Parvovirus Capsid Structures Required for Infection: Mutations Controlling Receptor Recognition and Protease Cleavages

Abstract

Parvovirus capsids are small but complex molecular machines responsible for undertaking many of the steps of cell infection, genome packing, and cell-tocell as well as host-to-host transfer. The details of parvovirus infection of cells are still not fully understood, but the processes must involve small changes in the capsid structure that allow the endocytosed virus to escape from the endosome, pass through the cell cytoplasm, and deliver the single-stranded DNA (ssDNA) genome to the nucleus, where viral replication occurs. Here, we examine capsid substitutions that eliminate canine parvovirus (CPV) infectivity and identify how those mutations changed the capsid structure or altered interactions with the infectious pathway. Amino acid substitutions on the exterior surface of the capsid (Gly299Lys/Ala300Lys) altered the binding of the capsid to transferrin receptor type 1 (TfR), particularly during virus dissociation from the receptor, but still allowed efficient entry into both feline and canine cells without successful infection. These substitutions likely control specific capsid structural changes resulting from TfR binding required for infection. A second set of changes on the interior surface of the capsid reduced viral infectivity by Ͼ100-fold and included two cysteine residues and neighboring residues. One of these substitutions, Cys270Ser, modulates a VP2 cleavage event found in ϳ10% of the capsid proteins that also was shown to alter capsid stability. A neighboring substitution, Pro272Lys, significantly reduced capsid assembly, while a Cys273Ser change appeared to alter capsid transport from the nucleus. These mutants reveal additional structural details that explain cell infection processes of parvovirus capsids.IMPORTANCE Parvoviruses are commonly found in both vertebrate and invertebrate animals and cause widespread disease. They are also being developed as oncolytic therapeutics and as gene therapy vectors. Most functions involved in infection or transduction are mediated by the viral capsid, but the structure-function correlates of the capsids and their constituent proteins are still incompletely understood, especially in relation to identifying capsid processes responsible for infection and release from the cell. Here, we characterize the functional effects of capsid protein mutations that result in the loss of virus infectivity, giving a better understanding of the portions of the capsid that mediate essential steps in successful infection pathways and how they contribute to viral infectivity.

Complex and Dynamic Interactions between Parvovirus Capsids, Transferrin Receptors, and Antibodies Control Cell Infection and Host Range

“…2B and D). This effect has also been previously observed in studies of a CPV-2 mutant with lysine substitutions of VP2 residues 299 and 300, which bound the feline TfR but did not infect cells (41).…”

“…1M), and a third variant contained all 10 surface substitutions in both loops 1 and 3. Wild-type VP2, Asn93Lys VP2, and the three mutant forms were prepared as VLPs by expression of VP2 in High Five cells and purified as described previously (41).…”

“…Protein purity for TfRs, scFv-Fcs, Fabs, VLPs, and parvovirus empty capsids was determined by silver staining samples run on 10% SDS-PAGE gels with a Pierce silver stain kit (Thermo Scientific) according to the manufacturer's protocol. Stability of mutant VLPs was determined by differential scanning fluorimetry of purified VLPs, as previously described (41). Three replicates were performed for each sample except for the N93K VLPs, where limited sample quantity allowed only one measurement.…”

“…The variant interactions resulting from virus and TfR differences may involve both the affinity of capsid binding to the TfR as well as additional structural interactions essential for virus infectivity of cells. This was seen most clearly in studies where we showed that some capsid variants bound efficiently to the TfR and were endocytosed into cells but did not result in infection (41). However, the details of the TfR-capsid interactions are not yet well understood, including the stoichiometry of receptor binding, the structures involved in attachment, and whether the number of bound receptors is important for successful infection.…”

Transferrin receptor binds virus capsid with dynamic motion

“…The interactions between CPV and TfR are intricate, as there appears to be no direct relationship between the affinity of TfR binding and the success of the infection; indeed, some receptor mutants bind with high affinity but do not mediate infection, whereas the binding of the infection-competent canine TfR has very low affinity (36,37). The capsids only engage 1 or a small number of TfRs on the surface of feline cells and also bind only small numbers of receptors when capsids are mixed with purified feline TfRs (17,36).…”