Human Cytomegalovirus Hijacks WD Repeat Domain 11 for Virion Assembly Compartment Formation and Virion Morphogenesis

Human cytomegalovirus (HCMV) has a large (∼235-kb) genome with over 200 predicted open reading frames and exploits numerous cellular factors to facilitate its replication. A key feature of HCMV-infected cells is the emergence of a distinctive membranous cytoplasmic compartment termed the virion assembly compartment (vAC). Here we report that host protein WD repeat domain 11 (WDR11) plays a key role in vAC formation and virion morphogenesis. We found that WDR11 was up-regulated at both mRNA and protein levels during HCMV infection. At the late stage of HCMV replication, WDR11 relocated to the vAC and co-localized with markers of the trans-Golgi network (TGN) and vAC. Depletion of WDR11 hindered HCMV-induced membrane reorganization of the Golgi and TGN, altered vAC formation, and impaired HCMV secondary envelopment and virion morphogenesis. Further, motifs critical for the localization of WDR11 in TGN were identified by alanine-scanning mutagenesis. Mutation of these motifs led to WDR11 mislocation outside of the TGN and loss of vAC formation. Taken together, these data indicate that host protein WDR11 is required for efficient viral replication at the stage of virion assembly, possibly by facilitating the remodeling of the endomembrane system for vAC formation and virion morphogenesis. Importance During the late phase of human cytomegalovirus (HCMV) infection, the endomembrane system is dramatically reorganized, resulting in the formation of a unique structure termed the virion assembly compartment (vAC), which is critical for the assembly of infectious virions. The mechanism of HCMV-induced vAC formation is still not fully understood. In this report, we identified a host factor, WDR11, that plays an important role in vAC formation. Our findings argue that WDR11 contributes to the relocation of the Golgi and trans-Golgi network to the vAC, a membrane reorganization process that appears to be required for efficient virion maturation. The present work provides new insights into the vAC formation and HCMV virion morphogenesis and a potential novel target for anti-viral treatment.

Dynamin Inhibitors Prevent the Establishment of the Cytomegalovirus Assembly Compartment in the Early Phase of Infection

Cytomegalovirus (CMV) infection initiates massive rearrangement of cytoplasmic organelles to generate assembly compartment (AC). The earliest events, the establishment of the preAC, are initiated in the early phase as an extensive reorganization of early endosomes (EEs), endosomal recycling compartment (ERC), trans-Golgi network (TGN), and the Golgi. Here, we demonstrate that dynamin inhibitors (Dynasore, Dyngo-4a, MiTMAB, and Dynole-34-2) block the establishment of the preAC in murine CMV (MCMV) infected cells. In this study, we extensively analyzed the effect of Dynasore on the Golgi reorganization sequence into the outer preAC. We also monitored the development of the inner preAC using a set of markers that define EEs (Rab5, Vps34, EEA1, and Hrs), the EE-ERC interface (Rab10), the ERC (Rab11, Arf6), three layers of the Golgi (GRASP65, GM130, Golgin97), and late endosomes (Lamp1). Dynasore inhibited the pericentriolar accumulation of all markers that display EE-ERC-TGN interface in the inner preAC and prevented Golgi unlinking and dislocation to the outer preAC. Furthermore, in pulse-chase experiments, we demonstrated that the presence of dynasore only during the early phase of MCMV infection (4–14 hpi) is sufficient to prevent not only AC formation but also the synthesis of late-phase proteins and virion production. Therefore, our results indicate that dynamin-2 acts as a part of the machinery required for AC generation and rearrangement of EE/ERC/Golgi membranes in the early phase of CMV infection.

Arf GTPases Are Required for the Establishment of the Pre-Assembly Compartment in the Early Phase of Cytomegalovirus Infection

Shortly after entering the cells, cytomegaloviruses (CMVs) initiate massive reorganization of cellular endocytic and secretory pathways, which results in the forming of the cytoplasmic virion assembly compartment (AC). We have previously shown that the formation of AC in murine CMV- (MCMV) infected cells begins in the early phase of infection (at 4–6 hpi) with the pre-AC establishment. Pre-AC comprises membranes derived from the endosomal recycling compartment, early endosomes, and the trans-Golgi network, which is surrounded by fragmented Golgi cisterns. To explore the importance of Arf GTPases in the biogenesis of the pre-AC, we infected Balb 3T3 cells with MCMV and analyzed the expression and intracellular localization of Arf proteins in the early phases (up to 16 hpi) of infection and the development of pre-AC in cells with a knockdown of Arf protein expression by small interfering RNAs (siRNAs). Herein, we show that even in the early phase, MCMVs cause massive reorganization of the Arf system of the host cells and induce the over-recruitment of Arf proteins onto the membranes of pre-AC. Knockdown of Arf1, Arf3, Arf4, or Arf6 impaired the establishment of pre-AC. However, the knockdown of Arf1 and Arf6 also abolished the establishment of infection. Our study demonstrates that Arf GTPases are required for different steps of early cytomegalovirus infection, including the establishment of the pre-AC.

