The herpes simplex virus (HSV) virion host shutoff (Vhs) protein is an endoribonuclease that accelerates decay of many host and viral mRNAs. Purified Vhs does not distinguish mRNAs from nonmessenger RNAs and cuts target RNAs at many sites, yet within infected cells it is targeted to mRNAs and cleaves those mRNAs at preferred sites including, for some, regions of translation initiation. This targeting may result in part from Vhs binding to the translation initiation factor eIF4H; in particular, several mutations in Vhs that abrogate its binding to eIF4H also abolish its mRNA-degradative activity, even though the mutant proteins retain endonuclease activity. To further investigate the role of eIF4H in Vhs activity, HeLa cells were depleted of eIF4H or other proteins by transfection with small interfering RNAs (siRNAs) 48 h prior to infection or mock infection in the presence of actinomycin D. Cellular mRNA levels were then assayed 5 h after infection. In cells transfected with an siRNA for the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase, wild-type HSV infection reduced -actin mRNA levels to between 20 and 30% of those in mock-infected cells, indicative of a normal Vhs activity. In contrast, in cells transfected with any of three eIF4H siRNAs, -actin mRNA levels were indistinguishable in infected and mock-infected cells, suggesting that eIF4H depletion impeded Vhsmediated degradation. Depletion of the related factor eIF4B did not affect Vhs activity. The data suggest that eIF4H binding is required for Vhs-induced degradation of many mRNAs, perhaps by targeting Vhs to mRNAs and to preferred sites within mRNAs.The herpes simplex virus (HSV) virion host shutoff (Vhs) protein (UL41) is an endoribonuclease (9,14,83,84,87) that is a minor structural component of virions (57,58,67) and affects the half-lives of many host and viral mRNAs
bRibosomal proteins S4 and S5 participate in the decoding and assembly processes on the ribosome and the interaction with specific antibiotic inhibitors of translation. Many of the characterized mutations affecting these proteins decrease the accuracy of translation, leading to a ribosomal-ambiguity phenotype. Structural analyses of ribosomal complexes indicate that the tRNA selection pathway involves a transition between the closed and open conformations of the 30S ribosomal subunit and requires disruption of the interface between the S4 and S5 proteins. In agreement with this observation, several of the mutations that promote miscoding alter residues located at the S4-S5 interface. Here, the Escherichia coli rpsD and rpsE genes encoding the S4 and S5 proteins were targeted for mutagenesis and screened for accuracy-altering mutations. While a majority of the 38 mutant proteins recovered decrease the accuracy of translation, error-restrictive mutations were also recovered; only a minority of the mutant proteins affected rRNA processing, ribosome assembly, or interactions with antibiotics. Several of the mutations affect residues at the S4-S5 interface. These include five nonsense mutations that generate C-terminal truncations of S4. These truncations are predicted to destabilize the S4-S5 interface and, consistent with the domain closure model, all have ribosomal-ambiguity phenotypes. A substantial number of the mutations alter distant locations and conceivably affect tRNA selection through indirect effects on the S4-S5 interface or by altering interactions with adjacent ribosomal proteins and 16S rRNA. C ellular protein synthesis systems translate mRNAs quickly and with high accuracy. The accuracy of the decoding process can, however, be altered by agents such as the antibiotic streptomycin that promote miscoding and by mutations in rRNA, ribosomal proteins, or translation factors (1). Among the first such accuracy mutants to be characterized were some of the streptomycin-resistant Escherichia coli strains carrying alterations in ribosomal protein S12 (2). Subsequently, E. coli mutants carrying altered ribosomal protein S4 or S5 were isolated that supported increased levels of miscoding (3-5). Since those early studies, many other mutants have been isolated that affect the accuracy of decoding and carry alterations in different components of the translation machinery (1, 4).The determination of high-resolution structures of ribosomes has offered structural interpretations of the effects of some accuracy-altering ribosomal mutations (6). X-ray crystallography of ribosomal complexes has shown that conserved RNA elements of the decoding center use a shape-sensing mechanism to monitor base pairing between the A site codon and the anticodon of incoming aminoacyl-tRNA. Successful interaction of a ternary complex of EF-Tu-GTP-aminoacyl-tRNA with mRNA in the decoding center triggers a series of conformational rearrangements of the head and shoulder domains of the 30S subunit, ultimately resulting in the formation of a ...
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