The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

Abstract

Mammalian adenoviruses (AdVs) comprise more than ~350 types including over 100 human (HAdVs) and just three mouse AdVs (MAdVs). While most HAdVs initiate infection by high affinity/avidity binding of their fiber knob (FK) protein to either coxsackievirus AdV receptor (CAR), CD46 or desmoglein (DSG)-2, MAdV-1 (M1) infection requires arginine-glycine-aspartate (RGD) binding integrins. To identify the receptors mediating MAdV infection we generated five novel reporter viruses for MAdV-1/-2/-3 (M1, M2, M3) transducing permissive murine (m) CMT-93 cells, but not B16 mouse melanoma cells expressing mCAR, human (h) CD46 or hDSG-2. Recombinant M1 or M3 FKs cross-blocked M1 and M3 but not M2 infections. Profiling of murine and human cells expressing RGD-binding integrins suggested that αvβ6 and αvβ8 heterodimers are associated with M1 and M3 infections. Ectopic expression of mβ6 in B16 cells strongly enhanced M1 and M3 binding, infection, and progeny production comparable with mαvβ6-positive CMT-93 cells, whereas mβ8 expressing cells were more permissive to M1 than M3. Anti-integrin antibodies potently blocked M1 and M3 binding and infection of CMT-93 cells and hαvβ8-positive M000216 cells. Soluble integrin αvβ6, and synthetic peptides containing the RGDLXXL sequence derived from FK-M1, FK-M3 and foot and mouth disease virus coat protein strongly interfered with M1/M3 infections, in agreement with high affinity interactions of FK-M1/FK-M3 with αvβ6/αvβ8, determined by surface plasmon resonance measurements. Molecular docking simulations of ternary complexes revealed a bent conformation of RGDLXXL-containing FK-M3 peptides on the subunit interface of αvβ6/β8, where the distal leucine residue dips into a hydrophobic pocket of β6/8, the arginine residue ionically engages αv aspartate215, and the aspartate residue coordinates a divalent cation in αvβ6/β8. Together, the RGDLXXL-bearing FKs are part of an essential mechanism for M1/M3 infection engaging murine and human αvβ6/8 integrins. These integrins are highly conserved in other mammals, and may favour cross-species virus transmission.

Adenovirus entry: Stability, uncoating, and nuclear import

“…Interestingly, MAdV-1 and -3 evolved a dual integrin binding motif, RGD, and LXXL (L for leucine and X for any amino acid). This motif has higher affinity for extended open integrins engaged in cytoskeletal interaction, and lower affinity for conformationally closed integrins that are not constrained by the cytoskeleton (Bieri et al, 2021;Campbell & Humphries, 2011).…”

“…This binding occurs through the major capsid protein hexon (Cassany et al, 2015;Trotman et al, 2001). A locking factor, or the lack of critical ubiquitination providing a positive signal for dismantling, precludes the NPC-docked virion from disassembly (Bauer et al, 2021). The E3 ubiquitin ligase MIB1 primes the virion for disassembly at the NPC by recruiting the disassembly machinery (2), or targeting the locking factor for proteasomal degradation (3).…”

“…This reaction renders the viral DNA in complex with the condensing protein VII a nuclear import substrate recognized by nuclear import factors (6). Remarkably, insufficient ubiquitination of protein V increases the misdelivery of viral DNA to the cytoplasm, instead of the nucleus, as shown by the all lysine to arginine HAdV-C5 mutant (Bauer et al, 2021) (Dohner & Sodeik, 2005;Greber & Way, 2006;Scherer et al, 2020;Wang et al, 2018;Welte, 2004;Witte et al, 2018). HAdVs use motor proteins for bidirectional trafficking on microtubules to the nucleus after endosomal escape (Engelke et al, 2011;Gazzola et al, 2009).…”

“…Both laboratories excluded an involvement of MIB1 in steps upstream of the NPC by assessing viral endocytosis, protein VI exposure (a surrogate for endosomal rupture), and transport to the nucleus (Bauer et al, 2019). They also used chemical inhibitors, such as LMB to block the attachment of virions to the NPC (Strunze et al, 2005), and showed that LMB was effective also in the absence of MIB1 (Bauer et al, 2019(Bauer et al, , 2021Sarbanes et al, 2021). Furthermore, both laboratories ectopically expressed catalytically inactive MIB1 in MIB1-KO HeLa or Hap1 cells and demonstrated that the ubiquitination activity of MIB1 was crucial for virion uncoating at the NPC.…”

“…To identify viral targets of MIB1 ubiquitination, Bauer, Gomez-Gonzalez et al used differential ubiquitin pulldown assays with a diglycine-specific antibody from normal and MIB1-KO cells, followed by quantitative mass spectrometry (Bauer et al, 2021). They found that four lysine residues of protein V were specifically ubiquitinated in normal, but not in MIB1-KO, cells.…”