Virus Structure

Capsid Structure, Function, and Variation

Parvoviruses infect cells after binding receptors, and enter cells by endocytosis, passing from the endocome to the  is a host range variant of a feline virus which acquired the ability to infect dogs through changes in its capsid protein. Canine and feline viruses both use the feline transferrin receptor (TfR) to infect feline cells, and here we show that CPV infects canine cells through its ability to specifically bind the canine TfR. Receptor binding on host cells at 37ºC only partially correlated with the host ranges of the viruses, and an intermediate virus strain (CPV type-2) bound to higher levels on cells than did either the feline panleukopenia virus or a later strain of CPV. During the process of adaptation to dogs that later variant strain of CPV gained the ability to both more efficiently use the canine TfR for infection, and also showed reduced binding to feline and canine cells compared to CPV type-2. Differences on the top and the side of the three fold spike of the capsid surface controlled specific TfR and other receptor binding, and also determined the infection properties of the viruses.

Capsid_Structure_smallThe structure of the CPV capsid determined by cryoEM, showing the raised regions around the threefold axes of symmetry, which are the binding sites of the TfRs.

Canine parvovirus (CPV) and feline panleukopenia virus (FPV)capsids bind the transferrin receptors (TfR) of their hosts and use those receptors to infect cells. The binding is host specific, as FPV binds only to the feline TfR, while CPV binds to both the canine and feline TfRs. The host-specific binding of the capsids is controlled by a small number of sequences that cluster in a raised region of the capsid, the threefold spike. To define the TfR structure that interacts with the virus, we introduced changes into the apical domain of the feline or canine receptor or prepared chimeras between them, and tested those altered receptors for their abilities to bind FPV or CPV capsids. Most changes in the apical domain of the feline receptor did not affect binding. However, substitution of Leu221 in the feline TfR with serine or aspartic acid stopped the receptor binding to both FPV and CPV capsids, and when Leu221 was replaced with Lys the receptor still bound CPV but bound poorly to FPV. Analysis of recombinants between the feline and canine TfRs showed that sequences controlling virus-specific binding were also within the apical domain. However, more than one sequence difference between those receptors was required to endow the feline TfR with the CPV-specific binding of the canine TfR. Single changes within the canine TfR which removed a single amino acid insertion or eliminated a glycosylation site gave that receptor the ability to bind FPV. Binding of capsids to some mutant receptors did not result in infection, suggesting a specific role for the receptor in cell infection by the viruses.

Fig2 JVI
The binding of the capsids of CPV or FPV to TfRs from different hosts – from cat, raccoon, dog, black backed jackal, or human – those show different affinities of binding based on variation of the TfR or the capsid. From Callaway et al., 2018.
We have examined the structures of the viral capsids for small changes that may have biological roles. Those include tryptophan fluorescence, bis-ANS binding, and sensitivity to protease treatment. We show the effect of heating on the intrinsic tryptophan fluorescence of CPV capsids. There is a linear decline in fluorescence due to thermal quenching, but no significant effect of the heating on the capsid structure until the particle falls apart around 60-70ºC.

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Publications

Dunbar, C.A., Callaway, H.M., Parrish, C.R., Jarrold, M.F. Probing Fragment Antibody Binding to Canine Parvovirus Capsids with Charge Detection Mass Spectrometry. Journal of the American Chemical Society, Accepted.

Callaway, H.M., Welsch, K., Weichert, W., Allison, A.B., Hafenstein, S.L., Huang, K., Iketani, S., Parrish, C.R. (2018). Complex and dynamic interactions between parvovirus capsids, transferrin receptors and antibodies control cell infection and host range. Journal of Virology, 92 no. 13 e00460-18

Callaway, H.M., Feng, K.H., Lee, D.W., Allison, A.B., Pinard, M., McKenna, R., Agbandje-McKenna, M., Hafenstein, S., Parrish, C.R. (2016) Parvovirus capsid structures required for infection: mutations controlling receptor recognition and protease cleavages. Journal of Virology, 91:e01871-16

Nelson, C.D.S., Minkinen, E., Bergkvist, M., Hoelzer, K., Fisher, M., Bothner, B.P., Parrish, C.R. (2008). Detecting small changes and additional peptides in the canine parvovirus capsid structure. Journal of Virology 82:10397-10407

Nelson, C.D.S., Palermo L.M., Hafenstein, S.L., Parrish, C.R. (2007). Different mechanisms of antibody-mediated neutralization of parvoviruses revealed using the Fab fragments of monoclonal antibodies. Virology 361:283-293.

Palermo, L. M., S. L. Hafenstein, and C. R. Parrish. (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 80:8482-8492.

Hueffer, K., Parrish, C.R. (2003). Parvovirus host range, cell tropism and evolution. Current Opinion in Microbiology. 6:392-398.

Govindasamy, L., Hueffer, K, Parrish, C.R., Agbandje-McKenna, M.(2003). The structures of host range controlling regions of the capsids of canine and feline parvoviruses and mutants. Journal of Virology. 77:12211-12221.

Simpson, A.A., Hebert, B., Sullivan, G.M., Parrish, C.R., Zadori, Z., Tjissen, P. and Rossmann, M.G. (2002). The structure of porcine parvovirus: comparison with related viruses. Journal of Molecular Biology 315: 1189-1198.

Simpson A.A., Chandrasekar V., Hebert B., Sullivan G.M, Rossmann M.G., Parrish C.R. (2000). Host range and variability of calcium binding by surface loops in the capsids of canine and feline parvoviruses. Journalof Molecular Biology 300:597-610.