Structural and biochemical studies of HCMV gH/gL/gO and Pentamer reveal mutually exclusive cell entry complexes.

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading viral cause of birth defects after congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are key targets of the human humoral response against HCMV and are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively. We expressed and characterized soluble forms of gH/gL, gH/gL/gO, and Pentamer. Mass spectrometry and mutagenesis analysis revealed that gL-Cys144 forms disulfide bonds with gO-Cys351 in gH/gL/gO and with UL128-Cys162 in the Pentamer. Notably, Pentamer harboring the UL128-Cys162Ser/gL-Cys144Ser mutations had impaired syncytia formation and reduced interference of HCMV entry into epithelial cells. Electron microscopy analysis showed that HCMV gH/gL resembles HSV gH/gL and that gO and UL128/UL130/UL131A bind to the same site at the gH/gL N terminus. These data are consistent with gH/gL/gO and Pentamer forming mutually exclusive cell entry complexes and reveal the overall location of gH/gL-, gH/gL/gO-, and Pentamer-specific neutralizing antibody binding sites. Our results provide, to our knowledge, the first structural view of gH/gL/gO and Pentamer supporting the development of vaccines and antibody therapeutics against HCMV.

Human cytomegalovirus escapes a naturally occurring neutralizing antibody by incorporating it into assembling virions.

Human cytomegalovirus (CMV) is a common but difficult to treat infection of immunocompromised patients. MSL-109 is a human monoclonal IgG isolated from a CMV seropositive individual that recognizes the viral glycoprotein H (gH) surface antigen complexes that mediate entry. Although MSL-109 blocks CMV infection in vitro, it lacked sufficient efficacy in human trials, and CMV isolated from treated patients suggested the evolution of MSL-109 resistance. To understand how CMV escapes MSL-109, we characterized a MSL-109-resistant CMV strain. Our results elucidate a nongenetic escape mechanism in which the antibody is selectively taken up by infected cells and incorporated into assembling virions in a dose-dependent manner. The resistant virus then utilizes the Fc domain of the incorporated antibody to infect naive nonimmune cells. This resistance mechanism may explain the clinical failure of MSL-109, illustrate a general mechanism of viral antibody escape, and inform antiviral vaccine and therapeutic development.

From target selection to the minimum acceptable biological effect level for human study: use of mechanism-based PK/PD modeling to design safe and efficacious biologics.

In this paper, two applications of mechanism-based modeling are presented with their utility from candidate selection to first-in-human dosage selection. The first example is for a monoclonal antibody against a cytomegalovirus glycoprotein complex, which involves an antibody binding model and a viral load model. The model was used as part of a feasibility analysis prior to antibody generation, setting the specifications for the affinity needed to achieve a desired level of clinical efficacy. The second example is a pharmacokinetic-pharmacodynamic model based on a single-dose pharmacology study in cynomolgus monkey using data on pharmacokinetics, receptor occupancy, and the dynamics of target cell depletion and recovery. The model was used to estimate the MABEL, here defined as the minimum acceptable biological effect level against which a dose is selected for a first-in-human study. From these applications, we demonstrate that mechanism-based PK/PD binding models are useful for predicting human response to biologics compounds. Especially, such models have the ability to integrate preclinical and clinical, in vitro and in vivo information and facilitate rational decision making during various stages of drug discovery and translational research.

High-content screening to distinguish between attachment and post-attachment steps of human cytomegalovirus entry into fibroblasts and epithelial cells.

Human cytomegalovirus (HCMV) enters cells through a complex pathway involving the interaction of multiple viral glycoproteins and cellular receptors. While HCMV clinical isolates enter a wide range of cell types, entry has historically been studied using a laboratory strain of virus that can only infect fibroblasts. Herein, we have constructed a HCMV reporter strain that contains GFP fused to the abundant tegument protein pp65 to allow for the direct visualization of virus attachment and entry. Furthermore, the UL131 gene of this strain was restored to clinical isolate sequence to expand our studies of entry into physiologically relevant epithelial cell types. Using the HCMV-GFP reporter virus, we developed an image-based assay and screened a library containing 65,000 compounds for the inhibition of virus entry into fibroblasts. In addition to assessing the effect on virus entry, automated image analysis provided information on compound toxicity and whether the compounds acted as attachment or post-attachment inhibitors. To identify therapeutically viable inhibitors capable of blocking entry in multiple cell types, the inhibitors were screened further for their ability to inhibit virus entry into epithelial cells. Compounds were identified that were able to inhibit virus entry into both cell types at either attachment or post-attachment steps.

