**1. Introduction**

Herpesviruses follow a complex assembly pathway in which viral DNA is first replicated and encapsidated in the nucleus, followed by the acquisition of a mature virion envelope within cytoplasmic membranes. The nuclear membrane, the protein lattice known as the lamina underlying the nuclear membrane, and chromatin represent significant barriers to the egress of subviral particles from the nucleus. To breach these physical barriers, herpesviruses have evolved mechanisms that include both viral and cellular functions to modify nuclear structures, such as modification of the nuclear lamina that allows access of capsids to the inner nuclear membrane (INM) and facilitates budding into the perinuclear space (PNS). While several components of the viral nuclear egress complex (NEC) that mediate the process of the capsid nuclear exit are conserved between members of the herpesvirus family, cellular functions that contribute to nuclear egress appear to vary among the α, β, and γ subfamilies presumably because of unique host cell responses that follow infection by viruses from each family. Even though different members of the herpesvirus family utilize different cellular functions in transit of their capsids from the nucleus to the cytoplasm, the conservation of the core components of the nuclear egress machinery of these viruses makes the core NEC an attractive target for the development of antiviral drugs to inhibit herpesvirus replication [1].

## **2. Human Cytomegalovirus Replication Cycle**

Like other herpesviruses, viral DNA replication occurs in the nucleus, while acquisition of the mature envelope occurs in the cytoplasm, but unlike α-herpesviruses, the

**Citation:** Sanchez, V.; Britt, W. Human Cytomegalovirus Egress: Overcoming Barriers and Co-Opting Cellular Functions. *Viruses* **2022**, *14*, 15. https://doi.org/10.3390/ v14010015

Academic Editor: Donald M. Coen

Received: 8 October 2021 Accepted: 16 December 2021 Published: 22 December 2021

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HCMV life cycle is protracted and progeny virions are produced over a period of days, as shown in Figure 1. Nonetheless, major steps of the nuclear phase of herpesvirus infections are similar between the different virus families. After entry of HCMV into the host cell, the viral genomes are deposited into the nucleus at PML nuclear domains and circularize. Expression of IE (immediate-early) and E (early) genes is initiated, and viral proteins accumulate at these viral pre-replication sites to stimulate rolling circle replication of the input genomes. As infection proceeds and depending on the multiplicity of infection, cells can contain several replication foci that coalesce into larger replication compartments (RC) containing the replication machinery and early-late proteins required for encapsidation of the viral genome. Concurrent with expansion of the RCs, host DNA is marginalized, and nuclear morphology is altered [2–6] (Figure 1). Although herpesvirus infection-induced changes in nuclear size have been noted by many investigators, advances in microscopy have facilitated accurate measurements of the increases in nuclear volume and overall surface area and of changes in substructures such as the nuclear envelope. For example, Aho et al. measured an average nuclear volume of 260 µm<sup>3</sup> in HSV-infected cells versus 170 µm<sup>3</sup> in uninfected controls [5]. The surface area of the nuclei of HSV-infected cells also increased to 260 µm<sup>2</sup> compared to 176 µm<sup>2</sup> for uninfected cells [5]. In HCMV-infected cells, nuclei are markedly larger than uninfected cells, and the surface area of the nucleus is further increased by the presence of NE infoldings [4,7,8]. Like other herpesviruses, viral DNA replication occurs in the nucleus, while acquisition of the mature envelope occurs in the cytoplasm, but unlike α-herpesviruses, the HCMV life cycle is protracted and progeny virions are produced over a period of days, as shown in Figure 1. Nonetheless, major steps of the nuclear phase of herpesvirus infections are similar between the different virus families. After entry of HCMV into the host cell, the viral genomes are deposited into the nucleus at PML nuclear domains and circularize. Expression of IE (immediate-early) and E (early) genes is initiated, and viral proteins accumulate at these viral pre-replication sites to stimulate rolling circle replication of the input genomes. As infection proceeds and depending on the multiplicity of infection, cells can contain several replication foci that coalesce into larger replication compartments (RC) containing the replication machinery and early-late proteins required for encapsidation of the viral genome. Concurrent with expansion of the RCs, host DNA is marginalized, and nuclear morphology is altered [2–6] (Figure 1). Although herpesvirus infection-induced changes in nuclear size have been noted by many investigators, advances in microscopy have facilitated accurate measurements of the increases in nuclear volume and overall surface area and of changes in substructures such as the nuclear envelope. For example, Aho et al. measured an average nuclear volume of 260 µm3 in HSV-infected cells versus 170 µm3 in uninfected controls [5]. The surface area of the nuclei of HSV-infected cells also increased to 260 µm2 compared to 176 µm2 for uninfected cells [5]. In HCMV-infected cells, nuclei are markedly larger than uninfected cells, and the surface area of the nucleus is further increased by the presence of NE infoldings [4,7,8].

