The interferon-inducible protein kinase PKR interacts with a number of small viral RNA species, including adenovirus VAI RNA and the Epstein-Barr virus-encoded RNA EBER-1. These RNAs bind to PKR and protect protein synthesis from inhibition by double-stranded RNA in the reticulocyte lysate system. Using a peptide phosphorylation assay we show here that EBER-1, like VAI, directly inhibits the activation of purified PKR. A second Epstein-Barr virus RNA, EBER-2, also regulates PKR. EBER-1, EBER-2 and VAI RNA exhibit mutually competitive binding to the native or recombinant enzyme, as assessed by U.V. crosslinking experiments and filter binding assays. The affinities of all three RNAs for PKR in vitro are similar (Kd = ca. 0.3 nM). Since this protein kinase has been proposed to exert a tumour suppressor function in vivo, the ability of EBER-1 to inhibit its activation suggests a role for this small RNA in cell transformation by Epstein-Barr virus.

Epstein-Barr virus encodes two small RNAs, EBER-1 and -2, that are abundantly expressed in latently infected cells. Recent evidence suggests a role for EBER-1 in regulation of translation since this RNA is able to prevent the inhibition of protein synthesis by double-stranded RNA in rabbit reticulocyte lysates. We show here that EBER-1 that has been synthesized in vitro forms a complex with the dsRNA-activated inhibitor of protein synthesis DAI, a protein kinase that specifically phosphorylates polypeptide chain initiation factor eIF-2. Gel retardation assays and UV crosslinking experiments indicate that complex formation is specific for EBER-1 and requires the presence of some secondary structure in the molecule. RNA competition studies show that EBER-1-DAI complex formation is not inhibited in the presence of other small RNA species, heparin or the synthetic double-stranded RNA, poly(I).poly(C). SDS gel analysis reveals the existence of two forms of the crosslinked complex, of 64-68kDa and 46-53kDa, both of which are recognized by anti-DAI antibodies in immunoprecipitation experiments. These data suggest that EBER-1 regulates protein synthesis through its ability to interact with DAI.

Hämoglobine bestehen aus zwei Komponenten: dem eisenhaltigen Häm lind Protein (Globin). Das Mol.-Gew. wurde in der Ultrazentrifuge zu 66-68000 ermittelt 1 . Auf dieses Mol.-Gew. bezogen, besteht die Partikel aus vier Fe-Atomen, vier Porphyrinringen und vier Peptidketten, von denen je zwei identisch vorliegen 2 . Die vier Harne sind für alle Ketten identisch. Die Konstitution dieses Anteils wurde von Hans Fischer ermittelt.Im folgenden wird über die vollständige chemische Struktur des Proteins der Hauptkomponente des normalen adulten Humanhämoglobins berichtet. Unsere früheren Arbeiten handelten über die Spaltung des Globins mit Trypsin 3 , Trennung der Spaltprodukte 4 , über eine Partialformel der a-5 und ß-Kette 6 sowie über die Lage der Sequenzlücken 7 .Zur vollständigen Strukturaufklärung mußten wir noch die Sequenzanalyse des sog. a-und /?-Cores sowie des Peptides HT all (62-90 der -Kette) ergänzen:Das -Core (bearbeitet von G. H.) wurde durch Reduktion der SH-bzw. der evtl. vorhandenen S-S-Gruppen nach Swan 8 bzw. Neurath 8 oder durch Reduktion und Carboxymethylierung mit Jodacetamid nach Anfinsen 9 vorbehandelt, der so modifizierte Kettenanteil anschließend chymotryptisch bzw. peptisch hydrolysiert und die über Dowex 1X2 10 isolierten Spaltprodukte der Sequenzanalyse unterworfen.Das ß-CoTe (bearbeitet von N. H.) wurde nach Oxydation der beiden SH-Gruppen mit Perameisensäure 11 mit Pepsin gespalten; die Bruchstücke wurden ebenfalls mittels Dowex 1X2 isoliert.

The subcellular distribution of the small Epstein-Barr virus-encoded RNAs EBER-1 and EBER-2 has been investigated by using a high-resolution in situ hybridization technique. The distribution prns in Raji cells of fluorescent oligodeoxynucleotides complementary to each RNA were detected by confocal laser nning microscopy. Both RNAs were found in the cytoplasm as well as in the nuclei of interphase cells. In contrast, use of the same technique indicated an exclusively nuclear location for cellular U2 RNA. In the cytoplasm distribution of the EBERs was similar to that of the double-stranded RNA-dependent protein kinase, to which these RNAs can bind, and was coincident with the rough endoplasmic reticulum. In cells undergoing mitosis the EBERs became localized around the chromosomes, whereas the protein kinase remained uniformly distributed in the cytoplasm. A cytoplasmic location for EBER-1 and EBER-2 in interphase ceils is consistent with the evidence for a role for these small RNAs in translational control.

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