The CD46 ectodomain participates in human cytomegalovirus infection of epithelial cells.

Human cytomegalovirus (HCMV) primary infections are typically asymptomatic in healthy individuals yet can cause increased morbidity and mortality in organ transplant recipients, AIDS patients, neonates, and the elderly. The successful, widespread dissemination of this virus among the population can be attributed in part to its wide cellular tropism and ability to establish life-long latency. HCMV infection is a multi-step process that requires numerous cellular and viral factors. The viral envelope consists of envelope protein complexes that interact with cellular factors; such interactions dictate virus recognition and attachment to different cell types, followed by fusion either at the cell membrane or within an endocytic vesicle. Several HCMV entry factors, including neuropilin-2 (Nrp2), THBD, CD147, OR14I1, and CD46, are characterized as participating in HCMV pentamer-specific entry of non-fibroblast cells such as epithelial, trophoblast, and endothelial cells, respectively. This study focuses on characterizing the structural elements of CD46 that impact HCMV infection. Infectivity studies of wild-type and CD46 knockout epithelial cells demonstrated that levels of CD46 expressed on the cell surface were directly related to HCMV infectivity. Overexpression of CD46 isomers BC1, BC2, and C2 enhanced infection. Further, CD46 knockout epithelial cells expressing CD46 deletion and chimeric molecules identified that the intact ectodomain was critical for rescue of HCMV infection in CD46 knockout cells. Collectively, these data support a model that the extracellular domain of CD46 participates in HCMV infection due to its surface expression.

Chemoproteomics reveals microbiota-derived aromatic monoamine agonists for GPRC5A.

The microbiota generates diverse metabolites to modulate host physiology and disease, but their protein targets and mechanisms of action have not been fully elucidated. To address this challenge, we explored microbiota-derived indole metabolites and developed photoaffinity chemical reporters for proteomic studies. We identified many potential indole metabolite-interacting proteins, including metabolic enzymes, transporters, immune sensors and G protein-coupled receptors. Notably, we discovered that aromatic monoamines can bind the orphan receptor GPRC5A and stimulate ß-arrestin recruitment. Metabolomic and functional profiling also revealed specific amino acid decarboxylase-expressing microbiota species that produce aromatic monoamine agonists for GPRC5A-ß-arrestin recruitment. Our analysis of synthetic aromatic monoamine derivatives identified 7-fluorotryptamine as a more potent agonist of GPRC5A. These results highlight the utility of chemoproteomics to identify microbiota metabolite-interacting proteins and the development of small-molecule agonists for orphan receptors.

Marburg and Ebola Virus Infections Elicit a Complex, Muted Inflammatory State in Bats.

The Marburg and Ebola filoviruses cause a severe, often fatal, disease in humans and nonhuman primates but have only subclinical effects in bats, including Egyptian rousettes, which are a natural reservoir of Marburg virus. A fundamental question is why these viruses are highly pathogenic in humans but fail to cause disease in bats. To address this question, we infected one cohort of Egyptian rousette bats with Marburg virus and another cohort with Ebola virus and harvested multiple tissues for mRNA expression analysis. While virus transcripts were found primarily in the liver, principal component analysis (PCA) revealed coordinated changes across multiple tissues. Gene signatures in kidney and liver pointed at induction of vasodilation, reduction in coagulation, and changes in the regulation of iron metabolism. Signatures of immune response detected in spleen and liver indicated a robust anti-inflammatory state signified by macrophages in the M2 state and an active T cell response. The evolutionary divergence between bats and humans of many responsive genes might provide a framework for understanding the differing outcomes upon infection by filoviruses. In this study, we outline multiple interconnected pathways that respond to infection by MARV and EBOV, providing insights into the complexity of the mechanisms that enable bats to resist the disease caused by filoviral infections. The results have the potential to aid in the development of new strategies to effectively mitigate and treat the disease caused by these viruses in humans.

Investigating N-arylpyrimidinamine (NAPA) compounds as early-stage inhibitors against human cytomegalovirus.