*Viruses* **2022**, *14*, x FOR PEER REVIEW 2 of 22

**2. Human Cytomegalovirus Replication Cycle** 

**Figure 1.** Timeline of viral gene expression and morphological changes in HCMV-infected cells. In the top panel, the kinetics of viral gene is shown. Immediate-early gene (yellow) expression is observed 1 h following infection of permissive human fibroblasts and within 24 h pi, cell rounding is **Figure 1.** Timeline of viral gene expression and morphological changes in HCMV-infected cells. In the top panel, the kinetics of viral gene is shown. Immediate-early gene (yellow) expression is observed 1 h following infection of permissive human fibroblasts and within 24 h pi, cell rounding is observed, concurrent with the expression of early gene (pink) products. Small replication foci containing IE and E proteins can be detected at this time point. By 48 h pi, cells begin to flatten and enlarge, and nuclear replication compartments can be seen along with expression of late gene (blue) products, including virion structural proteins. In the lower panel, the morphological changes that take place during a permissive infection of human fibroblast cell are illustrated. At 72 h pi, the enlarged nuclei begin to adopt the characteristic kidney shape, and the juxtanuclear assembly compartment (AC) becomes readily detected with microtubules emanating from the Golgi-derived MTOC.

Immediate-early (IE) viral gene products can be detected within 8–12 h post-infection (h pi) in nuclear pre-replication foci after HCMV infection of permissive cells in G0/G<sup>1</sup> [9–11] (Figure 1). Viral DNA replication begins by 24 h pi and plateaus by 72–96 h pi (Figure 1). The expression of virion structural genes is an early-late event, and many of the protein products of these viral genes can be detected by 48 h pi. Remodeling of the nuclear lamina and changes in nuclear morphology are detected during a similar time interval after infection. HCMV induces major reorganization of intracellular membranes and dysregulates membrane trafficking to generate a juxtanuclear membrane compartment that is required for efficient cytoplasmic envelopment and virus assembly [6,12–15]. The morphogenesis of the juxtanuclear site of cytoplasmic envelopment, also known as the assembly compartment (AC), begins at approximately 24 h pi when depolymerization of actin and changes in Golgi morphology are observed [12,13,15,16]. Virus particles exiting the nucleus acquire their mature envelope by budding into AC vesicles that contain virusencoded glycoproteins and that subsequently fuse with the plasma membrane, releasing virions into the extracellular space. The morphogenesis and function of the AC in virus assembly are areas of intense investigation and beyond the scope of this review; however, there are several structural features of the AC that are relevant to a discussion of nuclear egress [6]. First, the AC serves as a Golgi-nucleated microtubule organizing center [16]. The microtubules that extend from the AC become acetylated, a modification that imparts stability. Acetylation of the microtubules is associated with nuclear rotation, a phenomenon often associated with mechanical stress during cell migration and also during preparation for cell division [16]. Lastly, activity of the AC MTs and their associated motors is required for changes in nuclear morphology observed in HCMV-infected cells [6,16,17].

#### *Overview of Nuclear Egress of HCMV*

Following viral DNA replication and packaging of newly formed capsids, genomecontaining capsids must transit from RC and cross the nuclear envelope through a process of envelopment at the inner nuclear membrane (INM) and de-envelopment at the outer nuclear membrane (ONM) to reach sites of tegumentation and final envelopment in the cytoplasm (Figure 2). In addition to the nuclear membrane, several other barriers restrict the egress of capsids from the viral replication centers to the cytoplasmic AC, including the nuclear lamina and chromatin (Figure 2). Modification of these nuclear structures during herpesvirus egress are briefly reviewed below. *Viruses* **2022**, *14*, x FOR PEER REVIEW 4 of 22

**Figure 2.** Organization of nuclear envelope in normal cells. The nuclear envelope consists of a double bilayer, the inner nuclear membrane (INM) and outer nuclear membrane (ONM). These mem-**Figure 2.** Organization of nuclear envelope in normal cells. The nuclear envelope consists of a double

branes are connected at nuclear pores, a protein complex that permits diffusion and active transport

posed of KASH domain proteins (nesprins) anchored in the ONM and SUN proteins anchored in the INM. The lamina underlies the INM and is composed of intermediate filament proteins known as the lamins and is connected to the INM by proteins including the lamin B receptor (LBR) and LEM-domain proteins (Lap2-emerin-Man1). The lamina maintains nuclear shape and stability and serves as a scaffold for other nuclear proteins. BAF (Barrier-to-autointegration factor) binds LEMdomain proteins and chromatin. Phosphorylation of BAF during mitosis leads to its release from chromatin. Similarly, HP1 (heterochromatin protein 1) is a component of heterochromatin that links

