Virus-Cell Interactions

Parvovirus cell infection – from Cell Surface to the Nucleus

All parvoviruses require receptor-mediated endocytosis for cell infection. CPV is taken up rapidly into cells by clathrin-mediated endocytosis, as has been shown by expression of the Lys44Ala (K44A) dominant interfering mutant of dynamin, and by the co-localization of the incoming virus particles with GFP-labeled adaptor protein 2 (AP2) . Infections are also inhibited by treatment of cells with lysosomotropic agents, such as NH4Cl and bafilomycin A1, indicating that endosomal low pH is required for infection. Shortly after uptake CPV capsids co-localize in endosomes with transferrin. The uptake that occurs after TfR binding occurs very quickly, within about 30 seconds of receptor attachment.

After endocytosis, penetration of CPV capsids into the cytosol is a slow process. Antibodies against the TfR cytoplasmic tail reduced virus infection when injected into cells 4 h after inoculation, showing that many infecting capsids remain associated with the TfR in endocytic compartments for several hours after uptake. Antibodies against the intact CPV capsid injected into the cytoplasm also prevented virus infection when injected 4 or more hours after virus inoculation, indicating that capsid pass slowly out of the vesicle. After uptake capsids were detected in perinuclear compartments for several hours. The mechanism of escape from endocytic vesicles into the cytosol are still unknown, but may involve the VP1- unique region, which contains phospholipase A2 (PLA2) enzyme activity. However, there is no wholesale lysis of the endosomal vesicle as there is little transport of alpha-sarcin into the cytoplasm.

viral entry pathway

It is not clear how the capsid is transported from the site of release from the endosome to the nuclear pore. Although endosomal trafficking carries the virus to the vicinity of the nucleus, active transport mechanisms are likely to be required for the particles or the viral DNA to reach the nuclear pore. The cytoplasm contains a lattice-like mesh of microtubule (MT), actin, and intermediate filament networks which restrict the free diffusion of macromolecular complexes larger than 500 kDa. CPV capsids injected into the cytoplasm rapidly became localized in a perinuclear location, and the capsids mostly entered the nucleus only 3 to 6 h later.

Parvovirus capsids are resistant for inactivation at temperatures up to 65ºC, and by pHs between 3 and 11. However a number of milder treatments change the capsid structure to expose internal sequences and structures, including VP1 N-terminal region and the 3’ end of the viral DNA of CPV. The VP1 unique region could also be detected in vivo 1 to 3 hrs after Student in the labA cell expressing Rab5-GFP active mutant (green), showing the viral capsids (red) that are located within the expanded early endosomes, and this appeared to be correlated with nuclear transport. The 3’ end of the viral genome was also released from the capsids under similar conditions to those that released the VP1 unique region, and that was available as a template for DNA synthesis by added DNA polymerases.

A VP1 sequence (PAKRARRGYK) between residues 4 and 13 likely functions for nuclear transport and that sequence was also exposed on incoming infectious virions, as antibodies specific for the N-terminal unique region blocked infection when injected into cells before virus inoculation. In addition, some specific changes in the VP1 N-terminal basic sequence reduced the relative infectivity of the capsids.

Publications

Lyi, S.M., Tam, M.J., Parrish, C.R. (2014) Parvovirus particles and movement in the cellular cytoplasm and effects of the cytoskeleton. Virology 456-457:342-352.

Cureton, D.K., Harbison, C.E., Cocucci1, E., Parrish.C.R, and Kirchhausen, T. (2012) Limited receptor crosslinking promotes rapid diffusion of canine parvovirus into clathrin endocytic structures. Journal of Virology 86:5330-5340.

Harbison, C.E., Lyi, S.M., Weichert, W.S., Parrish, C.R. (2009). Early steps in cell infection by parvoviruses: host specific differences in cell receptor binding but similar endosomal trafficking. Journal of Virology 83:10504-10514.

Harbison, C.E., Chiorini, J.A., and Parrish, C.R. (2008) The parvovirus capsid odyssey: from the cell surface to the nucleus. Trends in Microbiology 16:208-214

Hafenstein, S., Palermo, L.M., Kostyuchenko, V.A., Xiao, C., Morais, M.C., Nelson, C.D., Bowman, V.D., Battisti, A.J., Chipman, P.R., Parrish, C.R., Rossmann, M.G.(2007). Asymmetric binding of transferrin receptor to parvovirus capsids. Proc Natl Acad Sci U S A. 104: 6585-9.

Palermo, L.M., Hafenstein, S.L., Parrish, C.R. (2006). Purified feline and canine transferrin receptors reveal complex interactions with the capsids of canine and feline parvoviruses that correspond to their host ranges. Journal of Virology 80:8482-92.

Palermo, L.M., Hueffer, K., Parrish, C.R. (2003). Residues in the apical domain of the feline and canine transferrin receptors receptor control host-specific binding and cell infection of canine and feline parvoviruses. Journal of Virology 77: 8915-8923.