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. 2000 Aug;74(15):6821–6831. doi: 10.1128/jvi.74.15.6821-6831.2000

FIG. 1.

FIG. 1

Insertion of the HA ORF of measles virus into the genome of recombinant PIV3. (A) Diagram (not to scale) of the 2,028-nt insert containing the complete ORF of the HA gene of measles virus. The insert contains, in 5′ to 3′ order, the following: a StuI site; nt 8602 to 8620 from the PIV3 antigenome, which consists of downstream noncoding sequence from the HN gene and its gene end signal; the conserved PIV3 intergenic (IG) trinucleotide; nt 6733 to 6805 from the PIV3 antigenome, which contains the HN gene start and upstream noncoding region; the measles virus HA ORF; PIV3 nt 8525 to 8597, which are downstream noncoding sequences from the HN gene; and a second StuI site. The construction is designed to, upon insertion, regenerate the PIV3 HN gene containing the StuI site and to place the measles virus ORF directly thereafter, flanked by the transcription signals and noncoding region of the PIV3 HN gene. The complete antigenome of PIV3 wild-type JS (rJS) with the introduced StuI site at nt 8600 in the 3′ noncoding region of the HN gene is illustrated in the middle diagram. The bottom diagram is the antigenome of PIV3 expressing the measles HA protein inserted into the StuI site. The HA cDNA used for this insertion came from a cDNA clone that had two amino acid differences from the wild-type Edmonston HA protein, indicated by the asterisks. (B) Diagram (not to scale) of the 1,926-nt insert containing the complete ORF of the measles virus HA gene, with a sequence confirmed to be identical to that of the Edmonston wild-type strain. The insert contains, in 5′ to 3′ order, the following: an AflII site; nt 3699 to 3731 from the PIV3 antigenome, which contains the P-M gene junction including the downstream noncoding sequence for the P gene, its gene end signal, the intergenic region, and the M gene start signal; three additional nonviral nucleotides (GCG); the complete HA ORF; PIV3 nt 3594 to 3623 from the downstream noncoding region of the P gene; and a second AflII site. Panel 1 illustrates the complete antigenome of the wild-type JS strain of PIV3 with the introduced AflII site in the 3′ noncoding region of the N gene before (top) and after (bottom) insertion of the measles HA ORF. Panel 2 illustrates the antigenome of the wild-type JS strain of PIV3 with the introduced AflII site in the 3′ noncoding region of the P gene before (top) and after (bottom) insertion of the measles HA ORF. Versions in the cp45L backbone differ only in the amino acid substitutions at positions 942, 992, and 1558 in the L protein and accompanying silent restriction enzyme markers (52, 53).