One of the strongest indications that binding to eIF4H is important for Vhs-induced decay is the observation that, to date, every Vhs mutation that abrogates its binding to eIF4H greatly reduces its ability to degrade mRNAs that are translated by cap-dependent scanning, even if the mutant protein retains endonuclease activity (14
). Conversely, every Vhs polypeptide that degrades scanned mRNAs binds eIF4H. While these data are very suggestive, one cannot exclude the possibility that the mutant Vhs polypeptides fail to degrade mRNAs not because they do not bind eIF4H but because they are defective in some other unknown function. To address this potential problem, we tested whether siRNA-mediated knockdown of the level of eIF4H prior to infection would affect the ability of wild-type Vhs to induce mRNA decay.
The first step was to check whether transfection of cells with various eIF4H siRNAs reduces the level of eIF4H protein. HeLa cells were transfected with three different eIF4H siRNAs, one directed against a sequence within the open reading frame and two against sequences in the 3′ untranslated region (Fig. ). Parallel cultures were transfected with siRNAs against eIF4B or the housekeeping enzyme GAPDH or mock transfected. Cell lysates were prepared 48 h after transfection and examined by SDS-PAGE and Western blotting to determine the levels of eIF4H and eIF4B protein (Fig. ). None of the siRNAs had an apparent effect upon cell number or morphology, as determined by light microscopy 48 h after transfection, nor did they affect the amount of 18S rRNA that could be recovered from the cultures (data not shown), further indicating that none of the siRNAs, or the transfection procedure itself, had a significant cytotoxic effect upon the cells over the first 48 h after transfection. Most importantly, each eIF4H siRNA significantly reduced the level of eIF4H relative to that in cells transfected with a GAPDH siRNA (Fig. ). Two of the siRNAs (139886 and 139887) did not appreciably affect the level of the related factor eIF4B. On this gel the lane for the third eIF4H siRNA (30064) was compressed, making it unclear whether transfection with this siRNA had induced a small decrease in the level of eIF4B. Examination of other gels from this or other transfections indicates that this was a gel artifact and that siRNA 30064 caused little, if any, decrease in the level of eIF4B (data not shown). Conversely, an eIF4B siRNA (217085) reduced the level of eIF4B but not that of eIF4H.
FIG. 2. Transfection with siRNAs depletes the levels of eIF4H or eIF4B. HeLa cells were transfected with no siRNA, with siRNAs for GAPDH or eIF4B, or with three different siRNAs for eIF4H as indicated above the gel lanes. Cell lysates were prepared 48 h after (more ...)
To examine the effect of eIF4H depletion upon Vhs activity, HeLa cells were infected 48 h after siRNA transfection with 20 PFU/cell of wild-type HSV-1 (KOS) or mock infected, both in the presence of 5 μg/ml of actinomycin D. Cytoplasmic RNAs were prepared 5 h later, and the levels of β-actin mRNA were determined by real-time quantitative reverse transcription-PCR using 18S rRNA as an internal control. Comparison of mRNA levels following infection or mock infection in the presence of actinomycin D is a standard assay for Vhs activity (20
). In cells transfected with no siRNA or an siRNA for GAPDH, wild-type HSV infection reduced the level of β-actin mRNA to between 20 and 30% of that in mock-infected cells (Fig. ), indicative of a normal Vhs activity. In contrast, in cells transfected with two of the three eIF4H siRNAs (139887 and 30064), β-actin mRNA levels were indistinguishable in infected and mock-infected cells, indicating that transfection with these siRNAs prevented detectable Vhs-mediated mRNA degradation. In cells transfected with the third siRNA (139886), the level of β-actin mRNA was 70% of that in mock-infected cells, indicating that, while Vhs-mediated degradation was not completely abolished, it was significantly impeded.
FIG. 3. siRNA-mediated depletion of eIF4H impedes Vhs-induced degradation of β-actin mRNA. HeLa cells were transfected with no siRNA, with an siRNA for GAPDH, or with three different siRNAs for eIF4H as indicated. The cells were infected 48 h after transfection (more ...)
Because of the functional similarities between eIF4H and eIF4B (28
), it was important to examine whether eIF4B also plays an important role in Vhs-mediated mRNA decay. The region of eIF4H shown by deletion mapping to be necessary and sufficient for binding Vhs overlaps the region of sequence homology shared by eIF4H and eIF4B (17
), and several point mutations in eIF4H which reduce binding to Vhs fall within this region of shared homology (Fig. ) (18
). In addition, purified eIF4H and eIF4B both stimulate the basal Vhs endonuclease activity that is observed in whole extracts of yeast that have been engineered to express Vhs, although neither fully reconstitutes the targeted cleavage of mRNAs that is observed for in vitro-translated Vhs in rabbit reticulocyte lysates (7
). The potential role of eIF4B in Vhs-mediated decay was examined in two ways. The first involved comparing the binding of mutant and wild-type Vhs polypeptides to eIF4H and eIF4B.
