The incredible bulk: Human cytomegalovirus tegument architectures uncovered by AI-empowered cryo-EM.

The compartmentalization of eukaryotic cells presents considerable challenges to the herpesvirus life cycle. The herpesvirus tegument, a bulky proteinaceous aggregate sandwiched between herpesviruses' capsid and envelope, is uniquely evolved to address these challenges, yet tegument structure and organization remain poorly characterized. We use deep-learning-enhanced cryogenic electron microscopy to investigate the tegument of human cytomegalovirus virions and noninfectious enveloped particles (NIEPs; a genome packaging-aborted state), revealing a portal-biased tegumentation scheme. We resolve atomic structures of portal vertex-associated tegument (PVAT) and identify multiple configurations of PVAT arising from layered reorganization of pUL77, pUL48 (large tegument protein), and pUL47 (inner tegument protein) assemblies. Analyses show that pUL77 seals the last-packaged viral genome end through electrostatic interactions, pUL77 and pUL48 harbor a head-linker-capsid-binding motif conducive to PVAT reconfiguration, and pUL47/48 dimers form 45-nm-long filaments extending from the portal vertex. These results provide a structural framework for understanding how herpesvirus tegument facilitates and evolves during processes spanning viral genome packaging to delivery.

Cryo-Electron Microscopy Structure and Interactions of the Human Cytomegalovirus gHgLgO Trimer with Platelet-Derived Growth Factor Receptor Alpha.

Human cytomegalovirus (HCMV) is a herpesvirus that produces disease in transplant patients and newborn children. Entry of HCMV into cells relies on gH/gL trimer (gHgLgO) and pentamer (gHgLUL128-131) complexes that bind cellular receptors. Here, we studied the structure and interactions of the HCMV trimer, formed by AD169 strain gH and gL and TR strain gO proteins, with the human platelet-derived growth factor receptor alpha (PDGFRα). Three trimer surfaces make extensive contacts with three PDGFRα N-terminal domains, causing PDGFRα to wrap around gO in a structure similar to a human hand, explaining the high-affinity interaction. gO is among the least conserved HCMV proteins, with 8 distinct genotypes. We observed high conservation of residues mediating gO-gL interactions but more extensive gO variability in the PDGFRα interface. Comparisons between our trimer structure and a previously determined structure composed of different subunit genotypes indicate that gO variability is accommodated by adjustments in the gO-PDGFRα interface. We identified two loops within gO that were disordered and apparently glycosylated, which could be deleted without disrupting PDGFRα binding. We also identified four gO residues that contact PDGFRα, which when mutated produced markedly reduced receptor binding. These residues fall within conserved contact sites of gO with PDGFRα and may represent key targets for anti-trimer neutralizing antibodies and HCMV vaccines. Finally, we observe that gO mutations distant from the gL interaction site impact trimer expression, suggesting that the intrinsic folding or stability of gO can impact the efficiency of trimer assembly. IMPORTANCE HCMV is a herpesvirus that infects a large percentage of the adult population and causes significant levels of disease in immunocompromised individuals and birth defects in the developing fetus. The virus encodes a complex protein machinery that coordinates infection of different cell types in the body, including a trimer formed of gH, gL, and gO subunits. Here, we studied the interactions of the HCMV trimer with its receptor on cells, the platelet derived growth factor receptor α (PDGFRα), to better understand how HCMV coordinates virus entry into cells. Our results add to our understanding of HCMV strain-specific differences and identify sites on the trimer that represent potential targets for therapeutic antibodies or vaccine development.

New Insights into Human Cytomegalovirus pUL52 Structure.

Human cytomegalovirus (HCMV) can cause serious diseases in immunocompromised patients. Current antiviral inhibitors all target the viral DNA polymerase. They have adverse effects, and prolonged treatment can select for drug resistance mutations. Thus, new drugs targeting other stages of replication are an urgent need. The terminase complex (pUL56-pUL89-pUL51) is highly specific, has no counterpart in the human organism, and thus represents a target of choice for new antivirals development. This complex is required for DNA processing and packaging. pUL52 was shown to be essential for the cleavage of concatemeric HCMV DNA and crucial for viral replication, but its functional domains are not yet identified. Polymorphism analysis was performed by sequencing UL52 from 61 HCMV naive strains and from 14 HCMV strains from patients treated with letermovir. Using sequence alignment and homology modeling, we identified conserved regions and potential functional motifs within the pUL52 sequence. Recombinant viruses were generated with specific serine or alanine substitutions in these putative patterns. Within conserved regions, we identified residues essential for viral replication probably involved in CXXC-like or zinc finger motifs. These results suggest that they are essential for pUL52 structure/function. Thus, these patterns represent potential targets for the development of new antivirals.

