Identification of functionally important domains of human cytomegalovirus gO that act after trimer binding to receptors.

Human cytomegalovirus (HCMV) entry involves trimer (gH/gL/gO) that interacts with PDGFRα in fibroblasts. Entry into epithelial and endothelial cells requires trimer, which binds unidentified receptors, and pentamer (gH/gL/UL128-131), which binds neuropilin-2. To identify functionally important domains in trimer, we screened an overlapping 20-mer gO peptide library and identified two sets of peptides: 19/20 (a.a. 235-267) and 32/33 (a.a. 404-436) that could block virus entry. Soluble trimer containing wild type gO blocked HCMV entry, whereas soluble trimers with the 19/20 or 32/33 sequences mutated did not block entry. Interestingly, the mutant trimers retained the capacity to bind to cellular receptors including PDGFRα. Peptide 19/20 and 32/33 sequences formed a lobe extending from the surface of gO and an adjacent concave structure, respectively. Neither of these sets of sequences contacted PDGFRα. Instead, our data support a model in which the 19/20 and 32/33 trimer sequences function downstream of receptor binding, e.g. trafficking of HCMV into endosomes or binding to gB for entry fusion. We also screened for peptides that bound antibodies (Abs) in human sera, observing that peptides 20 and 26 bound Abs. These peptides engendered neutralizing Abs (NAbs) after immunization of rabbits and could pull out NAbs from human sera. Peptides 20 and 26 sequences represent the first NAb epitopes identified in trimer. These studies describe two important surfaces on gO defined by: i) peptides 19/20 and 32/33, which apparently act downstream of receptor binding and ii) peptide 26 that interacts with PDGFRα. Both these surfaces are targets of NAbs.

Distinct roles for the Charcot-Marie-Tooth disease-causing endosomal regulators Mtmr5 and Mtmr13 in axon radial sorting and Schwann cell myelination.

The form of Charcot-Marie-Tooth type 4B (CMT4B) disease caused by mutations in myotubularin-related 5 (MTMR5; also called SET binding factor 1, SBF1) shows a spectrum of axonal and demyelinating nerve phenotypes. This contrasts with the CMT4B subtypes caused by MTMR2 or MTMR13 (SBF2) mutations, which are characterized by myelin outfoldings and classic demyelination. Thus, it is unclear whether MTMR5 plays an analogous or distinct role from that of its homolog, MTMR13, in the peripheral nervous system (PNS). MTMR5 and MTMR13 are pseudophosphatases predicted to regulate endosomal trafficking by activating Rab GTPases and binding to the phosphoinositide 3-phosphatase MTMR2. In the mouse PNS, Mtmr2 was required to maintain wild-type levels of Mtmr5 and Mtmr13, suggesting that these factors function in discrete protein complexes. Genetic elimination of both Mtmr5 and Mtmr13 in mice led to perinatal lethality, indicating that the two proteins have partially redundant functions during embryogenesis. Loss of Mtmr5 in mice did not cause CMT4B-like myelin outfoldings. However, adult Mtmr5-/- mouse nerves contained fewer myelinated axons than control nerves, likely as a result of axon radial sorting defects. Consistently, Mtmr5 levels were highest during axon radial sorting and fell sharply after postnatal day seven. Our findings suggest that Mtmr5 and Mtmr13 ensure proper axon radial sorting and Schwann cell myelination, respectively, perhaps through their direct interactions with Mtmr2. This study enhances our understanding of the non-redundant roles of the endosomal regulators MTMR5 and MTMR13 during normal peripheral nerve development and disease.

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.

Proteoglycan 4 Reduces Neuroinflammation and Protects the Blood-Brain Barrier after Traumatic Brain Injury.

