Epstein-barr virus-specific RNA. II. Analysis of polyadenylated viral RNA in restringent, abortive, and prooductive infections.

The complexity and abundance of Epstein-Barr (EBV)-specific RNA in cell cultures restringently, abortively, and productively infected with EBV has been analyed by hybridization of the infected cell RNA with purified viral DNA. The data indicate the following. (i) Cultures containing productively infected cells contain viral RNA encoded by at least 45% of EBV DNA, and almost all of the species of viral RNA are present in the polyadenylated and polyribosomal RNA fractions. (ii) Restringently infected Namalwa and Raji cultures, which contain only intranuclear antigen, EBNA, and enhanced capacity for growth in vitro, contain EBV RNA encoded by at least 16 and 30% of the EBV DNA, respectively. The polyadenylated and polyribosomal RNA fractions of Raji and Namalwa cells are enriched for a class of EBV RNA encoded by approximately 5% of EBV DNA. The same EBV DNA sequences encode the polyadenylated and polyribosomal RNA of both Raji and Namalwa cells. (iii) After superinfection of Raji cultures with EBV (HR-1), the abortively infected cells contain RNA encoded by at least 41% of EBV DNA. The polyadenylated RNA of superinfected Raji cells is enriched for a class of EBV RNA encoded by approximately 20% of EBV HR-1 DNA. Summation hybridization experiments suggest that the polyadenylated RNA in superinfected Raji cells is encoded by the same DNA sequences as encode RNA present in Raji cells before superinfection, most of which is not polyadenylated. That the same EBV RNA sequences are present in the polyadenylated and polyribosomal fractions of two independently derived, restringently infected cell lines suggests that these RNAs may specify functions related to maintenance of the transformed state. The complexity of this class of RNA is adequate to specify a sequence of a least 5,000 amino acids. That only some RNA species are polyadenylated in restringent and abortive infection suggests that polyadenylation or whatever determines polyadenylation may play a role in the restricted expression of the EVB genome.     

Epstein-Barr virus gene expression in P3HR1-superinfected Raji cells.

The pattern of Epstein-Barr virus (EBV) RNAs expressed in Raji cells superinfected with P3HR1 EBV was examined. RNAs whose expression was of an immediate-early type (resistant to treatment of the cells with anisomycin) were identified. These RNAs, encoding the EBV reading frames BZLF1 and BRLF1, were probably expressed from defective virus within the P3HR1 preparation, and some of them were responsible for the induction of the EBV productive cycle in the Raji cells. The structures of the B95-8 RNAs equivalent to the anisomycin-resistant RNAs were determined. The RNA encoding the BZLF1 reading frame contained two splices which extended and modified the reading frame from that previously described.     

Transfer of the Epstein-Barr virus genes coding for small RNAs to human lymphoid cells with a vector carrying a dominant selectable marker.

Epstein-Barr virus (EBV)-negative, Burkitt-like lymphoma-derived cells were transformed with a transducing vector (pSV2-gpt) containing the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase (XGPRT) and with a derivative of PSV2-gpt that carries the genes for the EBV-associated small RNAs on the EcoRI J fragment of B95-8 EBV DNA inserted at the unique EcoRI site (pJ-gpt). Cells transformed with PSV2-gpt and pJ-gpt express the E. coli gpt gene to approximately the same extent, judged by determinations of the XGPRT activity of cell extracts. Blot hybridisation experiments with restriction endonuclease-cleaved DNA from the transformants have revealed the presence of vector DNA sequences in the cells, at least some of which are most probably integrated into high mol. wt. chromosomal DNA. Northern blot hybridisation analysis of cytoplasmic RNA from pJ-gpt-transformed cells revealed the presence of an EcoRI J DNA complementary RNA species of the same size as the EBV DNA-encoded small RNAs found in EBV-transformed cells.     

