Analysis of the noncoding regions of measles virus strains in the Edmonston vaccine lineage

Abstract

The noncoding sequence of five Edmonston vaccine viruses (AIK-C, Moraten, Rubeovax, Schwarz, and Zagreb) and those of a low-passage Edmonston wild-type (wt) measles virus have been determined and compared. Twenty-one nucleotide positions were identified at which Edmonston wt and one or more vaccine strains differed. The location of some of these nucleotide substitutions suggests that they may influence the efficiency of mRNA synthesis, processing, and translation, as well as genome replication and encapsidation. Five nucleotide substitutions were conserved in all of the vaccine strains. Two of these were in the genomic 3'-terminal transcriptional control region and could affect RNA synthesis or encapsidation. Three were found within the 5'-untranslated region of the F mRNA, potentially altering translation control sequences. The remaining vaccine virus base changes were found in one to four vaccine strains. Their genomic localization suggests that some may modify cis-acting regulatory domains, including the Kozak consensus element of the P and M genes, the F gene-end signal, and the F mRNA 5'-untranslated sequence.

FIG. 1

MV genome map. The location of protein coding regions (white boxes: N, P, V, C, M, F, H, and L) and noncoding regions including the leader and trailer (black terminal boxes) and intergenic regions (shaded blue) are shown, along with specialized sequence motifs (2, 16, 19, 27, 41, 52). The TCRs at the 3′ end of the genomic RNA (leader TCR, nucleotides 1 to 107) and the 3′ end of the antigenomic RNA (trailer TCR, nucleotides 15785 to 15894) are enlarged below the genome map. Genomic RNA synthesis initiation is shown as open arrowheads in the enlargement of the leader and trailer TCR regions. Sequence motifs shown in the expanded TCR maps include the leader, trailer, GE and GS signals, the 16-base conserved terminal sequence (shown 3′ to 5′), the repeated B- and B'-box motif, the G(N)5 motif, the ATG codon for the N gene adjacent to the leader, and the L protein stop codon outside of the 5′ end of the trailer. The positions of GE/GS signals are designated with a combination of solid arrowhead (GS) and a black box (GE). The leader TCR GE/GS symbol lacks a GE box, and the trailer TCR GE/GS lacks a GS arrowhead to signify that these sequences may not function identically to GE/GS elements in the intergenic regions. The mRNA editing site is indicated below the V protein coding region (6). Part of the F gene mRNA 5′-untranslated nucleotide sequence is shown to illustrate the three in-frame AUG initiator codons (5). At the bottom of the figure, several symbols found in the TCR maps are defined.

FIG. 2

Sequence comparison of Edmonston wt and vaccine leader and trailer TCRs. Terminal 110 bases of genomic sequence (3′ to 5′) containing the leader TCR (nucleotides 1 to 107; part A) and trailer TCR (nucleotides 15785 to 15894; part B) of Edmonston wt are shown and labeled with genome nucleotide positions. Sequence motifs are illustrated on the sequence according to the key at the bottom of the figure. Relevant protein start or stop codons also are included. The sequence corresponding to the GE region in the leader TCR and the GS region of the trailer TCR are not shaded in gray to indicate that these sequence motifs may not function identically to their intergenic GE/GS counterparts. Illustrated below the wt sequence is the sequence comparison with vaccine strains. Nucleotide identity is given as a dot, and a typed nucleotide indicates disagreement with wt.

FIG. 3

Comparison of N/P and P/M intergenic regions of Edmonston wt and vaccine viruses. Description of this figure is as described for Fig. 2.

FIG. 4

Comparison of M/F intergenic regions from Edmonston wt and vaccine viruses. The description of this figure is similar to that for Fig. 2. One line of dots indicating homology to all vaccines is shown under the wt sequence if there were no base changes to report. The sequence shown in this figure extends beyond the noncoding intergenic region to show the position of the three F gene AUG codons. The second AUG codon is the predominately used initiation codon (5). Vaccine virus names are abbreviated as follows: AIK-C (AIK), Moraten and Schwarz (Mor/Sch) Rubeovax (Rubeo) and Zagreb (Zag).

FIG. 5

Comparison of F/H and H/L intergenic regions from Edmonston wt and vaccine viruses. The description of this figure parallels that for Fig. 2.

FIG. 6

Summary of noncoding region nucleotide substitutions. A genome map similar to Fig. 1 is shown along with the nucleotide positions that varied in vaccine strains. The relative position of the GE/GS signals is shown by an arrow head plus the nucleotide position corresponding to the central A residue in the conserved GAA motif. Boundaries of the noncoding regions are indicated at the ends of the brackets containing the nucleotide substitutions. Nucleotides highlighted in gray depict changes from the Edmonston wt sequence. An asterisk above a column of nucleotides indicates that a base change occurred in all vaccine strains. The dashed box outlining groups of three nucleotides shows positions where Rubeovax differs from Moraten and Schwarz.

FIG. 7

Vaccine nucleotide substitutions in cis-acting sequence motifs. (A) Alignment of MV TCRs containing the GE/GS signal. Similarity between the signals and a consensus sequence is outlined by the box (yellow). The leader TCR GE and trailer TCR GS regions are presented in lower case to indicate that these sequences may not function like the corresponding sequences of the intergenic GE/GS elements and that these sequences were not considered in evaluating the consensus. Below the consensus are the two GE/GS sequences that contained vaccine virus base substitutions. The nucleotide substitution is highlighted by a blue box. (B) Summary of the base changes found in the leader and trailer TCRs. The description of the leader and trailer TCRs can be found in Fig. 1. Nucleotide changes are illustrated below the leader and trailer TCRs. Base changes are highlighted in blue. (C) Comparison of the Kozak element (24) found in wt MV mRNAs. Variation in vaccine virus P and M gene Kozak sequences is shown below the wt sequences.

FIG. 8

Alignment of the Edmonston wt leader (minus strand) and trailer (plus strand). The alignment illustrates the homology between the terminal 16 nucleotides at the 3′ end of the leader and trailer, as well as the B-box regions. The motifs identified in the figure are described in the legend to Fig. 1. Vaccine virus leader TCR base changes are shown above the wt sequence, and vaccine trailer TCR changes are shown below.