Neuroinflammation and dysfunction of the blood-brain barrier (BBB) are two prominent mechanisms of secondary injury in neurotrauma. It has been suggested that Toll-like receptors (TLRs) play important roles in initiating and propagating neuroinflammation resulting from traumatic brain injury (TBI), but potential beneficial effects of targeting these receptors in TBI have not been broadly studied. Here, we investigated the effect of targeting TLRs with proteoglycan 4 (PRG4) on post-traumatic neuroinflammation and BBB function. PRG4 is a mucinous glycoprotein with strong anti-inflammatory properties, exerting its biological effects by interfering with TLR2/4 signaling. In addition, PRG4 has the ability to inhibit activation of cluster of differentiation 44 (CD44), a cell-surface glycoprotein playing an important role in inflammation. Using the controlled cortical impact model of TBI in rats, we showed a rapid and prolonged upregulation of message for TLR2/4 and CD44 in the injured cortex. In the in vitro model of the BBB, recombinant human PRG4 (rhPRG4) crossed the endothelial monolayers through a high-capacity, saturable transport system. In rats sustaining TBI, PRG4 delivery to the brain was enhanced by post-traumatic increase in BBB permeability. rhPRG4 injected intravenously at 1 h post-TBI potently inhibited post-traumatic activation of nuclear factor kappa B and extracellular signal-regulated kinases 1/2, the two major signal transduction pathways associated with TLR2/4 and CD44, and curtailed the post-traumatic influx of monocytes. In addition, PRG4 restored normal BBB function after TBI by preventing the post-traumatic loss of tight junction protein claudin 5 and reduced neuronal death. Our observations provide support for therapeutic strategies targeting TLRs in TBI.

HCMV trimer- and pentamer-specific antibodies synergize for virus neutralization but do not correlate with congenital transmission.

Human cytomegalovirus (HCMV) causes substantial disease in transplant patients and harms the development of the nervous system in babies infected in utero. Thus, there is a major focus on developing safe and effective HCMV vaccines. Evidence has been presented that a major target of neutralizing antibodies (NAbs) is the HCMV pentamer glycoprotein gH/gL/UL128-131. In some studies, most of the NAbs in animal or human sera were found to recognize the pentamer, which mediates HCMV entry into endothelial and epithelial cells. It was also reported that pentamer-specific antibodies correlate with protection against transmission from mothers to babies. One problem with the studies on pentamer-specific NAbs to date has been that the studies did not compare the pentamer to the other major form of gH/gL, the gH/gL/gO trimer, which is essential for entry into all cell types. Here, we demonstrate that both trimer and pentamer NAbs are frequently found in human transplant patients' and pregnant mothers' sera. Depletion of human sera with trimer caused reductions in NAbs similar to that observed following depletion with the pentamer. The trimer- and pentamer-specific antibodies acted in a synergistic fashion to neutralize HCMV and also to prevent virus cell-to-cell spread. Importantly, there was no correlation between the titers of trimer- and pentamer-specific NAbs and transmission of HCMV from mothers to babies. Therefore, both the trimer and pentamer are important targets of NAbs. Nevertheless, these antibodies do not protect against transmission of HCMV from mothers to babies.

The Human Cytomegalovirus Trimer and Pentamer Promote Sequential Steps in Entry into Epithelial and Endothelial Cells at Cell Surfaces and Endosomes.

Human cytomegalovirus (HCMV) infects a wide variety of human cell types by different entry pathways that involve distinct envelope glycoprotein complexes that include gH/gL, a trimer complex consisting of gHgL/gO, and a pentamer complex consisting of gH/gL/UL128/UL130/UL131. We characterized the effects of soluble forms of these proteins on HCMV entry. Soluble trimer and pentamer blocked entry of HCMV into epithelial and endothelial cells, whereas soluble gH/gL did not. Trimer inhibited HCMV entry into fibroblast cells, but pentamer and gH/gL did not. Both trimer and pentamer bound to the surfaces of fibroblasts and epithelial cells, whereas gH/gL did not bind to either cell type. Cell surface binding of trimer and pentamer did not involve heparin sulfate moieties. The ability of soluble trimer to block entry of HCMV into epithelial cells did not involve platelet-derived growth factor PDGFRα, which has been reported as a trimer receptor for fibroblasts. Soluble trimer reduced the amount of virus particles that could be adsorbed onto the surface of epithelial cells, whereas soluble pentamer had no effect on virus adsorption. However, soluble pentamer reduced the ability of virus particles to exit from early endosomes into the cytoplasm and then travel to the nucleus. These studies support a model in which both the trimer and pentamer are required for HCMV entry into epithelial and endothelial cells, with trimer interacting with cell surface receptors other than PDGFR and pentamer acting later in the entry pathway to promote egress from endosomes.IMPORTANCE HCMV infects nearly 80% of the world's population and causes significant morbidity and mortality. The current antiviral agents used to treat HCMV infections are prone to resistance and can be toxic to patients, and there is no current vaccine against HCMV available. The data in this report will lead to a better understanding of how essential HCMV envelope glycoproteins function during infection of biologically important cell types and will have significant implications for understanding HCMV pathogenesis for developing new therapeutics.

