Nonenveloped virus penetration of the limiting membrane represents a decisive step in virus infection. Host factors facilitate this process by imparting conformational changes to the virus to initiate membrane penetration. Although multiple host factors likely work together to mediate this process, until now, only individual cellular components responsible for these remodeling events have been identified; more complex mechanisms remain undefined. In the present study, we pinpoint a network of ER-resident proteins that act on Py distinctly and coordinately to facilitate infection. They do so by modifying the viral disulfide bonds to promote its ER membrane penetration. We also identify specific viral cysteines that play pivotal roles in this process.
Using the siRNA knockdown strategy, we demonstrate that individually downregulating ERp57, ERp72, or PDI decreased infection, while simultaneously downregulating any two of these proteins attenuated infection more severely. The fact that infection was not blocked completely is likely due to incomplete downregulation of the PDI family proteins. Additionally, compensation by other PDI family members may contribute to this effect. Under none of the knockdown conditions did we observe induction of significant ER stress, suggesting that the decrease in virus infection is not due to nonspecific effects. Instead, these results point to the possibility that the PDI family members engage Py directly. Although knockdown of PDI in a human cell line was shown previously to block Py infection (6
), whether PDI interacts with Py directly is not known.
For SV40, downregulation of ERp57 and PDI attenuated infection (20
), consistent with our observation for Py. However, different results using two independent siRNAs to downregulate ERp72 were observed: one enhanced infection, while the other did not have an effect (20
). For Py, downregulating ERp72 using two different siRNAs decreased infection. The reason for this discrepancy is not clear, but it may be due to the different disulfide bond arrangements in the two viruses. In addition, the PDI-like protein ERp29 does not appear to act on SV40 (20
), in contrast to its established role in Py infection (11
). As ERp29 extrudes Py's VP1 C-terminal arm to initiate viral disassembly (11
), the precise disassembly mechanisms in Py and SV40 may be different.
To test the hypothesis that ERp57, PDI, and ERp72 affect Py directly, we expressed and purified the three proteins to homogeneity. When incubated with Py, these proteins individually induced the formation of virus-derived products consisting of VP1 monomer, trimer, pentamer, and higher oligomers. VP1 monomer is likely generated when the five disulfide bonds within a pentamer (formed between C19 of one monomer and C114 of another monomer) are disrupted. Interestingly, a double band pattern corresponding to the size of the monomer was observed. This pattern could be explained by the formation of a new intramonomer disulfide bond within a subset of the monomers, causing a different mobility pattern. This doublet pattern is more obvious for the ERp57- and ERp72-mediated reactions than the PDI-triggered reaction. Precisely how this new intramonomer disulfide bond is formed is unknown. One possibility is that a free cysteine in VP1 of the native Py is used to form a new disulfide bond. Alternatively, a new free cysteine generated in VP1 after reduction may possibly catalyze intramonomer disulfide bond formation. Why PDI-mediated reduction of Py did not generate the doublet pattern as efficiently as the ERp72-dependent reaction is not clear.
How is a trimer, but not a dimer or a tetramer, generated? In principle, the trimers could be formed from three monomers within a pentamer that are disulfide bonded to each other. Alternatively, given our findings that suggest the presence of interpentamer disulfide bonds, the trimers could be formed from two monomers from a pentamer (linked to each other by a disulfide bond), with one of the monomers forming an interpentamer disulfide bond with a monomer from an adjacent pentamer.
During disassembly by the PDI family proteins, transient viral intermediates may form that expose certain disulfide bonds while rendering others less exposed. This creates a situation in which certain disulfide bonds are preferentially disrupted over others. Indeed, the intrinsic asymmetric property of Py—with 12 pentamers surrounded by 5 pentamers and 60 pentamers surrounded by 6 pentamers—favors the presence of nonequivalent disulfide bonds during viral disassembly. Although it is not entirely clear why trimers, but not tetramers or dimers, were generated in our experiments, this finding suggests that disulfide bonds within a trimer are likely less exposed than a tetramer or dimer during the course of disassembly, thereby rendering the disulfide bonds linking the trimers less easy to disrupt.
How are VP1 pentamers and higher oligomers liberated from the intact virus? One possibility is that, as the C19-C114 disulfide bonds within a pentamer clamp the invading VP1 C-terminal arm in place (24
), disruption of this bond loosens the C-terminal arm, thereby generating VP1 pentamers and higher oligomers. Alternatively, interpentamer disulfide bonds may be disrupted to produce these species. The X-ray structure of Py, which did not indicate the presence of interpentamer disulfide bonds (24
), did not include information on the VP1 N-terminal C11 and C15 residues. Our present analyses suggest that these residues stabilize interpentamer interactions for a subset of the pentamers, possibly by forming interpentamer disulfide bonds (see below).
