Binding Site on the Transferrin Receptor for the Parvovirus Capsid and Effects of Altered Affinity on Cell Uptake and Infection

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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.

Section: Discussionmentioning

confidence: 74%

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

Callaway

Feng

Lee

et al. 2017

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“…To determine the functionality of eGFP-tagged feline TfR, virus binding and uptake assays were performed with CRFK or TRVb fTfR-eGFP cells as previously described (12). Briefly, cells were detached from the plate, washed in PBS containing 1% ovalbumin (Sigma-Aldrich), and incubated in solution with 10 g/ml of Alexa 647 CPV capsids for the indicated time intervals at 37°C.…”

Section: Cells and Virusesmentioning

confidence: 99%

“…The butterfly-shaped receptor ectodomain spans ϳ11 nm and consists of three subdomains (23). Tf binds to residues in the membrane-proximal protease-like domain and the central helical domain (4), while CPV capsids contact residues in the membrane distal apical domain (12,32). Structural modeling of CPV-TfR interactions suggested that a single capsid can bind up to 24 TfRs, but biochemical and cryo-electron microscopic analysis indicated that CPV capsids bind fewer than 7 TfR ectodomains in solution (14).…”

mentioning

confidence: 99%

Limited Transferrin Receptor Clustering Allows Rapid Diffusion of Canine Parvovirus into Clathrin Endocytic Structures

Cureton

Harbison

Cocucci

et al. 2012

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b Viral pathogens usurp cell surface receptors to access clathrin endocytic structures, yet the mechanisms of virus incorporation into these structures remain incompletely understood. Here we used fluorescence microscopy to directly visualize the association of single canine parvovirus (CPV) capsids with cellular transferrin receptors (TfR) on the surfaces of live feline cells and to monitor how these CPV-TfR complexes access endocytic structures. We found that most capsids associated with fewer than five TfRs and that ϳ25% of TfR-bound capsids laterally diffused into assembling clathrin-coated pits less than 30 s after attachment. Capsids that did not encounter a coated pit dissociated from the cell surface with a half-life of ϳ30 s. Together, our results show how CPV exploits the natural mechanism of TfR endocytosis to engage the clathrin endocytic pathway and reveal that the low affinity of capsids for feline TfRs limits the residence time of capsids on the cell surface and thus the efficiency of virus internalization.

“…Infection of NLFK, A72, or Cf2Th cells was assessed using standard methods for 50% tissue culture infective dose (TCID 50 ) assays (34). Cell binding was analyzed using purified virus capsids at 10 g/ml incubated in a solution for 60 min at 37°C with NLFK or Cf2Th cells, or with TRVb cells transfected with a plasmid expressing the feline, canine, or raccoon TfR (6). Virus capsids were detected with MAb 2 and an Alexa 488-conjugated goat anti-mouse antibody (Sigma, St. Louis, MO), and binding was quantified using a Guava EasyCyte Plus flow cytometer (Millipore).…”

Section: Evolutionary Analysismentioning

confidence: 99%

Role of Multiple Hosts in the Cross-Species Transmission and Emergence of a Pandemic Parvovirus

Allison

Harbison

Pagán

et al. 2012

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121

Understanding the mechanisms of cross-species virus transmission is critical to anticipating emerging infectious diseases. Canine parvovirus type 2 (CPV-2) emerged as a variant of a feline parvovirus when it acquired mutations that allowed binding to the canine transferrin receptor type 1 (TfR). However, CPV-2 was soon replaced by a variant virus (CPV-2a) that differed in antigenicity and receptor binding. Here we show that the emergence of CPV involved an additional host range variant virus that has circulated undetected in raccoons for at least 24 years, with transfers to and from dogs. Raccoon virus capsids showed little binding to the canine TfR, showed little infection of canine cells, and had altered antigenic structures. Remarkably, in capsid protein (VP2) phylogenies, most raccoon viruses fell as evolutionary intermediates between the CPV-2 and CPV-2a strains, suggesting that passage through raccoons assisted in the evolution of CPV-2a. This highlights the potential role of alternative hosts in viral emergence.