Virus-host protein interactions as footprints of human cytomegalovirus replication

Introduction

Human cytomegalovirus (HCMV) is a ubiquitous pathogen, with a seroprevalence that exceeds 80% worldwide [1]. As a β-herpesvirus, HCMV establishes life-long latent infections in myeloid cells. Reactivation from these latent reservoirs can lead to disease in the immunocompromised or cause severe birth defects. There is currently no available vaccine and there are limited antiviral therapeutics. Knowledge of interfaces that mediate virus-host interactions is critical for understanding the biology and pathogenesis of infection, as well as for discovering targets for therapeutic intervention.

HCMV is an enveloped, double-stranded DNA (dsDNA) virus that replicates in the nucleus (Figure 1). As an obligate parasite, HCMV relies on the host to provide precursors and much of the machinery to produce its proteins, replicate its genome, and form its lipid envelope. HCMV accomplishes this through a remarkably long replication cycle of four to five days, underpinned by a global transformation of the host cell proteome, metabolism, and organelle morphology (reviewed in [2,3]). Meanwhile, viral factors must disable host intrinsic defense pathways and prevent cytokine production.

Herpesviruses such as HCMV have coevolved with vertebrates over millions of years, leading to a myriad of virus-host interactions that occur through several core interfaces: protein-protein interactions (PPIs), post-translational modifications (PTMs) and non-coding RNA-based regulation. Here, we provide an overview of the current knowledge of critical virus-host interactions that mediate different stages of the HCMV lytic replication cycle, and highlight advanced technologies that can further our understanding of HCMV biology and pathogenesis.

Viral Entry and the Initiation of Host-Virus Interactions

Host-virus interactions during HCMV infection commence upon binding and entry of the virus into the host cell. Virus attachment and entry are achieved via protein interactions occurring between glycoproteins found on the viral envelope and cellular proteins embedded in the host plasma membrane. Initial virion-cell attachment is mediated by an interaction between the viral glycoproteins gM/gN or gB with heparin sulfate proteoglycans exposed on the cell surface [4–6]. After virion attachment to the host cell, the mechanism of entry and the complement of virus and host proteins involved is cell-type dependent.

HCMV contains trimeric and pentameric glycoprotein complexes within its viral envelope that are involved in cell-type dependent receptor binding and entry processes. Viral entry into fibroblast cells requires gB and the gH/gL/gO trimeric complex and proceeds via gB-mediated fusion with the plasma membrane [7–10]. In addition to direct fusion with the plasma membrane, macropinocytosis has been proposed as the entry mechanism for HCMV into fibroblasts [11,12]. PDGFRα has been shown to interact with gO and serve as an HCMV receptor for either mechanism of entry into fibroblasts [13–19]. In contrast, HCMV entry into endothelial and epithelial cells requires the trimeric complex, as well as the gH/gL/UL128-UL131 pentameric complex [18,20–23]. Virions enter epithelial and endothelial cells via endocytosis followed by endosomal acidification [24]. Neuropilin-2 (Nrp2) and OR14I1 have been identified as receptors for endothelial and epithelial cell entry [13,15]. In addition to PDGFRα, Nrp2, and OR14I1, several other cell surface proteins have been proposed to act as receptors or co-receptors for HCMV entry, including CD46 [25], CD90 [11,12,26], CD147 [27], integrins [28,29], EGFR [15,28,30,31], and annexin II [32,33]. The internalization of some of these co-receptors, such as the CD63-dependent internalization of integrin beta 1 [34], was observed to support virus production, suggesting a mechanism to prevent virion reattachment to the plasma membrane during egress from the cell.

Given the importance of viral entry for initiating the cascade of events resulting in the production of new virus progeny, several host proteins have been found to act in host defense by specifically interfering with the viral entry process. For example, galectin-9 (Gal-9) has been shown to become upregulated in the plasma of patients experiencing HCMV reactivation. Gal-9 binds to the HCMV virion, likely through interaction with the trimeric complex, and acts to inhibit viral fusion with both epithelial and fibroblast cells [35].

