Lipid rafts are small, heterogeneous, cholesterol- and glycosphingolipid-enriched domains that play important roles in cellular processes such as membrane signaling and trafficking (31
). They are also involved in multiple stages of the virus life cycle, such as attachment, internalization, uncoating, protein transport, assembly, and budding (53
). Several pieces of evidence indicate that B19V exploits lipid rafts during the process of entry: (i) colocalization of B19V and its receptor globoside (Gb4Cer) with the lipid raft marker GM1 (), (ii) a shift of flotillin migration toward nonraft fractions in infected cells (), and (iii) inhibition of B19V infection following lipid raft disruption (). The fact that lipid raft disruption had no effect a few hours after infection indicates that plasma membrane rafts are important for the infectious entry/trafficking of B19V and not for later steps. The exact mechanism by which these membrane microdomains contribute to virus entry is not known. Preliminary results indicate that lipid raft disruption does not inhibit virus attachment (data not shown). Gb4Cer is required but is not sufficient for B19V infection. Other receptors have been shown to be important in the process of viral entry (12
). Thus, lipid rafts may act as platforms for the concentration of virus receptors/coreceptors required for B19V infection, as has been shown for other viruses (55
Clathrin-dependent endocytosis is the default uptake mechanism for parvoviruses (3
). To date, only adeno-associated virus 5 (AAV-5) and porcine parvovirus use additional routes based on caveolae and macropinocytosis, respectively (2
). Since caveolae are integral parts of some lipid rafts and B19V associates with rafts, a possibility of caveola-mediated internalization of B19V into UT7/Epo cells could be envisioned. Although Gb4Cer was associated with both caveolar and noncaveolar rafts, B19V associated preferentially with noncaveolar rafts (). By means of electron microscopy, B19V was recurrently observed in clathrin-coated invaginations and vesicles (); however, viruses in flask-shaped invaginations characteristic of caveolae were not observed. While at the attachment step B19V did not colocalize with transferrin, which is internalized through clathrin-mediated endocytosis (26
), extensive colocalization was observed during the internalization step (). In contrast to the slow internalization by caveolae, cargo internalized by clathrin-mediated endocytosis is quickly delivered to early endosomes (39
). Consistent with a rapid internalization mechanism, immunofluorescence pictures taken 5 min p.i. confirmed the presence of B19V capsids in early endosomes (). Therefore, although B19V interacts with plasma membrane rafts, internalization occurs by clathrin-mediated endocytosis and does not involve caveolae. This mechanism of internalization based on lipid rafts and clathrin has been observed in other viruses (19
We have found that BafA1 and NH4
Cl inhibited viral infection by blocking viral escape, resulting in the accumulation of viral particles in the degradative lysosomes. However, CQ, which also alkalinizes the endosomes, enhances the infection. We have shown previously that CQ enhances B19V infection in UT7, HepG2, and primary bone marrow mononuclear cells (8
). The case of HepG2 cells is particularly striking. These cells are considered nonpermissive for B19V infection (10
); however, in the presence of CQ, HepG2 cells support B19V infection (8
). The reason for this enhancement can be explained, at least in part, by the particular effects of CQ on endosomal vesicles. CQ causes vesicle swelling and hampers the fusion of endosomes and lysosomes, preventing the transfer of endocytosed material to the degradative lysosomal compartment (28
). Because of these particular properties, CQ is frequently used in transfection experiments to increase transduction efficiency (32
). We confirmed that CQ, but not BafA1, induced the vacuolization of endocytic vesicles () and prevented the transfer of the incoming capsids to the degradative lysosomes (). We could also confirm that CQ, but not BafA1, prevented the degradation of incoming B19V DNA (). The capsids retained in vacuolated prelysosomal vesicles would profit by progressively escaping into the cytosol.
Although the effect of CQ on endosomal vesicles is independent of the cell type, only B19V, and no other parvovirus, can benefit (4
). All parvoviruses studied to date exploit endosomal acidification for capsid modifications required for subsequent steps, primarily the externalization of N-VP1 and its constitutive PLA2
domain, which is thought to be required for endosomal escape (35
). In addition, nuclear localization signal (NLS) sequences have been identified in the N-VP1 proteins from some parvoviruses, which might assist in the transport of capsids toward the nucleus (57
). We have recently shown that B19V is unique among parvoviruses in that N-VP1 becomes externalized upon receptor binding (7
). Therefore, in contrast to other parvoviruses, B19V would not depend on low endosomal pH for this critical conformational rearrangement. However, B19V would require acidification to facilitate the process of endosomal escape, for example, by promoting interactions between the PLA2
domain of N-VP1 and the endocytic membranes.
At increasing postinternalization times, incoming capsids appeared dispersed throughout the cytoplasm and did not colocalize with endocytic markers, the ER, caveolin-containing vesicles, the Golgi apparatus, or recycling vesicles, suggesting that they had escaped into the cytosol. The PLA2
activity of N-VP1 is thought to play a role in endosomal escape; however, the mechanism is not known. Adenovirus is able to release coendocytosed dextrans of different sizes, implying a mechanism of escape based on endosomal disruption. Human rhinovirus 2 is able to release only small dextrans, suggesting a less invasive mechanism of escape based on pore formation (43
). The escape mechanism of canine parvovirus (CPV) has been studied by cointernalization of alpha-sarcin, where no disruption of endosomes was found (41
). In other studies, however, CPV was able to release rhodamine-labeled dextrans of 3 kDa but not of 10 kDa (52
). However, the effect was evident only 20 h postinfection, while endosomal escape of CPV occurs earlier. In addition, a parvovirus capsid would not be able to escape through a pore that selectively allows the escape of dextrans of 3 kDa but not of 10 kDa. In the case of B19V, there was no detectable leakage of dextrans of 3 kDa at any time or in the presence of CQ ( and ). Two possibilities can be envisaged: either capsids permeabilize only a minor amount of endocytic vesicles or capsids escape through a yet unidentified mechanism, which does not involve membrane damage.
Viral capsids ( and ) or viral DNA () was not detected inside the nucleus, even after the onset of viral transcription (). Thus, B19V is inefficiently imported into the nucleus, making it difficult to study this step of the infection. In clear contrast, in CQ-treated cells, B19V DNA was efficiently imported into the nucleus, reaching a plateau by 3 h postinfection (). At this time, more than half of the total viral DNA was found associated with the nuclear fraction (). Examination of the incoming nuclear DNA confirmed that the viral DNA was not associated with capsids, which remained extranuclear (, , and ). In CQ-treated cells, the number of intact capsids decreased significantly (). Since the viral DNA remained stable () and a large proportion was found in the nucleus ( and ), the decrease in the number of intact capsids cannot be attributed to degradation, as was the case for untreated cells. These results would suggest either that the viral DNA is imported into the nucleus from intact capsids that are immediately uncoated or that uncoating takes place prior to the import of the DNA into the nucleus. The second possibility seems more plausible, since no capsids were found in the nucleus at any time point.
Our studies provide the first insight into the early steps of B19V infection and reveal mechanisms involved in virus uptake, endocytic trafficking, and nuclear import. This study also outlines novel questions that warrant further investigation, such as the precise role of lipid rafts in the process of virus entry, the mechanism by which B19V escapes from endosomes without detectable permeabilization/damage, and the pathway involved in the nuclear import of viral DNA.