Evidence for Tethering of Human Cytomegalovirus Genomes to Host Chromosomes

Abstract

Tethering of viral genomes to host chromosomes has been recognized in a variety of DNA and RNA viruses. It can occur during both the productive cycle and latent infection and may impact viral genomes in manifold ways including their protection, localization, transcription, replication, integration, and segregation. Tethering is typically accomplished by dedicated viral proteins that simultaneously associate with both the viral genome and cellular chromatin via nucleic acid, histone and/or non-histone protein interactions. Some of the most prominent tethering proteins have been identified in DNA viruses establishing sustained latent infections, including members of the papillomaviruses and herpesviruses. Herpesvirus particles have linear genomes that circularize in infected cell nuclei and usually persist as extrachromosomal episomes. In several γ-herpesviruses, tethering facilitates the nuclear retention and faithful segregation of viral episomes during cell division, thus contributing to persistence of these viruses in the absence of infectious particle production. However, it has not been studied whether the genomes of human Cytomegalovirus (hCMV), the prototypical β-herpesvirus, are tethered to host chromosomes. Here we provide evidence by fluorescence in situ hybridization that hCMV genomes associate with the surface of human mitotic chromosomes following infection of both non-permissive myeloid and permissive fibroblast cells. This chromosome association occurs at lower frequency in the absence of the immediate-early 1 (IE1) proteins, which bind to histones and have been implicated in the maintenance of hCMV episomes. Our findings point to a mechanism of hCMV genome maintenance through mitosis and suggest a supporting but non-essential role of IE1 in this process.

Human Cytomegalovirus Genomes Survive Mitosis via the IE19 Chromatin-Tethering Domain

“…In the context of the 72-kDa IE1 protein, the major protein encoded by UL123, the CTD associates with the acidic patch of nucleosomes and mediates the colocalization of IE1 with condensed cellular chromosomes similar to KSHV LANA (37,38). While the UL123 CTD has long been hypothesized as a viral genome tether (33,36,37,39,40), the function of this domain has never been tested in mitotic cells. Like the tethering proteins described above, IE1 dimerizes.…”

Rethinking human cytomegalovirus latency reservoir

“…One of HCMV's immediate early proteins, IE1, encoded by the UL123 gene, was shown to have a chromatin tethering domain (CTD 17 ). This domain was shown to have a supporting role in the noncovalent tethering of the viral genome to the cellular chromosomes during mitosis during the first few days after infection of fibroblasts 18 . This was further supported by findings that a splice variant of the UL123, IE19, which contains the CTD, is important for viral genome maintenance during mitosis when infection occurs in cells that are in the S‐phase of the cell cycle 19 .…”

“…This domain was shown to have a supporting role in the noncovalent tethering of the viral genome to the cellular chromosomes during mitosis during the first few days after infection of fibroblasts. 18 This was further supported by findings that a splice variant of the UL123, IE19, which contains the CTD, is important for viral genome maintenance during mitosis when infection occurs in cells that are in the S-phase of the cell cycle. 19 Another IE1 isoform, IE1x4, a product of a transcript initiating within exon 4 of the UL123 gene, was suggested to mediate the maintenance of the viral genome during latent infection of HSCs.…”

“…Moreover, it was reported that ectopically expressed IE1x4 is diffuse within the cytoplasm and does not localize to mitotic chromosomes 19 . Importantly, in both primary CD34 + HSCs and in the KG‐1 CD34 + cell line, following long times after initial infection, there was a substantial drop in the level of viral genomes 18,20 . Therefore, while several lines of evidence support a role for the IE1 CTD in the maintenance of the viral genome when cells are infected during S‐phase, strong data are lacking to support a long‐term viral genome maintenance function in latent HCMV infection.…”

Critical Role for the Human Cytomegalovirus Major Immediate Early Proteins in Recruitment of RNA Polymerase II and H3K27Ac To an Enhancer-Like Element in Ori Lyt

“…IE1-72 regulates the viral epigenome by antagonizing de-acetylation of histones bound to the MIEP and by modulating the nucleosome organization of the HCMV genome ( 15 , 16 ). In addition, IE1-72 tethers the viral genome to cellular chromatin through interaction with an acidic patch on the nucleosome surface formed by histones H2A and H2B, although this tethering is dispensable for viral replication and regulation of viral gene expression ( 17 – 20 ).…”