Selective targeting of ligand-dependent and -independent signaling by GPCR conformation-specific anti-US28 intrabodies.

While various GPCRs, including US28, display constitutive, ligand-independent activity, it remains to be established whether ligand-dependent and -independent active conformations differ and can be selectively modulated. Previously, the agonist-bound conformation of US28 was stabilized and its structure was solved using the anti-US28 nanobody Nb7. Here we report the recognition of the constitutively active, apo-conformation of US28 by another nanobody VUN103. While the Nb7 intrabody selectively inhibits ligand-induced signaling, the VUN103 intrabody blocks constitutive signaling, indicating the existence of distinct US28 conformational states. By displacing Gαq protein, VUN103 prevents US28 signaling and reduces tumor spheroids growth. Overall, nanobodies specific for distinct GPCR conformational states, i.e. apo- and agonist-bound, can selectively target and discern functional consequences of ligand-dependent versus independent signaling.

Structural basis for genome packaging, retention, and ejection in human cytomegalovirus.

How the human cytomegalovirus (HCMV) genome-the largest among human herpesviruses-is packaged, retained, and ejected remains unclear. We present the in situ structures of the symmetry-mismatched portal and the capsid vertex-specific components (CVSCs) of HCMV. The 5-fold symmetric 10-helix anchor-uncommon among known portals-contacts the portal-encircling DNA, which is presumed to squeeze the portal as the genome packaging proceeds. We surmise that the 10-helix anchor dampens this action to delay the portal reaching a "head-full" packaging state, thus facilitating the large genome to be packaged. The 6-fold symmetric turret, latched via a coiled coil to a helix from a major capsid protein, supports the portal to retain the packaged genome. CVSCs at the penton vertices-presumed to increase inner capsid pressure-display a low stoichiometry, which would aid genome retention. We also demonstrate that the portal and capsid undergo conformational changes to facilitate genome ejection after viral cell entry.

Role of Envelope Glycoprotein Complexes in Cell-Associated Spread of Human Cytomegalovirus.

The role of viral envelope glycoproteins, particularly the accessory proteins of trimeric and pentameric gH/gL-complexes, in cell-associated spread of human cytomegalovirus (HCMV) is unclear. We aimed to investigate their contribution in the context of HCMV variants that grow in a strictly cell-associated manner. In the genome of Merlin pAL1502, the glycoproteins gB, gH, gL, gM, and gN were deleted by introducing stop codons, and the mutants were analyzed for viral growth. Merlin and recent HCMV isolates were compared by quantitative immunoblotting for expression of accessory proteins of the trimeric and pentameric gH/gL-complexes, gO and pUL128. Isolates were treated with siRNAs against gO and pUL128 and analyzed regarding focal growth and release of infectious virus. All five tested glycoproteins were essential for growth of Merlin pAL1502. Compared with this model virus, higher gO levels were measured in recent isolates of HCMV, and its knockdown decreased viral growth. Knockdown of pUL128 abrogated the strict cell-association and led to release of infectivity, which allowed cell-free transfer to epithelial cells where the virus grew again strictly cell-associated. We conclude that both trimer and pentamer contribute to cell-associated spread of recent clinical HCMV isolates and downregulation of pentamer can release infectious virus into the supernatant.

Rat and human cytomegalovirus ORF116 encodes a virion envelope glycoprotein required for infectivity.

