Maintenance and replication of the human cytomegalovirus genome during latency.

Human cytomegalovirus (HCMV) can establish latent infection in hematopoietic progenitor cells (HPCs) or CD14 (+) monocytes. While circularized viral genomes are observed during latency, how viral genomes persist or which viral factors contribute to genome maintenance and/or replication is unclear. Previously, we identified a HCMV cis-acting viral maintenance element (TR element) and showed that HCMV IE1 exon 4 mRNA is expressed in latently infected HPCs. We now show that a smaller IE1 protein species (IE1x4) is expressed in latently infected HPCs. IE1x4 protein expression is required for viral genome persistence and maintenance and replication of a TR element containing plasmid (pTR). Both IE1x4 and the cellular transcription factor Sp1 interact with the TR, and inhibition of Sp1 binding abrogates pTR amplification. Further, IE1x4 interacts with Topoisomerase IIß (TOPOIIß), whose activity is required for pTR amplification. These results identify a HCMV latency-specific factor that promotes viral chromosome maintenance and replication.

Cis and trans acting factors involved in human cytomegalovirus experimental and natural latent infection of CD14 (+) monocytes and CD34 (+) cells.

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. Formaldehyde Assisted Isolation of Regulatory Elements (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.

Regulation of viral and cellular gene expression by Kaposi's sarcoma-associated herpesvirus polyadenylated nuclear RNA.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the cause of Kaposi's sarcoma and body cavity lymphoma. In cell culture, KSHV results in a latent infection, and lytic reactivation is usually induced with the expression of K-Rta or by treatment with phorbol 12-myristate 13-acetate (TPA) and/or n-butyrate. Lytic infection is marked by the activation of the entire viral genomic transcription cascade and the production of infectious virus. KSHV-infected cells express a highly abundant, long, noncoding transcript referred to as polyadenylated nuclear RNA (PAN RNA). PAN RNA interacts with specific demethylases and physically binds to the KSHV genome to mediate activation of viral gene expression. A recombinant BACmid lacking the PAN RNA locus fails to express K-Rta and does not produce virus. We now show that the lack of PAN RNA expression results in the failure of the initiation of the entire KSHV transcription program. In addition to previous findings of an interaction with demethylases, we show that PAN RNA binds to protein components of Polycomb repression complex 2 (PRC2). RNA-Seq analysis using cell lines that express PAN RNA shows that transcription involving the expression of proteins involved in cell cycle, immune response, and inflammation is dysregulated. Expression of PAN RNA in various cell types results in an enhanced growth phenotype, higher cell densities, and increased survival compared to control cells. Also, PAN RNA expression mediates a decrease in the production of inflammatory cytokines. These data support a role for PAN RNA as a major global regulator of viral and cellular gene expression.