Structural characterization of the PCV2d genotype at 3.3 Å resolution reveals differences to PCV2a and PCV2b genotypes, a tetranucleotide, and an N-terminus near the icosahedral 3-fold axes

Abstract

17Porcine circovirus 2 (PCV2) is a T=1 non-enveloped icosahedral virus that has a major impact on the 18 swine industry as an agent of porcine circovirus associate disease. PCV2 capsid protein sequences 19 have been employed by others to provide a temporal description of the emerging genotypes. PCV2a 20 is believed to be the earliest genotype and responsible for giving rise to PCV2b, which gives rise to 21 PCV2d. The underlying mechanism responsible for the emerging genotypes is not understood. To 22 determine if a change in the PCV2d capsid accompanies the emergence of this genotype, we 23 determined the cryo-electron microscopy image reconstruction of PCV2d VLP at 3.3 Å resolution and 24 compared it to the previously reported PCV2a and PCV2b structures. Differences between the CD 25 and GH loops identify structural changes that accompany the emergence of PCV2b from PCV2a, and 26 PCV2d from PCV2b. We also model additional amino acids for the N-terminus near the icosahedral 27 3-fold axes of symmetry and a tetranucleotide between the 5-and 2-fold axes of symmetry. To 28 interpret the sequence diversity that defines the PCV2 genotypes on a structural platform we have 29 performed structure-based sequence comparison. Our analysis demonstrates that each genotype 30 possesses a unique set of amino acids located on the surface of the capsid that experience a high 31 degree of substitution. These substitutions may be a response to the PCV2 vaccination program. The 32 structural difference between PCV2a, b and d genotypes indicate that it is important to determine the 33 PCV2 capsid structure as the virus evolves into different genotypes. 34 35 3 Importance 36PCV2 is a significant epidemic agricultural pathogen that is the causative agent of a variety of swine 37 illnesses. PCV2 infections have significant economic impact in the swine industry and must be 38 controlled by vaccination. Outbreaks in farms vaccinated for PCV2 suggest that improvements to the 39 current vaccination programs are needed. Better understanding of the assembly, structure, replication 40 and evolution of these viruses is necessary for production of improved vaccines. The ability of PCV2 41 to rapidly shift genotypes suggests that expression systems capable of rapidly producing large 42 quantities of virus-like particles should be pursued. To these ends we have established a mammalian 43 cell-based virus-like particle expression system and performed high resolution structural studies of a 44 new PCV2 genotype. Differences between the structure of this genotype and earlier genotypes 45 demonstrate that it is important to study the PCV2 structure as it shifts genotypes. 46 47 Key Words: 48 Circovirus, virus-like particle (VLP), cryo-electron microscopy, virus evolution, capsid structure, 49 vaccine development 50 51 52Circoviruses are small nonenveloped icosahedral viruses that contain a circular, covalently 53 closed, single stranded DNA (ssDNA) genome. Circoviruses have been identified in a variety of 54 species and are known to cause infection...