Human cytomegalovirus (CMV) is a ubiquitous ß-herpesvirus that establishes latent asymptomatic infections in healthy individuals but can cause serious infections in immunocompromised people, resulting in increased risk of morbidity and mortality. The current FDA-approved CMV drugs target late stages of the CMV life-cycle. While these drugs are effective in most cases, they have serious drawbacks, including poor oral bioavailability, dose-limiting toxicity, and a low barrier to resistance. Given the clinical relevance of CMV-associated diseases, novel therapies are needed. Thus, a novel class of compounds that inhibits the early stages of the CMV life-cycle was identified and found to block infection of different strains in physiologically relevant cell types. This class of compounds, N-arylpyrimidinamine (NAPA), demonstrated potent anti-CMV activity against ganciclovir-sensitive and -resistant strains in in vitro replication assays, a selectivity index >30, and favorable in vitro ADME properties. Mechanism of action studies demonstrated that NAPA compounds inhibit an early step of virus infection. NAPA compounds are specific inhibitors of cytomegaloviruses and exhibited limited anti-viral activity against other herpesviruses. Collectively, we have identified a novel class of CMV inhibitor that effectively limits viral infection and proliferation.

Anti-infective bile acids bind and inactivate a Salmonella virulence regulator.

Bile acids are prominent host and microbiota metabolites that modulate host immunity and microbial pathogenesis. However, the mechanisms by which bile acids suppress microbial virulence are not clear. To identify the direct protein targets of bile acids in bacterial pathogens, we performed activity-guided chemical proteomic studies. In Salmonella enterica serovar Typhimurium, chenodeoxycholic acid (CDCA) most effectively inhibited the expression of virulence genes and invasion of epithelial cells and interacted with many proteins. Notably, we discovered that CDCA can directly bind and inhibit the function of HilD, an important transcriptional regulator of S. Typhimurium virulence and pathogenesis. Our characterization of bile acid-resistant HilD mutants in vitro and in S. Typhimurium infection models suggests that HilD is one of the key protein targets of anti-infective bile acids. This study highlights the utility of chemical proteomics to identify the direct protein targets of microbiota metabolites for mechanistic studies in bacterial pathogens.

Development of broadly neutralizing antibodies targeting the cytomegalovirus subdominant antigen gH.

Human cytomegalovirus (HCMV) is a ß-herpesvirus that increases morbidity and mortality in immunocompromised individuals including transplant recipients and newborns. New anti-HCMV therapies are an urgent medical need for diverse patient populations. HCMV infection of a broad range of host tissues is dependent on the gH/gL/gO trimer and gH/gL/UL28/UL130/UL131A pentamer complexes on the viral envelope. We sought to develop safe and effective therapeutics against HCMV by generating broadly-neutralizing, human monoclonal antibodies (mAbs) from VelocImmune® mice immunized with gH/gL cDNA. Following high-throughput binding and neutralization screening assays, 11 neutralizing antibodies were identified with unique CDR3 regions and a high-affinity (KD 1.4-65 nM) to the pentamer complex. The antibodies bound to distinct regions within Domains 1 and 2 of gH and effectively neutralized diverse clinical strains in physiologically relevant cell types including epithelial cells, trophoblasts, and monocytes. Importantly, combined adminstration of mAbs with ganciclovir, an FDA approved antiviral, greatly limited virus dissemination. Our work identifies several anti-gH/gL mAbs and sheds light on gH neutralizing epitopes that can guide future vaccine strategies.

Valspodar limits human cytomegalovirus infection and dissemination.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that establishes a life-long infection affecting up to 80% of the US population. HCMV periodically reactivates leading to enhanced morbidity and mortality in immunosuppressed patients causing a range of complications including organ transplant failure and cognitive disorders in neonates. Therapeutic options for HCMV are limited to a handful of antivirals that target late stages of the virus life cycle and efficacy is often challenged by the emergence of mutations that confer resistance. In addition, these antiviral therapies may have adverse reactions including neutropenia in newborns and an increase in adverse cardiac events in HSCT patients. These findings highlight the need to develop novel therapeutics that target different steps of the viral life cycle. To this end, we screened a small molecule library against ion transporters to identify new antivirals against the early steps of virus infection. We identified valspodar, a 2nd-generation ABC transporter inhibitor, that limits HCMV infection as demonstrated by the decrease in IE2 expression of virus infected cells. Cells treated with increasing concentrations of valspodar over a 9-day period show minimal cytotoxicity. Importantly, valspodar limits HCMV plaque numbers in comparison to DMSO controls demonstrating its ability to inhibit viral dissemination. Collectively, valspodar represents a potential new anti-HCMV therapeutic that limits virus infection by likely targeting a host factor. Further, the data suggest that specific ABC transporters may participate in the HCMV life-cycle.

