Reassessing the organization of the UL42-UL43 region of the human cytomegalovirus strain AD169 genome

Abstract

A polymorphism in the UL42-UL43 region of the human cytomegalovirus genome has been characterized by nucleotide sequence analysis, revealing a 929-bp insertion following nt 54,612 relative to the published strain AD169-UK genome sequence (M.S. Chee et al., 1990, Curr. Top. Microbiol Immunol. 154, 125-170). Although AD169-UK exhibited polymorphism in this genomic region, other CMV strains (Towne, Toledo, and AD169-ATCC) carried only the newly characterized longer form. The additional sequence altered the assignment of UL42 and UL43 open reading frames. UL42 decreased in size from 157 to 125 codons, retaining 76 of the previously reported carboxyl terminal codons, and UL43 increased in size from 187 to 423 codons, retaining 185 of the previously reported amino terminal codons. This additional sequence makes UL43 a more conserved betaherpesvirus US22 family member. Only AD169-UK exhibited restriction fragment length polymorphism in this region, suggesting that a deletion occurred during the propagation of this strain in cell culture. The additional sequence should be considered a bona fide part of the cytomegalovirus genome and the AD169 genome size should be corrected to 230,283 bp.

Fig. 1

Size variation in the HindIII M region of CMV strain AD169-UK compared to AD169-ATCC, Towne, and Toledo. (A) Electrophoretically separated (0.5% agarose gel) HindIII-digested DNAs from AD169-ATCC (Lot 16), AD169-UK, AD169-ATCC (Lot 2), AD169-ATCC (Lot 4), and AD169-ATCC (Lot 22) were visualized by staining with ethidium bromide (lanes 1 – 5) and by subsequent DNA blot hybridization (lanes 5 – 10) with a fluorescein-11-dUTP (Amersham) random-primed labeled pON2128 probe detected by ECL (Amersham). Lots 2 and 4 of AD169 are the earliest stocks grown from material that was deposited with ATCC by the Rowe laboratory. Lot 16 represents material obtained from ATCC by our laboratory in 1987, and Lot 22, which has sustained 80 to 90 passages in culture, is a more recent (1995) preparation. Although early passages of AD169-UK were not examined, the strain we obtained from H. Browne (Cambridge) generated a pattern previously reported (Oram et al., 1982). This strain had been obtained from the Rowe laboratory in 1960 in its 14th passage and was passaged 54 times by Stern and colleagues (Elek and Stern, 1974) and was provided to J. Oram and H. Browne by J. Booth (St. Georges Hospital Medical School, London). (B) Electrophoretically separated (0.7% agarose gel) HindIII-digested (lanes 1 – 4) or XbaI-digested (lanes 5 – 8) strain Toledo, Towne, AD169-ATCC (Lot 16), and AD169-UK DNA fragments hybridized with a fluorescein-11-dUTP random-primed labeled pCM1017 probe (Fleckenstein et al., 1982) and detected by ECL. Note that Towne lacks the XbaI R/T site present at nt 56,067 in the AD169 genome and therefore exhibits a 9.5-kbp fragment instead of XbaI fragments Rm (6 kbp) and T (3.5 kbp) that are observed in the other strains. Size markers are given on the left and AD169 genome restriction fragment names (with subscript m indicating the modified fragments identified here) are indicated to the right of each set of lanes.

Fig. 2

Nucleotide sequence arrangement and predicted aa sequence of UL42_mod and UL43_mod. (A) The top line shows the CMV strain AD169 genome with the thickened blocks representing repeated sequences (Mocarski, 1995). A HindIII map of the prototype genome is shown with the region carried by pCM1017 (nt 25921 to 64521) and the region carried by pCM1049 (nt 51805 to 84864) depicted by thickened lines below and above the HindIII map, respectively (Fleckenstein et al., 1982). The expanded region shows the published ORF arrangement of the region between nt 54,144 and 55,164 on the AD169 genome (Chee et al., 1990) and the modified region containing a 929-bp insert at nt 54,612 that alters the predicted size and aa sequence of UL42 and UL43. The plasmid clone pON2202, which carries 1599 bp of sequence aligning with a HindIII site at 54,144 and a HpaI site at 54,813 within UL43 (Chee et al., 1990). (B) Deduced aa sequence of UL43_mod and alignment with aa 136 to 597 of murine CMV M43 (Rawlinson et al., 1996). Areas with US22 family motifs (I – IV) are shown above. Identical aa are boxed, conserved aa are shaded, and gaps are indicated by a dash. Identity across all four UL43 homologs (UL43_mod, M43, U25 of HHV-6, and U25 of HHV-7) are denoted by an asterisk and identity in three of the four homologs along with a conservative substitution in the fourth are denoted by a dot below the aa. (C) Deduced aa sequence of UL42_mod and alignment with murine CMV M42 (Rawlinson et al., 1996). The GenBank Accession No. for the 1602-bp region (including the complete HindIII and HpaI sites) is AF01963.

Fig. 2

Nucleotide sequence arrangement and predicted aa sequence of UL42_mod and UL43_mod. (A) The top line shows the CMV strain AD169 genome with the thickened blocks representing repeated sequences (Mocarski, 1995). A HindIII map of the prototype genome is shown with the region carried by pCM1017 (nt 25921 to 64521) and the region carried by pCM1049 (nt 51805 to 84864) depicted by thickened lines below and above the HindIII map, respectively (Fleckenstein et al., 1982). The expanded region shows the published ORF arrangement of the region between nt 54,144 and 55,164 on the AD169 genome (Chee et al., 1990) and the modified region containing a 929-bp insert at nt 54,612 that alters the predicted size and aa sequence of UL42 and UL43. The plasmid clone pON2202, which carries 1599 bp of sequence aligning with a HindIII site at 54,144 and a HpaI site at 54,813 within UL43 (Chee et al., 1990). (B) Deduced aa sequence of UL43_mod and alignment with aa 136 to 597 of murine CMV M43 (Rawlinson et al., 1996). Areas with US22 family motifs (I – IV) are shown above. Identical aa are boxed, conserved aa are shaded, and gaps are indicated by a dash. Identity across all four UL43 homologs (UL43_mod, M43, U25 of HHV-6, and U25 of HHV-7) are denoted by an asterisk and identity in three of the four homologs along with a conservative substitution in the fourth are denoted by a dot below the aa. (C) Deduced aa sequence of UL42_mod and alignment with murine CMV M42 (Rawlinson et al., 1996). The GenBank Accession No. for the 1602-bp region (including the complete HindIII and HpaI sites) is AF01963.