Functional exchangeability of the nuclear localization signal (NLS) of capsid protein between PCV1 and PCV2 in vitro: Implications for the role of NLS in viral replication

Abstract

Background: Porcine circovirus type 2 (PCV2) is believed to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS). It is supposed that capsid protein of PCV may contribute to replication control via interaction between Cap and Rep in the nucleoplasm. In this study, we described the construction and in vitro characterization of NLS-exchanged PCV DNA clones based on a PMWS-associated PCV2b isolate from China to determine the role of ORF2 NLS in PCV replication.

Results: The PCV1, PCV2, PCV2-NLS1 and PCV1-NLS2 DNA clone were generated by ligating a copy of respective genome in tandem with a partial duplication. The PCV2-NLS1 and PCV1-NLS2 DNA clone contained a chimeric genome in which the ORF2 NLS was exchanged. The four DNA clones were all confirmed to be infectious in vitro when transfected into PK-15 cells, as PCV capsid protein were expressed in approximately 10-20% of the transfected cells. The in vitro growth characteristics of the DNA clones were then determined and compared. All the recovered progeny viruses gave rise to increasing infectious titers during passages and were genetically stable by genomic sequencing. The chimeric PCV1-NLS2 and PCV2-NLS1 viruses had the final titers of about 104.2 and 103.8 TCID50/ml, which were significantly lower than that of PCV1 and PCV2 (105.6 and 105.0 TCID50/ml, respectively). When the ORF2 NLS exchanged, the mutant PCV2 (PCV2-NLS1) still replicated less efficiently and showed lower infectious titer than did PCV1 mutant (PCV1-NLS2), which was consistent with the distinction between wild type PCV1 and PCV2.

Conclusions: Recovery of the chimeiric PCV1-NLS2 and PCV2-NLS1 progeny viruses indicate that the nuclear localization signal sequence of capsid protein are functionally exchangeable between PCV1 and PCV2 with respect to the role of nuclear importing and propagation. The findings also reveal that ORF2 NLS play an accessory role in the replication of PCV. However, we found that ORF2 NLS was not responsible for the distinction of in vitro growth characteristic between PCV1 and PCV2. Further studies are required to determine the in vivo viral replication and pathogenicity of the NLS chimeric DNA clones.

Figure 1

Schematic construction of the infectious DNA clones. (A) The PCV1 DNA clone was constructed by ligating a duplicated fragment (915-1740 nt) to the Kpn I-digested PCV1 genome in pUC-18. The chimeric PCV1-NLS2 DNA clone was constructed replacing the ORF2 NLS sequence in PCV1 genomic backbone with NLS of PCV2 ORF2. (B) The PCV2 DNA clone was constructed by ligating a fragment (1419-1745 bp) to the EcoR I-digested PCV2 genome in pUC-18. The chimeric PCV2-NLS1 was constructed by replacing the PCV2 ORF2 NLS with PCV1 ORF2 NLS. The partial duplications were indicated by dashed. Positions of primers and the directions of ORF were marked by arrows and the orientations of the triangles, respectively.

Figure 2

The DNA clones were infectious when transfected in PK-15 cells in vitro. The cells were stained with polyclonal antibody to the PCV1 (A and B) or PCV2 (C and D) capsid protein. (A) PK-15 cells transfected with PCV1 DNA clone. (B) PK-15 cells transfected with chimeric PCV1-NLS2 DNA clone. (C) PK-15 cells transfected with PCV2 DNA clone. (D) PK-15 cells transfected with chimeric PCV2-NLS1 DNA clone. (E and F) Mock-transfected PK-15 cells with pUC-18.

Figure 3

In vitro viability of progeny viruses after initial transfection of PK-15 cells with DNA clones. Synchronized PK-15 cells were transfected with 20 μg of PCV1, PCV2, PCV1-NLS2 and PCV2-NLS1 DNA clone followed by twenty-six serial passages. The DNA clones were capable of producing infectious and viable virions in vitro, and all the progeny viruses gave rise to gradually increasing infectious titers during passages and propagated stably after 16 to 20 passages. The infectious titers were determined at each passage according to the Reed-Muench method.

Figure 4

One-step growth curve of PCV1, PCV2, PCV1-NLS2 and PCV2-NLS1 viruses. Synchronized PK-15 cells were infected with viruses all at an MOI of 0.1. The infectious titers for all viruses were similarly lower than 10^1.8 TCID₅₀/ml after initial infection, but gradually increased from 12 to 96 hours. PCV1 propagated much more efficiently in vitro with a significantly higher titer of 10^4.3 TCID₅₀/ml by 96 h post-infection. The infectious titers of viruses were determined by IFA with Reed-Muench method.

Figure 5

Synchronized PK-15 cells were infected with viruses all at an MOI of 0.1. (A)The virus loads for PCV1, PCV2, PCV1-NLS2 and PCV2-NLS1 in cell cultures at each time point were determined by real-time PCR based on PCV1 or PCV2 ORF2 gene. (B) Kinetics of viral capsid protein (28 kD) biosynthesis of progeny viruses from infected PK-15 cells at different time points were confirmed by western blot analysis with polyclonal antibodies to PCV1 or PCV2 capsid protein.