Type‐I interferons suppress microglial production of the lymphoid chemokine, CXCL13

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108063/1/glia22692.pd

Complexity of the microglial activation pathways that drive innate host responses during lethal alphavirus encephalitis in mice

Microglia express multiple TLRs (Toll-like receptors) and provide important host defence against viruses that invade the CNS (central nervous system). Although prior studies show these cells become activated during experimental alphavirus encephalitis in mice to generate cytokines and chemokines that influence virus replication, tissue inflammation and neuronal survival, the specific PRRs (pattern recognition receptors) and signalling intermediates controlling microglial activation in this setting remain unknown. To investigate these questions directly in vivo, mice ablated of specific TLR signalling molecules were challenged with NSV (neuroadapted Sindbis virus) and CNS viral titres, inflammatory responses and clinical outcomes followed over time. To approach this problem specifically in microglia, the effects of NSV on primary cells derived from the brains of wild-type and mutant animals were characterized in vitro. From the standpoint of the virus, microglial activation required viral uncoating and an intact viral genome; inactivated virus particles did not elicit measurable microglial responses. At the level of the target cell, NSV triggered multiple PRRs in microglia to produce a broad range of inflammatory mediators via non-overlapping signalling pathways. In vivo, disease survival was surprisingly independent of TLR-driven responses, but still required production of type-I IFN (interferon) to control CNS virus replication. Interestingly, the ER (endoplasmic reticulum) protein UNC93b1 facilitated host survival independent of its known effects on endosomal TLR signalling. Taken together, these data show that alphaviruses activate microglia via multiple PRRs, highlighting the complexity of the signalling networks by which CNS host responses are elicited by these infections

A Chaperone-Activated Nonenveloped Virus Perforates the Physiologically Relevant Endoplasmic Reticulum Membrane▿

The nonenveloped polyomavirus (Py) traffics from the plasma membrane to the endoplasmic reticulum (ER), where it penetrates the ER membrane, allowing the viral genome to reach the nucleus to cause infection. The mechanism of membrane penetration for Py, and for other nonenveloped viruses, remains poorly characterized. We showed previously that the ER chaperone ERp29 alters the conformation of Py coat protein VP1, enabling the virus to interact with membranes. Here, we developed a membrane perforation assay and showed that the ERp29-activated Py perforates the physiologically relevant ER membrane, an event that likely initiates viral penetration. Biochemical analysis revealed that the internal protein VP2 is exposed in the activated viral particle. Accordingly, we demonstrate that VP2 binds to, integrates into, and perforates the ER membrane; the other internal protein, VP3, binds to and integrates into the ER membrane but is not sufficient for perforation. Our data thus link the activity of a cellular factor on a nonenveloped virus to the membrane perforation event and identify a viral component that mediates this process

The C-Terminal Domain of ERp29 Mediates Polyomavirus Binding, Unfolding, and Infection▿

Penetration of the endoplasmic reticulum (ER) membrane by polyomavirus (PyV) is a decisive step in virus entry. We showed previously that the ER-resident factor ERp29 induces the local unfolding of PyV to initiate the ER membrane penetration process. ERp29 contains an N-terminal thioredoxin domain (NTD) that mediates its dimerization and a novel C-terminal all-helical domain (CTD) whose function is unclear. The NTD-mediated dimerization of ERp29 is critical for its unfolding activity; whether the CTD plays any role in PyV unfolding is unknown. We now show that three hydrophobic residues within the last helix of the ERp29 CTD that were individually mutated to either lysine or alanine abolished ERp29's ability to stimulate PyV unfolding and infection. This effect was not due to global misfolding of the mutant proteins, as they dimerize and do not form aggregates or display increased protease sensitivity. Moreover, the mutant proteins stimulated secretion of the secretory protein thyroglobulin with an efficiency similar to that of wild-type ERp29. Using a cross-linking coimmunoprecipitation assay, we found that the physical interaction of the ERp29 CTD mutants with PyV is inefficient. Our data thus demonstrate that the ERp29 CTD plays a crucial role in PyV unfolding and infection, likely by serving as part of a substrate-binding domain

Dimerization of ERp29, a PDI-like Protein, Is Essential for Its Diverse Functions

Protein disulfide isomerase (PDI)-like proteins act as oxido-reductases and chaperones in the endoplasmic reticulum (ER). How oligomerization of the PDI-like proteins control these activities is unknown. Here we show that dimerization of ERp29, a PDI-like protein, regulates its protein unfolding and escort activities. We have demonstrated previously that ERp29 induces the local unfolding of polyomavirus in the ER, a step required for viral infection. We now find that, in contrast to wild-type ERp29, a mutant ERp29 (D42A) that dimerizes inefficiently is unable to unfold polyomavirus or stimulate infection. A compensatory mutation that partially restores dimerization to the mutant ERp29 (G37D/D42A) rescues ERp29 activity. These results indicate that dimerization of ERp29 is crucial for its protein unfolding function. ERp29 was also suggested to act as an escort factor by binding to the secretory protein thyroglobulin (Tg) in the ER, thereby facilitating its secretion. We show that this escort function likewise depends on ERp29 dimerization. Thus our data demonstrate that dimerization of a PDI-like protein acts to regulate its diverse ER activities