Viruses promote their own survival and proliferation in the host cell by hijacking cellular machineries that normally serve physiological functions. For example, during entry, many nonenveloped viruses co-opt the properties or activities of various host receptors, proteases, or chaperones to facilitate viral penetration across a host limiting membrane and gain access to the cytosol (24
). Defining the mechanisms by which viruses divert normal physiological processes to achieve pathological ends is fundamental to understanding the nature of the host-pathogen interaction. Here, we characterized a host chaperone, ERp29, which is co-opted by the MPyV to facilitate its penetration across the ER membrane. The results of this study demonstrate that the hydrophobic residues in the last α-helix of ERp29 CTD (i.e., L240, I246, and L247 in helix α9) are critical to ERp29's pathogen-related activities, likely due to a role for the CTD in substrate binding.
Although differences in the structure of the rat ERp29 CTD as determined by nuclear magnetic resonance (9
) and the crystal structures of Wind (11
) and human ERp29 (submitted recently to the Protein Data Bank [accession number 2qc7]) make the specific relative orientation of helices α8 and α9 uncertain, all of these structures at least indicate that ERp29 L240 (Wind L232) is surface exposed, suggesting that it is available to interact with a substrate or potential cofactors. ERp29 I246 and L247 (Wind I238 and L239, respectively) are likely buried within the helix bundle; their mutations may affect the positioning of L240 or the local integrity of helix α9 and thereby indirectly affect the interaction of ERp29 with PyV or a cofactor. Intriguingly, ERp29 residue L244 (Wind L236), which is not required for PyV unfolding, is exposed on the opposite surface of the CTD from L240, suggesting that PyV binds exclusively to one side of the ERp29 CTD. As we demonstrated previously that the C-terminal arms of the VP1 pentamers are exposed in an ERp29-dependent manner (12
), we suspect that helix α9 of the ERp29 CTD likely makes contact with this region of the viral capsid. Of note, ERp29 helix α9 does not appear to mediate the binding and secretion of Tg, indicating that the mechanisms by which ERp29 engages its physiological and foreign substrates are distinct.
It remains unclear whether one CTD of the ERp29 dimer binds to PyV or whether the two CTDs of dimeric ERp29 simultaneously act on the virus. Additionally, whether ERp29 interacts with PyV using only its CTD or in cooperation with the NTD is a key question. Interestingly, Wind has been shown to bind to its substrate Pipe via its NTD and helix α8/α9 of the CTD (1
). Thus, while a major function of the ERp29 NTD is to mediate dimerization of ERp29, which we showed previously is required for ERp29 activity (16
), this domain may play an additional role in binding to PyV. Future studies will further clarify the substrate-binding roles of the ERp29 NTD and CTD.
ERp29 is one of several proteins in the PDI family of ER chaperones that are manipulated by viruses and other pathogens. For example, in addition to ERp29 being required by PyV, downregulation of canonical PDI inhibits PyV infection (8
). Downregulation of PDI and the PDI-like protein ERp72 affects the infection of cells by the related PyV simian virus 40 (SV40) in opposing manners, presumably through mechanisms related to their protein-folding and isomerase activities (18
). Furthermore, the PDI-like protein ERp57 promotes the disulfide bond rearrangements of the SV40 capsid in the ER, an event that likely initiates SV40 uncoating (18
). Bacterial toxins, such as cholera toxin, also hijack PDI-related proteins during their intracellular trafficking (6
). Why PDI-like proteins are uniquely targeted by pathogens and pathogenic factors is not known. These ER chaperones display a myriad of cellular activities. They function normally to ensure the proper folding of newly synthesized proteins by catalyzing disulfide bond formation and isomerization reactions. Furthermore, the PDI-like proteins participate in the ER quality control process, in which they act to refold misfolded substrates or to unfold them in preparation for removal from the ER (4
). Hence, the ability of PDI-like proteins to perform a variety of functions coupled with the diversity of substrates on which they act likely contribute to the tendency of PDI-like proteins to be co-opted by viruses and toxins during entry.