A Human In Vitro Model to Study Adenoviral Receptors and Virus Cell Interactions

Abstract

To develop adenoviral cell- or tissue-specific gene delivery, understanding of the infection mechanisms of adenoviruses is crucial. Several adenoviral attachment proteins such as CD46, CAR and sialic acid have been identified and studied. However, most receptor studies were performed on non-human cells. Combining our reporter gene-tagged adenovirus library with an in vitro human gene knockout model, we performed a systematic analysis of receptor usage comparing different adenoviruses side-by-side. The CRISPR/Cas9 system was used to knockout CD46 and CAR in the human lung epithelial carcinoma cell line A549. Knockout cells were infected with 22 luciferase-expressing adenoviruses derived from adenovirus species B, C, D and E. HAdV-B16, -B21 and -B50 from species B1 as well as HAdV-B34 and -B35 were found to be CD46-dependent. HAdV-C5 and HAdV-E4 from species E were found to be CAR-dependent. Regarding cell entry of HAdV-B3 and -B14 and all species D viruses, both CAR and CD46 play a role, and here, other receptors or attachment structures may also be important since transductions were reduced but not completely inhibited. The established human knockout cell model enables the identification of the most applicable adenovirus types for gene therapy and to further understand adenovirus infection biology.

Adenovirus type 34 and HVR1-deleted Adenovirus type 5 do not bind to PF4: clearing the path towards vectors without thrombosis risk

“…Incubation of Ad5 or Ad69 particles with PF4 increased VP uptake in A549 cells by around 2 to 3 fold in average, while PF4 did not influence Ad34 internalization (figure 3d). PF4 effect on infectivity was not significantly altered by deletion of CAR (primary receptor of Ad5 and Ad69 ( 19 )) or CD46 (primary receptor of Ad34 and Ad69 ( 19 )) in target cells (figure 3d). In order to test whether PF4 enhanced productive infections by Ad5 and Ad69 or only abortive internalizations, we measured Ad-driven luciferase expression in infected cells and detected an increase for both Ads and in all tested cell lines (figure 3e).…”

“…The A549-ΔCAR and A549-ΔCD46 cell lines were constructed by deleting respectively the CAR and CD46 receptors using CRISPR-Cas9 ( 19 ). Cells were cultivated with Dulbecco’s Modified Eagle Medium (DMEM, Pan-Biotech; for 293, A549-WT, A549-CAR and A549-ΔCD46 cells) or Roswell Park Memorial Institute 1640 Medium (RPMI, Pan-Biotech; for SKOV-3 cells), each supplemented with 10% Fetal Bovine Serum (FBS, Pan-Biotech) and Penicillin-Streptomycin (P/S, Pan-Biotech) at 37°C under an atmosphere with 5% CO 2 .…”

Adenovirus receptors on antigen‐presenting cells of the skin

“…As the name suggests, CAR is an attachment molecule for coxsackieviruses (subgroup B) and some human and nonhuman (e.g., canine type 2) AdVs, with a notable exception of species B HAdVs (Bergelson et al., 1997; Roelvink et al., 1998; Soudais et al., 2000). Of note, the use of CAR has recently been implicated in species E HAdV uptake (Tsoukas et al., 2022). AdV attachment to high affinity receptors is typically, but not exclusively, mediated by the fiber knob (Louis et al., 1994).…”

HEHR: Homing Endonuclease-Mediated Homologous Recombination for Efficient Adenovirus Genome Engineering

“…We have previously developed high-throughput adenoviral genome direct cloning via linear–linear homologous recombination (LLHR) and genome modification via linear–circular homologous recombination (LCHR) ( Figure 1 ) [ 6 , 7 ]. With this strategy, various new adenovirus vectors have been generated for receptor studies, as oncolytic viruses for tumor treatment, as gene delivery vector for gene therapy, as well as novel immunotherapeutic strategies to combat severe adenovirus infection in immunocompromised hosts [ 8 , 9 , 10 , 11 , 12 ].…”