Epstein-Barr virus RNA. V. Viral RNA in a restringently infected, growth-transformed cell line

Abstract

A continuous lymphoblastoid cell line, IB-4, was established by infection and growth transformation of normal neonatal B lymphocytes with the B95-8 isolate of Epstein-Barr virus (EBV). The IB-4 cells contained the intranuclear antigen, EBNA, but not early antigen, EA. The fragments produced by the digestion of intracellular episomal viral DNA (density, 1.700 to 1.720 g/cm3) with EcoRI restriction endonuclease were identical in size to the A, B, C, E, F, G, and H fragments of virion DNA. As expected from the previous observation that episomal intracellular DNA is circular, the fragment containing the rightward terminal sequences of EBV DNA in IB-4 cells was larger than the corresponding fragment of linear viral DNA, probably as a consequence of covalent linkage to the leftward terminal fragment. Also, two fragments, EcoRI-I and -J, which were adjacent to each other in the virion DNA, were absent from the intracellular DNA. The labeled EcoRI-J of viral DNA hybridized instead to a new fragment equal in size to EcoRI-I and -J combined. Analysis of viral RNA in IB-4 cells showed that RNAs encoded by more than 30% of the viral DNA comprised approximately 0.06% of the nuclear RNA, whereas RNAs encoded by 20% and 10% of the viral DNA comprised approximately 0.06% and 0.003% of the polyadenylated and polyribosomal RNAs, respectively. Viral mRNA (polyribosomal RNA) was encoded by DNA which mapped at 0.05 x 10(8) to 0.36 x 10(8) daltons and to a lesser extent by DNAs which mapped at 0.62 x 10(8) to 0.67 x 10(8), 0.70 x 10(8) to 0.73 x 10(8), and 1.13 x 10(8) to 1.15 x 10(8) daltons in the B95-8 genome. The most agundant nuclear viral RNAs were encoded primarily by DNA which mapped at the same loci; but RNAs encoded by many other fragments of viral DNA could also be detected among nuclear RNAs. Viral mRNA(s) (polyribosomal) was encoded by about 40% of the internal reiteration and by 25% of the BamHI-H fragments which mapped from 0.32 x 10(8) to 0.36 x 10(8) daltons, nuclear RNAs were encoded by at least 57% of the internal reiteration and 40% of BamHI-H. These data indicate that there is selective accumulation of some viral RNAs within the nucleus of IB-4 cells and that there is selective post-transcriptional processing of these RNAs. Finer mapping of the DNA which encodes mRNA (polyribosomal) in IB-4 cells indicated that some of this DNA is deleted in the DNA of the P3 HR-1 virus, the only isolate of EBV which cannot initiate growth transformation. These data, therefore, support the hypothesis that expression of this region of EBV genome is important for growth transformation or for the maintenance of restrigent infection.

Deregulated c‐myc expression in epstein‐barr‐virus‐immortalized b‐cells induces altered growth properties and surface phenotype but not tumorigenicity

“…The EBV-immortalized cord-blood cell line, IB4 (King et al, 1980), was cultured at 5% CO, in RPMI 1640 supplemented with 10% foetal calf serum (FCS), 2 m~ L-glutamine, 100 IU/ml penicillin and 100 pgfml streptomycin. The concentration of hygromycin B required to prevent cell growth was determined by adding serial dilutions of the drug to cells and staining with Trypan blue 10 days later.…”

Variations Among Isolates of Epstein‐barr Virus*

“…5 7 In order to determine whether P3 HR-1 DNA has the BarnHI X and H sequences, which are transcribed in the IB-4 cells, labeled P3 HR-1 DNA was hybridized to blots of fragments of a Hind 111 digest of BnmHI X and of a HinfI digest of BarnHI H. P3 HR-1 DNA lacks the sequences to the right of the Hind 111 cut site in BamHl X and the sequences of the adjacent 0.4 X lo6 d fragment of BarnHI H. Both of these DNA fragments encode polyribosomal RNA in restringently infected growth-transformed IB-4 cells. 57 Two lines of evidence indicate that the DNA of P3 HR-1 virus is heterogeneous in its organization. While some molecules have only the small deletion of part of BamHI X and H, other molecules have much more extensive deletions.…”

Expression of Epstein‐Barr virus‐encoded proteins in nasopharyngeal carcinoma

“…, 1983). The same sequences are also transcribed in EBV-infected normal nasopharyngeal epithelium (Tugwood et al, 1987) and in EBV-transformed B cells (Tugwood et al, 1987;Arrand and Rymo, 1982;van Santen et al, 1981;King et al, 1980). Transcription of the EcoRI E fragment has also been detected in some NPC tumors (Tugwood et al, 1987).…”