The nuclear envelope consists of a double bilayer, the INM and the ONM, as well as the underlying lamina (Figure 2). The ONM is contiguous with the endoplasmic reticulum (ER), thus allowing lateral diffusion of membrane-bound proteins destined for localization within nuclear membranes. In addition, since the ER is the main site of phospholipid synthesis, the continuity of the ONM with the ER allows for nuclear membrane expansion by lateral diffusion of lipids, although local synthesis and storage of lipids at the INM have been described [18]. In fact, local synthesis of phospholipids has been shown to contribute to the formation of NM invaginations known as the nucleoplasmic reticulum (NR) [19]. One potential function proposed for the NR is to facilitate communication from the nuclear periphery to inner domains of the nucleus. Local phospholipid accumulation in the INM has also been shown to be regulated by host cell functions contained within the endosomal sorting complex required for transport III (ESCRT-III), a complex of proteins that has a critical role in vesicle formation, abscission during cytokinesis, and the budding of enveloped viruses [20,21]. Proteins within this complex have been shown to contribute

condensed chromatin to the INM through LBR.

**3. Interphase Nuclear Architecture** 

bilayer, the inner nuclear membrane (INM) and outer nuclear membrane (ONM). These membranes are connected at nuclear pores, a protein complex that permits diffusion and active transport of molecules across the envelope. The space between the ONM and INM, the perinuclear space (PNS), is traversed by the LINC (linker of nucleoskeleton and cytoskeleton) complex that is composed of KASH domain proteins (nesprins) anchored in the ONM and SUN proteins anchored in the INM. The lamina underlies the INM and is composed of intermediate filament proteins known as the lamins and is connected to the INM by proteins including the lamin B receptor (LBR) and LEM-domain proteins (Lap2-emerin-Man1). The lamina maintains nuclear shape and stability and serves as a scaffold for other nuclear proteins. BAF (Barrier-to-autointegration factor) binds LEM-domain proteins and chromatin. Phosphorylation of BAF during mitosis leads to its release from chromatin. Similarly, HP1 (heterochromatin protein 1) is a component of heterochromatin that links condensed chromatin to the INM through LBR.

#### **3. Interphase Nuclear Architecture**

The nuclear envelope consists of a double bilayer, the INM and the ONM, as well as the underlying lamina (Figure 2). The ONM is contiguous with the endoplasmic reticulum (ER), thus allowing lateral diffusion of membrane-bound proteins destined for localization within nuclear membranes. In addition, since the ER is the main site of phospholipid synthesis, the continuity of the ONM with the ER allows for nuclear membrane expansion by lateral diffusion of lipids, although local synthesis and storage of lipids at the INM have been described [18]. In fact, local synthesis of phospholipids has been shown to contribute to the formation of NM invaginations known as the nucleoplasmic reticulum (NR) [19]. One potential function proposed for the NR is to facilitate communication from the nuclear periphery to inner domains of the nucleus. Local phospholipid accumulation in the INM has also been shown to be regulated by host cell functions contained within the endosomal sorting complex required for transport III (ESCRT-III), a complex of proteins that has a critical role in vesicle formation, abscission during cytokinesis, and the budding of enveloped viruses [20,21]. Proteins within this complex have been shown to contribute to the regulation of INM proliferation and repair during mitosis, resealing nuclear ruptures in migrating cells, and, most recently, nuclear egress of herpesvirus particles [20–23].

Nuclear permeability is maintained by nuclear pores that serve as junctions between the INM and ONM (Figure 2). Pore complexes are large, multi-subunit structures 110 MDa in size (~800 Å wide) that allow nucleocytoplasmic transit of macromolecules such as RNAs and proteins in an energy-dependent manner [24]. Smaller molecules (<40 kDa) can diffuse through pore channels without ATP consumption. The intermembrane or perinuclear space (PNS) between the INM and ONM spans approximately 20–40 nM and contains components of the LINC (linker of nucleoskeleton and cytoskeleton) complex (Figure 2). LINC complexes function as mechanosensors relaying signals to and from the nuclear environment and cytoplasm [25]. They are composed of a trimer of SUN-domain (Sad1 and UNC-84) proteins anchored in the INM that are in turn bound to a trimer of nesprins (KASH domain proteins) anchored in the ONM. Thus, the LINC complex extends from the INM and PNS into the cytoplasm (Figure 2). Nesprins bind to cytoskeletal elements such as tubulin, and thus the LINC complex provides a bridge to the karyoskeleton both structurally and for intracellular signaling. For example, the LINC complex has been implicated in the regulation of transcription in response to changes in the extracellular matrix [26]. The LINC complex is also important for nuclear positioning [27–30].

