The herpes simplex virus 1 protein kinase US3 is required for protection from apoptosis induced by the virus

Abstract

An earlier report showed that a disabled mutant lacking both copies of the major regulatory gene (alpha4) of herpes simplex virus 1 induced DNA degradation characteristic of apoptosis in infected cells, whereas the wild-type virus protected cells from apoptosis induced by thermal shock. More extensive analyses of the disabled mutant revealed a second mutation which disabled US3, a viral gene encoding a protein kinase known to phosphorylate serine/threonine within a specific arginine-rich consensus sequence. Analyses of cells infected with a viral mutant carrying a wild-type alpha4 gene but from which the US3 gene had been deleted showed that it induced fragmentation of cellular DNA, whereas a recombinant virus in which the deleted sequences of the US3 gene had been restored did not cause the cellular DNA to fragment. These results point to the protein kinase encoded by the US3 gene as the principal viral product required to block apoptosis.

Figure 1

Map of HSV-1 genome and summary of results. (A) The HSV-1 genome is represented in its circular form. Cosmids pBC1004, pBC1009, and pBC1008 are indicated as A, B, and C, respectively. The termini of the cosmid sequences carry a nucleotide number according to McGeoch et al. (9, 10). (B) Recombinant viruses were obtained by homologous recombination with either plasmid or cosmid DNA. The ∗ denotes a virus that was generated by recombination with the α4 gene sequence resident in the E5 cell line. The results are expressed as the ratio of viral plaque isolates that protected from virus-induced apoptosis to total isolates tested.

Figure 2

Photograph of an agarose gel containing electrophoretically separated DNA fragments and stained with ethidium bromide. Vero cells were mock-infected (lane 1) or infected with HSV-1(F) (lane 2), HSV-1 d120 mutant (lane 3), HSV-1 120FR mutant (lane 4), or HSV-1 120KR mutant (lane 5). At 30 hr after infection, 2 × 10⁶ cells per sample were collected, rinsed with PBS, lysed in a solution containing 10 mM Tris⋅HCl at pH 8.0, 10 mM EDTA, and 0.5% Triton X-100, and centrifuged at 12,000 rpm for 25 min in an Eppendorf microcentrifuge to pellet chromosomal DNA. Supernatant fluids were digested with 0.1 mg of RNase A per ml at 37°C for 1 hr and then for 2 hr with 1 mg of proteinase K per ml at 50°C in the presence of 1% sodium dodecyl sulfate (SDS), extracted with phenol and chloroform, and precipitated in cold ethanol and subjected to electrophoresis on horizontal 1.5% agarose gels containing 5 μg of ethidium bromide per ml. DNA was visualized by UV light transillumination. Photographs were taken with the aid of a computer-assisted image processor (Eagle Eye II; Stratagene).

Figure 3

Photograph of an agarose gel containing electrophoretically separated DNA fragments and stained with ethidium bromide. Vero cells were mock-infected (lane 1), infected with HSV-1 120FR mutant (lane 2), wild-type HSV-1(F) (lane 3), HSV-1 R325 mutant (lane 4), or HSV-1 R7041 mutant (lane 5). Cells were harvested and processed as described in the legend to Fig. 2.

Figure 4

Photograph of an agarose gel containing electrophoretically separated DNA fragments and stained with ethidium bromide. Vero cells were infected with HSV-1(F) (lane 1), HSV-1 120FR mutant (lane 2), HSV-1 R7041 mutant (lane 3), HSV-1 R7306 mutant (lane 4), or double-infected with HSV-1 120FR and R7041 mutants (lane 5), HSV-1 d120 and 120FR mutants (lane 6), or HSV-1 120FR and R7306 mutants (lane 7). Cells were harvested and processed as described in the legend to Fig. 2.

Figure 5

BHK-C13 cells were infected with 10 pfu of indicated viruses per cell. At 13 hr after infection the cells were incubated in phosphate-free medium for 1 hr and then labeled in the same medium supplemented with ³²P_i for 4 hr as described earlier (5). Cell lysates were electrophoretically separated in SDS/polyacrylamide gels and electrically transferred onto nitrocellulose filters. Filters were exposed to film or allowed to react with rabbit polyclonal antibody to the U_L34 protein as described earlier (5).

Figure 6

Photograph of electrophoretically separated proteins from HEp-2 cells either mock-infected or infected with 10 pfu of indicated virus per cell and harvested 6 or 18 hr after infection. A total of 2 × 10⁵ cells per sample were disrupted in SDS and 2-mercaptoethanol-containing buffer, boiled for 3 min, and electrophoretically separated on an SDS/N,N′-diallyltartardiamide/9.5% polyacrylamide gel at 4°C. The denatured, electrophoretically separated polypeptides were electrically transferred to a nitrocellulose sheet, where they were allowed to react with an anti-ICP4 monoclonal antibody (H640).