The role of repetitive DNA sequences in the size variation of Epstein--Barr virus (EBV) nuclear antigens, and the identification of different EBV isolates using RFLP and PCR analysis

Abstract

The six Epstein-Barr virus (EBV) nuclear antigen proteins show characteristic size variations between different virus isolates; this is a feature that has been used to identify the source of virus isolates in epidemiological studies (Ebnotyping). We have now studied the correlation between restriction fragment length polymorphisms (RFLPs) within exons coding for the EBNAs and the molecular masses of the respective proteins. The B95-8 EBV strain was used as the prototype virus. The variation in apparent molecular mass of EBNA-1, -3 and -6 correlated positively with the size of RFLP coding for repeat sequences in these polypeptides. For EBNA-2, no correlation between apparent molecular mass and length of the repetitive sequences was found. The EBNA-4 protein showed virtually no variation in apparent molecular mass and RFLP size across the repeat sequence. Based on the strong correlation between apparent molecular mass and RFLP size for EBNA-6, we developed an EBNA-6 PCR assay that discriminated between different isolates of EBV. This assay offers the advantage of EBV characterization using uncultured material (e.g. throat washings, blood or biopsies), thus avoiding the selection against poorly transforming strains that occurs during establishment of lymphoblastoid cell lines required for Ebnotyping at the protein level.

Reduction in RNA Levels Rather than Retardation of Translation Is Responsible for the Inhibition of Major Histocompatibility Complex Class I Antigen Presentation by the Glutamic Acid-Rich Repeat of Herpesvirus Saimiri Open Reading Frame 73

“…This is intriguing, given the different amino acid contents and proposed mechanisms of action of these distinct repeats. Moreover, large amino acid repeat domains are present in the two HVS strains sequenced (2,14) and in all EBV and KSHV virus isolates studied to date (16,18), perhaps suggesting there is a requirement for larger repeats to be effective in vivo or indeed indicating additional functionality yet to be mapped to the large repeat domains.…”

Cloning and Mutagenesis of the Murine Gammaherpesvirus 68 Genome as an Infectious Bacterial Artificial Chromosome

“…Size variation due to alterations in repeat structures also occurs in other gammaherpesviruses. For example, the molecular masses of some EBV nuclear antigen proteins vary considerably between virus isolates due to variation in the number of the repetitive sequences (10). Whether the number of repeats in the MHV-68 WT genome remains constant under physiological conditions is not known (9).…”

Diversity of naturally occurring Epstein-Barr virus revealed by nucleotide sequence polymorphism in hypervariable domains in the BamHI K and N subgenomic regions.

“…(A) BamHI E ; (B) BamHI Z ; (C) BamHI N ; and (D) BamHI K. Lanes are labelled as follows : M, molecular mass markers ; 1, B95-8 ; 2, EB176 ; 3, EB185 ; 4, P3HR-1 ; 5, AW-Ramos ; 6, AG876 ; 7, Namalwa ; 8, Daudi ; 9, Raji ; 10, water control. Falk et al, 1995 ;Chen et al, 1996) and post-PCR RFLP assays (Bhatia et al, 1996). The discriminatory ranges of these procedures are finite, however, so sequencing is often required to examine further heterogeneity within the amplified fragments (Buisson et al, 1994 ;Aitken et al, 1994 ;Miller et al, 1994 ;Sandvej et al, 1994 ;Busson et al, 1995 ;Bhatia et al, 1996 ;Gutie!…”

“…Other than these dichotomies, EBV can be differentiated according to size polymorphisms, depending on the number of internal repeats in the BamHI E, H, I, K and N regions (Lung et al, 1988 ;Miller et al, 1994 ;Falk et al, 1995). A variety of natural EBV variants have been discovered, and inter-person transmissions traced (Lung et al, 1991 ;Cen et al, 1991 ;Alfieri et al, 1996 ;Falk et al, 1997).…”