Epstein-Barr virus gene expression in interferon-treated cells. Implications for the regulation of protein synthesis and the antiviral state

This paper presents data on the effects of interferon treatment on Epstein-Barr virus (EBV) gene expression in latently infected Daudi Burkitt's lymphoma cells, and reviews the possible role of viral gene products in the regulation of translation. In Daudi cells the main virally coded RNAs are the small untranslated RNAs EBER-1 and EBER-2, two mRNAs for the DNA binding protein EBNA-1, and a number of small RNAs containing sequences from the BamHI W repeat region of the viral genome. Interferon treatment does not change the qualitative pattern of EBV gene expression but decreases the levels of the EBNA-1 mRNAs. The chromatographic behaviour of EBV-encoded RNAs on CF11-cellulose indicates that many contain double-stranded regions; these RNAs co-purify with RNA that activates the interferon-induced, dsRNA-sensitive protein kinase DAI. Computer analysis indicates that the exons transcribed from the BamHI W repeats have the potential for formation of very stable secondary structures. Many viruses can counteract the inhibition of protein synthesis mediated by the DAI-catalysed phosphorylation of initiation factor eIF-2 and our data suggest that the small RNA EBER-1 may fulfil this function in the EBV system. During the infection and immortalization of B lymphocytes by EBV the synthesis of large amounts of EBER-1 RNA might thus allow the virus to circumvent one of the interferon-mediated mechanisms of host cell defence.     

CpG-methylation silences the activity of the RNA polymerase III transcribed EBER-1 promoter of Epstein-Barr virus

CpG-methylation blocks the activity of RNA polymerase II transcribed promoters in most cases. In contrast, the role of DNA methylation in the regulation of RNA polymerase III transcribed promoters is less clarified. There are two untranslated viral RNAs (EBER-1 and EBER-2) in most malignant cells carrying latent Epstein-Barr virus (EBV) genomes. We found that in vitro methylation blocked binding of the cellular proteins c-Myc and ATF to the 5'-region of the EBER-1 gene, and silenced the expression of the EBER-1 and EBER-2 genes, transcribed by RNA polymerase III, in transfected cells.     

Multiple domains of EBER 1, an Epstein-Barr virus noncoding RNA, recruit human ribosomal protein L22

EBER 1, a small noncoding viral RNA abundantly expressed in all cells transformed by Epstein-Barr virus (EBV), has been shown to associate with the human ribosomal protein L22. Here we present in vitro binding studies using purified RNAs and recombinant proteins. Electrophoretic mobility-shift assays (EMSAs) show that recombinant L22 (rL22) and maltose-binding protein (MBP)-tagged L22 protein bind EBER 1 in vitro, both forming three specific protein-dependent mobility shifts. Use of a mixture of rL22 and MBP-L22 indicates that these three shifts contain one, two, or three L22 proteins per EBER 1 molecule. EMSAs performed with EBER 1 deletion constructs and EBER 1 stem-loops inserted into a nonbinding RNA, HSUR 3, identify stem-loops I, III, and IV as L22 binding sites. The existence of multiple L22 binding sites on EBER 1 inside cells is demonstrated by in vivo UV cross-linking. Our results are discussed with respect to the function of EBER 1 in EBV-infected human B cells.     

Interactions of heterogeneous nuclear ribonucleoprotein D-like protein JKTBP and its domains with high-affinity binding sites

JKTBP proteins consisting of two canonical RNA binding domains (RBDs) and a glycine-rich carboxyl domain are nucleocytoplasmic shuttling proteins. We studied in vivo and in vitro interactions between JKTBP and RNA. UV cross-linking experiments on HL-60 cells indicated that following RNA synthesis inhibition by actinomycin D, JKTBP1 accumulated in the cytoplasam is bound to poly(A)(+) RNAs. Recombinant JKTBP1 protein blots could bind poly(A)(+) RNAs, but not poly(A)(-) RNAs. For examination of RNA binding specificity of JKTBP, we enriched high binding sites from pools of 20 nt random sequence-containing RNAs by a selection/amplification method. After eight rounds of a selection and amplification, >20 sequences for each of JKTBPs 1 and 2 were identified. Their consensus high-affinity site was ACUAGC. Approximate K(d)s of JKTBPs 2 and 1 were estimated to be 6-12 nM for the selected sequences by filter binding assays. JKTBP deletion analysis indicated that not individual RBDs, both RBDs and the N-terminal 15 amino acids of the carboxyl domain are required for sequence-specific and high-affinity binding. These results indicate that JKTBP is a sequence-specific RNA binding protein differing from the related heterogeneous nuclear ribonucleoproteins A1 and D.     

