Ex Vivo and In Vivo CD46 Receptor Utilization by Species D Human Adenovirus Serotype 26 (HAdV26).

Human adenovirus serotype 26 (Ad26) is used as a gene-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and HIV-1. However, its primary receptor portfolio remains controversial, potentially including sialic acid, coxsackie and adenovirus receptor (CAR), integrins, and CD46. We and others have shown that Ad26 can use CD46, but these observations were questioned on the basis of the inability to cocrystallize Ad26 fiber with CD46. Recent work demonstrated that Ad26 binds CD46 with its hexon protein rather than its fiber. We examined the functional consequences of Ad26 for infection in vitro and in vivo. Ectopic expression of human CD46 on Chinese hamster ovary cells increased Ad26 infection significantly. Deletion of the complement control protein domain CCP1 or CCP2 or the serine-threonine-proline (STP) region of CD46 reduced infection. Comparing wild-type and sialic acid-deficient CHO cells, we show that the usage of CD46 is independent of its sialylation status. Ad26 transduction was increased in CD46 transgenic mice after intramuscular (i.m.) injection but not after intranasal (i.n.) administration. Ad26 transduction was 10-fold lower than Ad5 transduction after intratumoral (i.t.) injection of CD46-expressing tumors. Ad26 transduction of liver was 1,000-fold lower than that ofAd5 after intravenous (i.v.) injection. These data demonstrate the use of CD46 by Ad26 in certain situations but also show that the receptor has little consequence by other routes of administration. Finally, i.v. injection of high doses of Ad26 into CD46 mice induced release of liver enzymes into the bloodstream and reduced white blood cell counts but did not induce thrombocytopenia. This suggests that Ad26 virions do not induce direct clotting side effects seen during coronavirus disease 2019 (COVID-19) vaccination with this serotype of adenovirus. IMPORTANCE The human species D Ad26 is being investigated as a low-seroprevalence vector for oncolytic virotherapy and gene-based vaccination against HIV-1 and SARS-CoV-2. However, there is debate in the literature about its tropism and receptor utilization, which directly influence its efficiency for certain applications. This work was aimed at determining which receptor(s) this virus uses for infection and its role in virus biology, vaccine efficacy, and, importantly, vaccine safety.

Single-particle EM reveals plasticity of interactions between the adenovirus penton base and integrin αVß3.

Human adenoviruses are double-stranded DNA viruses responsible for numerous infections, some of which can be fatal. Furthermore, adenoviruses are currently used in clinical trials as vectors for gene therapy applications. Although initial binding of adenoviruses to host attachment receptors has been extensively characterized, the interactions with the entry receptor (integrins) remain poorly understood at the structural level. We characterized the interactions between the adenovirus 9 penton base subunit and αVß3 integrin using fluorescence correlation spectroscopy and single-particle electron microscopy to understand the mechanisms underlying virus internalization and infection. Our results indicate that the penton base subunit can bind integrins with high affinity and in several different orientations. These outcomes correlate with the requirement of the pentameric penton base to simultaneously bind several integrins to enable their clustering and promote virus entry into the host cell.

In vivo protein crystallization opens new routes in structural biology.

Protein crystallization in cells has been observed several times in nature. However, owing to their small size these crystals have not yet been used for X-ray crystallographic analysis. We prepared nano-sized in vivo-grown crystals of Trypanosoma brucei enzymes and applied the emerging method of free-electron laser-based serial femtosecond crystallography to record interpretable diffraction data. This combined approach will open new opportunities in structural systems biology.

Structure of adenovirus type 21 knob in complex with CD46 reveals key differences in receptor contacts among species B adenoviruses.

The complement regulation protein CD46 is the primary attachment receptor for most species B adenoviruses (Ads). However, significant variability exists in sequence and structure among species B Ads in the CD46-binding regions, correlating with differences in affinity. Here, we report a structure-function analysis of the interaction of the species B Ad21 knob with the two N-terminal repeats SCR1 and SCR2 of CD46, CD46-D2. We have determined the structures of the Ad21 knob in its unliganded form as well as in complex with CD46-D2, and we compare the interactions with those observed for the Ad11 knob-CD46-D2 complex. Surface plasmon resonance measurements demonstrate that the affinity of Ad21 knobs for CD46-D2 is 22-fold lower than that of the Ad11 knob. The superposition of the Ad21 and Ad11 knob structures in complex with CD46-D2 reveals a substantially different binding mode, providing an explanation for the weaker binding affinity of the Ad21 knob for its receptor. A critical difference in both complex structures is that a key interaction point, the DG loop, protrudes more in the Ad21 knob than in the Ad11 knob. Therefore, the protruding DG loop does not allow CD46-D2 to approach the core of the Ad21 knob as closely as in the Ad11 knob-CD46-D2 complex. In addition, the engagement of CD46-D2 induces a conformational change in the DG loop in the Ad21 knob but not in the Ad11 knob. Our results contribute to a more profound understanding of the CD46-binding mechanism of species B Ads and have relevance for the design of more efficient gene delivery vectors.

Viral attachment strategies: the many faces of adenoviruses.

The engagement of a cell-surface receptor by a virus is the first step in a complex process that culminates in the infection of the cell. Human adenoviruses are important pathogens that, depending on serotype, can utilize an impressive number of cellular receptors for attachment. Several structures of adenovirus attachment proteins in complex with their cognate ligands have been determined, enhancing our understanding of the underlying molecular recognition processes. Moreover, two new receptors for adenoviruses have also recently been identified. Using adenoviruses as an example, we examine the different attachment strategies that highly similar viral proteins within a single virus family can employ to engage different protein and carbohydrate receptors. For many viruses, consequences of such differences are altered tropism and pathogenicity. A better understanding of the parameters that determine specificity will therefore help to establish links between receptor tropism, spread, and disease. Moreover, it has relevance for the design and use of adenovirus-based gene delivery vehicles with improved properties and for the development of new antiviral strategies.