3.2. Genome organization and viral proteins functional
analysis
The full genome of a Korean PCV4 strain (E115) was 1,770 nucleotides in
length, showing 98.5% to 98.9% similarity to three PCV4 strains
available at GenBank (MT015686, MK986820, MK948416). Based on the
ORFfinder results, the genome of E115 strain generated in this study was
predicted to contain two common ORFs encoding for putative
replication-associated protein
(Rep) and putative capsid (Cap) protein and four other short ORFs
oriented in the different directions (Figure 2A). All of the putative
ORFs identified in E115 strain were also detected in the genome of
previous known PCV4 strains (Supplementary Figure S1). A palindrome
stem-loop structure with the
conserved nonanucleotide
(CAGTATTAC) was observed (Figures 2B- 2C). In detail, a 11- nucleotide
of loop structure was flanked by two reversed complement regions of 16
nucleotides each (Figure 2B). Additionally, the putative Rep and Cap
genes were separated by two intergenic regions of 111 and 81 nucleotides
in length at the 5’ and 3’, respectively (Figure 2A and Supplementary
Table S1).
Further analysis results (Figure 3) indicated that the putative Rep of
E115 strain contained (i) the endonuclease domain with three motifs,
(ii) the helicase domain of superfamily 3 (SF3) containing three Walker
motifs and (iii) other unknown conserved motifs which are normally
observed in Circoviridae in particular and in CRESS DNA viruses
in general as previously described (Delwart & Li, 2012; Kazlauskas et
al., 2018; Krupovic et al., 2020; Ye, Berg, Fossum, Wallgren, &
Blomstrom, 2018).
Of the putative Cap, a nuclear localization signal (NLS) was predicted
in the N-terminus of the putative Cap of E115 strain (Figure 4). Based
on the alignment with other members of Circovirus genus, Figure 4
showed that the arginine-rich region of E115 strain was aligned with the
basic motifs of the experimentally confirmed NLS of PCV1, PCV2 and PCV3
(Liu, Tikoo, & Babiuk, 2001; Mou, Wang, Pan, & Chen, 2019; Shuai et
al., 2008). Additionally, motif screening (Figure 5) indicated that the
putative Cap of E115 strain contained several tyrosine-based Y-x-x-φ
motifs and P-x-x-P motifs (x represents any amino acid and φ denotes a
large hydrophobic residue of either F, I, L or V) which were related to
clathrin-mediated endocytosis (Sobhy, 2016).
Of the other four putative ORF3- ORF6 of E115 strain, only ORF3 protein
contained homologous regions with a recently identified ORF5 protein of
PCV2 (Lv, Guo, Xu, Wang, & Zhang, 2015). Widely comparing with
homologous putative proteins of some members of genus Circovirus ,
it was found that they shared some short, conserved linear peptides of
W-A-S-[PL]-[DG]-M, G-G-M-x-[TI], W-M-[TI]-[IF]-M-A-G
and [MG]-M-[TI]-C (Figure 6). Interestingly, based on LMDIPrep
results, the above last three linear peptides at the C-terminal region
were predicted to interact with PDZ domain (Supplementary data S3-S4).