TrkB-enhancer facilitates functional recovery after traumatic brain injury.

Brain-derived neurotrophic factor (BDNF), a key player in regulating synaptic strength and learning, is dysregulated following traumatic brain injury (TBI), suggesting that stimulation of BDNF signaling pathways may facilitate functional recovery. This study investigates whether CN2097, a peptidomimetic ligand which targets the synaptic scaffold protein, postsynaptic density protein 95, to enhance downstream signaling of tropomyosin-related kinase B, a receptor for BDNF, can improve neurological function after TBI. Moderate to severe TBI elicits neuroinflammation and c-Jun-N-terminal kinase (JNK) activation, which is associated with memory deficits. Here we demonstrate that CN2097 significantly reduces the post-traumatic synthesis of proinflammatory mediators and inhibits the post-traumatic activation of JNK in a rodent model of TBI. The recordings of field excitatory post-synaptic potentials in the hippocampal CA1 subfield demonstrate that TBI inhibits the expression of long-term potentiation (LTP) evoked by high-frequency stimulation of Schaffer collaterals, and that CN2097 attenuates this LTP impairment. Lastly, we demonstrate that CN2097 significantly improves the complex auditory processing deficits, which are impaired after injury. The multifunctionality of CN2097 strongly suggests that CN2097 could be highly efficacious in targeting complex secondary injury processes resulting from neurotrauma.

Schwann cell-specific deletion of the endosomal PI 3-kinase Vps34 leads to delayed radial sorting of axons, arrested myelination, and abnormal ErbB2-ErbB3 tyrosine kinase signaling.

The PI 3-kinase Vps34 (Pik3c3) synthesizes phosphatidylinositol 3-phosphate (PI3P), a lipid critical for both endosomal membrane traffic and macroautophagy. Human genetics have implicated PI3P dysregulation, and endosomal trafficking in general, as a recurring cause of demyelinating Charcot-Marie-Tooth (CMT) peripheral neuropathy. Here, we investigated the role of Vps34, and PI3P, in mouse Schwann cells by selectively deleting Vps34 in this cell type. Vps34-Schwann cell knockout (Vps34SCKO ) mice show severe hypomyelination in peripheral nerves. Vps34-/- Schwann cells interact abnormally with axons, and there is a delay in radial sorting, a process by which large axons are selected for myelination. Upon reaching the promyelinating stage, Vps34-/- Schwann cells are significantly impaired in the elaboration of myelin. Nerves from Vps34SCKO mice contain elevated levels of the LC3 and p62 proteins, indicating impaired autophagy. However, in the light of recent demonstrations that autophagy is dispensable for myelination, it is unlikely that hypomyelination in Vps34SCKO mice is caused by impaired autophagy. Endosomal trafficking is also disturbed in Vps34-/- Schwann cells. We investigated the activation of the ErbB2/3 receptor tyrosine kinases in Vps34SCKO nerves, as these proteins, which play essential roles in Schwann cell myelination, are known to traffic through endosomes. In Vps34SCKO nerves, ErbB3 was hyperphosphorylated on a tyrosine known to be phosphorylated in response to neuregulin 1 exposure. ErbB2 protein levels were also decreased during myelination. Our findings suggest that the loss of Vps34 alters the trafficking of ErbB2/3 through endosomes. Abnormal ErbB2/3 signaling to downstream targets may contribute to the hypomyelination observed in Vps34SCKO mice.