Control of VP16 translation by the herpes simplex virus type 1 immediate-early protein ICP27

Abstract

Herpes simplex virus (HSV) ICP27 is an essential and multifunctional regulator of gene expression that modulates the synthesis and maturation of viral and cellular mRNAs. Processes that are affected by ICP27 include transcription, pre-mRNA splicing, polyadenylation, and nuclear RNA export. We have examined how ICP27 influences the expression of the essential HSV tegument protein and transactivator of immediate-early gene expression VP16. We monitored the effects of ICP27 on the levels, nuclear export, and polyribosomal association of VP16 mRNA and on the amount and stability of VP16 protein. Deletion of ICP27 reduced the levels of VP16 mRNA without altering its nuclear export or the stability of the encoded protein. However, the translational yield of the VP16 mRNA produced in the absence of ICP27 was reduced 9- to 80-fold relative to that for wild-type infection, suggesting a defect in translation. In the absence of ICP27, the majority of cytoplasmic VP16 mRNA was not associated with actively translating polyribosomes but instead cosedimented with 40S ribosomal subunits, indicating that the translational defect is likely at the level of initiation. These effects were mRNA specific, as polyribosomal analysis of two cellular transcripts (glyceraldehyde-3-phosphate dehydrogenase and beta-actin) and two early HSV transcripts (thymidine kinase and ICP8) indicated that ICP27 is not required for efficient translation of these mRNAs. Thus, we have uncovered a novel mRNA-specific translational regulatory function of ICP27.

FIG. 1.

VP16 mRNA is predominantly cytoplasmic in the absence of ICP27. Vero cells were either mock infected or infected with the indicated viruses at a multiplicity of 10. At 12 h postinfection, postmitochondrial supernatants were prepared from the cells and RNA was extracted either from this cytoplasmic (C) fraction or from the pelleted nuclei (the nuclear [N] fraction). RNAs from equivalent numbers of cells were analyzed by Northern blotting for the VP16 transcript or the U3 snoRNA.

FIG. 2.

Comparison of VP16 protein and RNA levels in infected cells containing or lacking ICP27. Vero cells were mock infected or infected in duplicate with the KOS1.1 or d27-1 virus at a multiplicity of 10. At the indicated times postinfection, protein was harvested from one dish and total RNA was isolated from the duplicate. VP16 protein (A) and RNA (B) were analyzed by Western blotting and Northern blotting, respectively. Levels of VP16 protein and RNA were quantified and plotted as a function of time.

FIG. 3.

VP16 protein stability is unaltered in the absence of ICP27. Vero cells were mock infected or infected with HSV KOS1.1 or d27-1 at a multiplicity of 10 for 12 h to allow VP16 to accumulate. Cycloheximide (CHX) was added to the culture medium at a concentration of 100 μg per milliliter to arrest further protein synthesis, and total protein was harvested directly into SDS-PAGE lysis buffer at the indicated times following addition of cycloheximide. VP16 protein was detected by Western blotting as described in Materials and Methods. Note that the volume loaded for the d27-1 samples was 4 times that loaded for the KOS1.1 samples (20 and 5 μl, respectively), in order to detect sufficient quantities of VP16 to note changes in the levels following CHX addition.

FIG. 4.

VP16 mRNA is predominantly associated with 40S ribosomal subunits in cells infected with viruses lacking ICP27. Postmitochondrial supernatants were prepared from mock-infected, KOS1.1-infected, or d27-1-infected Vero cells and fractionated on sucrose gradients. (A) UV absorbance profiles at 254 nm of the 20 fractions collected from each gradient. The top of the gradient is to the left (fraction 1), and the bottom is to the right (fraction 20). The peak in fractions 5 to 8 corresponds to ribosomal subunits and monoribosomes. Note that since the absorbance of the gradient was not monitored continuously, the individual ribosomal subunit peaks are not resolved in these profiles. (B) Agarose gel electrophoresis of RNA isolated from the sucrose gradient fractions. Half of the RNA isolated from each fraction was electrophoresed on a 1% denaturing agarose gel and stained with ethidium bromide to visualize the 5S, 18S, and 28S rRNAs (indicated to the right). (C) Distribution of VP16 transcripts across the sucrose gradients as determined by Northern blotting. (D) The VP16 bands in panel C were quantified, and the percentage of total RNA present in each fraction was graphed.

FIG. 5.

Phosphorylation of eIF2α is not increased in cells infected with d27-1. Vero cells were either mock infected, infected for 12 h with the indicated virus, or treated with thapsigargin (TG) for 1 h. The cells were harvested for SDS-PAGE, and the content of eIF2α or phospho-Ser51-eIF2α was determined by Western blot analysis.

FIG.6.

Translational efficiency of two cellular RNAs is not impaired in HSV-infected cells in the absence of ICP27. Postmitochondrial supernatants from uninfected Vero cells or from cells infected with d27-1 for 12 h at a multiplicity of 10 were analyzed by sedimentation through sucrose gradients as in Fig. 4. (A) UV absorbance profiles at 254 nm of the 20 fractions collected from each gradient. Fraction 1 represents the top of the gradient, and fraction 20 represents the bottom. (B) Agarose gel electrophoresis of RNA isolated from the sucrose gradient fractions. Half of the RNA isolated from each fraction was electrophoresed on a 1% denaturing agarose gel and stained with ethidium bromide to visualize the 5S, 18S, and 28S rRNAs (indicated to the right). (C and D) The distribution of the GAPDH (C) and β-actin (D) transcripts across each gradient was determined by Northern blot analysis. The transcript bands were quantified, and the percentage of total RNA present in each fraction was graphed and is shown below the Northern blots.

FIG. 7.

Polyribosomal distributions of VP16, TK, and ICP8 mRNAs at early and late times postinfection. Vero cells were infected with HSV KOS1.1 or d27-1 at a multiplicity of 10. At 6 or 12 h postinfection, postmitochondrial supernatants were prepared and fractionated on 10 to 50% sucrose gradients such that polysomes sedimented in fractions 15 to 18. Following fractionation, RNA in the pellet was recovered by resuspending in H₂O. The distributions of VP16, TK, and ICP8 mRNA in the gradient fractions and the pellet (last fraction) were determined by Northern blot analysis. The RNA in each fraction was quantified, and a graph of the percentage of total RNA in each fraction is shown.