RNA aptamers binding the double-stranded RNA-binding domain

Specific RNA recognition of proteins containing the double-strand RNA-binding domain (dsRBD) is essential for several biological pathways such as ADAR-mediated adenosine deamination, localization of RNAs by Staufen, or RNA cleavage by RNAse III. Structural analysis has demonstrated the lack of base-specific interactions of dsRBDs with either a perfect RNA duplex or an RNA hairpin. We therefore asked whether in vitro selections performed in parallel with individual dsRBDs could yield RNAs that are specifically recognized by the dsRBD on which they were selected . To this end, SELEX experiments were performed using either the second dsRBD of the RNA-editing enzyme ADAR1 or the second dsRBD of Xlrbpa, a homolog of TRBP that is involved in RISC formation. Several RNA families with high binding capacities for dsRBDs were isolated from either SELEX experiment, but no discrimination of these RNAs by different dsRBDs could be detected. The selected RNAs are highly structured, and binding regions map to two neighboring stem-loops that presumably form stacked helices and are interrupted by mismatches and bulges. Despite the lack of selective binding of SELEX RNAs to individual dsRBDS, selected RNAs can efficiently interfere with RNA editing in vivo.     

Metabolism of pre-messenger RNA splicing cofactors: modification of U6 RNA is dependent on its interaction with U4 RNA.

The requirements for the formation of pseudouridine (psi) in U4 and U6 RNAs, cofactors in the splicing of pre-messenger RNA, were investigated in vitro using HeLa nuclear (NE) and cytoplasmic (S100) extracts. Maximal psi formation for both RNAs was extract order-dependent. Maximal psi formation in U4 RNA required incubation in S100 followed by the addition of NE, paralleling the in vivo maturation pathway of U4 RNA. In contrast, maximal formation of psi in U6 RNA required incubation in NE followed by the addition of S100 extract. Since U6 RNA does not exit the nucleus in vivo the contribution of S100 was investigated. In experiments where the extracts were treated with micrococcal nuclease to digest endogenous snRNAs, the efficient formation of psi in U6 RNA was dependent on the presence of U4 RNA, but not in U5 RNA or tRNA. When mutant U4 RNAs that inhibit or strengthen the interaction between U4 RNA, and U6 RNA were substituted for wild-type U4 RNA, the results confirmed the need for the interaction between these two RNAs for psi formation in U6 RNA. U6 RNA isolated from glycerol gradients after incubation in extracts had four times as much psi when associated with U4 RNA.     

The heterogeneous nuclear ribonucleoproteins I and K interact with a subset of the ro ribonucleoprotein-associated Y RNAs in vitro and in vivo

The hY RNAs are a group of four small cytoplasmic RNAs of unknown function that are stably associated with at least two proteins, Ro60 and La, to form Ro ribonucleoprotein complexes. Here we show that the heterogeneous nuclear ribonucleoproteins (hnRNP) I and K are able to associate with a subset of hY RNAs in vitro and demonstrate these interactions to occur also in vivo in a yeast three-hybrid system. Experiments performed in vitro and in vivo with deletion mutants of hY1 RNA revealed its pyrimidine-rich central loop to be involved in interactions with both hnRNP I and K and clearly showed their binding sites to be different from the Ro60 binding site. Both hY1 and hY3 RNAs coprecipitated with hnRNP I in immunoprecipitation experiments performed with HeLa S100 extracts and cell extracts from COS-1 cells transiently transfected with VSV-G-tagged hnRNP-I, respectively. Furthermore, both anti-Ro60 and anti-La antibodies coprecipitated hnRNP I, whereas coprecipitation of hnRNP K was not observed. Taken together, these data strongly suggest that hnRNP I is a stable component of a subpopulation of Ro RNPs, whereas hnRNP K may be transiently bound or interact only with (rare) Y RNAs that are devoid of Ro60 and La. Given that functions related to translation regulation have been assigned to both proteins and also to La, our findings may provide novel clues toward understanding the role of Y RNAs and their respective RNP complexes.