Endosomal Phosphatidylinositol-3-Phosphate-Associated Functions Are Dispensable for Establishment of the Cytomegalovirus Pre-Assembly Compartment but Essential for the Virus Growth

Murine cytomegalovirus (MCMV) initiates the stepwise establishment of the pre-assembly compartment (pre-AC) in the early phase of infection by the expansion of the early endosome (EE)/endosomal recycling compartment (ERC) interface and relocation of the Golgi complex. We depleted Vps34-derived phosphatidylinositol-3-phosphate (PI(3)P) at EEs by VPS34-IN1 and inhibited PI(3)P-associated functions by overexpression of 2xFYVE- and p40PX PI(3)P-binding modules to assess the role of PI(3)P-dependent EE domains in the pre-AC biogenesis. We monitored the accumulation of Rab10 and Evectin-2 in the inner pre-AC and the relocation of GM130-positive cis-Golgi organelles to the outer pre-AC by confocal microscopy. Although PI(3)P- and Vps34-positive endosomes build a substantial part of pre-AC, the PI(3)P depletion and the inhibition of PI(3)P-associated functions did not prevent the establishment of infection and progression through the early phase. The PI(3)P depletion in uninfected and MCMV-infected cells rapidly dispersed PI(3)P-bond proteins and reorganized EEs, including ablation of EE-to-ERC transport and relocation of Rab11 endosomes. The PI(3)P depletion one hour before pre-AC initiation and overexpression of 2xFYVE and p40PX domains neither prevented Rab10- and Evectin-2 accumulation, nor Golgi unlinking and relocation. These data demonstrate that PI(3)P-dependent functions, including the Rab11-dependent EE-to-ERC route, are dispensable for pre-AC initiation. Nevertheless, the virus growth was drastically reduced in PI(3)P-depleted cells, indicating that PI(3)P-associated functions are essential for the late phase of infection.

Human cytomegalovirus exploits TACC3 to control microtubule dynamics and late stages of infection

While it is well established that microtubules (MTs) facilitate various stages of virus replication, how viruses actively control MT dynamics and functions remains less-well understood. Recent work has begun to reveal how several viruses exploit End-Binding (EB) proteins and their associated microtubule plus-end tracking proteins (+TIPs), in particular to enable loading of viral particles onto MTs for retrograde transport during early stages of infection. But distinct from other viruses studied to date, at mid-to-late stages of its unusually protracted replication cycle human cytomegalovirus (HCMV) increases the expression of all three EB family members. This occurs coincident with the formation of a unique structure termed the Assembly Compartment (AC), which serves as a Golgi-derived MT organizing center. Together, the AC and distinct EB proteins enable HCMV to increase the formation of dynamic and acetylated microtubule subsets to regulate distinct aspects of the viral replication cycle. Here, we reveal that HCMV also exploits EB-independent +TIP pathways by specifically increasing the expression of Transforming Acidic Coiled Coil protein 3 (TACC3) to recruit the MT polymerase, chTOG from initial sites of MT nucleation in the AC out into the cytosol, thereby increasing dynamic MT growth. Preventing TACC3 increases or depleting chTOG impaired MT polymerization, resulting in defects in early versus late endosome organization in and around the AC as well as defects in viral trafficking and spread. Our findings provide the first example of a virus that actively exploits EB-independent +TIP pathways to regulate MT dynamics and control late stages of virus replication. Importance Diverse viruses rely on host cell microtubule networks in order to transport viral particles within the dense cytoplasmic environment and to control the broader architecture of the cell to facilitate their replication. Yet precisely how viruses regulate the dynamic behavior and function of microtubule filaments remains poorly defined. We recently showed that the Assembly Compartment (AC) formed by human cytomegalovirus (HCMV) acts as a Golgi-derived microtubule organizing center. Here, we show that at mid-to-late stages of infection, HCMV increases the expression of Transforming Acidic Coiled Coil protein 3 (TACC3) in order to control the localization of the microtubule polymerase, chTOG. This in turn enables HCMV to generate dynamic microtubule subsets that organize endocytic vesicles in and around the AC and facilitate the transport of new viral particles released into the cytosol. Our findings reveal the first instance of viral targeting of TACC3 to control microtubule dynamics and virus spread.

The SARS-CoV-2 nucleocapsid protein associates with the replication organelles before viral assembly at the Golgi/ERGIC and lysosome-mediated egress

Despite being the target of extensive research efforts due to the COVID-19 pandemic, relatively little is known about the dynamics of SARS-CoV-2 replication within cells. We investigate and characterise the tightly orchestrated sequence of events during different stages of the infection cycle by visualising the spatiotemporal dynamics of the four structural proteins of SARS-CoV-2 at high resolution. The nucleoprotein is expressed first and accumulates around folded ER membranes in convoluted layers that connect to viral RNA replication foci. We find that of the three transmembrane proteins, the membrane protein appears at the Golgi apparatus/ERGIC before the spike and envelope proteins. Relocation of the lysosome marker LAMP1 towards the assembly compartment and its detection in transport vesicles of viral proteins confirm an important role of lysosomes in SARS-CoV-2 egress. These data provide new insights into the spatiotemporal regulation of SARS-CoV-2 assembly, and refine current understanding of SARS-CoV-2 replication.

mSphere of Influence: Viruses—Pathogens or Expert Cell Biologists?