Herpesviruses encode multiple glycoproteins required for different stages of viral attachment, fusion, and envelopment. The protein encoded by the human cytomegalovirus (HCMV) open reading frame UL116 forms a stable complex with glycoprotein H that is incorporated into virions. However, the function of this complex remains unknown. Herein, we characterize R116, the rat CMV (RCMV) putative homolog of UL116. Two R116 transcripts were identified in fibroblasts with three proteins expressed with molecular weights of 42, 58, and 82 kDa. R116 is N-glycosylated, expressed with late viral gene kinetics, and is incorporated into the virion envelope. RCMV lacking R116 failed to result in productive infection of fibroblasts and siRNA knockdown of R116 substantially reduced RCMV infectivity. Complementation in trans of an R116-deficient virus restored ability of the virus to infect fibroblasts. Finally, UL116 knockdown also decreased HCMV infectivity indicating that R116 and UL116 both contribute to viral infectivity.

Structures of HCMV Trimer reveal the basis for receptor recognition and cell entry.

Human cytomegalovirus (HCMV) infects the majority of the human population and represents the leading viral cause of congenital birth defects. HCMV utilizes the glycoproteins gHgLgO (Trimer) to bind to platelet-derived growth factor receptor alpha (PDGFRα) and transforming growth factor beta receptor 3 (TGFßR3) to gain entry into multiple cell types. This complex is targeted by potent neutralizing antibodies and represents an important candidate for therapeutics against HCMV. Here, we determine three cryogenic electron microscopy (cryo-EM) structures of the trimer and the details of its interactions with four binding partners: the receptor proteins PDGFRα and TGFßR3 as well as two broadly neutralizing antibodies. Trimer binding to PDGFRα and TGFßR3 is mutually exclusive, suggesting that they function as independent entry receptors. In addition, Trimer-PDGFRα interaction has an inhibitory effect on PDGFRα signaling. Our results provide a framework for understanding HCMV receptor engagement, neutralization, and the development of anti-viral strategies against HCMV.

Recognition of a highly conserved glycoprotein B epitope by a bivalent antibody neutralizing HCMV at a post-attachment step.

Human cytomegalovirus (HCMV) is one of the main causative agents of congenital viral infection in neonates. HCMV infection also causes serious morbidity and mortality among organ transplant patients. Glycoprotein B (gB) is a major target for HCMV neutralizing antibodies, yet the underlying neutralization mechanisms remain largely unknown. Here we report that 3-25, a gB-specific monoclonal antibody previously isolated from a healthy HCMV-positive donor, efficiently neutralized 14 HCMV strains in both ARPE-19 cells and MRC-5 cells. The core epitope of 3-25 was mapped to a highly conserved linear epitope on antigenic domain 2 (AD-2) of gB. A 1.8 Å crystal structure of 3-25 Fab in complex with the peptide epitope revealed the molecular determinants of 3-25 binding to gB at atomic resolution. Negative-staining electron microscopy (EM) 3D reconstruction of 3-25 Fab in complex with de-glycosylated postfusion gB showed that 3-25 Fab fully occupied the gB trimer at the N-terminus with flexible binding angles. Functionally, 3-25 efficiently inhibited HCMV infection at a post-attachment step by interfering with viral membrane fusion, and restricted post-infection viral spreading in ARPE-19 cells. Interestingly, bivalency was required for HCMV neutralization by AD-2 specific antibody 3-25 but not the AD-4 specific antibody LJP538. In contrast, bivalency was not required for HCMV binding by both antibodies. Taken together, our results reveal the structural basis of gB recognition by 3-25 and demonstrate that inhibition of viral membrane fusion and a requirement of bivalency may be common for gB AD-2 specific neutralizing antibody.

Human cytomegalovirus protein pUL36: A dual cell death pathway inhibitor.

Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of intrinsic, innate, and adaptive viral immune evasion. Here, we employed multiplexed tandem mass tag-based proteomics to characterize host proteins targeted for degradation late during HCMV infection. This approach revealed that mixed lineage kinase domain-like protein (MLKL), a key terminal mediator of cellular necroptosis, was rapidly and persistently degraded by the minimally passaged HCMV strain Merlin but not the extensively passaged strain AD169. The strain Merlin viral inhibitor of apoptosis pUL36 was necessary and sufficient both to degrade MLKL and to inhibit necroptosis. Furthermore, mutation of pUL36 Cys131 abrogated MLKL degradation and restored necroptosis. As the same residue is also required for pUL36-mediated inhibition of apoptosis by preventing proteolytic activation of procaspase-8, we define pUL36 as a multifunctional inhibitor of both apoptotic and necroptotic cell death.