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and appear to be critical for miRNA-target mRNA recognition (Jackson and
Standart 2007).
The major functional consequence of miRNA biogenesis is alteration of protein
concentration through either translation inhibition or cleavage of mRNA. It is
important to note that operationally miRNA-mediated inhibition of gene expression
is not necessarily an all-or-nothing mechanism. Rather, the present consensus is that
miRNAs allows a fine tuning of gene expression (Bartel 2004). miRNAs may be
seen as a means by which the cell or virus can achieve a spectrum of gene expres-
sion levels appropriate to differentiation or developmental states, proliferative
signals, and most recently in response to infection. As such, it is not difficult to
imagine a role for viral miRNAs, particularly in those viruses that find persistence
and/or latency part of their life cycle.
Location and Conservation of HCMV miRNAs
Mapping of HCMV miRNAs
Using a small RNA cloning and sequencing approach, a total number of 14 mature
HCMV miRNAs were identified, which arise from 11 pre-miRNAs (Fig. 1) (Dunn
et al., 2005: Grey et al., 2005; Pfeffer et al., 2005). In order to correlate our previous
work (Dunn et al. 2003), where we functionally profiled the HCMV genome by
constructing a deletion mutant library, with the discovery of HCMV miRNAs, we
have mapped the HCMV miRNAs to the genome of the HCMV Towne (HCMV
Towne-BAC) strain that was cloned as a bacterial artificial chromosome (Fig. 3).
Our annotation of open reading frames (ORFs) within the HCMV Towne-BAC
reveals that five HCMV pre-miRNAs are intergenic, four are found within ORFs and
two partially overlap annotated ORFs. The vector for bacterial propagation of the
Towne-BAC replaces ORFs US1-US12. Additionally, a well-characterized sponta-
neous deletion of UL150 has occurred on passaging of Towne in tissue culture. To
map miRNAs in these regions, we have deferred to other publications for their anno-
tation (Grey et al. 2005; Pfeffer et al. 2005). From the map and data generated by the
deletion mutant library, we have determined that six of the miRNAs (mir-US4, mir-
US5-1, mir-US5-2, mir US25-2, mir-US33 and mir-UL148D) are not essential for
growth in tissue culture. These six miRNAs along with their associated ORFs have
been deleted from the viral genome with no apparent resulting defect in viral replica-
tion in tissue culture. The possibility remains, however, that these miRNA targeted
the ORFs in which they are found, and that concomitant deletion of both the miRNA
and its target ORF precluded any phenotype we may have observed if only the
miRNA had been disrupted. The two deletions that did result in a phenotype (UL70
and UL114) are in ORFs whose gene products are known to be important for viral
infection (Pari and Anders 1993; Courcelle et al. 2001). This precludes determining
the contribution that the deletion of miRNAs in these ORFs makes to viral growth