ABSTRACT Chelsey C. Spriggs works in the field of DNA viral entry with a specific interest in virus-host interactions. In this mSphere of Influence article, she reflects on how two papers, “The HCMV assembly compartment is a dynamic Golgi-derived MTOC that controls nuclear rotation and virus spread” (D. J. Procter, A. Banerjee, M. Nukui, K. Kruse, et al., Dev Cell 45:83–100.e7, 2018, https://doi.org/10.1016/j.devcel.2018.03.010) and “Cytoplasmic control of intranuclear polarity by human cytomegalovirus” (D. J. Procter, C. Furey, A. G. Garza-Gongora, S. T. Kosak, D. Walsh, Nature 587:109–114, 2020, https://doi.org/10.1038/s41586-020-2714-x), impacted her research by reinforcing the scientific value in using viruses to understand cell biology.

Localization of the WD repeat-containing protein 5 to the Virion Assembly Compartment Facilitates Human Cytomegalovirus Assembly

We previously reported that human cytomegalovirus (HCMV) utilizes the cellular protein WD repeat-containing protein 5 (WDR5) to facilitate capsid nuclear egress. Here, we further show that HCMV infection results in WDR5 localization in a juxtanuclear region, and that its localization to this cellular site is associated with viral replication and late viral gene expression. Furthermore, WDR5 accumulated in the virion assembly compartment (vAC) and co-localized with vAC markers of gamma-tubulin (γ-tubulin), early endosomes, and viral vAC marker proteins pp65, pp28, and glycoprotein B (gB). WDR5 co-immunoprecipitated with multiple virion proteins, including MCP, pp150, pp65, pIRS1, and pTRS1, which may explain WDR5 accumulation in the vAC during infection. WDR5 fractionated with virions either in the presence or absence of Triton X-100 and was present in purified viral particles, suggesting that WDR5 was incorporated into HCMV virions. Thus, WDR5 localized to the vAC and was incorporated into virions, raising the possibility that in addition to capsid nuclear egress, WDR5 could also participate in cytoplasmic HCMV virion morphogenesis. Importance Human cytomegalovirus (HCMV) has a large (∼235-kb) genome that contains over 170 ORFs and exploits numerous cellular factors to facilitate its replication. In the late phase of HCMV infection cytoplasmic membranes are reorganized to establish the virion assembly compartment (vAC), which has been shown to necessary for efficient assembly of progeny virions. We previously reported that WDR5 facilitates HCMV nuclear egress. Here, we show that WDR5 is localized to the vAC and incorporated into virions, perhaps contributing to efficient virion maturation. Thus, findings in this study identified a potential role for WDR5 in HCMV assembly in the cytoplasmic phase of virion morphogenesis.

Human Cytomegalovirus Envelope Protein gpUL132 Regulates Infectious Virus Production through Formation of the Viral Assembly Compartment

ABSTRACT The human cytomegalovirus (HCMV) UL132 open reading frame encodes a 270-amino-acid type I envelope glycoprotein, gpUL132. The deletion of UL132 (ΔUL132) from the HCMV genome results in a pronounced deficit in virus yield, with an approximately 2-log decrease in the production of infectious virus compared to the wild-type (WT) virus. Characterization of the ΔUL132 mutant virus indicated that it was less infectious with a high particle-to-infectious unit ratio and an altered composition of virion proteins compared to the WT virus. In addition, the viral assembly compartment (AC) failed to form in cells infected with the ΔUL132 mutant virus. The expression of gpUL132 in trans rescued the defects in the morphogenesis of the AC in cells infected with the ΔUL132 mutant virus and in infectious virus production. Furthermore, using cell lines expressing chimeric proteins, we demonstrated that the cytosolic domain of gpUL132 was sufficient to rescue AC formation and WT levels of virus production. Progeny virions from ΔUL132-infected cells expressing the cytosolic domain of gpUL132 exhibited particle-to-infectious unit ratios similar to those of the WT virus. Together, our findings argue that gpUL132 is essential for HCMV AC formation and the efficient production of infectious particles, thus highlighting the importance of this envelope protein for the virus-induced reorganization of intracellular membranes and AC formation in the assembly of infectious virus. IMPORTANCE Following infection of permissive cells, human cytomegalovirus (HCMV) induces the reorganization of intracellular membranes resulting in the formation of a distinctive membranous compartment in the cytoplasm of infected cells. This compartment has been designated the viral assembly compartment (AC) and is thought to be a site for cytoplasmic virion assembly and envelopment. In this study, we have demonstrated that a single virion envelope glycoprotein is essential for AC formation in infected cells, and in its absence, there is a significant decrease in the production of infectious virions. These findings are consistent with those from other studies that have demonstrated the importance of host cell proteins in the formation of the AC and demonstrate a critical role of a single virion protein in AC formation and the efficient assembly of infectious virus.