These studies demonstrate that the Vhs protein of HSV interacts with the cellular translation initiation factor eIF4H in vitro, in yeast, and in mammalian cells. The biologic importance of this interaction is suggested by the observation that several point mutations in Vhs, which abrogate its ability to induce mRNA decay, also abolish its ability to bind eIF4H.
Although in vivo Vhs is targeted to mRNAs and perhaps to regions of translation initiation, the Vhs-dependent nuclease that is observed in extracts of partially purified virions is much less specific (44
). Our observation that Vhs binds eIF4H suggests a mechanism for targeting the Vhs activity. eIF4H shares a region of sequence homology with eIF4B and appears to be functionally similar in that both proteins stimulate the RNA helicase of eIF4A, possibly by increasing its processivity (34
). Along with eIF4E and eIF4G, eIF4A is a component of the tripartite cap binding complex eIF4F (13
). Thus, eIF4H appears to act at an early stage of cap-dependent translation initiation to help unwind mRNA secondary structure and facilitate scanning by the small ribosomal subunit (15
). The data suggest a model in which binding to eIF4H somehow targets Vhs to mRNAs, as opposed to non-mRNAs, and to regions of translation initiation (Fig. ).
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, (more ...)
While this model is attractive in outline, many of its details remain to be developed and tested. Although the model is based upon known functions of eIF4H and other cellular translation factors, it remains to be proven that binding to eIF4H actually leads to the localization of Vhs near regions of translation initiation. An implication of the model is that targeting of Vhs may be dependent not only on its interaction with eIF4H but also on a series of protein-protein and protein-RNA interactions involving other translation factors. Thus, Vhs binds eIF4H, which in turn interacts functionally, and perhaps physically, with eIF4A. eIF4A binds eIF4G, which interacts with eIF4E, which binds the mRNA cap. Whether all or only some of these interactions are required for efficient targeting of Vhs, or whether other protein-protein or protein-RNA interactions are also required, remains to be determined. In addition, it is interesting to speculate whether the Vhs-eIF4H interaction inhibits the normal activity of eIF4H. If this is the case, Vhs may affect gene expression both by degrading mRNA and by inhibiting the rate of translation initiation.
These results also are significant because of the light they shed upon mechanisms of eukaryotic mRNA decay in general. In eukaryotes, the regulated degradation of many mRNAs is dependent upon their being translated (14
). The observation that a viral mRNA degradation protein binds a cellular translation initiation factor provides a novel example of one way in which this linkage between mRNA decay and translation can be accomplished.
Although it is clear that Vhs induces mRNA turnover, it is uncertain whether it is itself a nuclease or, instead, somehow activates a cellular enzyme. Data suggesting that Vhs is an RNase include the observation that the Vhs homologues of alpha herpesviruses share sequence homologies and a number of key conserved residues with a family of mammalian, yeast, bacterial, and phage nucleases. For several of these nucleases, point mutations that alter key conserved residues abrogate the nuclease activity, and mutations that alter the corresponding residues of Vhs inactivate its ability to induce mRNA decay (Everly et al., unpublished). While these results are suggestive, the Vhs protein has not been purified and definitively proven to have nuclease activity. However, recent progress has been made in this area by taking advantage of the interaction between Vhs and eIF4H. In our laboratory, initial attempts to express Vhs in bacteria resulted in the production of Vhs protein that was insoluble, except in buffers containing high concentrations of guanidine hydrochloride or urea. However, coexpression of Vhs and a GST-eIF4H fusion protein resulted in the formation of soluble complexes of Vhs–GST-eIF4H that could be isolated by binding to glutathione-Sepharose and subsequent ion exchange chromatography. Complexes containing the wild-type Vhs protein were found to have RNase activity, while complexes containing either of two mutant forms of Vhs, which lack mRNA-degradative activity in mammalian cells but still bind eIF4H, lacked detectable nuclease activity (Everly et al., unpublished). The results indicate that Vhs indeed is an RNase, either by itself or as a complex with eIF4H. Interestingly, Lu and coworkers recently reported that extracts from yeast that expressed wild-type Vhs lacked detectable RNase activity but that RNase activity was observed after the extracts were supplemented with rabbit reticulocyte lysates (22
). This observation is consistent with the possibility that one or more mammalian factors, such as eIF4H, are required to activate the nuclease activity of Vhs. However, a number of alternative interpretations exist. Additional characterization of purified Vhs and Vhs-eIF4H complex are required.
At present it is unclear how many times the Vhs nuclease cleaves an mRNA. Cleavage at a single site near the 5′ end would be sufficient to inhibit further cap-dependent translation, while cellular 5′-to-3′ and 3′-to-5′ exonucleases exist which, in theory, could degrade the resulting fragments. Alternatively, Vhs may cleave mRNAs multiple times at sites that are progressively closer to the 3′ end. Data from Elgadi and coworkers suggest that this may occur in rabbit reticulocyte lysates containing in vitro-translated Vhs (7
). Whether it occurs in vivo remains to be determined.
The present results suggest obvious models for the role of eIF4H in Vhs-mediated degradation of mRNAs that are undergoing cap-dependent translation. However, Vhs also has been shown to induce endonuclease cleavage of mRNAs downstream from a picornavirus IRES in vitro (8
). Whether eIF4H is required for Vhs-directed decay of IRES-containing mRNAs is unclear. At present, it is unknown whether eIF4H is required for IRES-dependent translation. However, with the notable exception of eIF4E, many of the initiation factors that are required for cap-dependent translation also are required for initiation from a picornavirus IRES (16
). If eIF4H is involved in initiation from picornavirus IRESs, it may play a similar role in the Vhs-mediated degradation of these mRNAs and those undergoing cap-dependent translation. However, Lu and coworkers recently reported that a Vhs polypeptide containing the T214I point mutation induces a residual amount of IRES-directed endonuclease activity in the rabbit reticulocyte in vitro degradation system (23
). Interestingly, this mutation greatly diminishes binding of Vhs to eIF4H (Fig. ). One possibility is that the T214I polypeptide retains a residual amount of eIF4H binding activity, which is sufficient for the residual amount of IRES-directed cleavage that is observed in vitro. Alternatively, Vhs may recognize IRES elements directly or through a cellular factor other than eIF4H. These and other questions are under investigation.