MicroRNAs (miRNAs) are an extensive class of noncoding genes that regulate gene expression through posttranscriptional repression. Given the potential for large viral genomes to encode these transcripts, we examined the human cytomegalovirus AD169 genome for miRNAs using a bioinformatics approach. We identified 406 potential stem-loops, of which 110 were conserved between chimpanzee cytomegalovirus and several strains of human cytomegalovirus. Of these conserved stem-loops, 13 exhibited a significant score using the MiRscan algorithm. Examination of total RNA from human cytomegalovirus-infected cells demonstrated that 5 of the 13 predicted miRNAs were expressed during infection. These studies demonstrate that human cytomegalovirus encodes multiple conserved miRNAs and suggest that human cytomegalovirus may utilize an miRNA strategy to regulate cellular and viral gene function.MicroRNAs (miRNAs) are a large class of noncoding RNAs involved in posttranscriptional regulation through RNA interference. A number of recent studies have identified virally encoded miRNAs using either biochemical cloning strategies, bioinformatics, or a combination of the two approaches (1, 2, 4, 7-9). In this study an alternative bioinformatics approach based on comparative conservation between predicted stemloop sequences of human cytomegalovirus (HCMV) and chimpanzee cytomegalovirus (CCMV) was used to predict candidate miRNAs. Expression of predicted miRNAs during HCMV infection was then assessed by Northern blot analysis. Our bioinformatics approach utilized a computer algorithm called Stem-loop Finder (SLF; Combimatrix) to predict potential RNA transcripts from the HCMV genome that could form stem-loop secondary structures. The algorithm uses free energy calculations to determine the theoretical stability of the base pairing within the stem region, including pairing between G · U bases, while maintaining a maximum and minimum length between the complementary base pairing to determine loop size. A scoring matrix then weights beneficial or detrimental folding structures and attributes a cumulative score to each potential stem-loop sequence. Analysis of the HCMV genome using SLF identified 406 potential stem-loop sequences. Previously identified miRNAs are often extensively conserved between many different species (6). Consequently, we hypothesized that functionally important miRNAs expressed by HCMV would be conserved between HCMV and closely related viruses, such as CCMV. The sequence of each of the 406 candidate SLF-derived HCMV stem-loop transcripts was compared with the CCMV genome for potential homology. A minimum score of 60% homology with CCMV was used to select stem-loop sequences for further analysis. Our preliminary studies determined that this level of homology was sufficiently stringent to identify significant sequence conservation without exclusion of any potential miRNAs. Of the 406 sequences analyzed, 110 potential stem-loop sequences scored higher than 60% homology.The 110 HCMV stem-loop sequences selected using the cri...