Initiation of Host Immune Responses and HCMV Immune Evasion Mechanisms

Following entry, virus capsids utilize the host cytoskeletal machinery for trafficking to the nucleus, where virus gene expression and genome replication occur [36,37]. The injection of the viral genome into the nucleus activates host intrinsic immune responses. The viral genome becomes exposed to nuclear DNA sensors, such as IFI16, which distinguish the HCMV dsDNA from host chromatin and induce antiviral responses upon binding to the viral DNA [38–42]. IFI16 was shown to both promote cytokine production and inhibit viral gene expression [38,39,42]. In response to host intrinsic immune responses, HCMV acquired a number of viral immune evasion mechanisms to suppress immune and inflammatory pathways. This is accomplished through the early actions of tegument proteins—a cargo of viral proteins packaged into virions along with the capsid—and the later actions of de novo synthesized viral proteins, in particular the immediate-early proteins. For example, the tegument protein pp65 (pUL83) binds to the pyrin domain of IFI16, inhibiting its oligomerization and intrinsic immune functions [39,43]. Similarly, several HCMV proteins—pUL31, pUL42, and pUL83—inhibit cytoplasmic DNA sensing and immune signaling by cyclic GMP-AMP synthase (cGAS) [44–46].

HCMV suppresses various host intrinsic and innate immune response pathways. HCMV gene products antagonize protein kinase R-dependent translation shutoff [47–49], MAVS and cGAS-STING immune signaling [44,45,50–53], and JAK-STAT immune signaling [54–59]. As another viral immune evasion strategy, HCMV encodes an IL-10 mimic to inhibit an inflammatory response [60]. One exception to this broad inhibition of immune signaling is the NF-κB pathway. NF-κB signaling and type I IFN is activated upon viral entry [61–65], but, perhaps in a counter-intuitive fashion, NF-κB transcription factors support virus replication by activating immediate-early genes [66–68]. As such, HCMV promotes expression of NF-κB subunits early in infection [69]. However, later in infection, NF-κB signaling is inhibited at several steps along the pathway. NF-κB signaling transduction and downstream expression of interferon-stimulated genes (ISGs) is inhibited by HCMV proteins IE2 [70–72], pUL26 [73–76], and pUL44 [77].

Orthogonal to the aforementioned suppression of immediate immune responses, HCMV acquired means to hide the infection status of its host cell from immune effector cells. HCMV encodes protein and miRNA to inhibit several steps in the MHC-I antigen presentation pathway to prevent T cell- and natural killer (NK) cell-mediated lysis. Viral pUS3 and pUS6 prevent trafficking of mature MHC-I to the plasma membrane [78,79], while pUS2 and pUS11 promote outright degradation of MHC-I [80,81]. Degradation of MHC-like receptors MICA and MICB is induced upon infection to prevent NK-dependent lysis: MICA is targeted by viral proteins [82–86] and MICB by viral miRNAs [87,88]. HCMV further promotes surface exposure of NK inhibitory receptors by enabling HLA-E translocation [89] and encoding an MHC-I surrogate [90,91]. This suite of mechanisms allows HCMV to antagonize both cell-intrinsic and -extrinsic host defense pathways.

Virus-Host Interactions Affecting Viral Gene Expression and Genome Replication

The HCMV genome is naked while encapsidated, but becomes chromatinized via the addition of histones after its deposition into host nuclei [92–95]. In the same manner as host chromatin, viral chromatin must be remodeled to promote gene expression, which is dictated by the addition or removal of histone post-translational modifications (PTMs) [92,94,96–102]. Herpesvirus genes are expressed in a “temporal cascade,” whereby the first set of viral genes, the immediate-early (IE) genes, drive the subsequent expression of delayed-early (DE) and late (L) genes (Figure 1). The initial deposition of histones results in a “pre-immediate-early” repression of the major immediate-early promoter (MIEP) [94]. Viral and host proteins compete for this key regulatory node. Upstream of the MIEP are various transcription factor (TF) binding sites for viral proteins, as well as host proteins [66–68,103–107]. While NF-κB TFs activate the MIEP, some host restriction factors bind and repress the MIEP [96,98–100,102,108–111].

