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.