Together, the lamina, the nuclear pores, the LINC complex, spectrin, actin, and the LEM-domain proteins, which are involved in tethering chromatin to the NE, all contribute to maintaining nuclear architecture in interphase cells [31,32] (Figure 2). Regulation of components of the nucleoskeleton effect changes in nuclear structure and function during the cell cycle, during stress responses, and in response to extracellular cues that trigger cell migration. The dynamic nature of nuclear architecture is essential for homeostasis and genome maintenance. Like other viruses that replicate in the nucleus, herpesviruses take advantage of the plasticity of nuclear structure to create an environment that favors the replication of the viral genome over that of the host without compromising the integrity of the nuclear compartment prematurely in order to maximize virus production.

#### **4. Herpesviruses Circumvent Nuclear Barriers**

Perhaps the most well understood step in the nuclear egress of α, β, and γ herpesviruses is the transit of the viral capsid through the INM into the perinuclear space (PNS) and in particular, the role of viral proteins in this step of the assembly pathway. Budding of membrane-bound capsids from the INM into the PNS, alternatively termed primary envelopment, has been most been well studied in the α-herpesviruses herpes simplex virus (HSV) and pseudorabies virus (PRV). Primary envelopment of α-herpesviruses is dependent on the interaction between a heterodimer of pUL31, a nuclear phosphoprotein, and pUL34, a type II membrane protein inserted into the INM, both of which are required for budding of the capsid into the PNS [33,34]. Together these viral proteins constitute the nuclear egress complex (NEC). Interactions between these core NEC proteins and formation of the heterodimer have been argued to lead to the recruitment of other viral proteins, including viral kinases and host cell proteins that are required for efficient passage through the INM. The NEC is conserved in all families of herpesviruses. In the prototypic β-herpesvirus, HCMV, the NEC consists of the pUL53 and pUL50, orthologs of the HSV pUL31 and pUL34 respectively, and a heterodimer of HCMV pUL53 and pUL50 has also been shown to contribute to the recruitment of viral and cellular proteins that contribute to nuclear egress [35,36]. Similarly, the NEC of the γ-herpesvirus EBV has been identified and is represented by the HSV pUL31 and pUL34 orthologs, BFLF2 and BFRF1, respectively [37–39]. A further description of the function of the HCMV NEC will be provided in greater detail in the following sections.

In the case of PRV, the interaction between the orthologs of HSV pUL31 and pUL34 is sufficient for vesiculation of the INM and formation of particles resembling L particles of α-herpesviruses in transfected cells in the absence of expression of other PRV encoded proteins, arguing that interactions between the NEC and the PRV capsid are not required for budding of particles at the INM [40]. Furthermore, purified pUL31 and pUL34 of HSV and the PRV orthologues can vesiculate membranes in cell-free systems, strongly suggesting that the minimal NEC of α-herpesviruses is sufficient for budding from lipid-containing membranes [40–43]. Although these observations could be interpreted as evidence that other viral and host cell functions are non-essential for herpesvirus nuclear egress, multiple studies have shown that efficient nuclear egress leading to WT levels of virus replication requires additional viral proteins together with the viral NEC. Furthermore, it could be argued that cellular proteins can functionally complement or even partially replace viral functions encoded by non-NEC viral proteins. Consistent with this possibility is the finding that although the deletion of the PRV-encoded protein kinase pUS3 led to large invaginations of the INM and to an increased number of primary enveloped capsids in the PNS in PRV-infected cells, this deletion had a marginal effect on the production of infectious virus [34,44], suggesting that other redundant functions compensate for the loss of US3. Consistent with this possibility, protein kinase C (PKC) was argued to disrupt the nuclear lamina during nuclear egress of murine CMV, whereas more recent findings have argued for a more prominent role of the conserved herpesvirus protein kinase (CHPK) of HCMV, pUL97, in this step in nuclear egress [45,46]. A number of other host cell functions have been proposed to contribute to budding of the herpesvirus capsid at the INM, including emerin and p53, and deletion or decreased expression of these cellular functions in some cases has been shown to result in a quantifiable change in nuclear egress and the production of infectious virus (Table 1) [35,47,48]. Thus, current data suggest that herpesviruses have usurped physiologic processes to facilitate efficient nuclear egress and, ultimately, virus replication. In the following sections, we will attempt to update the current understanding of the role of cellular and viral proteins in HCMV nuclear egress, often in the context of the more well-studied and well-understood models of the nuclear egress of α-herpesviruses.


**Table 1.** Viral and cellular factors involved in nuclear egress.
