VP2 Cleavage and the Leucine Ring at the Base of the Fivefold Cylinder Control pH-Dependent Externalization of both the VP1 N Terminus and the Genome of Minute Virus of Mice

Abstract

Cylindrical projections surrounding the fivefold-symmetry axes in minute virus of mice (MVM) harbor central pores that penetrate through the virion shell. In newly released DNA-containing particles, these pores contain residues 28 to 38 belonging to a single copy of VP2, disposed so that its extreme N-terminal domain projects outside the particle. Virions are metastable, initially sequestering internally the N termini of all copies of the minor capsid protein, VP1, that is essential for entry. This VP1 domain can be externalized in vitro in response to limited heating, and we show here that the efficiency of this transition is greatly enhanced by proteolysis of VP2 N termini to yield VP3. This step also renders the VP1 rearrangement pH dependent, indicating that VP2 cleavage is a maturation step required to prime subsequent emergence of the VP1 "entry" domain. The tightest constriction within the cylinder is created by VP2 leucine 172, the five symmetry-related copies of which form a portal that resembles an iris diaphragm across the base of the pore. In MVMp, threonine substitution at this position, L172T, yields infectious particles following transfection at 37°C, but these can initiate infection only at 32°C, and this process can be blocked by exposing virions to a cellular factor(s) at 37°C during the first 8 h after entry. At 32°C, the mutant particle is highly infectious, and it remains stable prior to VP2 cleavage or following cleavage at pH 5.5 or below. However, upon exposure to neutral pH following VP2 cleavage, its VP1-specific sequences and genome are extruded even at room temperature, underscoring the significance of the VP2 cleavage step for MVM particle dynamics. Viral particles function as entry "nanomachines" that not only protect the genome from environmental assaults encountered during transmission, but also recognize and respond to a succession of specific cellular signals that allow them to navigate complex entry portals in their host cell and ultimately deliver their nucleic acid to the appropriate cellular replication compartment. How nonenveloped viruses achieve this odyssey is generally poorly understood, but in several virus families, a virion protein that is known to be essential for membrane penetration is subject to a specific proteolytic cleavage (6, 8). This allows the particle to exist in a "metastable" state, where the lowest energy form of the cleaved product is sequestered by the energy barrier between the two forms (19). During entry, these viruses encounter some form of catalyst, such as low pH or an interaction with a specific receptor, which releases the metastable configuration, allowing the de novo exposure of sequences required for membrane penetration. A variety of "penetration proteins" have been identified, including the N-myristoylated VP4 protein of poliovirus (3, 4, 17), the membrane lytic 1 protein of reovirus (7), and protein VI of adenovirus (42). Here, we provide evidence for an activation cleavage step in the maturation of virions of the murine parvovirus minu...

Visualization of the Externalized VP2 N Termini of Infectious Human Parvovirus B19

“…Proteolytic cleavage of the externalized N terminus converts VP2 into the amino-terminal-shortened VP3 (9,12,37,51,57), a maturation step that has been suggested to prime the virions of the genus Parvovirus for VP1u exposure during cellular trafficking (11). Such maturation cleavage does not occur in B19, possibly because VP1u is always external.…”

Toll-Like Receptor 9 in Plasmacytoid Dendritic Cells Fails To Detect Parvoviruses

“…We have shown that a large portion of the MVMp virions is excluded from either LAMP2-or TLR9-containing vesicles in RAW cells, suggesting that a majority of MVMp traffics away from the TLR9 signaling endosomes in pDCs. In addition, the MVMp capsid is extremely stable, capable of withstanding severe environmental conditions, including low pH (7,16). Thus, it is possible that the capsids remain impenetrable within the endolysosome, thereby restricting genomic DNA access for TLR9.…”

Structurally Mapping the Diverse Phenotype of Adeno-Associated Virus Serotype 4

“…The protrusions surrounding the threefold axis create valley-like depressions between the two-and threefold axes, as observed in Aleutian mink disease virus and B19. The cylindrical channel at the fivefold axis of the parvovirus capsid, which is implicated in packaging of the parvovirus genome and the phospholipase activity of the VP1 unique region (4,16,17), is conserved.…”