4.2. The genomic characterization and genetic relationships of PCV4
Upon detection of a novel virus in animals suffering from a certain illness, it is common to urge epidemiological and pathological studies (Faccini et al., 2017; Franzo et al., 2018). In this study, in addition to providing supportive evidence for the active circulation of PCV4, attempts were made to describe the genetic structure as well as to investigate the potential biological important proteins encoded by PCV4 which might play roles in the replication process and/or pathogenesis of PCV4 in pigs.
Of the genomic organization, the previous study described in brief that PCV4 represents a new circovirus species, having a typical origin of replication of conserved nonanucleotide stem-loop motif (Zhang et al., 2020). In this study, besides the palindrome stem-loop structure of the origin of replication in the 5’- intergenic region (Figure 2B), more genetic features were elucidated from the nucleotide sequences of E115 strain as well as three previous PCV4 strains so far. It was found that the circular genome contains several overlapping ORFs in different orientations and the existence of two intergenic regions in between genes encoding the Rep and Cap proteins (Figure 2A, Supplementary Figure S1). These features were also observed in viruses belonging toCircovirus genus (Li et al., 2010). Combining the results of genetic structure analyses (Figure 2) and phylogenetic classification (Figure 7), the study agreed with the results obtained in previous publications (Tian et al., 2020; Zhang et al., 2020) and that E115 and other three strains of PCV4 belonged to the genus Circovirus but phylogenetically differed from three porcine circoviruses of PCV1, PCV2 and PCV3.
Of the functional analysis, this study found essential elements known so far for DNA replication of circoviruses (Figures 2B, 3, 4), such as: the NLS mediating nuclear targeting of the viral genome (Heath, Williamson, & Rybicki, 2006), the structure at the origin of DNA replication (Cheung, 2004), endonuclease and helicase domains of Rep (Faurez, Dory, Grasland, & Jestin, 2009; Mankertz & Hillenbrand, 2001). Additionally, the presence of tyrosine-based Y-x-x-φ and P-x-x-P motifs in the Cap of PCV4 (Figure 5) might be involved in the entry process of this virus into the host cell. That inference was made by evidence that the Y-x-x-φ were observed in a variety of viruses including SARS-CoV, HCV, EBV or SIV viruses that contribute to the viral penetration (Hraber et al., 2020; Karamichali et al., 2017; Minakshi & Padhan, 2014). Another effect of P-x-x-P motif in Nef protein of HIV was demonstrated to interact with SH3-domains that related to the increase of virus replication (Saksela, Cheng, & Baltimore, 1995). Lastly, in this study, several linear peptides in the putative ORF3 protein of PCV4 were observed that are likely conserved in other putative homologous proteins of viruses belonging to family Circoviridae (Figure 5). Functional analysis predicted that these peptides might interact with the PDZ domains, an important regions associated with cell signaling pathways (Lee & Zheng, 2010). Proteins containing PDZ domains were demonstrated as target of several different viruses (James & Roberts, 2016; Javier & Rice, 2011). Therefore, it was possible to imply that the putative ORF3 protein of PCV4 could alter the cell signaling pathway during the virus replication. All of the above-mentioned results of genetic analyses provided not only details about genomic structure and organization of a newly described PCV4, but also supported that the virus contains essential elements for the replication process.