Low pH-Dependent Endosomal Processing of the Incoming Parvovirus Minute Virus of Mice Virion Leads to Externalization of the VP1 N-Terminal Sequence (N-VP1), N-VP2 Cleavage, and Uncoating of the Full-Length Genome

Abstract

Minute virus of mice (MVM) enters the host cell via receptor-mediated endocytosis. Although endosomal processing is required, its role remains uncertain. In particular, the effect of low endosomal pH on capsid configuration and nuclear delivery of the viral genome is unclear. We have followed the progression and structural transitions of DNA full-virus capsids (FC) and empty capsids (EC) containing the VP1 and VP2 structural proteins and of VP2-only virus-like particles (VLP) during the endosomal trafficking. Three capsid rearrangements were detected in FC: externalization of the VP1 N-terminal sequence (N-VP1), cleavage of the exposed VP2 N-terminal sequence (N-VP2), and uncoating of the full-length genome. All three capsid modifications occurred simultaneously, starting as early as 30 min after internalization, and all of them were blocked by raising the endosomal pH. In particles lacking viral single-stranded DNA (EC and VLP), the N-VP2 was not exposed and thus it was not cleaved. However, the EC did externalize N-VP1 with kinetics similar to those of FC. The bulk of all the incoming particles (FC, EC, and VLP) accumulated in lysosomes without signs of lysosomal membrane destabilization. Inside lysosomes, capsid degradation was not detected, although the uncoated DNA of FC was slowly degraded. Interestingly, at any time postinfection, the amount of structural proteins of the incoming virions accumulating in the nuclear fraction was negligible. These results indicate that during the early endosomal trafficking, the MVM particles are structurally modified by low-pH-dependent mechanisms. Regardless of the structural transitions and protein composition, the majority of the entering viral particles and genomes end in lysosomes, limiting the efficiency of MVM nuclear translocation.

Bovine parvovirus uses clathrin-mediated endocytosis for cell entry

“…Thus, acidification can be essential for viruses which internalize within CCVs (Damm & Pelkmans, 2006;Marsh & Helenius, 2006;Smith & Helenius, 2004). Studies of parvovirus entry reported that minute virus of mice, adeno-associated virus and CPV internalization require endosomal acidification, and the endosomal acidic environment may induce capsid conformational changes vital for viral release from endosomes to the cytoplasm (Basak & Compans, 1989;Douar et al, 2001;Mani et al, 2006). Blocking acidification of endosomes is a way of suggesting involvement of the clathrin system in cargo entry.…”

“…Previously, reported studies have demonstrated that the clathrin-dependent endocytic pathway is the primary route for uptake for some members of the family Parvoviridae into cells (Douar et al, 2001;Mani et al, 2006;Ros et al, 2002;Vendeville et al, 2009). To determine BPV entry dependence on clathrin, CHPZ, a cationic amphiphilic drug that inhibits the formation of clathrin-coated pits at the plasma membrane was used.…”

Clathrin-Associated Endocytosis as a Route of Entry into Cells for Parvoviruses

“…The relatively rapid endocytic uptake of parvoviruses appears to be followed by slower traffic along the endocytic compartments toward the nucleus. The endosomal pathway undertaken by parvoviruses appears to be complex and depends on the virus, its concentration, and likely the cell type (Dorsch et al, 2002;Mani et al, 2006;Sonntag et al, 2006;Suikkanen et al, 2003;Yuan & Parrish, 2001). Conformational alterations in capsid structure probably occur in the endosomal compartment facilitating uncoating and transport to the nucleus.…”

“…It has been widely used as a probe to study acid-mediated viral trafficking using a variety of virus models. Entry pathway studies of mouse polyomavirus (Liebl et al, 2006), feline calicivirus (Stuart & Brown, 2006), bovine viral diarrhea virus (Lecot et al, 2005), AAV (Bartlett et al, 2000), MVM (Mani et al, 2006;Ros et al, 2002), mouse hepatitis virus type 2 (Pu & Zhang, 2008), poliovirus , baculovirus (Long et al, 2006), hepatitis C virus (Blanchard et al, 2006), influenza virus (Guinea & Carrasco, 1995;Sieczkarski et al, 2003), HIV-1 (Fredericksen et al, 2002), and human rhinovirus 14 (Bayer et al, 1999) have analyzed the effects of Baf A1 on virus trafficking and examined the role of endosomal acidification in a route that leads to viral replication. For studies of parvovirus entry Baf A1 may be an inhibitor preferred over chlorpromazine because chlorpromazine is not only an inhibitor of clathrin lattice processing, but is also an inhibitor of phospholipase A2.…”

“…Chemical inhibitors are assumed to block the entry pathway at certain specific points and incomplete virus replication in the presence of inhibitor is presumed to indicate viral penetration using the pathway blocked by the drug. Commonly used drugs are chlorpromazine (prevents assembly and disassembly of clathrin lattices at the cell surface and on endosomes by inhibiting clathrin polymerization which blocks assembly of coated pits and is also a phospholipase A2 inhibitor, Blanchard et al, 2006); bafilamycin A1 (inhibits vacuolar proton ATPases inhibiting acidification of endosomes by blocking transport of protons into the vesicle, Clague et al, 1994;Drose & Altendorf, 1997;Wassmer et al, 2005); ammonium chloride (penetrates into the endosome increasing endosomal pH, Jin et al, 2002;Liebl et al, 2006); chloroquine (blocks assembly of clathrin coated pits and raises endosomal pH, Mani et al, 2006;Ros et al, 2002). Brefeldin A has no effect on the early endosome but inhibits vesicle transport and early-to-late endosome transition (Clague et al, 1994;Nebenfuhr et al, 2002).…”