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Human adenovirus type 36 (HAdV-D36) has been putatively linked to obesity in animals and has been associated with obesity in humans in some but not all studies. Despite extensive epidemiological research there is limited information about its receptor profile. We investigated the receptor portfolio of HAdV-D36 using a combined structural biology and virology approach. The HAdV-D36 fiber knob domain (FK), which mediates the primary attachment of many HAdVs to host cells, has a significantly elongated DG loop that alters known binding interfaces for established adenovirus receptors such as the coxsackie- and adenovirus receptor (CAR) and CD46. Our data suggest that HAdV-D36 attaches to host cells using a versatile receptor pool comprising sialic acid-containing glycans and CAR. Sialic acids are recognized at the same binding site used by other HAdVs of species D such as HAdV-D37. Using glycan microarrays, we demonstrate that HAdV-D36 displays a binding preference for glycans containing a rare sialic acid variant, 4-O,5-N-diacetylneuraminic acid, over the more common 5-N-acetylneuraminic acid. To date, this sialic acid variant has not been detected in humans, although it can be synthesized by various animal species, including a range of domestic and livestock animals. Taken together, our results indicate that HAdV-D36 has evolved to recognize a specialized set of primary attachment receptors that are different from known HAdV types and coincides with a unique host range and pathogenicity profile.

Mammalian α-defensins are a family of abundant effector peptides of the mucosal innate immune system. Although primarily considered to be antimicrobial, α-defensins can increase rather than block infection by certain prominent bacterial and viral pathogens in cell culture and in vivo. We have shown previously that exposure of mouse and human adenoviruses (HAdVs) to α-defensins is able to overcome competitive inhibitors that block cell binding, leading us to hypothesize a defensin-mediated binding mechanism that is independent of known viral receptors. To test this hypothesis, we used genetic approaches to demonstrate that none of several primary receptors nor integrin co-receptors are needed for human α-defensin-mediated binding of HAdV to cells; however, infection remains integrin dependent. Thus, our studies have revealed a novel pathway for HAdV binding to cells that bypasses viral primary receptors. We speculate that this pathway functions in parallel with receptor-mediated entry and contributes to α-defensin-enhanced infection of susceptible cells. Remarkably, we also found that in the presence of α-defensins, HAdV tropism is expanded to non-susceptible cells, even when viruses are exposed to a mixture of both susceptible and non-susceptible cells. Therefore, we propose that in the presence of sufficient concentrations of α-defensins, such as in the lung or gut, integrin expression rather than primary receptor expression will dictate HAdV tropism in vivo. In summary, α-defensins may contribute to tissue tropism not only through the neutralization of susceptible viruses but also by allowing certain defensin-resistant viruses to bind to cells independently of previously described mechanisms.

Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackieadenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon’s hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development.

Enteric adenovirus types F40 and 41 (EAdVs) are a leading cause of diarrhea and diarrhea-associated death in young children and have recently been proposed to cause acute hepatitis in children. EAdVs have a unique capsid architecture and exhibit — unlike other human adenoviruses — a relatively strict tropism for gastrointestinal tissues with, to date, understudied infection mechanism and unknown target cells. In this study, we turn to potentially limiting host factors by comparing EAdV entry in cell lines with respiratory and intestinal origin by cellular perturbation, virus particle tracking, and transmission electron microscopy. Our analyses highlight kinetic advantages for EAdVs in duodenal HuTu80 cell infection and reveal a larger fraction of mobile particles, faster virus uptake, and infectious particle entry in intestinal cells. Moreover, EAdVs display a dependence on clathrin- and dynamin-dependent pathways in intestinal cells. Detailed knowledge of virus entry routes and host factor requirements is essential to understanding pathogenesis and developing new countermeasures. Hence, this study provides novel insights into the entry mechanisms of a medically important virus with emerging tropism in a cell line originating from a relevant tissue. IMPORTANCE Enteric adenoviruses have historically been difficult to grow in cell culture, which has resulted in lack of knowledge of host factors and pathways required for infection of these medically relevant viruses. Previous studies in non-intestinal cell lines showed slow infection kinetics and generated comparatively low virus yields compared to other adenovirus types. We suggest duodenum-derived HuTu80 cells as a superior cell line for studies to complement efforts using complex intestinal tissue models. We show that viral host cell factors required for virus entry differ between cell lines from distinct origins and demonstrate the importance of clathrin-mediated endocytosis.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by rich deposits of extracellular matrix (ECM), affecting the pathophysiology of the disease. Here, we identified galectin 4 (gal 4) as a cancer cell produced protein deposited into the ECM of PDAC tumors and detected high circulating levels of gal 4 in PDAC patients. In orthotopic transplantation experiments we observed increased infiltration of T-cells and prolonged survival in immunocompetent mice transplanted with cancer cells with reduced expression of gal 4. Increased survival was not observed in immunodeficient RAG1-/- mice, demonstrating that the effect was mediated by the adaptive immune system. Furthermore, by performing single-cell RNA-sequencing we found that the myeloid compartment and cancer-associated fibroblast (CAF) subtypes were altered in the transplanted tumors. Reduced gal 4 expression was associated with higher proportion of myofibroblastic CAFs and reduced numbers of inflammatory CAFs. We also found higher proportions of M1 macrophages, T-cells and antigen presenting dendritic cells in tumors with reduced gal 4 expression. Using a co-culture system, we observed that extracellular gal 4 induced apoptosis in T-cells by binding N-glycosylation residues on CD3 epsilon/delta. Hence, we show that gal 4 is involved in immune evasion and identify gal 4 as a promising drug target for overcoming immunosuppression in PDAC. 

