mRNA decay during herpesvirus infections: interaction between a putative viral nuclease and a cellular translation factor

Abstract

During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating mRNA decay, it helps determine the levels and kinetics of viral and cellular gene expression. In vivo, Vhs shows a strong preference for mRNAs, as opposed to non-mRNAs, and degrades the 5' end of mRNAs prior to the 3' end. In contrast, partially purified Vhs is not restricted to mRNAs and causes cleavage of target RNAs at various sites throughout the molecule. To explain this discrepancy, we searched for cellular proteins that interact with Vhs using the Saccharomyces cerevisiae two-hybrid system. Vhs was found to interact with the human translation initiation factor, eIF4H. This interaction was verified by glutathione S-transferase pull-down experiments and by coimmunoprecipitation of Vhs and epitope-tagged eIF4H from extracts of mammalian cells. The interaction was abolished by several point mutations in Vhs that abrogate its ability to degrade mRNAs in vivo. The results suggest that Vhs is a viral mRNA degradation factor that is targeted to mRNAs, and to regions of translation initiation, through an interaction with eIF4H.

FIG. 1

Sequences of eIF4H and two cellular Vhs binding proteins. The published sequence of eIF4H is shown, along with the deduced sequences of two Vhs binding proteins identified in the yeast two-hybrid screen. Amino acids that are identical to those of eIF4H are shaded grey. Asterisks indicate the absence of an amino acid and were introduced to facilitate alignment of eIF4H, Vhs binding protein 1, and Vhs binding protein 2.

FIG. 2

Binding of Vhs to a GST-eIF4H_i fusion protein in vitro. GST or a GST-eIF4H_i was bound to glutathione-Sepharose 4B and incubated with rabbit reticulocyte lysates containing in vitro-translated Vhs (A) or a cytoplasmic extract from HSV-1 infected cells (B). Bound proteins were eluted with 10 mM glutathione and analyzed by SDS -PAGE. In vitro-translated proteins were detected by autoradiography, while Vhs from infected cells was detected by Western blotting. In both panels, lane 1 contains the starting material, lane 2 contains proteins that bound to GST, and lane 3 contains proteins that bound to GST-eIF4H_i.

FIG. 3

Coimmunoprecipitation of Vhs and epitope-tagged eIF4H from mammalian cells. (A and B) Vero cells were infected with the recombinant vaccinia virus vTF7-39 and transfected with plasmids expressing HA-tagged eIF4H_i (lanes 1); Vhs (lanes 2); HA-tagged eIF4H_i and Vhs (lanes 3); or HA-tagged eIF4H_i, HA-tagged eIF4H, and Vhs (lane 4, panel A). Cell extracts were prepared and immunoprecipitated (upper gels) using Vhs-specific antiserum (A) or HA-specific monoclonal antibody (B). Immunoprecipitates (IP) were analyzed for coprecipitated proteins by Western blotting (upper gels) using the reciprocal antibody—either the HA-specific monoclonal antibody (A) or Vhs-specific antiserum (B). To check for protein expression, an aliquot of the cell lysate was analyzed directly by Western blotting (lower gels) using HA-specific monoclonal antibody (A) or Vhs-specific antiserum (B).

FIG. 4

Strategy of the recombination-based yeast two-hybrid assay. Yeast strain Y190 was transformed with pACT2-4H and the large SmaI fragment of pAS2-Vhs. Transformations also included an EcoRI-XbaI fragment from another plasmid containing the entire mutant Vhs allele whose binding to eIF4H was to be tested. Within the yeast, recombination between the SmaI fragment of pAS2-Vhs and the fragment containing the mutant Vhs gene resulted in reconstitution of a pAS2-Vhs plasmid containing the Vhs mutation. Transformants were plated on SD medium containing 35 mM 3-AT and lacking tryptophan, leucine, and histidine to select for yeast that grew because transcription of the Gal4-responsive his3 gene had been activated. Transformations that contained a Vhs allele encoding a protein that bound eIF4H gave rise to 200 to 300 colonies on the selective medium, while control transformations containing just pACT2-4H and the large SmaI fragment of pAS2-Vhs gave rise to at most 5 to 10 colonies.

FIG. 5

eIF4H binding by wild-type and mutant Vhs. (A) The structures of the wild-type Vhs protein and various mutants. For deletion mutants, the Vhs residues included in the mutant protein are indicated. For each point mutant, the location of the altered residue is indicated by the vertical line above the bar representing the protein. The in vivo mRNA-degradative activity of each Vhs protein is shown in the column immediately to the right of the diagram. This was assayed in transfected Vero cells for all of the alleles and during virus infections for wild-type Vhs and the mutant T214I. The right-hand column indicates whether a Vhs protein binds (++) or does not (−) bind eIF4H in the conventional (superscript letter a) or recombination-based (superscript letter b) yeast two-hybrid system. (B) GST pull-down assays. Wild-type and mutant Vhs polypeptides were produced by in vitro translation and analyzed for the ability to bind a GST-eIF4H fusion protein as described for Fig. 2. Material that bound to GST-eIF4H and was eluted with 10 mM glutathione is shown in the upper gels (GST Pulldown). Aliquots of the total in vitro-translated material are shown in the lower gels (Input).

FIG. 6

Vhs binding by eIF4H mutants. (A) The structures of eIF4H mutants are shown, with residues shared by eIF4H and eIF4H_i shaded light grey and those unique to eIF4H_i shaded dark grey. At the right is the relative binding of each eIF4H protein to Vhs in the conventional yeast two-hybrid assay. Binding is expressed as the amount of lacZ activity resulting from the activation of a GAL4-dependent lacZ gene, where the activity induced by eIF4H_i is defined as 100%. (B) Wild-type Vhs was produced by in vitro translation and analyzed for the ability to bind various fusion proteins containing GST fused to mutant forms of eIF4H. Vhs bound by the various GST-eIF4H polypeptides and eluted with 10 mM glutathione is shown in the upper gel (GST Pulldown). A Coomassie-stained gel of the mutant GST-eIF4H proteins is shown below (Input).

FIG. 7

Model for Vhs targeting. eIF4H (4H) stimulates the RNA helicase and RNA-dependent ATPase activities of eIF4A (4A). eIF4A, in turn, is a component of the tripartite cap-binding complex eIF4F. The dashed arrow depicts the functional, and perhaps physical, interaction of eIF4H with eIF4A. Vhs is targeted to mRNAs, as opposed to non-mRNAs, and to regions of translation initiation through its interaction with eIF4H.