Cis and Trans Acting Factors Involved in Human Cytomegalovirus Experimental and Natural Latent Infection of CD14 (+) Monocytes and CD34 (+) Cells

Abstract

The parameters involved in human cytomegalovirus (HCMV) latent infection in CD14 (+) and CD34 (+) cells remain poorly identified. Using next generation sequencing we deduced the transcriptome of HCMV latently infected CD14 (+) and CD34 (+) cells in experimental as well as natural latency settings. The gene expression profile from natural infection in HCMV seropositive donors closely matched experimental latency models, and included two long non-coding RNAs (lncRNAs), RNA4.9 and RNA2.7 as well as the mRNAs encoding replication factors UL84 and UL44. Chromatin immunoprecipitation assays on experimentally infected CD14 (+) monocytes followed by next generation sequencing (ChIP-Seq) were employed to demonstrate both UL84 and UL44 proteins interacted with the latent viral genome and overlapped at 5 of the 8 loci identified. RNA4.9 interacts with components of the polycomb repression complex (PRC) as well as with the MIE promoter region where the enrichment of the repressive H3K27me3 mark suggests that this lncRNA represses transcription. F ormaldehyde A ssisted I solation of R egulatory E lements (FAIRE), which identifies nucleosome-depleted viral DNA, was used to confirm that latent mRNAs were associated with actively transcribed, FAIRE analysis also showed that the terminal repeat (TR) region of the latent viral genome is depleted of nucleosomes suggesting that this region may contain an element mediating viral genome maintenance. ChIP assays show that the viral TR region interacts with factors associated with the pre replication complex and a plasmid subclone containing the HCMV TR element persisted in latently infected CD14 (+) monocytes, strongly suggesting that the TR region mediates viral chromosome maintenance.

Author Summary

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus where infection is usually subclinical. HCMV initial infection is followed by the establishment of latency in CD34 (+) myeloid cells and CD14 (+) monocytes. Primary infection or reactivation from latency can be associated with significant morbidity and mortality can occur in immune compromised patients. Latency is marked by the persistence of the viral genome, lack of production of infectious virus and the expression of only a few previously recognized latency associated transcripts. Despite the significant interest in HCMV latent infection, little is known regarding the mechanism involved in establishment or maintenance of the viral chromosome. We have now identified the transacting factors present in latently infected CD14 (+) monocytes and CD34 (+) progenitor cells as well as identification of a region of the HCMV genome, the terminal repeat locus that mediates viral DNA maintenance. This is a major step toward understanding the mechanism of HCMV latent infection.