A prominent gene repression mechanism is driven by nuclear domain 10 (ND10), also known as PML bodies. These macromolecular structures activate both intrinsic and innate immune responses against herpesviruses. Components of ND10 are interferon-induced and can themselves promote induction of interferons [57,58,112,113]. ND10 recruits histone deacetylases and other repressive proteins to the HCMV genome to prevent gene expression [99,100,109,114]. The HCMV protein IE1 dissolves these structures to relieve transcriptional repression [115–117], which is accomplished by antagonizing SUMOylation of ND10 components PML and Sp100 [115,118–120]. In parallel with these deSUMOylation events, IE2 becomes SUMOylated to bolster its transactivation of viral promoters [121–123]. Regulation of this PTM is key to galvanizing early gene expression events.

As gene expression proceeds through the temporal cascade and genome synthesis begins, HCMV is regulating core host pathways to maximize replication. HCMV maintains a robust pool of dNTPs by antagonizing the dNTPase SAMHD1 [124–126]. HCMV prevents degradation of certain viral mRNAs through stem-loop secondary structures that recruit 3’ tail extension machinery [127]. A critical requirement for effective replication is the manipulation of the cell cycle. HCMV encodes a cell cycle sensor, pp150 (pUL32), that blocks IE gene expression in S/G2 until the cell returns to G1 [128,129]. Once in a permissive state, the serine/threonine protein kinase pUL97 and pUL21a inhibit progression into S-phase via degradation and inactivation of the anaphase-promoting complex (APC/C) [130–133]. HCMV proteins further inactivate tumor-suppressor proteins p53 [134–137] and Rb [138–142]. This stops cells at the G1/S boundary, which is believed to establish an S-phase proteome conducive to viral genome replication without allowing host DNA replication to compete for replication machinery and dNTPs. With favorable conditions present, the interaction between pUL44 and the host nucleolin protein establishes nuclear replication compartments for the synthesis of new genomes [143,144]. These genomes are then packaged into viral capsids and must egress from the nucleus.

Host-Virus Interactions in Metabolic Regulation

To promote productive infection, HCMV interfaces with host cell metabolic pathways to induce energetic and biosynthetic output necessary for the production of progeny virions. The specific metabolic program activated during HCMV infection consists of upregulated glycolysis, glutaminolysis, lipid biosynthesis, and oxidative phosphorylation [145–149]. These metabolic changes begin around 48 hours post infection and are necessary for productive virus replication [146,149–152].

The importance of cellular metabolism for HCMV replication is reflected by the diverse mechanisms acquired by the virus to alter metabolic processes during infection. For instance, glycolytic induction is achieved via a finely orchestrated modulation of signaling pathways, enzyme activity, and protein localization. The increases in glucose uptake and glycolysis during infection are linked to HCMV induction of the calmodulin-dependent kinase kinase (CaMKK) and AMP-activated protein kinase (AMPK) signaling pathways [152–155]. The induction of AMPK is achieved by HCMV protein pUL37x1-mediated targeting of the host protein viperin to the mitochondria [155,156]. At the mitochondria, viperin interacts with the mitochondrial trifunctional protein to decrease fatty acid oxidation, resulting in decreased ATP production, AMPK activation, and increased GLUT4 expression [155,156]. Mitochondrial localized viperin also results in induction and nuclear localization of ChREBP, which further increases GLUT4 expression [157]. GLUT4, a high-capacity glucose transporter, replaces the lower-capacity glucose transporter GLUT1 at the plasma membrane, resulting in increased glucose uptake [158,159].

