The human cytomegalovirus decathlon: Ten critical replication events provide opportunities for restriction

Abstract

Human cytomegalovirus (HCMV) is a ubiquitous human pathogen that can cause severe disease in immunocompromised individuals, transplant recipients, and to the developing foetus during pregnancy. There is no protective vaccine currently available, and with only a limited number of antiviral drug options, resistant strains are constantly emerging. Successful completion of HCMV replication is an elegant feat from a molecular perspective, with both host and viral processes required at various stages. Remarkably, HCMV and other herpesviruses have protracted replication cycles, large genomes, complex virion structure and complicated nuclear and cytoplasmic replication events. In this review, we outline the 10 essential stages the virus must navigate to successfully complete replication. As each individual event along the replication continuum poses as a potential barrier for restriction, these essential checkpoints represent potential targets for antiviral development.

Keywords: HCMV (human cytomegalovirus); antiviral therapeutic; herpes viral infection; viral replication; virion assembly.

FIGURE 1

For infection in fibroblasts, the glycoprotein trimer consisting of gH, gL and gO binds to PDGFRα. Fusion between the envelope and cell membrane is mediated by gB and the tegumented nucleocapsid is released into the cytoplasm. For infection of epithelial, endothelial, myeloid, and likely many other cell types, the glycoprotein pentamer consisting of gH, gL, UL128, UL130 and UL131A binds to either OR14I1 or NRP2 on the cell surface and induces endocytosis of the virion. Pentamer mediated entry is dependent on acidification of the endosome as well as gO. The fusion step is mediated by gB to release the tegumented capsid into the cytoplasm, and tegument proteins dissociate and begin to reprogram the host cell. The capsid traffics to the nucleus for genome deposition. PDGFRα, platelet derived growth factor receptor alpha; NRP2, neuropilin; gB, envelope glycoprotein B.

FIGURE 2

Inside the host cell nucleus, viral genomes circularise, and IE gene expression commences independently of viral protein synthesis. The MIEP is immediately upstream of the IE genes and acts as a hub for transcriptional activation or repression of the IE proteins by diverse host and viral factors. MIEP regulation also dictates the switch from lytic replication to latency and vice versa. The MIEP begins immediately upstream of the UL122/UL123 ORF and consists of the core (+1 to −40), enhancer (−40 to −550), unique (−550 to −750) and modulator (−750 to −1,140) regions. The IE1 and IE2 proteins are the main IE effectors which are encoded by the UL122/UL123 ORF by alternative splicing. IE1-72 and IE2-86 are multifunctional proteins that transactivate DE viral genes, remodel chromatin, disrupt interferon signalling, and inhibit apoptosis to create a conducive cellular environment for viral replication. IE, immediate early; MIEP, major immediate early promoter; ORF, open reading frame; DE, delayed early; crs, cis-repression sequence.

FIGURE 3

HCMV forms an intranuclear replication compartment for viral DNA replication and transcription. The UL112/UL113 proteins coalesce around viral genomes and together induce a phase separation. Nuclear viral proteins, including DNA replication machinery preferentially localises to the phase separated compartment, enhancing DNA replication and creating positive feedback. Over the course of infection, multiple small pre-RCs expand and coalesce into a single large structure that occupies most of the nuclear volume. RC, replication compartment.

FIGURE 4

Viral DNA replication proceeds by a rolling circle mechanism to produce linear concatemers. The tripartite helicase-primase complex composed of UL70, UL102 and UL105 separates the strands to form a replication fork, while the DNA polymerase (UL54) and processivity factor (UL44) synthesise the daughter strands by leading and lagging strand synthesis. The ssDNA binding protein UL57 forms filaments on ssDNA, which stimulates polymerase and primase activity. ssDNA, single stranded DNA.

FIGURE 5

HCMV late genes are expressed with comparable kinetic profiles, however, distinct mechanisms of transcriptional initiation exist. The first involves TATA binding by host TBP, recruitment of a canonical PIC and RNA pol II, and transcription initiation akin to the alpha-herpesviruses. The second involves IE2-based regulation of transcriptional initiation, by binding to a crs-like motif and recruiting the host PIC and pol II for gene transcription. It must be noted that IE2 is a multifunctional protein that likely influences viral gene activation, repression, and as an elongation barrier depending on the promoter sequence, bound transcription factors and local chromatin environment. The third mechanism is unique to the beta- and gamma-herpesviruses that encode a 6-member vPIC that binds to unconventional TATT promoter sequences and recruits host pol II for transcript elongation. RC, replication compartment; IE2, immediate early 2; TBP, TATA binding protein; PIC, pre-initiation complex; vPIC, viral pre-initiation complex; pol II, host RNA polymerase II complex.