Real-time analysis of antibody interactions with whole enveloped human cytomegalovirus using surface plasmon resonance.

Human cytomegalovirus (CMV) is a large enveloped virus that encodes multiple glycoproteins required for virus-cell binding and fusion. To assess the binding properties of antibodies with target glycoprotein in a natural context of infection, we investigated the feasibility of using the surface plasmon resonance (SPR) technique for studying the direct binding of antibodies with CMV virions. Direct immobilization of whole virions to sensor surface and a surface regeneration procedure allowed for quantitative and reproducible measurements of binding affinity and binding kinetics of antibody-whole virion interactions. The conformational and functional integrity of viral particles was not compromised by the regeneration condition as evaluated with antibodies recognizing conformational epitopes and by electron microscopy. Binding of an irrelevant antibody was not observed, indicating the high specificity of the method. A panel of anti-gB antibodies was measured and the binding affinities correlated fairly well with those determined by ELISA. These data demonstrated that the interaction of anti-gB antibody with whole virion of large enveloped CMV can be quantitatively studied using SPR. This method has been successfully applied for screening and selection of anti-CMV antibodies and can be potentially extended to study antibody-glycoprotein interactions of other related herpesviruses.

The glycoprotein B disintegrin-like domain binds beta 1 integrin to mediate cytomegalovirus entry.

Cellular integrins were identified as human cytomegalovirus (HCMV) entry receptors and signaling mediators in both fibroblasts and endothelial cells. The goal of these studies was to determine the mechanism by which HCMV binds to cellular integrins to mediate virus entry. HCMV envelope glycoprotein B (gB) has sequence similarity to the integrin-binding disintegrin-like domain found in the ADAM (a disintegrin and metalloprotease) family of proteins. To test the ability of this region to bind to cellular integrins, we generated a recombinant soluble version of the gB disintegrin-like domain (gB-DLD). The gB-DLD protein bound to human fibroblasts in a specific, dose-dependent and saturable manner that required the expression of an intact beta1 integrin ectodomain. Furthermore, a physical association between gB-DLD and beta1 integrin was demonstrated through in vitro pull-down assays. The function of this interaction was shown by the ability of cell-bound gB-DLD to efficiently block HCMV entry and the infectivity of multiple in vivo target cells. Additionally, rabbit polyclonal antibodies raised against gB-DLD neutralized HCMV infection. Mimicry of the ADAM family disintegrin-like domain by HCMV gB represents a novel mechanism for integrin engagement by a virus and reveals a unique therapeutic target for HCMV neutralization. The strong conservation of the DLD across beta- and gammaherpesviruses suggests that integrin recognition and utilization may be a more broadly conserved feature throughout the Herpesviridae.

Differential initiation of innate immune responses induced by human cytomegalovirus entry into fibroblast cells.

Infection of permissive fibroblasts with human CMV (HCMV, AD169) is accompanied by a robust activation of innate immune defense. In this study, we show that inflammatory cytokine (IC) secretion and activation of the type I IFN pathway (alphabeta IFN) are initiated through distinct mechanisms. HCMV is recognized by TLR2 leading to the NF-kappaB activation and IC secretion. However, the IFN response to HCMV is not a TLR2-dependent process, as a dominant negative TLR2 does not affect the antiviral response to infection. Additionally, bafilomycin, an endosomal acidification inhibitor, has no effect on HCMV-induced IFN responses suggesting that IFN signaling is independent of endosomal resident TLRs. By contrast, disruption of lipid rafts by depletion of cellular cholesterol inhibits both HCMV entry as well as IFN responses. Cholesterol depletion had no effect on the induction of ICs by HCMV, illustrating a biological distinction at the cellular level with the initiation of innate immune pathways. Furthermore, HCMV entry inhibitors block IFN responses but not IC signaling. In particular, blocking the interaction of HCMV with beta(1) integrin diminished IFN signaling, suggesting that this virus-cell interaction or subsequent downstream steps in the entry pathway are critical for downstream signal transduction events. These data show that HCMV entry and IFN signaling are coordinated processes that require cholesterol-rich microdomains, whereas IC signaling is activated through outright sensing via TLR2. These findings further highlight the complexity and sophistication of innate immune responses at the earliest points in HCMV infection.