Despite the finding that ERp57, PDI, and ERp72 can individually act on Py to form the virus-derived species, only ERp57 and PDI cooperate with ERp29 to extrude the VP1 C-terminal arm. We showed previously that this reaction generates a hydrophobic viral particle that binds and disrupts ER membrane integrity (11
), events that initiate virus penetration across the ER membrane. Although PDI can use its chaperone activity to unfold cholera toxin (CT) in the ER to initiate the toxin's translocation into the cytosol (3
), the catalytic, but not chaperone, activity of PDI (and ERp57) is required to assist ERp29 in unfolding the VP1 C-terminal arm. As there are two pentamer types in Py (12 five coordinated and 60 six coordinated) (9
), it is possible that the ERp57-PDI-ERp29 network acts on one of these two types, while ERp72 engages the other.
Our next key finding indicated that free viral cysteines play an important role in Py infection, as an alkylated virus cannot promote infection. Consistent with this observation, we found that while PDI and ERp72 function as reductases in engaging Py in vitro
, ERp57 acts as an isomerase, a reaction that requires free viral cysteines. We have yet to pinpoint the precise mechanism by which ERp57, PDI, and ERp72 act on Py in cells, because less than 5% of the total internalized Py reaches the ER from the cell surface (16
). However, the biochemical analyses, coupled with the infection result of the alkylated virus, point to a role of isomerization facilitated by viral cysteines as a potential critical reaction during infection. This idea is supported by our identification of a role for VP1's C11 and C15 residues in infection, as mutating C11 to A, C15 to A, or both residues to A decreased infection by 50%. These mutations did not significantly affect the global viral conformations, suggesting a specific role of these cysteines in infection. One interpretation of these data is that some of the C11 and C15 residues exist in the free and reduced state, enabling them to participate in isomerization.
Strikingly, using a nonreducing SDS-PAGE system, VP1 higher oligomers were generated when both C11 and C15 were mutated to A, but not when only a single cysteine was altered. This finding demonstrates that C11 and C15 stabilize interpentamer interactions for a subset of the pentamers, possibly by forming interpentamer disulfide bonds between some of the pentamers. If these residues stabilized all interpentameric interactions, a VP1 pentamer, but not a higher oligomer, would appear. Moreover, because mutating a single cysteine did not generate the higher oligomers, the potential interpentamer disulfide bonds likely exist between two C11 residues and two C15 residues. Py's C11 is the homolog of SV40 C9, which is implicated in C9-C9 interpentamer disulfide bonding (20
). Because Py contains two types of pentamers, certain pentamers will be situated in a different local environment than others. This structural asymmetry likely allows some C11 and C15 residues to be disulfide bonded while others are in the reduced state. This situation is similar to that of C104 in SV40's VP1, where it is thought to exist in both the reduced and disulfide-bonded states (20
While we cannot formally rule out the possibility that C11 and C15 are used to stabilize interpentamer contacts without forming interpentamer disulfide bonds, we consider this possibility unlikely based on our data provided using NEM-Py. Specifically, alkylation of these cysteine residues with NEM would presumably disrupt their ability to make non-disulfide-bond-mediated stabilizing contacts, thereby generating higher-order VP1 species. However, on nonreducing SDS-PAGE, we did not observe higher-order VP1 species containing only NEM-Py.
The double Py mutant (C11A-C15A) did not display a more pronounced decrease in infection than the single cysteine mutants. This could be because C11 and C15 are normally used to isomerize Py to generate the VP1 higher-oligomer intermediate that is critical for successful infection. Preformation of this intermediate in the double mutant thus renders these cysteine residues dispensable. However, the fact that the double mutant was nonetheless defective in infection indicates that this mutant harbors subtle structural alterations that do affect infection. The precise nature of this structural defect is not known, but it may be due to premature disassembly in the endolysosomes prior to arrival at the ER.
Although we have identified three additional PDI family members in the ER that engage and facilitate Py infection, whether additional ER components act on Py is not known. In this context, it is interesting that in the in vitro
trypsin digestion assay, ERp57 or PDI added in combination with a reduced ERp29-enriched ER lumenal extract (which lacks ERp57 and PDI) generated VP1b. The fact that reducing the ERp29-enriched lumenal extract was required in this reaction suggests that ERp29 in the extract must be in the reduced form to unfold Py (ERp29 contains one cysteine). Indeed, there are precedents for the redox state of a PDI protein controlling its chaperone-unfolding activity (27
). Alternatively, additional reductases/isomerases in the extract may be involved in triggering a penetration-competent viral particle. Further experiments are required to distinguish these possibilities.
In conclusion, our findings unveil a complex interplay between viral cysteine residues and host reductases, isomerases, and chaperones of the PDI family that act coordinately and distinctly on Py to facilitate infection. These reactions promote transport of the virus from the ER into the cytosol, a pivotal infection step. PDI family members not only engage endogenous cellular substrates (7
), they are often coopted by toxins and viruses during infection (3
). The vast number of members within this family (1
), coupled with their functional versatility, thus renders them attractive targets for pathogens during entry.