Example: AMDV has 95 aa less than CPV in VP1u and 61 aa more in the major VP. \* Including the VP1/2 common region.

Helix-turn-helix motifs are predicted to be present in the PLA2 domains of the VP1u for most viruses of the *Parvovirinae* (Figure 6). Despite low amino acid sequence identities for the PLA2 domains ranging from 30–50%, the predicted α-helices of the different viruses are well superposable. In addition to the PLA2 domain, some viruses may have other functional domains in their VP1u and/or VP1/2 common regions. Both HBoV1 and CPV possess α-helices near their C-termini (Figure 6b,c). CPV's α-helix in this region has high sequence similarity to the only α-helix in AMDV VP1u (Figure 6a). AAV2, B19, and PARV4 display α-helices near the N-terminus of VP1u (Figure 6d–f). For Parvovirus B19, the presence of a receptor-binding domain at the N terminus, which is predominantly α-helical, has been described before [6,38]. A recent study showed that AAV2 and other AAV serotypes are dependent on a G protein-coupled receptor, GPR108, for effective transduction and that the interaction is dictated by the VP1u region [39]. While the exact amino acids in VP1u for the interaction have not been determined, it is possible that the N-terminal region including the α-helix (aa 9–24) are involved (Figure 6d). PARV4, with its very long VP1u region, displays a substantial α-helical region near the N-terminus. Five α-helices (aa 15–121) were predicted, which could represent a receptor-binding domain (Figure 6f). To date, no receptor has been identified for PARV4. Due to the size, it is highly likely that most, if not all, of the VP1u region of PARV4 is localized on the exterior side of the capsid, similar to that of Parvovirus B19 [40], which would enable PARV4 to bind its receptor with its potential VP1u RBD.

**Figure 6.** Structural prediction for VP1u. Alphafold [35] predictions utilizing the primary sequences of (**a**) the AMDV VP1u and VR-VII regions, (**b**) the HBoV1 VP1u and VP1/2 common region, (**c**) the CPV VP1u, (**d**) the AAV2 VP1u and VP1/2 common region, (**e**) the B19 VP1u, and (**f**) the PARV4 VP1u. The helices of the PLA2 domains are colored magenta, the helices of (potential) receptor-binding domains (RBD) light green, the C-terminal helices dark red, and the basic regions blue. The N- and C-termini are indicated.

#### **4. Conclusions**

This study extends the available capsid structural atlas of the subfamily *Parvovirinae* to six of the ten genera (Figure 7). The newly added structures of AMDV and PARV4 will provide and add to the structural platform for functional annotation of these viruses. Currently, the cellular receptors and many steps of the viral life cycle for these viruses are unknown, and elucidation of the capsid structures may help to understand their disease mechanisms at a molecular level.

**Figure 7.** Cladogram of the *Parvovirinae* subfamily based on Penzes et al. [2]. The representative members of the genera for which capsid structures have been determined are CPV (*Protoparvovirus*), AMDV (*Amdoparvovirus*), HBoV1 (*Bocaparvovirus*), PARV4 (*Tetraparvovirus*), AAV2 (*Dependoparvovirus*), and B19 (*Erythroparvovirus*). Radially colored capsid surface representations (blue to red) are viewed along the two-fold axis and were generated using Chimera [25]. TBD: to be determined.

In the case of AMDV, similar to B19 and HBoV1, antibody-dependent enhancement (ADE) of infection was described as a form of entry to host cells for this virus, complicating vaccination strategies [41–44]. The capsid structure of AMDV may help to identify the epitopes of the antibodies, as in studies on other parvoviruses [45]. Alternatively, if ADE post-vaccination cannot be prevented, the capsid structures may help in the development of therapeutics directly targeting the capsid.

**Author Contributions:** Conceptualization, M.M., M.S.-V. and R.M.; methodology, R.L. and M.M.; validation, M.M. and R.M.; formal analysis, R.L. and M.M.; investigation, R.L., M.M. and A.J.Y.; resources, P.C., J.Q., M.S.-V. and X.F.; data curation, R.L. and M.M.; writing—original draft preparation, R.L. and M.M.; writing—review and editing, R.L., M.M., R.M., J.Q. and M.S.-V.; visualization, R.L. and M.M.; supervision, R.M.; project administration, R.M.; funding acquisition, and R.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was funded by the NIH grant R01 NIH GM082946 (to R.M.). Data collection at Florida State University through the Southeastern Consortium for Microscopy of Macro Molecular Machines (SECM4) was made possible by NIH grants S10OD018142-01, S10RR025080-01, and U24GM116788.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The PARV4 and AMDV cryo-EM-reconstructed density maps and models built for their capsids were deposited in the Electron Microscopy Data Bank (EMDB) with the accession numbers EMD-28522/PDB ID 8EP9 (PARV4), EMD-28514/PDB ID 8EP2 (AMDV VP1/2), and EMD-28514 (AMDV-VP2).

**Acknowledgments:** The authors wish to thank the late Mavis Agbandje-McKenna for initiating this research project and for her pioneering studies of parvovirus capsid structures. The authors also want to thank the UF-ICBR Electron Microscopy Core (RRID:SCR\_019146) for access to electron microscopes utilized for cryo-electron micrograph screening.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

