Biology Reference
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C. Sinzger et al., this volume). Although these passaged viruses have not succeeded as
vaccine candidates, they have been widely studied since they grow more rapidly, release
more cell-free virus, and generate higher yields in fibroblasts than do clinical strains.
Consequently, they are often termed laboratory strains. The improved replication of
laboratory strains in fibroblasts results in part from disruption of the glycoprotein com-
plex mentioned above. When the UL131 mutation was repaired in AD169, the repaired
virus grew to a reduced yield in fibroblasts (Wang and Shenk 2005a); a similar growth
defect was observed when pUL131 was provided to AD169 in trans (Adler et al. 2006).
It is not clear why an intact gH-gL-pUL128-pUL130-pUL131 complex inhibits the
replication of AD169 in fibroblasts.
In addition to numerous more subtle alterations, the genomes of these passaged
viruses have undergone major rearrangements, suffering large deletions and concomitant
duplications (Cha et al. 1996). Two laboratory strains have been sequenced: AD169
(Chee et al. 1990; Murphy et al. 2003b) and Towne (Dunn et al. 2003; Murphy et al.
2003b). Both viruses have independently lost a multigene segment and acquired a
repeated multigene sequence of nearly identical size. The sequence duplication most
likely serves to maintain a unit genome length for viral packaging. Even though the two
viruses were generated from different clinical isolates in different parts of the world, their
substitutions are very similar. AD169 lacks ORFs UL133-UL151 and carries a duplica-
tion of ORFs RL1-14 (and termed TRL1-14 and IRL1-14 to discriminate terminal and
internal copies of the repeat) (Fig. 1, AD169). The AD169 rearrangement resulted from
a recombination event between RL14 and an ORF in a different frame but within UL148
at one end and within the a and b sequences at the other end. Towne underwent a recom-
bination where sequences within UL1 and the a and b repeats were duplicated at the
expense of ORFs UL144-UL151. Both recombination events disrupted the ORFs that
allow for the synthesis of a functioning gH-gL-pUL128-pUL130-pUL131 complex
mentioned above, and this appears to be the result of a selective pressure to lose this
complex for efficient replication in fibroblasts.
It is noteworthy that Toledo has also, at least partially, adapted to more efficient
replication in fibroblasts. In this case, rather than undergoing a deletion/duplication
event, an approximately 15-kbp domain has been inverted (Cha et al. 1996). The
UL128 ORF was disrupted at one end of the inversion.
Protein-Coding ORFs
How many functional ORFs reside in the HCMV genome? Answering this question
is a daunting task. The first estimate came with the original sequence of AD169
(Chee et al. 1990). This annotation predicted that AD169 has the potential to
encode 208 ORFs, of which several are repeated (TRL1-14 and IRL1-14). An ORF
was considered a coding ORF if it encoded a polypeptide of 100 amino acids or
more and did not overlap a larger ORF across more than 60% of its length. A DNA
segment containing 19 additional ORFs was discovered in the Toledo clinical strain
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