Intracellular pathogens, which include viruses and some bacteria, typically disseminate through extracellular fluids before entering their target cells and beginning replication. While in the extracellular environment, pathogens can be intercepted by humoral immunity or by professional immune cells. However, immune surveillance is not always sufficient to prevent infection, and all cells need innate mechanisms to detect and disable pathogens.

Intracellular complement C3 activates innate immunity. Complement component C3 covalently attaches to pathogens in the extracellular space. Upon pathogen entry into the cytosol, the cell senses attached C3. Sensing of C3 triggers a dual sensor and effector response, involving mitochondrial antiviral signaling (MAVS)–dependent immune signaling and proteasome-mediated viral degradation.

We hypothesized that one method of pathogen detection may be to take advantage of the pathogen’s transition between extracellular and intracellular environments. Complement is a system of immune serum proteins with the ability to attach covalently to pathogens. We investigated whether this irreversible tagging of pathogens results in complement component C3 being carried into the cytosol during infection. Given that C3 should not otherwise be present inside the cell, we tested whether this could act as an invasion signal.

Antibodies and complement components C3 and C4, but not other serum proteins, were found to associate with adenovirus. During adenoviral infection, deposited C3 was carried into cells, resulting in potent nuclear factor–κB (NF-κB) activation. Activation of NF-κB by C3 required viral entry into the cytosol, and no activity was observed when C3-coated adenovirus was trapped in endosomes. In addition to NF-κB, C3 also activated the activating protein 1 (AP-1) and interferon regulatory factor 3 (IRF3)/IRF5/IRF7 transcription pathways. Induction of these signaling pathways resulted in robust cytokine secretion, including interferon-β. Cytosolic C3 sensing was dependent on a number of signaling hubs known to be involved in innate immunity. In addition to activating immune signaling, C3 targeted cytosolic adenovirus for rapid degradation via the AAA–adenosine triphosphatase (ATPase) valosin-containing protein (VCP) and the proteasome. This degradation pathway potently restricted viral infection.

C3-dependent intracellular sensing was widely conserved in mammals. Moreover, C3 activated NF-κB upon infection of diverse cell lines and primary cells including human lung cells, a physiologically relevant adenovirus target. Intracellular C3 also activated NF-κB in response to infection by diverse nonenveloped viruses—including papillomavirus, astrovirus, calicivirus, rhinovirus, poliovirus, coxsackievirus, enterovirus, and the facultative cytosolic bacteria Salmonella—but not enveloped respiratory syncytial virus. Picornaviruses were much less susceptible to complement sensing as a result of antagonism by their 3C protease, which cleaved C3 and prevented NF-κB activation and proteasome-mediated restriction. However, treatment with the 3C antagonist rupintrovir prevented 3C cleavage and restored full complement sensing of rhinovirus and poliovirus.

Complement mediates a potent intracellular immune response to nonenveloped viruses and cytosolic bacteria. The deposition and covalent attachment of C3 onto pathogens results in its translocation into cells during infection, in which it simultaneously induces an antiviral state and directs the degradation of viral particles. Intracellular complement immunity is highly effective against a range of pathogens …