence are each 6.3 kb in size. Line 2, expansion of specific domains of the genome showing gene arrangements within the expanded region. Line 4, schematic representation of one of two γ₁34.5 coding domains in recombinant R3616 in which the sequences between the BstEII and StuI restriction endonuclease sites had been deleted. Line 6, representation of a portion of the genome of the recombinant R7023 shown in the I_S arrangement. Line 7, schematic representation of the wild-type U_L23 gene encoding thymidine kinase and key restriction endonuclease sites present in R7023. The domain encoding the genes U_S8 through U_S12 as well as the reiterated sequences a′ and ac and the portion of the b′ sequence encoding one of the copies of the γ₁34.5, ORF O, and ORF P genes are absent. R7023 was the parent of R5103 schematically represented in line 9. In R5103, the coding domain between the BstEII and StuI restriction sites in the remaining copy of the γ₁34.5 gene was replaced with the E. coli lacZ gene represented in line 10. Line 13, schematic representation of the recombinant virus R5104 constructed by homologous recombination between plasmid pRB4999 and R5103 DNA. In the resulting recombinant, R5104 (line 14), the U_L23 gene was disrupted by the insertion of U_S10 and U_S11 driven by the α47 promoter. Lines 3, 5, 8, 11, 12, and 15 represent the predicted bands produced by restriction endonuclease digestion of viral DNAs and are shown as reference for the bands shown in Fig. 2. Abbreviations: B, BamHI; N, NcoI; Bs, BstEII; St, StuI; Sc, SacI; Bg, BglII.

FIG. 2

Autoradiographic images of electrophoretically separated digests of viral DNAs hybridized with specific probes. (A) Electrophoretically separated NcoI digests hybridized with ³²P-labeled nick-translated probe of the wild-type NcoI fragment encoding the γ₁34.5 gene cloned as pRB4974. Both wild-type HSV-1(F) and recombinant virus R7023 contained the wild-type γ₁34.5 gene and the radioactive probe hybridized with a 1.8-kb NcoI fragment (band B); in recombinant virus R3616, the NcoI fragment (band C) was approximately 800 bp smaller since it lacked the sequence between the BstEII and StuI sites encoding the γ₁34.5 gene. In recombinant viruses R5103 and R5104, the 1-kb γ₁34.5 sequence between BstEII and StuI was replaced by the 3.2-kb E. coli lacZ gene, yielding a 4-kb DNA fragment (band A). (B) Electrophoretically separated BamHI digests probed with the labeled pGEM 3Zf(+) vector containing approximately 400 bp of the lacZ sequence. The lacZ sequences in recombinant viruses R5103 and R5104 formed single 3.2-kb bands (band D). The probe failed to hybridize with digests of HSV-1(F), R7023, or R3616 DNA. (C) Autoradiogram of the electrophoretically separated fragments shown in panel B but stripped and hybridized with labeled plasmid pRB3982 carrying the BamHI Q DNA fragment in a pUC-9 vector. The probe hybridized with wild-type 3.58-kb BamHI Q fragments (band E) in digests of HSV-1(F), R7023, R3616, or R5103 consistent with the expected size of the wild-type BamHI Q fragment and with an additional band (band D) in digests of recombinants R5103 and R5104 corresponding to the hybridization of the lacZ sequence in the probe with the gene inserted in place of the γ₁34.5 gene. In lane 5, two R5104 bands of 3.4 kb (band F) and 2.1 kb (band G) hybridized with the BamHI Q fragment. The insertion of the α47-U_S11 chimeric gene into BglII site of the BamHI Q fragment introduced an additional BamHI site from the U_S11 gene as shown schematically in Fig. 1, line 15.

FIG. 3

Autoradiographic image of electrophoretically separated [³⁵S]methionine-labeled proteins prepared from lysates of SK-N-SH cells mock infected or virus infected. Replicate cultures of SK-N-SH cells were mock infected or infected with 10 PFU of HSV-1(F), R7023, R3616, R3617, R5103, or R5104. At 13.5 h after infection the medium was replaced with medium 199V lacking methionine but supplemented with 50 μCi of [³⁵S]methionine (specific activity, >1,000 Ci/mmol; Amersham) for 1 h. After labeling, the infected cells were rinsed, harvested, solubilized in an SDS-containing buffer, and electrophoretically separated on a denaturing 12% polyacrylamide gel cross-linked with DATD. The electrophoretically separated proteins were transferred to a nitrocellulose sheet and subjected to autoradiography.

FIG. 4

Autoradiographic image of PKR electrophoretically separated from immune complexes derived from labeled infected HeLa cells. Replicate HeLa cell cultures were mock infected or infected with R7023, R5103, or R5104 as described in Materials and Methods. At 18 h after infection, the cells were harvested and lysed, and immune precipitates obtained with the anti-PKR antibody (K-17; Santa Cruz Research) were electrophoretically separated on a 12% polyacrylamide gel, transferred to a nitrocellulose sheet, and subjected to autoradiography. The positions of the M_r-200,000, -90,000, and -67,000 proteins determined from the molecular weight markers (Pharmacia) are indicated on the left. The heavily labeled bands with an apparent M_r of 67,000 were identified as PKR (7).

FIG. 5

Dephosphorylation of eIF-2α by lysates of mock-infected and infected cells. (A) Autoradiographic image of purified, in vitro-labeled eIF-2α reacted with lysates of mock-infected cells or cells infected with wild-type or recombinant viruses for time intervals shown and then electrophoretically separated in a denaturing gel. The reaction was terminated by mixing with buffer containing SDS; the reaction times are indicated above the image. The arrow identifies the α subunit of eIF-2 and the M_r-39,000 protein which copurifies with eIF-2. The slowly migrating unlabeled band represents the β subunit of eIF-2. (B) Radioactivity contained in individual bands as a function of time of exposure to infected cell lysates.

FIG. 6

(A) Autoradiographic image of electrophoretically separated purified eIF-2 samples after reaction in the presence of [γ³²P]ATP with S10 fractions from cells mock-infected or infected with HSV-1(F), R7023, R3616, R5103, or R5104. The arrows indicate the position of the α subunit of eIF-2 and the M_r-39,000 protein unrelated to eIF-2. (B) Autoradiographic image of electrophoretically separated proteins contained in the eIF-2 kinase reactions carried out as described above. The samples were electrophoretically separated and run on a longer gel to increase the separation of the M_r-39,000 phosphoprotein from eIF-2α. The arrows indicate the position of the α subunit of eIF-2 and the unrelated M_r-39,000 phosphoprotein present in the purified eIF-2 samples.