Syncrip facilitates porcine parvovirus viral dna replication through the alternative splicing of ns1 mrna to promote ns2 mrna formation



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PPV.NS

Materials and methods
Cell culture and virus
PK-15 and HEK293T cells were purchased from the 
American Type Culture Collection (ATCC, Manas-
sas, VA, USA). These cells were cultured in Dulbecco 
modified eagle medium (DMEM) (Gebico, USA). All 
media were supplemented with 10% heat-inactivated 
fetal bovine serum (FBS; Sijiqing, Hangzhou, China) in 
5% CO
2
culture. PPV XY strain (Genbank: MK993540) 
was propagated in PK-15 cells. The crude PPV prepara-
tion was purified using ultracentrifugation over sucrose 
cushions (2 mL of 50% sucrose plus 2 mL of 20% sucrose) 
by Optima XPN-100 Ultracentrifugation with a SW 41 
Ti rotor, at 200 000 g for 2 h. Virus titers in the culture 
supernatants were determined by the Reed-Muench 
method [
28
].
Plasmid constructions and reactions
PPV open reading frame 1 (ORF1) coding NS1 protein 
and three deletion mutant fragments were constructed 
using the overlap PCR approach from PPV genome and 
cloned into the pCI-neo vector. SYNCRIP protein was 
constructed from PK-15 cDNA and sub-cloned into the 
pCDNA3.0 vector. All sequences were confirmed by 
sequencing analysis (Sangon Biotech, Shanghai, China). 
For the construction of NS2 knockout Y-PPV clone 
(Y-PPV
NS2−
), the three CTC in NS2 ORF of Y-PPV were 
mutated to TAG using the overlap PCR approach, as 
described previously [
29
].
CRISPR/Cas9 knockout cell: Lentiviral vector LentiC-
RISPR v2 with puro resistance gene was used to clone 
sgRNA sequences between BsmB I sites. The sgRNA 
sequences were used to knockout SYNCRIP: SYN-
CRIP-1: 5′-CTA TTG ACG TCT TCA TCA TAG GTA CC-3′; 
SYNCRIP-2: 5′-CAC CGA GAA TTC AAT GAA GACGG 
CGCA-3′.
pLenti-NS2 and pLenti-SYNCRIP constructs: Lentivi-
ral vector pCDH-EF1-MCS-T2A-Puro was used to clone 
optimized NS2 and SYNCRIP ORF between the restric-
tion sites Nhe I and Not I. The PCR primers used in this 
study are shown in Table 
1
.

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