Virus entry is a complex process that involves interactions with multiple cellular compartments, transport to and from them, and different sorting pathways. A productive infection by a virus requires a proper association with its receptors, internalization into the host cell, and the delivery of its genomic content to the site of replication, all of which can be influenced by the cell types that the virus infects (36
). Therefore, it is important to dissect the entry mechanism of a virus in its natural host cell type to gain the most relevant knowledge about the infectious course. In this study, we characterize several intracellular trafficking events of BKV in RPTE cells, the cell type in which the virus persists and reactivates in vivo (8
). Pharmacological experiments demonstrate that BKV infection requires a low-pH step, an intact microtuble network, and trafficking to the ER. A more-detailed time course reveals the kinetics of these events, as follows: BKV travels to an acidic compartment soon after internalization, which is followed by movement along the microtubule network, presumably within a vesicular compartment, to reach the ER (Fig. , steps A to C).
FIG. 8. Proposed model of BKV entry in RPTE cells. (A) BKV enters an acidic compartment following caveola-mediated endocytosis, where the low pH triggers conformational changes within the capsid, beginning the disassembly process. (B) BKV proceeds to the pH-neutral (more ...)
Disassembly is another important step during polyomavirus entry, as the highly structured virion needs to be dissociated to allow entry of the genome into the nucleus. We show that the extensive disulfide bond network in the BKV capsid starts to be disintegrated at about 8 to 12 h postinfection. Interestingly, at similar times, we begin to detect two distinct, smaller fragments of VP1 that likely represent specific proteolytic cleavage products. To our knowledge, this is the first time that cleavage of the capsid protein VP1 of a polyomavirus has been observed during the course of infection. At this time, we do not know the sites of cleavage, and the epitope that the P5G6 monoclonal antibody detects is not known. Furthermore, whether the rearrangement and cleavage of VP1 molecules lead to a productive infection remains to be determined.
The endocytic events associated with BKV trafficking in RPTE cells are far from being completely understood. BKV is seen within caveosome-like structures by EM analysis (10
), and the colocalization between labeled BKV and caveolin-1, a major component of the caveosome (39
), supports the idea that BKV passes through the caveosome en route to the nucleus. The caveosome is a pH-neutral organelle (43
); however, our results clearly show that BKV infection requires a low-pH step. In addition, colocalization between BKV and caveolin-1 peaks at ~4 h postinfection (39
), which is beyond the time of the low-pH requirement. Cross talk between the caveosome and the endosomal compartments has been observed in both polyomavirus and papillomavirus infections (42
). It is likely that an unidentified endosomal/lysosomal compartment may take part in BKV transport prior to the caveosome (Fig. , steps A and B). Furthermore, the role that low pH plays during BKV trafficking is still unknown. Treatment with NH4
Cl can completely block the rearrangement of the disulfide bond network within the BKV virion and VP1 cleavage. Therefore, it is likely that the low pH induces conformational changes within the capsid that lead to further virion disassembly and membrane penetration (Fig. , step A), as has been observed in the adenovirus endosomal escape process (55
). Alternatively, the acidic pH may be required for certain cellular proteases to function, as is the case for reovirus capsid disassembly (15
). It is also possible that NH4
Cl treatment traps the virus in a compartment that prevents it from further trafficking to the site of disassembly.
All the polyomaviruses that have been examined so far enter the ER compartment (10
); the BFA inhibition data of BKV infection are consistent with this. The ER Derlin family proteins have been implicated in the ER escape of SV40 and MPyV (29
). Our results show that, similar to SV40, Derlin-1 is important for BKV infection in RPTE cells. Moreover, we are able to demonstrate, for the first time, an interaction between the major capsid protein VP1 and Derlin-1. This strongly argues that Derlin-1 plays a direct role in facilitating the exit of BKV from the ER. Demonstration of an interaction between VP1 and Derlin-1 by coimmunoprecipitation during the course of infection, however, was not feasible due to the high background of VP1 adhering to the protein A/G agarose beads even with control serum (data not shown). The question that remains to be answered is whether an intact or partially disassembled virion is transported out of the ER. Derlin-1 has been implicated as part of a retrotranslocation channel (54
). The size of the channel, however, may not be large enough to accommodate the intact BKV virion (38
). For SV40 and MPyV, the current model proposes that oxidoreductases residing in the ER cause conformational changes in the capsid, which in turn lead to ER escape and membrane penetration (35
). Once the virus encounters the low-Ca2+
environment in the cytosol, further disassembly occurs to aid in genome delivery to the nucleus (48
). Whether this model also applies to BKV uncoating requires further investigation.
The involvement of the proteasome during BKV infection is also intriguing. A specific proteasome inhibitor, lactacystin, reduces the infectivity of BKV but does not block BKV capsid rearrangement or VP1 cleavage. Proteasome function has been associated with multiple entry events of different viruses, including endosomal penetration and nuclear translocation (7
), and the specific step at which it is required for BKV infection remains to be determined. Since Derlin-1 is closely associated with ERAD components (30
), it is possible that BKV may be transported to the proteasome from the ER via Derlin-1 (Fig. , step E). Alternatively, the effect of lactacystin on BKV infection may be indirect by impacting Derlin-1 function.
Our results show a unique pattern of BKV capsid protein rearrangement and cleavage. This observation raises several questions. First, the cellular location(s) of VP1 rearrangement and cleavage is not clear. BFA treatment results in an increased level of overall disulfide bond network breakdown and an accumulation of the VP1** molecule, suggesting that the ER is not the site of either event. Lactacystin treatment has a similar effect on capsid rearrangement and VP1 cleavage, and both drugs have an inhibitory effect on TAg expression. These results imply that the presence of either drug may reroute the virus to a pathway that is more favorable for capsid rearrangement (Fig. , step D). Second, specific cleavage products of VP1 are seen during infection (Fig. ), indicating that cleavage is not a random event. Identification of the cleavage sites on the VP1 molecules may help us elucidate the mechanism of capsid rearrangement. Third, what triggers the disassembly process remains elusive. Low pH certainly plays a significant role, but other signals such as the presence of host factors or certain cellular environments may also contribute. Our results indicate that the breakdown of the disulfide network and VP1 cleavage occur at similar times, and it would be interesting to examine whether these processes are interdependent. Finally, additional genetic and biochemical analyses are warranted to determine whether the capsid rearrangement process and/or VP1 cleavage detected by our assays represent part of an infectious or noninfectious pathway of BKV infection.
In conclusion, our results show that BKV entry in RPTE cells is a highly regulated process, engaging coordinated interactions between viral structures and cellular components. We demonstrate that BKV infection and capsid rearrangement involve low-pH activation, trafficking to the ER, and components of the ERAD pathway. Understanding these steps at the detailed molecular level will expand our insight on general virus entry mechanisms. Moreover, it may help identify important immune regulators, such as the innate immune receptors that are involved in BKV infection, and offer potential intervention targets for BKV-related diseases.