The herpes simplex virus type 1 U(S)11 protein interacts with protein kinase R in infected cells and requires a 30-amino-acid sequence adjacent to a kinase substrate domain

Abstract

The herpes simplex virus type 1 gamma(1)34.5 gene product precludes the host-mediated protein shutoff response induced by activated protein kinase R (PKR). Earlier studies demonstrated that recombinant viruses lacking the gamma(1)34.5 gene (Deltagamma(1)34.5) developed secondary mutations that allowed earlier U(S)11 expression and enabled continued protein synthesis. Further, in vitro studies demonstrated that a recombinant expressed U(S)11 protein binds PKR, blocks the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) by activated PKR, and, if provided prior to PKR activation, precluded PKR autophosphorylation. The present study furthers the hypothesis that early U(S)11 production precludes PKR-mediated host protein shutoff by demonstrating that (i) U(S)11 and PKR interact in the context of viral infection, (ii) this interaction is RNA dependent and requires a 30-amino-acid domain (amino acids 91 to 121) in the carboxyl domain of the U(S)11 protein, (iii) the proteins biochemically colocalize in the S100 ribosomal fraction, and (iv) there is a PKR substrate domain immediately adjacent to the binding domain. The results suggest that the U(S)11 interaction with PKR at the ribosome is RNA dependent and that the U(S)11 protein contains a substrate domain with homology to eIF-2alpha in close proximity to an essential binding domain.

FIG. 1.

U_S11 protein coimmunoprecipitates with PKR in infected HeLa cell cytoplasmic fractions. Replicate cell cultures of HeLa cells were either mock infected or infected with HSV-1 (F), R5103, or R5104 virus, and S10 cytoplasmic fractions were generated. (A) Photograph of an immunoblot of the starting supernatant material. The electrophoretically separated proteins from mock-, HSV-1 (F)-, R5103-, and R5104-infected-HeLa cell S10 fractions (lanes 1 to 4, respectively) were probed with an anti-U_S11 monoclonal antibody, and the relative amounts of U_S11 in the starting lysates are shown. (B) Photographic image of electrophoretically separated proteins after immunoprecipitation from the above S10 fractions using 1 μg of anti-PKR antiserum (K-17; Santa Cruz Biotechnology) or rabbit preimmune serum (R IgG; Southern Research Institute). S10 cytoplasmic fractions were precleared with protein A and then incubated either with 1 μg of anti-PKR antibody (K-17; Santa Cruz Biotechnology) or with 1 μg of the rabbit preimmune serum antibody overnight. The complexes were precipitated with protein A-Sepharose and washed three times, and the immunoprecipitated proteins were electrophoretically separated, transferred to nitrocellulose filters, and probed with an antibody to U_S11.

FIG. 2.

Fine mapping of the U_S11 protein domain required for PKR interaction using recombinant U_S11 truncation virus-infected HeLa cell cytoplasmic S10 fractions and PKR immunoprecipitation. (A) Redrawing of Roller's schematic depiction of the U_S11 truncation viruses and the amino acid deletions (29). Solid bars, N-terminal domains of the protein; hatched bars, C-terminal domains comprising tandemly repeated P-R-X amino acids. (B) Photographic depiction of immunoblots. (Top) Photograph of mock- and virus-infected HeLa cell cytoplasmic proteins after electrophoretic separation, transfer to a nitrocellulose membrane, and staining with a mouse anti-U_S11 monoclonal antibody. (Bottom) Immunoblot of the wild-type and mutant U_S11 proteins coimmunoprecipitated with 1 μg of rabbit anti-PKR antibody and then stained with an anti-U_S11 mouse monoclonal antibody. (C) Photograph of the immunoblots from panel B after they were reprobed with a chicken polyclonal antibody against U_S11 and after the proteins were detected using the alkaline phosphatase-conjugated secondary antibody used for panel B. The new antiserum is capable of detecting the mutant U_S11 from R4779 in which amino acids 5 to 35 have been deleted.

FIG. 3.

RNase A and T₁ incubation disrupts PKR-U_S11 interaction. Photograph of an immunoblot probed with an anti-U_S11 monoclonal antibody. PKR was immunoprecipitated from mock- and virus-infected-cell cytoplasmic lysates after either mock or RNase A and T₁ treatment. The immunoprecipitated proteins were washed, disrupted, electrophoretically separated by SDS-PAGE, and transferred to nitrocellulose membranes. The membrane was then developed using an anti-U_S11 monoclonal antibody and an antimouse peroxidase-conjugated secondary antibody and developed by enhanced chemiluminescence. IgG, immunoglobulin G.

FIG. 4.

Composite photograph of immunoblots of electrophoretically separated S100 ribosomal proteins from mock- or virus-infected HeLa cell after isotonic or high-salt (0.8 M KCl) washes. (Top) Demonstration of PKR in the S100 protein pellet following incubation in isotonic buffer (lanes 1 to 4) and the presence of PKR in the supernatant (lanes 13 to 16) as well as the ribosomal pellet (lanes 9 to 12) following incubation in high-salt-concentration buffer. The U_S11 protein is detectable only in the S100 ribosomal pellet (lanes 1 to 4) after incubation in isotonic buffer; however, following incubation in high-salt-concentration buffer Us11 is present in both the pellet (lanes 9 to 12) and supernatant (lanes 13 to 16).

FIG. 5.

Schematic representation of the HSV-1 (F) U_S11 amino acid sequence in single-letter code. The sequenced tryptic peptide constituting the site of phosphorylation by activated PKR is in boldface, and the residues phosphorylated by activated PKR are under-lined. The boxed residues represent the required 30-amino-acid PKR binding domain (amino acids 91 to 121).

FIG. 6.

Homology between the substrate site of the HSV-1 U_S11 protein and eIF-2α. Solid line, identical residues; dots, semiconservative changes. Residues phosphorylated by PKR are in boldface.