Adeno-associated viruses (AAVs) are being developed for gene delivery applications, with more than 100 ongoing clinical trials aimed at the treatment of monogenic diseases. In this study, the unique N-terminus of AAV capsid viral protein 1 (VP1u), containing a canonical group XIII PLA₂ enzyme domain, was observed to also exhibit proteolytic activity. This protease activity can target casein and gelatin, two standard substrates used for testing protease function but does not self-cleave in the context of the capsid or target globular proteins, for example, bovine serum albumin (BSA). However, heated BSA is susceptible to VP1u-mediated cleavage, suggesting that disordered proteins are substrates for this protease function. The protease activity is partially inhibited by divalent cation chelators ethylenediaminetetraacetic acid (EDTA) and ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA), and human alpha-2-macroglobulin (A2M), a non-specific protease inhibitor. Interestingly, both the bovine pancreatic (group VIIA) and bee venom (group III) PLA₂ enzymes also exhibit protease function against casein. This indicates that PLA₂ groups, including VP1u, have a protease function. Amino acid substitution of the PLA₂ catalytic motif (⁷⁶HD/AN) in the AAV2 VP1u resulted in attenuation of protease activity, suggesting that the protease and PLA₂ active sites are related. However, the amino acid substitution of histidine H38, which is not involved in PLA₂ function, to alanine, also affects protease activity, suggesting that the active site/mechanism of the PLA₂ and protease function are not identical.

Parvovirus B19 (B19V) is a human pathogen that is the causative agent of fifth disease in children. It is also known to cause hydrops in fetuses, anemia in AIDS patients, and transient aplastic crisis in patients with sickle cell disease. The unique N-terminus of Viral Protein 1 (VP1u) of parvoviruses, including B19V, exhibits phospholipase A₂ (PLA₂) activity, which is required for endosomal escape. Presented is the structural dynamics of B19V VP1u under conditions that mimic the pHs of cell entry and endosomal trafficking to the nucleus. Using circular dichroism spectroscopy, the receptor-binding domain of B19V VP1u is shown to exhibit an α-helical fold, whereas the PLA₂ domain exhibits a probable molten globule state, both of which are pH invariant. Differential scanning calorimetry performed at endosomal pHs shows that the melting temperature (T_m) of VP1u PLA₂ domain is tuned to body temperature (37 °C) at pH 7.4. In addition, PLA₂ assays performed at temperatures ranging from 25–45 °C show both a temperature and pH-dependent change in activity. We hypothesize that VP1u PLA₂ domain differences in T_m at differing pHs have enabled the virus to “switch on/off” the phospholipase activity during capsid trafficking. Furthermore, we propose the environment of the early endosome as the optimal condition for endosomal escape leading to B19V infection.

Parvoviruses are small, single-stranded DNA viruses with non-enveloped capsids. Determining the capsid structures provides a framework for annotating regions important to the viral life cycle. Aleutian mink disease virus (AMDV), a pathogen in minks, and human parvovirus 4 (PARV4), infecting humans, are parvoviruses belonging to the genera Amdoparvovirus and Tetraparvovirus, respectively. While Aleutian mink disease caused by AMDV is a major threat to mink farming, no clear clinical manifestations have been established following infection with PARV4 in humans. Here, the capsid structures of AMDV and PARV4 were determined via cryo-electron microscopy at 2.37 and 3.12 Å resolutions, respectively. Despite low amino acid sequence identities (10–30%) both viruses share the icosahedral nature of parvovirus capsids, with 60 viral proteins (VPs) assembling the capsid via two-, three-, and five-fold symmetry VP-related interactions, but display major structural variabilities in the surface loops when the capsid structures are superposed onto other parvoviruses. The capsid structures of AMDV and PARV4 will add to current knowledge of the structural platform for parvoviruses and permit future functional annotation of these viruses, which will help in understanding their infection mechanisms at a molecular level for the development of diagnostics and therapeutics.

Introduction: Parvovirus B19 (B19V) is a human pathogen, and the minor capsid protein of B19V possesses a unique N terminus called VP1u that plays a crucial role in the life cycle of the virus Objective: The objective of this study was to develop a method for domain segmentation of B19 VP1u using intein technology, particularly its receptor binding domain (RBD) and phospholipase A2 (PLA2 ) domain. Methods: RBD and PLA2 domains of VP1u were each fused to the DnaE split inteins derived from the Nostoc punctiforme. Each of these precursor proteins was expressed in E. coli. Combining the purified precursors in equal molar ratios resulted in the formation of full-length VP1u. Furthermore, Circular Dichroism (CD) spectroscopy and PLA2 assays were used to probe the structure and activity of the newly formed protein. Results: The CD spectrum of the full length VP1u confirmed the secondary structure of protein, while the PLA2 assay indicated minimal disruption in enzymatic activity. Conclusion: This method would allow for the selective incorporation of NMR-active isotopes into either of the VP1u domains, which can reduce signal overlap in NMR structural determination studies.