To date, binding of Vhs to eIF4B has been demonstrated only using a far-Western blotting assay in which soluble in vitro-translated Vhs bound to denatured eIF4B on a nitrocellulose membrane (7
). In the present study we examined the binding of Vhs to GST-eIF4B or GST-eIF4H in solution, focusing on wild-type Vhs and a collection of mutant Vhs polypeptides, each of which lacks in vivo mRNA-degradative activity and has been characterized previously with respect to binding eIF4H (see Fig. ) (14
). In this experiment, each binding reaction mixture contained an excess of GST-eIF4B or GST-eIF4H and a mixture of [35
S]methionine-labeled, in vitro-translated Vhs and eIF4B (Fig. and ).Control reactions showed that GST-eIF4B readily binds in vitro-translated eIF4B, as well as two forms of eIF4AII: the full-length 407-amino-acid polypeptide (labeled eIF4AII45
) and a truncated form (labeled eIF4AII41
) containing amino acids 38 through 407 of the full-length protein (Fig. , lane 7, middle panel). Previous studies showed that eIF4AII41
is produced during in vitro translation by initiation at an AUG codon that encodes methionine 38 of the full-length protein (18
). Binding of GST-eIF4B to in vitro-translated eIF4B reflected the fact that eIF4B exists as a dimer in vivo (28
) and provided an internal control for the amount of protein loaded onto the gel. GST-eIF4H differed from GST-eIF4B in that it did not interact appreciably with eIF4B (Fig. , lane 7, bottom panel). This was unsurprising because the region of homology shared by eIF4H and eIF4B does not contain the dimerization domain of eIF4B (18
). GST-eIF4H also differed from GST-eIF4B in that it bound the truncated form of eIF4AII (eIF4AII41
) more robustly than the full-length protein (eIF4AII45
) (Fig. , lane 7, bottom panel). In accord with previous results (18
), binding to eIF4AII45
was observed (data not shown), but only upon longer exposure of the gel.
FIG. 6. Summary of the structures and in vivo mRNA-degradative activities of wild-type and mutant Vhs polypeptides and their abilities to bind eIF4H and eIF4B. The Vhs polypeptide encoded by wild-type HSV-1(KOS) is represented by the solid rectangle in line 1, (more ...)
FIG. 4. Binding of wild-type and mutant Vhs polypeptides to GST-eIF4B and GST-eIF4H. (A) [35S]methionine-labeled wild-type polypeptide and the mutant Vhs polypeptides T214I and R435H were produced by in vitro transcription and translation, as was [35S]methionine-labeled (more ...)
FIG. 5. Binding of wild-type HSV-1(KOS) and mutant Vhs polypeptides to GST-eIF4B. [35S]methionine-labeled HSV-1(KOS) and mutant Vhs polypeptides were produced by in vitro transcription and translation, mixed with [35S]methionine-labeled in vitro-translated eIF4B, (more ...)
Analysis of the Vhs mutations revealed a number, such as D34N or D194N, that had no appreciable effect upon the amount of Vhs that bound to either GST-eIF4H or GST-eIF4B (Fig. , , and ) (17
). Many of these were point mutations that altered key conserved residues in the nuclease motif of Vhs (14
). Several other mutations—typified by R435H, K(89-489), and ΔSma—greatly reduced binding to both GST-eIF4H and GST-eIF4B (Fig. , , and ) (17
). However, a number of mutations had different effects upon Vhs binding to GST-eIF4H and GST-eIF4B (Fig. ). The alleles K(1-382) and K(1-453) contain nonsense mutations that result in truncated Vhs polypeptides containing the first 382 and 453 amino acids of the wild-type protein, respectively (Fig. ) (17
). These mutations abolished detectable binding to GST-eIF4H (Fig. ) (17
) but either had little [K(1-382)] or much less [K(1-453)] of an effect on binding to GST-eIF4B (Fig. and ). Similarly, the point mutations T211A and T214I greatly reduced Vhs binding to eIF4H (17
) but did not affect binding to eIF4B (Fig. and ). T214I was particularly informative. This mutant polypeptide lacks detectable in vivo mRNA-degradative activity for mRNAs that are translated by cap-dependent scanning (48
) but still cuts downstream of an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) in vitro (44
), indicating that it retains endonuclease activity. It fails to bind eIF4H (17
) but still binds eIF4B (Fig. and ), indicating not only that binding to eIF4B and eIF4H can be distinguished genetically but that binding of an active Vhs endonuclease to eIF4B is not sufficient for in vivo degradation of scanned mRNAs.
The second, and complementary, approach that was used to analyze the involvement of eIF4B in Vhs activity was to determine whether siRNA-mediated knockdown of the level of eIF4B inhibits Vhs-mediated degradation of β-actin mRNA. As with the experiment in Fig. , HeLa cells were transfected with no siRNA or with siRNAs for GAPDH or eIF4B (Fig. ). Transfection with this eIF4B siRNA (217085) significantly reduces the level of eIF4B while not affecting the level of eIF4H (Fig. ). At 48 h after transfection, the cells were infected with 20 PFU/cell of wild-type HSV-1(KOS) or mock infected, both in the presence of 5 μg/ml of actinomycin D. Cytoplasmic RNAs were analyzed 5 h later, and the relative levels of β-actin mRNA in infected and mock-infected cultures were taken as a measure of the extent of Vhs-mediated mRNA degradation (Fig. ). In cells transfected with the eIF4B siRNA, wild-type HSV infection reduced the level of β-actin mRNA to between 10 and 20% of that in mock-infected cells (Fig. ), an effect similar to that seen in cells transfected with no siRNA or a GAPDH siRNA and indicative of a normal Vhs activity. Thus, in contrast to the situation for eIF4H, significant siRNA-mediated depletion of the level of eIF4B did not impede Vhs-induced degradation of β-actin mRNA.
FIG. 7. siRNA-mediated depletion of eIF4B does not affect Vhs-induced degradation of β-actin mRNA. HeLa cells were transfected with no siRNA or with siRNAs for GAPDH or eIF4B as indicated. The cells were infected 48 h after transfection with 20 PFU/cell (more ...)