An Influenza Virus Hemagglutinin-Based Vaccine Platform Enables the Generation of Epitope Specific Human Cytomegalovirus Antibodies.

Human cytomegalovirus (CMV) is a highly prevalent pathogen with ~60%-90% seropositivity in adults. CMV can contribute to organ rejection in transplant recipients and is a major cause of birth defects in newborns. Currently, there are no approved vaccines against CMV. The epitope of a CMV neutralizing monoclonal antibody against a conserved region of the envelope protein gH provided the basis for a new CMV vaccine design. We exploited the influenza A virus as a vaccine platform due to the highly immunogenic head domain of its hemagglutinin envelope protein. Influenza A variants were engineered by reverse genetics to express the epitope of an anti-CMV gH neutralizing antibody that recognizes native gH into the hemagglutinin antigenic Sa site. We determined that the recombinant influenza variants expressing 7, 10, or 13 residues of the anti-gH neutralizing antibody epitope were recognized and neutralized by the anti-gH antibody 10C10. Mice vaccinated with the influenza/CMV chimeric viruses induced CMV-specific antibodies that recognized the native gH protein and inhibited virus infection. In fact, the influenza variants expressing 7-13 gH residues neutralized a CMV infection at ~60% following two immunizations with variants expressing the 13 residue gH peptide produced the highest levels of neutralization. Collectively, our study demonstrates that a variant influenza virus inserted with a gH peptide can generate a humoral response that limits a CMV infection.

CD46 facilitates entry and dissemination of human cytomegalovirus.

Human cytomegalovirus (CMV) causes a wide array of disease to diverse populations of immune-compromised individuals. Thus, a more comprehensive understanding of how CMV enters numerous host cell types is necessary to further delineate the complex nature of CMV pathogenesis and to develop targeted therapeutics. To that end, we establish a vaccination strategy utilizing membrane vesicles derived from epithelial cells to generate a library of monoclonal antibodies (mAbs) targeting cell surface proteins in their native conformation. A high-throughput inhibition assay is employed to screen these antibodies for their ability to limit infection, and mAbs targeting CD46 are identified. In addition, a significant reduction of viral proliferation in CD46-KO epithelial cells confirms a role for CD46 function in viral dissemination. Further, we demonstrate a CD46-dependent entry pathway of virus infection in trophoblasts, but not in fibroblasts, highlighting the complexity of CMV entry and identifying CD46 as an entry factor in congenital infection.

Functional screening for anti-CMV biologics identifies a broadly neutralizing epitope of an essential envelope protein.

The prototypic ß-herpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. The CMV envelope consists of various protein complexes that enable wide viral tropism. More specifically, the glycoprotein complex gH/gL/gO (gH-trimer) is required for infection of all cell types, while the gH/gL/UL128/130/131a (gH-pentamer) complex imparts specificity in infecting epithelial, endothelial and myeloid cells. Here we utilize state-of-the-art robotics and a high-throughput neutralization assay to screen and identify monoclonal antibodies (mAbs) targeting the gH glycoproteins that display broad-spectrum properties to inhibit virus infection and dissemination. Subsequent biochemical characterization reveals that the mAbs bind to gH-trimer and gH-pentamer complexes and identify the antibodies' epitope as an 'antigenic hot spot' critical for virus entry. The mAbs inhibit CMV infection at a post-attachment step by interacting with a highly conserved central alpha helix-rich domain. The platform described here provides the framework for development of effective CMV biologics and vaccine design strategies.

Relaxed tRNA specificity of the Staphylococcus aureus aspartyl-tRNA synthetase enables RNA-dependent asparagine biosynthesis.

The human pathogen Staphylococcus aureus is an asparagine prototroph despite its genome not encoding an asparagine synthetase. S. aureus does use an asparaginyl-tRNA synthetase (AsnRS) to directly ligate asparagine to tRNA(Asn). The S. aureus genome also codes for one aspartyl-tRNA synthetase (AspRS). Here we demonstrate the lone S. aureus aspartyl-tRNA synthetase has relaxed tRNA specificity and can be used with the amidotransferase GatCAB to synthesize asparagine on tRNA(Asn). S. aureus thus encodes both the direct and indirect routes for Asn-tRNA(Asn) formation while encoding only one aspartyl-tRNA synthetase. The presence of the indirect pathway explains how S. aureus synthesizes asparagine without either asparagine synthetase.