It has been recently demonstrated that, in spite of the activation of glycolysis during infection, HCMV also induces and requires mitochondrial respiration for energy production [148,149,160,161]. Upregulated oxidative phosphorylation during infection is partly achieved via increased transcription and translation of mitochondrial DNA genes, resulting in elevated electron transport chain protein abundances [160]. An HCMV non-coding RNA (ncRNAβ2.7) has been shown to contribute to increased ATP production by interacting with complex I of the electron transport chain to stabilize mitochondrial membrane potential [162]. Furthermore, the HCMV protein pUL13 has been demonstrated to target the mitochondria during infection, where it interacts with the mitochondrial contact site and cristae organizing system (MICOS) complex and modulates cristae ultrastructure to increase electron transport chain efficiency and ATP production [161].

A major source of biosynthetic output during HCMV infection relates to lipid biosynthesis, which is important for viral envelopment and cellular membrane remodeling. The increase in lipid biosynthesis is due to the activation and nuclear translocation of the SREBP and ChREBP transcription factors during infection, resulting in increased lipogenic gene expression [151,155,157,163]. This is achieved through the actions of the HCMV proteins pUL38 and pUL37x1. pUL38 contributes to SREBP activation by binding to and antagonizing the function of tuberous sclerosis protein complex (TSC1/2), resulting in mTOR activation and induction of SREBP processing [163,164], pUL37x1-mediated translocation of viperin to the mitochondria also induces nuclear localization of ChREBP, resulting in lipogenic gene transcription [155,157]. In addition to inducing ChREBP, pUL37x1 also localizes to the peroxisomes during infection [165,166]. There, it activates the peroxisome fission factor PEX11β resulting in increased peroxisome abundance [167]. This serves to increase plasmalogen synthesis, which is important for virion envelopment [166]. In addition to phospholipids, cholesterol is required during HCMV replication for inclusion in the viral envelope [168]. Virions depleted of cholesterol are unable to fuse with the plasma membrane of successive host cells [168]. HCMV primarily drives increased cellular cholesterol levels by regulating the abundance of cholesterol receptors and transporters at the plasma membrane. The levels of low-density lipoprotein scavenger receptors at the plasma membrane are increased by HCMV [169]. As this occurs, HCMV activates calpain-mediated cleavage of the cholesterol efflux transporter ABCA1 [170] and metalloproteinase-mediated cleavage of the low-density lipoprotein receptor protein LRP1 [168].

Remodeling of Subcellular Organization during Virion Assembly and Trafficking

The remainder of the HCMV replication cycle is characterized by a striking remodeling of organelle structure and location for supporting virus assembly processes. Hallmarks of HCMV-infected cells are the formation of a kidney bean-shaped nucleus with infoldings present at the nuclear periphery and the dynamic reorganization of host secretory membranes into the perinuclear viral assembly complex (vAC) [171] (Figure 1).

The nuclear egress of newly formed virus capsids requires the remodeling of the nuclear periphery via the formation of infoldings of the lamina. To accomplish this remodeling, capsids first associate with myosin 5A to move from replication compartments to the nuclear periphery via nuclear actin, which is both induced and bundled into thick filaments connecting replication compartments to the nuclear membrane [172,173]. Once at the nuclear periphery, capsids dock to the multimeric nuclear egress complex (NEC). The primary function of the NEC is to orchestrate phosphorylation of nuclear lamin proteins, resulting in disruption of the nuclear lamina and capsid egress to the cytoplasm [174]. The core components of the NEC are the viral proteins pUL50, pUL53, and pUL97 [174,175]. However, it has been shown that host proteins, including the structural proteins p32 [176,177], emerin [177], and WDR5 [178] and cellular kinases CDK1 [179] and PKC [179,180] are also recruited to the NEC and serve critical roles in NEC formation and lamina disruption. It was demonstrated that capsid nuclear egress can be inhibited by the host cell via acetylation of lamin B1, which protects the integrity of the nuclear periphery [181].

