Direct Evidence from Single-Cell Analysis that Human α-Defensins Block Adenovirus Uncoating To Neutralize Infection

Abstract

Human ␣-defensins are evolutionarily conserved effectors of the innate immune response with broadly acting antibacterial activity. Their role in antiviral immunity is less well understood. We previously showed that these antimicrobial peptides are potent inhibitors of human adenovirus infection. Based on biochemical studies and indirect evidence from confocal microscopy, we proposed that defensins bind to and stabilize the virus capsid and neutralize infection by preventing the release of the endosomalytic protein VI. To determine whether defensin action also restricts exposure of the viral genome, we developed a system to evaluate adenovirus uncoating during cell entry by monitoring the exposure of BrdU-labeled viral genomes. This assay allowed us to determine the kinetics of uncoating of virus particles in single cells. Using this assay, we now provide direct evidence that human ␣-defensins block adenovirus infection by preventing uncoating during cell entry.

The Human Alpha Defensin HD5 Neutralizes JC Polyomavirus Infection by Reducing Endoplasmic Reticulum Traffic and Stabilizing the Viral Capsid

“…Adenovirus escapes from endosomes after exposure of pVI and subsequent puncturing of the endosomal membrane (63). HD5 blocks exposure of pVI and results in an accumulation in endosomes and lysosomes (29,31). These data suggest that HD5 is acting like "glue," keeping the capsid intact and preventing genome release to the nucleus.…”

“…HD5-treated virions that did colocalize to the ER showed no evidence of VP2 exposure, suggesting that HD5 may also stabilize virions that do the reach the ER from host cell chaperones that drive the unfolding reaction. There is precedent in the literature for this, as other viruses have been shown to be stabilized by alpha defensins (29,47).…”

“…They have been shown recently to neutralize nonenveloped viral infection as well. Adenovirus infection is neutralized by blocking viral uncoating and subsequent release of the genome (29)(30)(31). Alpha defensins have also been shown to block papillomavirus infection by sequestering the virions in their en-docytic vesicles (32).…”

Integrin and Defensin Modulate the Mechanical Properties of Adenovirus

“…It was shown in heat-stability assays of adenovirus that defensin HD5 binding prevents release of the penton from the capsid (16). The subsequent release of the interior capsid protein VI, which is required for endosomal escape, is prevented, thus restricting infection (17). Whereas HD5 acts on the same crucial step in capsid disassembly as does integrin, it does not appear to modulate the stress distribution in the icosahedral shell but simply cements the penton into capsid.…”

“…Binding of defensins to the outer surface of adenovirus blocks endosomal escape and thermally stabilizes the virion. Whereas cell attachment and internalization are unaffected by binding of defensins, stabilization of the virus prevents release of the endosomolytic protein VI and exposure of the internal core viral DNA; this prevents subsequent steps in the virus life cycle, including endosome escape, nuclear localization, and ultimately replication of adenovirus in the host cell (15)(16)(17). Thus, binding of defensin and integrin to adenovirus has opposing effects on capsid disassembly and infectivity.…”