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Activation of the IKK/NF-κB signalling pathway is a hallmark of human cytomegalovirus (HCMV) infection. However, its role in regulating major immediate-early promoter (MIEP)-dependent transcription and HCMV replication remains controversial. This study uses a combination of genetic approaches to investigate the effects of cell culture conditions on the importance of virus-induced NF-κB activation during the infection of endothelial cells or fibroblasts. Adenoviral-mediated expression of a dominant-negative mutant of IKK2 kinase (dnIKK2) in human umbilical vein endothelial cells resulted in a strong reduction of IκBα degradation and NF-κB activation following infection with an HCMV clinical isolate. Viral replication was impaired in dnIKK2-expressing cells that were growth-arrested before infection, but not in replicating cells. The inhibitory effect of dnIKK2 was independent from the virus strain and the cell type used, because replication of the laboratory AD169 strain was impaired as well in dnIKK2-expressing quiescent fibroblasts. Moreover, progressive disruption of NF-κB response elements within the MIEP in recombinant HCMV viruses derived from the clinical isolate prevented their replication in quiescent cells but not in actively growing cells. These results demonstrate an essential role of virus-induced IKK/NF-κB activity to trigger both viral IE gene expression and productive replication in quiescent cells. [ABSTRACT FROM AUTHOR]

Our basic understanding of how viruses infect, replicate, and cause disease has been largely derived from genetic approaches in which viral sequences have been mutated and the consequences of those mutations determined. In the herpesvirus field, deletions or insertions that preclude expression of specific viral proteins or remove critical cis elements have been invaluable in identifying their overall functions. We are now ready to move to a new level of detail—mapping functional domains within viral proteins or defining cis elements to the nucleotide level. This level of detail will require mutagenesis on a new scale, with recombinant viruses containing mutations within a given locus perhaps numbering in the hundreds. Mutagenesis on this scale would be greatly facilitated by more rapid methods of recombinant virus construction. In this report, we adapted a technology employing Tn7-mediated site-specific transposition [J. Virol. 67 (1993) 4566] as a rapid and highly reliable method to introduce novel sequences into bacmid-cloned herpesvirus genomes. We show that recombinant viruses can be rapidly created and that a deletion of the human cytomegalovirus (HCMV) essential gene ie2 can be complemented in cis by reintroduction, via transposition, of an ie2 cDNA; detailed mutagenesis of the complementing ie2 gene can now follow. Tn7-mediated transposition should accelerate greatly the pace at which recombinant herpesviruses can be constructed and, thus, facilitate the use of recombinant viruses for detailed mutagenic studies of both cis- and trans-acting genetic elements. [Copyright &y& Elsevier]

Virally encoded microRNAs (miRNAs) have recently been discovered in herpesviruses. However, their biological roles are mostly unknown. We developed an algorithm for the prediction of miRNA targets and applied it to human cytomegalovirus miRNAs, resulting in the identification of the major histocompatibility complex class I-related chain B (MICB) gene as a top candidate target of hcmv-miR-UL112. MICB is a stress-induced ligand of the natural killer (NK) cell activating receptor NKG2D and is critical for the NK cell killing of virus-infected cells and tumor cells. We show that hcmv-miR-UL112 specifically down-regulates MICB expression during viral infection, leading to decreased binding of NKG2D and reduced killing by NK cells. Our results reveal a miRNA-based immunoevasion mechanism that appears to be exploited by human cytomegalovirus. [ABSTRACT FROM AUTHOR]