The work presented in here stems from our long time interest in determining the function of caveolae and their SDs, which is currently unknown. Although, caveolae have been involved in a plethora of cellular functions, to date there is no specific assay for caveolar function, significantly hampering the task of determining the role of the SD.
Early morphological data have suggested that the SDs might play a barrier role, as they seem to oppose the access of cargo such as cationized ferritin [31
] or albumin[32
] to the lumen of caveolae, whereas allowing the access of HRP. Thus, the SDs might modulate caveolae endocytosis, thought to mediate the uptake of cargoes such as serum albumin [33
], integrins [35
], toxins (i.e. cholera toxin) or viruses (i.e. polyoma viruses such as SV40).
SV40 virus binds to cell surface receptors such as GM1 ganglioside [24
] and MHC class I molecules [19
] from where, the virus translocates to preformed membrane invaginations such as those of the caveolae/lipid raft pathway which mediate its internalization. At high concentrations, by clustering GM1 the virus induces the formation of tubular invaginations, which will mediate the virus internalization in caveolae independent manner [24
]. Both pathways converge into the same endosomes from where the virus journeys to the endoplasmic reticulum, cytoplasm and the nucleus where it will replicate (establishing a so-called productive infection). Both lipid raft containing endocytosis pathways (i.e.
caveolae dependent and independent) have been demonstrated to be essential for viral penetration and establishment of productive infection. To note, the virus is able to enter mammalian cells via other uptake pathways, but these pathways do not result in productive infection. Thus, SV40 infectivity at low viral concentrations is a good test for whether the caveolae SDs have any impact on viral entry via caveolae.
De novo expression of PV1 has been shown to induce the formation of caveolae SDs in endothelial cells and fibroblasts in culture. Taking advantage of PV1 ability to induce SD formation, we have expressed it in CV-1 cells, and asked whether it has an effect on SV40 infectivity. Indeed, our data clearly show that expression of PV1 in CV-1 cells significantly reduces SV40 infectivity at low viral concentrations, whereas it does not have an effect at high viral doses. Unfortunately, we could not do these experiments in endothelial cells, the only cell type known to express PV1 and SDs physiologically. While SV40 is able to enter endothelial cells with very low efficiency due to low levels of GM1 expression in these cells, this rarely, if ever, results in productive infection (our unpublished data).
Mechanistically, we were able to rule out the down regulation of cell surface receptors such as GM1 and MHC class I upon PV1 expression, to be responsible for decreased infectivity. Moreover, PV1 expression did not reduce the binding of fluorescent VP1 VLP (a non-infectious SV40 surrogate) to the cell surface, bringing further support to this idea. Interestingly, PV1 did not reduce the internalization of fluorescent VP1 VLP at either low or high dose. However, testing the internalization of VLPs by our flow cytometry assay proved to be challenging due to high avidity of the pentavalent VLPs for their cell surface receptors precluding reliable removal of the non-internalized surface bound VLPs.
These data suggest that either PV1 is by blocking a minor pathway that cannot be easily detected by the flow cytometry assay we have employed or PV1 acts at a latter time in the infective pathway.
In summary we have found conditions by which SV40 infectivity can be drastically reduced in conditions of low viral concentration. These data are significant as the low viral concentration is a norm for initial infection.
- Expression of PV1 in Cv-1 cells reduces SV40 virus infectivity at low viral titer
- PV1 expression does not reduce the cell surface levels of SV40 receptors GM1 ganglioside and MHC class I molecules
- PV1 expression does not inhibit binding and internalization of SV40 VP-1 virus like particles