The Fiber Knob Protein of Human Adenovirus Type 49 Mediates Highly Efficient and Promiscuous Infection of Cancer Cell Lines Using a Novel Cell Entry Mechanism

Abstract

The human adenovirus (HAdV) phylogenetic tree is diverse, divided across seven species and comprising over 100 individual types. Species D HAdV are rarely isolated with low rates of preexisting immunity, making them appealing for therapeutic applications. Several species D vectors have been developed as vaccines against infectious diseases, where they induce robust immunity in preclinical models and early phase clinical trials. However, many aspects of the basic virology of species D HAdV, including their basic receptor usage and means of cell entry, remain understudied. Here, we investigated HAdV-D49, which previously has been studied for vaccine and vascular gene transfer applications. We generated a pseudotyped HAdV-C5 presenting the HAdV-D49 fiber knob protein (HAdV-C5/D49K). This pseudotyped vector was efficient at infecting cells devoid of all known HAdV receptors, indicating HAdV-D49 uses an unidentified cellular receptor. Conversely, a pseudotyped vector presenting the fiber knob protein of the closely related HAdV-D30 (HAdV-C5/D30K), differing in four amino acids from HAdV-D49, failed to demonstrate the same tropism. These four amino acid changes resulted in a change in isoelectric point of the knob protein, with HAdV-D49K possessing a basic apical region compared to a more acidic region in HAdV-D30K. Structurally and biologically we demonstrate that HAdV-D49 knob protein is unable to engage CD46, while potential interaction with coxsackievirus and adenovirus receptor (CAR) is extremely limited by extension of the DG loop. HAdV-C5/49K efficiently transduced cancer cell lines of pancreatic, breast, lung, esophageal, and ovarian origin, indicating it may have potential for oncolytic virotherapy applications, especially for difficult to transduce tumor types.IMPORTANCE Adenoviruses are powerful tools experimentally and clinically. To maximize efficacy, the development of serotypes with low preexisting levels of immunity in the population is desirable. Consequently, attention has focused on those derived from species D, which have proven robust vaccine platforms. This widespread usage is despite limited knowledge in their basic biology and cellular tropism. We investigated the tropism of HAdV-D49, demonstrating that it uses a novel cell entry mechanism that bypasses all known HAdV receptors. We demonstrate, biologically, that a pseudotyped HAdV-C5/D49K vector efficiently transduces a wide range of cell lines, including those presenting no known adenovirus receptor. Structural investigation suggests that this broad tropism is the result of a highly basic electrostatic surface potential, since a homologous pseudotyped vector with a more acidic surface potential, HAdV-C5/D30K, does not display a similar pantropism. Therefore, HAdV-C5/D49K may form a powerful vector for therapeutic applications capable of infecting difficult to transduce cells.

Keywords: adenoviruses; anticancer therapy; oncolytic viruses; surface receptor.

FIG 1

HAdV-C5/D49K infection is not dependent upon CAR or CD46. Transduction assays were performed in Chinese hamster ovary (CHO) cells stably expressing CAR (A), CHO-BC1 cells stably expressing human CD46 isoform BC1 (B), or CHO-K1 cells, which do not express any known adenovirus fiber knob receptors (C and D). Cells were infected with 5,000 viral particles per cell of replication-deficient HAdV-C5, HAdV-C5/B35K, or HAdV-C5/49K expressing a GFP transgene (A to C) or HAdV-C5, HAdV-C5/49K, or HAdV-C5/49F expressing luciferase (D). n = 3; error is expressed ± the standard deviations (SD).

FIG 2

HAdV-C5/49K transduction is not dependent upon HSPGs, sialic acid bearing glycans or Desmoglein 2 (DSG2). Transduction assays were performed in CHO-K1 (A) or SKOV-3 (B) cells with or without heparinase pretreatment. As a positive control, HAdV-C5 assays were performed also in the presence of 10 μg/ml of FX. Transduction assays were performed with the indicated viral vectors in CHO-K1 (C) or SKOV-3 (D) cells that had been pretreated with neuraminidase to cleave cell surface sialic acid. Cells were infected with 5,000 viral particles per cell of replication-deficient HAdV-C5, HAdV-C5/B35K, or HAdV-C5/49K expressing a GFP transgene. n = 3; error is expressed ± the SD. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001 (based on a nonparametric Mann-Whitney test).

FIG 3

HAdV-D49K interacts with CAR but is not dependent upon it as an entry receptor. (A) SPR was used to detect potential interactions between HAdV-D49K or HAdV-D49.KO1.K and CAR, CD46, and DSG2. (B) Antibody binding inhibition assays were used to assess the ability of recombinant HAdV-D49K to inhibit antibody binding to CHO-CAR or CHO-BC1 cells. (C and D) Blocking of HAdV-C5-mediated transduction was studied by preincubation of CHO-CAR cells with HAdV-C5K (C) or HAdV-D49K (D). (E and F) Blocking of HAdV-C5/D49K-mediated transduction was studied by preincubation of CHO-CAR cells with HAdV-C5K (E) or HAdV-D49K (F). (G and H) Blocking of HAdV-C5/D49K-mediated transduction was studied by preincubation of CHO-K1 cells with HAdV-C5K (G) or HAdV-D49K (H). Cells were infected with 5,000 viral particles per cell of replication-deficient HAdV-C5 or HAdV-C5/D49K expressing a luciferase transgene, with or without blockade by 20 μg of recombinant HAdV-C5 or HAdV-D49 fiber knob protein. n = 3; error is expressed ± the SD. *, P < 0.05; **, P < 0.01 (based on a nonparametric Mann-Whitney test).

FIG 4

HAdV-D49K differs from HAdV-D30K in only three surface-exposed amino acids but demonstrates radically altered cellular tropism. (A) Clustal Ω sequence alignment (numbering is based on the whole fiber sequence) of HAdV-D30K and HAdV-D49K. (B and C) Viewed from the apex down the 3-fold axis, as if toward the viral capsid, the crystal structures of HAdV-D30K (B) and HAdV-D49K (C) reveal that three of these residues are surface exposed. These residues can be seen projecting into the solvent from loops on the apex of HAdV-D30K (D) and HAdV-D49K (E). Residue numbers and names correspond to the fiber knob protein depicted in that frame. Sticks representing residues belonging to HAdV-D30K and HAdV-D49K are seen in pink and green, respectively. (F and G) Transduction assays were performed to assess tropism of HAdV-C5/D30K, HAdV-C5/D49K, and HAdV-C5/D49.KO1.K in CHO-CAR cells (F) and CHO-K1 cells (G). Cells were infected with 5,000 viral particles per cell of replication-deficient HAdV-C5 or HAdV-C5/D49K expressing a luciferase transgene, with or without blockade by 20 μg of recombinant HAdV-C5 or HAdV-D49 fiber knob protein.

FIG 5

The residue differences between HAdV-D30K and HAdV-D49K affect the surface electrostatic potential of the fiber knob. The calculated pIs of HAdV-D30K and HAdV-D49K differ as a result of the residue changes, which are shown as green sticks as they occur in that fiber knob protein. The calculated electrostatic surface potential at pH 7.35 is projected on a ramp from –10 mV to +10 mV (red to blue). HAdV-D49K has much more basic potential around the apex, where the residue substitutions are located.