Herpes simplex virus virion host shutoff protein is stimulated by translation initiation factors eIF4B and eIF4H

Abstract

The virion host shutoff protein (vhs) of herpes simplex virus triggers accelerated degradation of cellular and viral mRNAs while sparing other cytoplasmic RNA species. Previous work has shown that vhs forms a complex with translation initiation factor eIF4H, which displays detectable RNase activity in the absence of other viral or host proteins. However, the contributions of eIF4H and other host factors to the activity and mRNA targeting properties of vhs have not yet been directly examined. An earlier report from our laboratory demonstrated that rabbit reticulocyte lysate (RRL) contains one or more factors that strongly stimulate the RNase activity of vhs produced in Saccharomyces cerevisiae. We report here that such yeast extracts display significant vhs-dependent RNase activity in the absence of mammalian factors. This activity differs from that displayed by vhs generated in RRL in that it is not targeted to the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). Activity was strongly enhanced by the addition of RRL, eIF4H, or the related translation factor eIF4B. RRL also reconstituted strong targeting to the EMCV IRES, resulting in a major change in the RNA cleavage pattern. In contrast, eIF4H and eIF4B did not reconstitute IRES-directed targeting. These data indicate that eIF4B and 4H stimulate the nuclease activity of vhs, and they provide evidence that additional mammalian factors are required for targeting to the EMCV IRES.

FIG. 1.

Extracts of yeast expressing 2.1vhs contain endoribonuclease activity that is enhanced by one or more mammalian cofactors. (A and B) pCITE-1 and SRP-α RNA substrates, respectively, showing the sites of initial cleavage events by vhs translated in RRL. The EMCV IRES on pCITE-1 is indicated. (C and D) Analysis of nuclease activity on pCITE-1 and SRP-α RNA substrates, respectively. Internally labeled pCITE-1 and SRP-α RNA was added to control RRL (RRL), RRL pretranslated with vhs (RRLvhs), extracts of yeast harboring the empty expression vector either alone (EV) or mixed with RRL (EV+RRL), and extracts of yeast expressing 2.1vhs either alone (2.1vhs) or mixed with RRL (2.1vhs+RRL). RNA was extracted at the indicated time points, resolved on a 1.2% agarose-2% formaldehyde gel, and transferred to a Gene Screen Plus membrane, and the RNA signal was detected by autoradiography. The solid triangle indicates the previously described 600-nt degradation product corresponding to the EMCV IRES. The position and size of the markers are indicated in nucleotides at the left. (E and F) Quantification of nuclease activity on pCITE-1 and SRP-α RNAs, respectively. The quantity of full-length RNA in panels C and D and in at least two other experiments was determined using phosphorimager analysis and plotted against the time points indicated. The error bars indicate the standard deviation of each time point calculated from at least three independent experiments.

FIG. 2.

Effect of the D215N vhs mutation on the RNase nuclease activity of yeast extracts. (A) Nuclease activity of cell extracts. Internally labeled pCITE-1 RNA was added to extracts of yeast harboring the empty expression vector (EV), 2.1 vhs expression vector (2.1vhs), or 2.1 expression vector bearing the D215N mutation (D215N), either alone or supplemented with RRL. In addition, a portion of the RNA was incubated in RRL (RRL). Samples were withdrawn at the indicated times, and RNA was extracted and analyzed as described in the legend for Fig. 1. (B) The data obtained in the experiment shown in panel A were quantified by phosphorimager analysis. (C) Western blot analysis of yeast extracts. Aliquots of the extracts used for panel A (each containing 20 μg of protein) were subjected to electrophoresis through an SDS-10% polyacrylamide gel, transferred to a nitrocellulose membrane, and then analyzed by Western blotting using a polyclonal rabbit anti-vhs antiserum (AE328).

FIG. 3.

Analysis of purified proteins. Partially purified eIF4H, eIF4A, eIF4B, and cdc34Δ₂₀₉ (5 μg of each) were resolved on an SDS-12% PAGE gel and stained with Coomassie brilliant blue. The positions and sizes of the protein markers are indicated in kilodaltons on the left.

FIG. 4.

eIF4H stimulates the endoribonuclease activity of vhs produced in yeast. (A and B) Analysis of nuclease activity on pCITE-1 and SRP-α RNA substrates, respectively. Internally labeled pCITE-1 and SRP-α RNAs were added to control RRL (RRL), eIF4H mixed with vhs assay buffer (eIF4H), control extracts alone, mixed with RRL (+RRL), or mixed with eIF4H (+eIF4H), and yeast extracts containing 2.1vhs alone, mixed with RRL (+RRL), or mixed with eIF4H (+eIF4H). eIF4H was used at a concentration of 7.7 μM. Samples were processed as described in the legend for Fig. 1. Symbols and brackets are as described in the legend for Fig. 1. The solid triangle indicates the 600-nt fragment. The open square and triangles indicate additional ca. 900- and 1,200-nt degradation products, respectively. The positions and sizes of the RNA markers are indicated in nucleotides at the left. (C and D) Quantification of nuclease activity on pCITE-1 and SRP-α RNAs, respectively. The quantity of full-length RNA in panels A and B and at least two other experiments was determined using phosphorimager analysis and plotted against time points indicated. The error bars indicate the standard deviation of each time point calculated from at least three independent experiments.