Coxsackievirus A24 variant (CVA24v) is responsible for several outbreaks and two pandemics of the highly contagious eye infection acute hemorrhagic conjunctivitis (AHC). Currently, neither prevention (vaccines) nor treatments (antivirals) are available for combating this disease. CVA24v attaches to cells by binding Neu5Ac-containing glycans on the surface of cells which facilitates entry. Previously, we have demonstrated that pentavalent Neu5Ac conjugates attenuate CVA24v infection of human corneal epithelial (HCE) cells. In this study, we report on the structure-based design of three classes of divalent Neu5Ac conjugates, with varying spacer lengths, and their effect on CVA24v transduction in HCE cells. In relative terms, the most efficient class of divalent Neu5Ac conjugates are more efficient than the pentavalent Neu5Ac conjugates previously reported.

The family Adenoviridae includes non-enveloped viruses with linear dsDNA genomes of 25-48 kb and medium-sized icosahedral capsids. Adenoviruses have been discovered in vertebrates from fish to humans. The family is divided into six genera, each of which is more common in certain animal groups. The outcome of infection may vary from subclinical to lethal disease. This is a summary of the ICTV Report on the family Adenoviridae, which is available at ictv.global/report/adenoviridae.

The 2016 Zika virus (ZIKV) epidemic illustrates the impact of flaviviruses as emerging human pathogens. For unknown reasons, ZIKV replicates more efficiently in neural progenitor cells (NPCs) than in postmitotic neurons. Here, we identified host factors used by ZIKV using the NCI-60 library of cell lines and COMPARE analysis, and cross-analyzed this library with two other libraries of host factors with importance for ZIKV infection. We identified BAF45b, a subunit of the BAF (Brg1/Brm-associated factors) protein complexes that regulate differentiation of NPCs to post-mitotic neurons. ZIKV (and other flaviviruses) infected HAP1 cells deficient in expression of BAF45b and other BAF subunits less efficiently than wildtype (WT) HAP1 cells. We concluded that subunits of the BAF complex are important for infection of ZIKV and other flavivirus. Given their function in cell and tissue differentiation, such regulators may be important determinants of tropism and pathogenesis of arthropod-borne flaviviruses.

Coxsackievirus A24 variant (CVA24v) is the primary causative agent of the highly contagious eye infection designated acute hemorrhagic conjunctivitis (AHC). It is solely responsible for two pandemics and several recurring outbreaks of the disease over the last decades, thus affecting millions of individuals throughout the world. To date, no antiviral agents or vaccines are available for combating this disease, and treatment is mainly supportive. CVA24v utilizes Neu5Ac-containing glycans as attachment receptors facilitating entry into host cells. We have previously reported that pentavalent Neu5Ac conjugates based on a glucose-scaffold inhibit CVA24v infection of human corneal epithelial cells. In this study, we report on the design and synthesis of scaffold-replaced pentavalent Neu5Ac conjugates and their effect on CVA24v cell transduction and the use of cryogenic electron microscopy (cryo-EM) to study the binding of these multivalent conjugates to CVA24v. The results presented here provide insights into the development of Neu5Ac-based inhibitors of CVA24v and, most significantly, the first application of cryo-EM to study the binding of a multivalent ligand to a lectin.

Preexisting immune responses toward adenoviral vectors limit the use of a vector based on particular serotypes and its clinical applicability for gene therapy and/or vaccination. Therefore, there is a significant interest in vectorizing novel adenoviral types that have low seroprevalence in the human population. Here, we describe the discovery and vectorization of a chimeric human adenovirus, which we call HAdV-20-42-42. Full-genome sequencing revealed that this virus is closely related to human serotype 42, except for the penton base, which is derived from serotype 20. The HAdV-20-42-42 vector could be propagated stably to high titers on existing E1-complementing packaging cell lines. Receptor-binding studies revealed that the vector utilized both CAR and CD46 as receptors for cell entry. Furthermore, the HAdV-20-42-42 vector was potent in transducing human and murine cardiovascular cells and tissues, irrespective of the presence of blood coagulation factor X. In vivo characterizations demonstrate that when delivered intravenously (i.v.) in mice, HAdV-20-42-42 mainly targeted the lungs, liver, and spleen and triggered robust inflammatory immune responses. Finally, we demonstrate that potent T-cell responses against vector-delivered antigens could be induced upon intramuscular vaccination in mice. In summary, from the data obtained we conclude that HAdV-20-42-42 provides a valuable addition to the portfolio of adenoviral vectors available to develop efficacious products in the fields of gene therapy and vaccination.