Direct stimulation of translation by the multifunctional herpesvirus ICP27 protein

Abstract

Herpes simplex virus type 1 (HSV-1) ICP27 protein is an essential regulator of viral gene expression with roles at various levels of RNA metabolism in the nucleus. Using the tethered function assay, we showed a cytoplasmic activity for ICP27 in directly enhancing mRNA translation in vivo in the absence of other viral factors. The region of ICP27 required for translational stimulation maps to the C terminus. Furthermore, in infected cells, ICP27 is associated with polyribosomes, indicating a function in translation during the lytic cycle.

FIG. 1.

ICP27 is associated with polyribosomes in infected cells. Cytoplasmic extracts of BHK cells either mock infected or infected with wt or M15 mutant virus were fractionated on 10% to 50% sucrose gradients, and fractions were examined by Western blotting using a mixture of anti-ICP27 1119 and 1113 monoclonal antibodies (Goodwin Institute for Cancer Research, Plantation, Fla.) or anti-PABP 10E10 antibody (9), kindly supplied by G. Dreyfuss. (A) UV absorbance profile (254 nm) of gradient fractions from mock-infected cells; the positions of polysomes, 80S monosomes, 60S and 40S ribosomal subunits, and mRNPs are indicated. (B) Western blot analysis of gradient fractions following mock infection or infection with the indicated viruses.

FIG. 2.

ICP27 stimulates translation in the tethered function assay. (A) Xenopus oocytes expressing MS2, MS2-U1A, MS2-PABP, or MS2-ICP27 were injected with a luciferase reporter mRNA containing MS2 coat protein binding sites (Luc-MS2) within its 3′ untranslated region and a β-galactosidase internal control mRNA (10). Shown are normalized luciferase/β-galactosidase activity ratios, with that of MS2 coat protein alone set as 1. The data represent the average stimulations from at least three independent experiments with standard errors. (B) mRNAs encoding MS2 coat protein or an MS2-ICP27 coat protein fusion were injected with β-galactosidase mRNA and either the reporter mRNA used for panel A (Luc-MS2; black bars) or a reporter lacking MS2 coat protein binding sites (Luc-ΔMS2; white bars [10]). The data represent the averages from three independent experiments with standard errors. (C) Oocytes expressing MS2 coat protein alone or the MS2-ICP27 fusion were injected with the Luc-MS2 and β-galactosidase reporter mRNAs (black and gray bars, respectively). Total RNA was extracted either immediately or after 12 h of incubation. Quantitative reverse transcription-PCR was performed with primers directed against the reporter mRNAs, using a TaqMan PCR machine (Applied Biosystems) linked to ABI Prism software. The threshold cycle shown is the number of amplification cycles required to obtain a detectable product in the linear range of the assay. The data represent the averages from three independent experiments with standard errors.

FIG. 3.

The ICP27 C terminus is required to stimulate translation. (A) Top, schematic diagram of ICP27 showing the locations of the RGG box (amino acids 138 to 152) and putative zinc finger region (amino acids 468 to 508). Bottom, horizontal bars represent sequences of the various ICP27 truncation mutants used. Gaps represent regions absent in the mutants, and locations of point mutations in the M15 (P465L, G466E) and M16 (C488L) mutants are shown by vertical bars. Deletions in the mutants are as follows: d1-2, amino acids 12 to 63; d4-5, amino acids 139 to 153; Δ407, amino acids 408 to 512. (B) and (C) MS2 coat protein or the indicated MS2-ICP27 fusion proteins were examined using the tethered function assay. The results are averages from at least three independent experiments with standard errors. (D) Xenopus oocytes were injected with RNA encoding MS2 fused to wild-type or mutants of ICP27 as indicated. Fusion protein expression was analyzed by [³⁵S]methionine labeling in vivo and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein extracts (lanes 1 to 5) or by translation in vitro (lanes 6 and 7). The upper and lower arrows mark the positions of full-length and truncated proteins, respectively.