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. 2003 Mar;23(6):2192-201.
doi: 10.1128/MCB.23.6.2192-2201.2003.

Genetic diversity: frameshift mechanisms alter coding of a gene (Epstein-Barr virus LF3 gene) that contains multiple 102-base-pair direct sequence repeats

Shao-An Xue  1 , M D Jones, Qi-Long Lu, J M Middeldorp, Beverly E Griffin
Affiliations

Genetic diversity: frameshift mechanisms alter coding of a gene (Epstein-Barr virus LF3 gene) that contains multiple 102-base-pair direct sequence repeats

Shao-An Xue et al. Mol Cell Biol. 2003 Mar.

Abstract

Frameshift mutations provide recognized mechanisms for changing the coding potential of an organism. Here, multiple frameshifts are identified in repetitive sequences within an Epstein-Barr virus unspliced early gene, LF3, which is associated with the viral replicative cycle and also transcriptionally expressed in many virally associated tumors. On the DNA strand encoding LF3, there are three open reading frames, only one of which contains an initiation codon. Most (>95%) of the gene consists of numerous (>20, varying with cell source) GC-rich copies of a 102-bp direct repeat (called IR 4) flanked by small unique sequences. LF3 may express a protein if its initiation and termination codons reside in the same reading frame, but this is not always the case. Frameshifting events, occurring in short runs of pyrimidines (mainly C residues) in the repeats, give rise to mutations which may provide a mechanism for escape of an LF3 function from host surveillance. Sequence studies link these frameshifts to DNA replication errors. Notably, the number of sites in LF3 at which such mutations can occur permits a very large amount of diversity in this gene. Our data also suggest a second degeneracy mechanism within the protein itself, which influences its stability and may reflect a host defense mechanism. LF3 thus provides a potentially important model for studying the quest for supremacy between a virus and its host.

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Figures

FIG. 1.
FIG. 1.
Schematic diagram of the structure of the LF3 early gene from EBV (not to scale). Within the LF3 coding region on the viral genome there are three open reading frames, only one with an initiation codon (ATG), and several termination codons, all in the same reading frame (see Fig. 2), the first of which (TGA) is shown. LF3 encodes an unspliced transcript with a single polyadenylation (AATAAA) signal. The gene has short unique sequences at its 3′ and 5′ ends (indicated by heavy solid lines) and contains numerous copies of 102-bp repeats (IR 4), as indicated. Within each repeat lie pyrimidine-rich sequences (two sets of which, at the 5′ and 3′ ends, are given) that are subject to frameshifting events during DNA replication, producing mutations, as noted. The number of repeats may vary (as indicated) according to the cell source, as do the oligopyrimidine sequences, and, depending upon the sequences at the latter slippage-prone regions, initiation and termination codons may or may not lie in the same reading frame.
FIG. 2.
FIG. 2.
Predicted translation products from LF3 transcripts. The sequence shown is that corresponding to the stable mature LF3 RNAs from different sources (see Table 1), which generate protein data (as given) that are predicted from particular sequences obtained by combining those identified in the framshift variable regions at the 5′ and 3′ ends of the genome (bold type). Deletions generated by frameshifting events at the 5′ and 3′ ends are indicated (−). Given here (dashed line between arrowheads) are two complete and one partial (0.7) IR 4 repeat sequences as found in different LF3 genes. Also shown (underlined) are the PstI (CTGCAG) restriction sites which allow single repeat copies, the source of previous sequence data (5, 11, 23, 30, 43), to be excised from the gene, and the first termination codon (TGA, also see Fig. 1) and the polyadenylation (AATAAA) signal. Notably, multiple termination codons (in italics) are all in the same coding frame, and the single AATAAA is used for maturation of all LF3 transcripts. As indicated (patterns 2 to 5), the initiation (ATG) and termination codons may not lie in the same reading frame. Depending upon the reading frame translated (as determined by 5′- and 3′-end combination patterns, designated 1 to 6, see Table 1), the LF3 protein in two of three cases would contain aspartic acid-proline (DP) regions, which are predictably sensitive to enzyme degradation at acidic pH conditions.
FIG. 3.
FIG. 3.
Slippage events in internal IR 4 repeats. Partial sequence profiles (see Fig. 2) in 102-bp (blue plaque) and 103-bp (white plaque) recombinant DNA repeats from Daudi-ICRF isolates. C6 and C7 sequences (bold type) were obtained from the same cell isolate and were the only sequence alterations found in the Daudi repeats, whether from chemically induced or uninduced cells.
FIG. 4.
FIG. 4.
Western blots of LF3 proteins. (A) LF3 polypeptides from cytoplasmic extracts of cells propagated in the absence (−) or presence (+) of the chemical inducing agents tetradecanoyl phorbol acetate and n-butyrate and identified with a rabbit polyclonal antibody from M-ABA cells (29). Sizes of protein markers are given on the right. Arrows indicate sizes predicted for full-length proteins from different sources or degraded residues with different numbers of repetitive (about 34 amino acids) sequences. No polypeptide ladders were observed in three cases, Daudi-100K, BL74, and C15, but in the last, the product was roughly half its expected size. No bands of the expected size were observed in Raji and M-81 cells (as predicted, Table 1 and Fig. 2). The data for M-ABA were taken from a separate Western blot. (B) Proteins from cytoplasmic extracts of Daudi-ICRF or P3HR-1 uninduced (−) and induced (+) cells (panels 2 and 4, respectively) stained with polypeptide antibody PR or PP, as noted, or with the corresponding preimmune serum samples (panels 1 and 3). Positions of protein size markers (lane M) are indicated on the right of each panel, and integrals suggesting loss of a repeat polypeptide are indicated on the left. In the case of Daudi cells, induction was required for protein detection, whereas this was not so with P3HR-1.
FIG. 5.
FIG. 5.
Immunostaining of LF3 proteins. (A) Cells propagated without (−) or with (+) chemical inducing agents and stained with preimmune serum (vertical column 1) or with antibody (columns 2 and 3) from recombinant M-ABA polypeptide (29). Only in P3HR-1 (horizontal panels 6) were stained cells observed without chemical induction (see also Fig. 4). By this technique, all other isolates examined (except the B95-8 control and Raji), including M81, show small numbers of positively (mainly cytoplasmic) stained cells following chemical induction. Preimmune serum samples gave negative results. (B) Cytoplasmic and nuclear staining of LF3 protein in P3HR-1 induced cells, photographed by confocal microscopy. Panel a shows a cell with cytoplasmic staining, and panel b shows cells with nuclear staining. These data demonstrate that the protein can reside at two cellular sites and belongs to the early antigen-diffuse family of EBV proteins. Examination of larger fields of cells (not shown) indicated that cytoplasmic staining predominated. Similar conclusions were reached from Western blot data on Daudi-ICRF and M-ABA cell isolates.
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