Once capsids egress from the nucleus, virion envelopment and egress from the cell commence. The processes of virion envelopment at the vAC and cellular egress are not fully understood; however, there is accumulating evidence that the virus utilizes the host exosome machinery for these processes [182,183]. Additionally, it has been demonstrated that virion tegument and envelope proteins possess diverse means for trafficking to the vAC. The envelope glycoprotein gB has been shown to interact with the host secretory protein PACS-1 for retrograde transport from endosomes to the Golgi [184,185]. In order for this to occur, viral protein pUL35 binds and inactivates sorting nexin SNX5 to regulate retrograde shuttling [186]. In contrast, gM localization to the vAC requires host RAB11-GTPase effector protein FIP4 [187]. Viral tegument protein pUL99 traffics to the vAC using the ESCRT pathway, while pUL32 traffics via an interaction with BicD1, a protein involved in dynein-mediated microtubule transport [188–190].

The final processes of virion assembly and cellular egress require global organelle remodeling and repurposing of host machinery that together serve as a platform for supporting virus production. The vAC contains early endosomes at the center, surrounded by fragmented Golgi networks that form concentric rings where viral capsids obtain their tegument layer and envelope [171,191–194]. Although the full panel of host proteins involved in vAC formation are still being identified, crucial roles for the ER chaperone BiP/GRP78 [195,196], SNARE proteins [182,197–199], GTPases [200,201], and the v-ATPase proton pump [202] have been demonstrated, supporting that host endosome and secretory pathway machinery is repurposed to function at the vAC during HCMV infection. Furthermore, viral miRNAs target secretory pathway proteins, which is necessary for vAC formation [203].

Beyond its translocation to the vAC, fragmentation of the Golgi is necessary for proper vAC formation. This is achieved via virus-mediated phosphorylation of the cellular Grasp65 protein, which functions in regulating Golgi structural integrity [204]. Phosphorylation of Grasp65 typically occurs during cellular mitosis and results in Golgi fragmentation. Therefore, this represents an instance of HCMV supporting replication by inducing a process that typically occurs in uninfected cells. At the vAC, Golgi fragments function in microtubule nucleation [205,206]. The vAC was previously shown to form around a microtubule organizing center (MTOC) via a microtubule-dependent process [171,189]. It has now been demonstrated that the microtubule end binding protein EB3 is phosphorylated by the host kinase CDK and virus kinase pUL97, resulting in its stabilization. It is then recruited to the vAC where it nucleates Golgi-derived microtubules that are rapidly acetylated and stabilized, resulting in nuclear rotation and vAC formation [205,206].

Perspective: Methods for Investigating Virus-Host Interactions and Future Directions

As highlighted above, HCMV infection dramatically alters the composition and function of the cellular proteome in a temporal manner. A range of biochemical assays have been used to gain insights into dynamic virus-host protein interactions, viewed either through the perspective of single or multiple proteins of interest (Figure 2). Immunoaffinity purification coupled with mass spectrometry (IP-MS) [161,167,207] and yeast-two hybrid screens (Y2H) [117,121,208] have proved valuable for investigations focused on specific virus or host proteins of interest. Global omic techniques have allowed researchers to acquire a holistic understanding of host cell remodeling by determining: temporal changes in the transcriptome and proteome of an infected cell [209,210]; changes in organellar proteomes through spatial proteomics [211,212]; regulation of protein functions via PTM states through PTM-enrichment methods [181,213,214]; and global protein-protein interaction dynamics through thermal-proximity coaggregation assays (TPCA) [34]. This has been augmented by the integration and computational mining of these datasets [167,215,216]. Despite the depth of information that can be garnered from these approaches, only a handful of PTM types have been investigated during infection and targeted or global omic investigations have been performed in limited cell types and upon infection with few viral strains. Another consideration is that many of these studies obtain information that is averaged across cell populations, thereby being impacted by cell-to-cell heterogeneity. Single-cell technologies [217] are poised to fill this niche, providing a complementary toolset for virus-host interaction studies in HCMV and beyond.