Structural insights into the interaction between adenovirus C5 hexon and human lactoferrin.

Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus 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. IMPORTANCE: Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study's cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon's hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5's mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.

Human AdV-20-42-42, a Promising Novel Adenoviral Vector for Gene Therapy and Vaccine Product Development.

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. IMPORTANCE Adenoviral vectors are under investigation for a broad range of therapeutic indications in diverse fields, such as oncology and gene therapy, as well as for vaccination both for human and veterinary use. A wealth of data shows that preexisting immune responses may limit the use of a vector. Particularly in the current climate of global pandemic, there is a need to expand the toolbox with novel adenoviral vectors for vaccine development. Our data demonstrate that we have successfully vectorized a novel adenovirus type candidate with low seroprevalence. The cell transduction data and antigen-specific immune responses induced in vivo demonstrate that this vector is highly promising for the development of gene therapy and vaccine products.

BAF45b Is Required for Efficient Zika Virus Infection of HAP1 Cells.

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.

Characterizing the cellular attachment receptor for Langat virus.

Tick-borne encephalitis infections have increased the last 30 years. The mortality associated to this viral infection is 0.5 to 30% with a risk of permanent neurological sequelae, however, no therapeutic is currently available. The first steps of virus-cell interaction, such as attachment and entry, are of importance to understand pathogenesis and tropism. Several molecules have been shown to interact with tick-borne encephalitis virus (TBEV) at the plasma membrane surface, yet, no studies have proven that these are specific entry receptors. In this study, we set out to characterize the cellular attachment receptor(s) for TBEV using the naturally attenuated member of the TBEV complex, Langat virus (LGTV), as a model. Inhibiting or cleaving different molecules from the surface of A549 cells, combined with inhibition assays using peptide extracts from high LGTV binding cells, revealed that LGTV attachment to host cells is dependent on plasma membrane proteins, but not on glycans or glycolipids, and suggested that LGTV might use different cellular attachment factors on different cell types. Based on this, we developed a transcriptomic approach to generate a list of candidate attachment and entry receptors. Our findings shed light on the first step of the flavivirus life-cycle and provide candidate receptors that might serve as a starting point for future functional studies to identify the specific attachment and/or entry receptor for LGTV and TBEV.