Epidermal growth factor receptor is not required for human cytomegalovirus entry or signaling.

Human cytomegalovirus (HCMV) can bind, fuse, and initiate gene expression in a diverse range of vertebrate cell types. This broad cellular tropism suggests that multiple receptors and/or universally distributed receptors mediate HCMV entry. Our laboratory has recently discovered that certain beta1 and beta3 integrin heterodimers are critical mediators of HCMV entry into permissive fibroblasts (A. L. Feire, H. Koss, and T. Compton, Proc. Natl. Acad. Sci. USA 101:15470-15475, 2004). It has also been reported that epidermal growth factor receptor (EGFR) is necessary for HCMV-mediated signaling and entry (X. Wang, S. M. Huong, M. L. Chiu, N. Raab-Traub, and E. E. Huang, Nature 424:456-461, 2003). Integrins are known to signal synergistically with growth factor receptors, and this coordination was recently reported for EGFR and beta3 integrins in the context of HCMV entry (X. Wang, D. Y. Huang, S. M. Huong, and E. S. Huang, Nat. Med. 11:515-521, 2005). However, EGFR-negative cell lines, such as hematopoietic cells, are known to be infected by HCMV. Therefore, we wished to confirm a role for EGFR in HCMV entry and then examine any interaction between beta1 integrins and EGFR during the entry process. Surprisingly, we were unable to detect any role for EGFR in the process of HCMV entry into fibroblast, epithelial, or endothelial cell lines. Additionally, HCMV did not activate the EGFR kinase in fibroblast cell lines. We first examined HCMV entry into two EGFR-positive or -negative cell lines but observed no increase in entry when EGFR was expressed to high levels. Physically blocking EGFR with a neutralizing antibody in fibroblast, epithelial, or endothelial cell lines or blocking EGFR kinase signaling with a chemical inhibitor in fibroblast cells did not inhibit virus entry. Lastly, we were unable to detect phosphorylation of EGFR in fibroblasts cells in response to HCMV stimulation. Our findings demonstrate that EGFR does not play a significant role in HCMV entry or signaling. These results suggest that specific integrin heterodimers either act alone as the primary entry receptors or interact in conjunction with an additional receptor(s), other than EGFR, to facilitate virus entry.

Cellular integrins function as entry receptors for human cytomegalovirus via a highly conserved disintegrin-like domain.

Human cytomegalovirus (HCMV) is capable of manifesting disease in nearly every organ system in immunocompromised patients. This broad pathogenic tropism correlates with the ability of the virus to infect all tested vertebrate cell types in vitro, a characteristic that has made receptor identification extremely difficult. During virus entry, HCMV induces cellular morphological changes and signaling cascades consistent with engagement of cellular integrins; however, HCMV structural proteins do not possess the widely used RGD integrin-binding motif. We identified an integrin-binding disintegrin-like domain within HCMV envelope glycoprotein B, a protein required for virus entry and fusion throughout the Herpesviridae. Accepted receptor criteria are met through the use of function-blocking integrin Abs, beta1 integrin knockout mouse fibroblasts, and glycoprotein B disintegrin-like peptides, all of which support a critical role for alpha2beta1, alpha6beta1, and alphaVbeta3 integrins as HCMV entry receptors and signaling mediators acting during the penetration stage of the entry pathway. Strikingly, the glycoprotein B disintegrin-like domain is conserved in many human and animal herpesviruses, suggesting that integrins may support entry across this medically important virus family.