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EBV gene expression in an NPC-related tumour
1Department of Virology, Royal Postgraduate Medical School, London, UK.
Abstract:
A nasopharyngeal carcinoma tumour (designated C15) propagated in nude mice has been used to generate a large cDNA library that we have analysed for Epstein-Barr virus (EBV) gene expression. No gross alterations exist in viral DNA from C15 relative to other human isolates and the large deletion present in the B95-8 'prototype' viral strain established in marmoset cells is not found; C15 contains no linear virion DNA. In the cDNA library, of the six EBV nuclear antigens (EBNAs) expressed in latently infected B-lymphocytes, only clones for EBNA-1 are found. These data are confirmed by immunoblotting. Sequence analysis shows the EBNA-1 mRNA splicing pattern in the carcinoma to differ from that observed in B-lymphocytes. Further, contrary to observations with B-cell lines, most viral transcription in the tumour is localized onto the 'rightmost' region of the conventional EBV physical map. Transcripts identified corresponding to known genes include those for the latent membrane protein (LMP), the alkaline DNA exonuclease and probably the terminal protein; major transcripts are also derived from the BamHI D fragment and the region deleted in B95-8 EBV DNA. Novel transcripts have also been identified that proceed in an anti-sense direction to genes encoding functions associated with replication, such as the viral DNA polymerase. They contain a large, hitherto unidentified, open reading frame in the viral genome that is complementary to the putative function known as BALF3 and a smaller open reading frame complementary to BALF5 (the DNA polymerase gene). From the present studies we can conclude that: (i) EBV transcription patterns in the epithelial cells vary markedly from those identified previously in B-cells, reflecting differential use of promoters or splicing patterns. (ii) Transcription is tightly regulated and restricted in the C15 tumour with many latent genes, notably EBNAs 2-6, being 'switched off.' (iii) A family of cytoplasmic RNAs are transcribed in an antisense direction to a number of existing open reading frames in the EBV genome. (iv) There are a number of mutations in C15 transcripts relative to the B95-8 genome, some of which could result in amino acid alterations in proteins.
Insights
Epstein-Barr virus (EBV) gene expression in nasopharyngeal carcinoma differs significantly from B-cells, with restricted transcription and novel antisense RNAs identified. This suggests distinct viral regulatory mechanisms in epithelial tumors.
Area of Science:
- Virology
- Molecular Biology
- Oncology
Background:
- Epstein-Barr virus (EBV) is associated with various human cancers, including nasopharyngeal carcinoma (NPC).
- Understanding EBV gene expression in epithelial cells is crucial for comprehending its oncogenic mechanisms.
- Previous studies primarily focused on EBV in B-lymphocytes, revealing distinct expression patterns.
Purpose of the Study:
- To analyze Epstein-Barr virus (EBV) gene expression in a nasopharyngeal carcinoma (NPC) tumor (C15).
- To compare EBV transcription patterns in epithelial cells with those in B-lymphocytes.
- To identify novel viral transcripts and regulatory mechanisms in NPC.
Main Methods:
- Generation of a large cDNA library from a human NPC tumor (C15) propagated in nude mice.
- Analysis of EBV gene expression using cDNA library screening and immunoblotting.
- Sequence analysis of viral DNA and mRNA transcripts, including splicing patterns and open reading frames.
Main Results:
- EBV DNA in C15 showed no gross alterations compared to other isolates and lacked the deletion found in B95-8 strain.
- Only EBV nuclear antigen-1 (EBNA-1) clones were detected, with distinct mRNA splicing patterns compared to B-cells.
- Viral transcription was predominantly localized to the rightmost region of the EBV genome, with novel antisense transcripts identified.
Conclusions:
- EBV transcription patterns in NPC epithelial cells differ markedly from those in B-cells, indicating differential promoter use or splicing.
- Viral transcription in the C15 tumor is tightly regulated and restricted, with many latent genes (EBNAs 2-6) switched off.
- Novel antisense RNAs transcribed against EBV genes and mutations in C15 transcripts suggest complex viral regulation and potential protein alterations.