FIGURE 6

The HCMV vAC is a cytoplasmic virus factory where virion cargo accumulates to enable tegumentation and secondary envelopment. The vAC is characterised by concentric rings of host-derived organelles, with endosome membranes surrounded by a Golgi ring, and the ER loosely associated around the periphery. The structure is adjacent to the enlarged kidney-shaped nucleus and functions as a Golgi-derived MTOC. The cytoskeleton is central to vAC formation and function, and is connected to the nucleus through polarised LINC complexes. LINC complexes provide a bridge between the nucleoskeleton and cytoskeleton that allows the vAC to exert control over nuclear morphology, rotation, and internal organisation. ER, endoplasmic reticulum; EE, early endosome; MT, microtubule; MTOC, microtubule organising centre; LINC, linker of nucleoskeleton and cytoskeleton; vAC, viral assembly compartment; RC, replication compartment.

FIGURE 7

HCMV capsid subunits self-assemble around the scaffold in the nucleus. Once the spherical procapsid is fully assembled, divergence in capsid maturation pathways can occur. If the procapsid angularises to the icosahedral form before the scaffold is removed a B capsid is formed that cannot undergo subsequent maturation. Next, the protease cleaves the scaffold to release it from the interior of the procapsid, and it is ejected through enlarged hexon pores. If angularisation occurs after this step, but before terminase complex engagement, an empty A capsid is formed. Mature C capsids are formed when the terminase complex successfully engages the capsid and delivers a genome. The terminase complex provides energy for genome translocation through ATP hydrolysis, and cleaves unit length genomes from the newly replicated concatemers by endonuclease activity. TRM, tripartite terminase complex.

FIGURE 8

Mature capsids traverse both nuclear membranes to enter the cytoplasm for subsequent maturation. The NEC consisting of UL50 and UL53 acts as an organisational hub on the INM to recruit host and viral proteins to facilitate this step. Additionally, the nuclear lamina poses a physical barrier for exiting capsids. The lamins are phosphorylated by the viral kinase UL97 and subsequently depolymerise. C capsids travel along nuclear actin filaments to the nuclear membrane where they undergo envelopment at the INM mediated by the NEC, and subsequent fusion with the ONM to release the nascent capsid into the cytoplasm. NEC, nuclear egress complex; INM, inner nuclear membrane; ONM, outer nuclear membrane; RC, replication compartment; PIN1, peptidyl-prolyl cis-trans isomerase NIMA-interacting 1.

FIGURE 9

Cytoplasmic envelopment of nascent virions occurs on MVB membranes or short cisternae within the vAC. In fibroblasts and likely epithelial cells, envelopment is mediated by multiple envelope glycoproteins and the membrane associated tegument proteins UL71 and UL99. The site of final envelopment as well as the virion envelope are enriched in exosome markers that indicates a common membrane origin. In endothelial cells, final envelopment also occurs on MVB membranes. However, these are not enriched in exosome markers, but rather contain Golgi and autophagic markers. Additionally, the viral proteins UL135 and UL136 are important for envelopment in endothelial cells, but are entirely dispensable in fibroblasts. Note: UL71 and UL99 are included in the diagram for endothelial cell envelopment, as they are well conserved in clinical strains. However, they have not been directly assayed in endothelial cells. MVB, multivesicular body; gB, envelope glycoprotein B.

FIGURE 10

Following envelopment, MVBs containing enveloped virions and ILVs traffic to the PM, fuse, and release virions and exosomes into the extracellular space through a bulk release mechanism. Simultaneously, virions that undergo envelopment on individual vesicles are thought to fuse directly with the PM to release single virions. Few regulators of virion egress have been characterised to date, however, many host proteins linked to exosome secretion have been associated with viral growth with involvement in egress. Cell type specific divergence, as well as cell-to-cell spread mechanisms, may exist in contrast to the schematic outlined. MVB, multivesicular body; vAC, viral assembly compartment.

FIGURE 11

HCMV lytic replication proceeds sequentially through 10 checkpoints, with completion of each essential for production of infectious virions. These are 1) entry, 2) early gene expression, 3) RC biogenesis, 4) viral genome replication, 5) late gene expression, 6) vAC biogenesis, 7) capsid assembly and maturation, 8) nuclear egress, 9) envelopment, and 10) cellular egress. IE, immediate early; DE, delayed early; DNA pol, viral DNA polymerase complex; RC, replication compartment;, PIC, pre-initiation complex; vPIC, viral pre-initiation complex; pol II, RNA polymerase II complex; TRM, tripartite terminase complex; NEC, nuclear egress complex; vAC, viral assembly compartment.