Six strains of human cytomegalovirus have been sequenced, including two laboratory strains (AD169 and Towne) that have been extensively passaged in fibroblasts and four clinical isolates that have been passaged to a limited extent in the laboratory (Toledo, FIX, PH, and TR). All of the sequenced viral genomes have been cloned as infectious bacterial artificial chromosomes. A total of 252 ORFs with the potential to encode proteins have been identified that are conserved in all four clinical isolates of the virus. Multiple sequence alignments revealed substantial variation in the amino acid sequences encoded by many of the conserved ORFs

A quantitative algorithm was developed and applied to predict target genes of microRNAs encoded by herpesviruses. Although there is almost no conservation among microRNAs of different herpesvirus subfamilies, a common pattern of regulation emerged. The algorithm predicts that herpes simplex virus 1, human cytomegalovirus, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus all employ microRNAs to suppress expression of their own genes, including their immediate-early genes. In the case of human cytomegalovirus, a virus-coded microRNA, miR-112-1, was predicted to target the viral immediate-early protein 1 mRNA. To test this prediction, mutant viruses were generated that were unable to express the microRNA, or encoded an immediate-early 1 mRNA lacking its target site. Analysis of RNA and protein within infected cells demonstrated that miR-UL112-1 inhibits expression of the major immediate-early protein. We propose that herpesviruses use microRNA-mediated suppression of immediate-early genes as part of their strategy to enter and maintain latency.miRNAs ͉ reactivation ͉ immune evasion M icroRNAs (miRNAs) are 20-23-nucleotide RNA molecules that bind to mRNA targets, generally within their 3Ј untranslated region (3Ј UTR), and interfere with their translation (1, 2). Viruses have co-evolved with cellular miRNAs and many encode their own miRNAs (3). Every herpesvirus genome that has been examined has been found to encode multiple miRNAs, including Epstein-Barr virus (EBV) (4 -6), human cytomegalovirus (HCMV) (4, 7, 8), herpes simplex virus 1 (HSV-1) (9, 10), and Kaposi's sarcoma-associated herpesvirus (KSHV) (4,6,11,12). These miRNAs can potentially function during lytic replication and latency. Lytic replication proceeds in a coordinated three-phase cascade: immediate-early (IE), early and late. IE products prepare the cell for infection and propagate the cascade. Early gene products support replication of viral DNA, and DNA replication is, in turn, a prerequisite for full activation of the late genes that encode the structural proteins of the virus. During latency, the virus is quiescent. A limited subset of the viral genome is expressed, but, importantly, the virus has the potential to reactivate and reenter the lytic cycle. Although the molecular mechanisms of reactivation are not understood, it is widely assumed that the lytic cascade is reinitiated with the expression of IE genes. Ectopic expression of a single IE protein has been shown to reactivate HSV-1 (13, 14), EBV (15), or KSHV (16,17) in cell culture models of latency.Whereas none or only a few protein-coding genes are expressed, multiple miRNAs are transcribed during latency. The HSV-1 miR-LAT lies within one of the latency associated transcripts (LATs), the only viral RNAs known to be expressed in latency. EBV and KSHV miRNAs also are expressed during latent infection. Because they are nonimmunogenic, miRNAs should be optimal agents for suppression of anti-viral responses and to modify behaviors of latently infected cells, and recent reports ...