FIG. 5.

The enhancement of the RNase activity of vhs expressed in yeast is specific to eIF4H. (A and B) Analysis of nuclease activity on pCITE-1 and SRP-α RNAs, respectively. Internally labeled pCITE-1 and SRP-α RNAs were added to yeast extracts containing 2.1vhs either alone, mixed with eIF4H (+eIF4H), or mixed with cdc34Δ₂₀₉ (+cdc34). The RNA substrates were also added to eIF4H or cdc34Δ₂₀₉ mixed with 20 μg of BSA and 1 μg of total yeast RNA per time point. eIF4H and cdc34Δ₂₀₉ were used at a concentration of 7.7 μM. Samples were processed as described in the legend for Fig. 1. The positions and sizes of the RNA markers in nucleotides are indicated at the left.

FIG. 6.

eIF4H does not enhance the RNase activity of yeast expressing inactive (D215N) vhs. Internally labeled pCITE-1 RNA was added to extracts of yeast harboring the empty expression vector (EV), 2.1 vhs expression vector (2.1vhs), or 2.1 expression vector bearing the D215N mutation (D215N), either alone or supplemented with eIF4H. In addition, a portion of the RNA was incubated with eIF4H alone. Samples were withdrawn at the indicated times, and the RNA was extracted and analyzed as described in the legend for Fig. 1. (B) The data obtained in the experiment shown in panel A were quantified by phosphorimager analysis.

FIG. 7.

eIF4H does not restore efficient IRES-directed targeting to yeast-expressed vhs. (A) Analysis of nuclease activity on pCITE-1 RNA. Internally labeled pCITE-1 RNA was added to control RRL (RRL) and yeast extracts containing 2.1vhs in the presence or absence of RRL or eIF4H (2.1vhs, 2.1vhs+RRL, and 2.1vhs+eIF4H). Samples were processed as described in the legend for Fig. 1. (B) Diagram of pCITE-1 RNA, indicating the positions of hybridization of oligonucleotides ORD17 and JPP-E specific for the 5′ and 3′ ends of the RNA, respectively. Also indicated are the RNA fragments produced upon initial cleavage of the RNA by vhs translated in RRL. (C and D) Northern blot analysis of nuclease activity on pCITE-1 RNA using oligonucleotide probes for the 5′ and 3′ ends, respectively, of pCITE-1 RNA. Unlabeled pCITE-1 RNA was added to control RRL (RRL), RRL containing pretranslated vhs (RRLvhs), and yeast extracts containing 2.1vhs, either alone (2.1vhs), mixed with RRL (2.1vhs+RRL), or mixed with eIF4H (2.1vhs+eIF4H). RNA was extracted at the indicated time points, resolved on an agarose-formaldehyde gel, transferred to a Gene Screen Plus membrane, and analyzed by Northern blot analysis. Symbols are as described in the legend for Fig. 3. The closed square indicates the position of the 1,800-nt fragment. The positions and sizes of the RNA markers in nucleotides are at the left.

FIG. 8.

Region of greatest similarity between eIF4H and eIF4B. Sequence similarity between residues 33 to 117 of eIF4H (accession no. NP_114381) and residues 87 to 147 of eIF4B (accession no. CAA39265) was determined using the Blast 2 sequence alignment program (65). The residues shown between the two sequences are conserved, and the residues from the same family are indicated with a +. There are 43% identity and 68% similarity between eIF4H and eIF4B in this region.

FIG. 9.

eIF4B interacts in vitro with vhs. (A) Far Western analysis of interactions between vhs and the proteins indicated. Eight micrograms of each protein was resolved on an SDS-12% PAGE gel, transferred to a nitrocellulose membrane, and incubated with RRL containing pretranslated ³⁵S-labeled vhs, and the interaction was detected by autoradiography. (B) Analysis of purified proteins. After transfer of proteins to the membrane, the SDS-PAGE gel was stained for residual protein with Coomassie brilliant blue. The positions and sizes of the protein markers (in kilodaltons) are indicated on the left.

FIG. 10.

eIF4B stimulates the endoribonuclease activity of the vhs protein produced in yeast. (A and B) Analysis of nuclease activity on pCITE-1 and SRP-α RNAs, respectively. Internally labeled pCITE-1 and SRP-α RNAs were added to extracts of yeast containing the empty expression vector either alone (EV), mixed with eIF4H (+eIF4H), mixed with eIF4A (+eIF4A), or mixed with eIF4B (+eIF4B) and yeast extracts containing 2.1vhs either alone (2.1vhs), mixed with eIF4H (+eIF4H), mixed with eIF4A (+eIF4A), or mixed with eIF4B (+eIF4B). The concentration of purified proteins used was as follows: eIF4H, 2.1 μM; eIF4B, 2.1 μM; eIF4A, 7.6 μM. Purified proteins were mixed with buffer B and 20 μg of BSA and 1 μg of total yeast RNA per time point. Symbols are as described in the legend for Fig. 3. The positions and sizes of the RNA markers in nucleotides are at the left. (C and D) Quantification of nuclease activity on pCITE-1 and SRP-α RNA substrates, respectively. The quantities of full-length RNA in panels A and B and two other experiments were determined using phosphorimager analysis and plotted against the time points indicated. The error bars indicate the standard deviation of each time point calculated from at least three independent experiments.