Effect Of Nuclear Localization Signal Of Porcine Circovirus ORF2 Protein On Viral Replication And The Immunogenicity Of Recombinant Viruses

Porcine circovirus (PCV), which belongs to the family Circoviridae, genus Circovirus, is one of the smallest animal viruses with unenveloped, single-stranded circular genome and a size of 17 nm in diameter. Two species of PCV, PCV1 and PCV2, have been characterized. PCV1 is considered to be non-pathogenic to pigs by experimental inoculation and was circulating widely in swine population in the world. PCV2 has been shown to be the causative agent of post-weaning multisystemic wasting syndrome (PMWS) of pigs. The affected pigs show progressive weight loss, respiratory and enlarged lymph nodes and characteristic microscopic lesions including granulomatous interstitial pneumonia, lymphadenopathy, hepatitis, nephritis and pancreatitis. PMWS has been a big threat to the swine industry worldwide. Three major open reading frames (ORF), ORF1, ORF2 and ORF3, are oriented in opposite directions in the genome of PCVs. ORF2 is highly variable between PCV1 and PCV2 (less than 60% homology) and encodes the only structural capsid (Cap) protein that contains the dominant immunological regions. It was reported that disruption or loss of nuclear localization of Cap might result in a reduced level of ssDNA in geminiviruses, which might result in low level of viral replication. Therefore, it is generally believed that PCV Cap is not merely involved in encapsidation and might contribute to replication control by way of interactions between Cap and Rep in the nucleoplasm. Mutagenic studies on PCV2 ORF2 could be helpful to understanding the mechenism of viral replication, pathogenesis and development of vaccines. The present study was aimed to investigate the status of PCV2 infection in swine population in southeastern China by ELISA and PCR based on PCV2 ORF2, the genetic diversity of PCV2 strains from southeastern China originating from PMWS cases, and the effects of nuclear localization signals (NLS) of PCV ORF2 protein on viral replication in vitro and in vivo using the recombinant PCV viruses by reverse genetic approaches.The prevalence of PCV2 infection in swine herds in southeastern China was investigated by ELISA and PCR. Seroprevalence of PCV2 in samples collected from 89 swine herds was significantly higher by ELISA in post-weaning (54.1%) and growing piglets (49.9%) than that of suckling pigs (33.3%) with an average rate of 46.0%(819/1779). Seventy-eight cases out of 159 diseased pigs from these herds were PCV2 positive by PCR. To provide new insights into the extent of genetic heterogeneity of PCV2 isolates in southeastern China, the ORF2 genes of 27 isolates from the area from January 2004 to March 2007 were sequenced and aligned. While closely related to each other with identity of 98.0%-100%, these isolates displayed lower homologies to those from other regions of China or to some foreign isolates. Alignment of deduced amino acid sequences of capsid protein identified two major hypervariable regions (positions 53-91 and 185-215) in isolates obtained in this study, which were within or close to the putative epitope domains. The substitutions consequently resulted in higher hydrophilicity of the epitope region (positions 47-85). Phylogenetic analysis revealed two clusters of 48 isolates including those from Genbank:the larger cluster I consisting of two subgroups and cluster II containing most of foreign isolates owing to the residue substitutions in epitope domains (amino acid positions 80,86,88 and 91). While the subgroup Ib contained all the isolates with ORF2 of 705 bp in length, the 27 isolates we sequenced were clustered exclusively in subgroup la together with some other Chinese strains. We conclude that PCV2 isolates prevailing in southeastern China were genetically different from those of other countries.Since PCV2 ORF2 protein was synthesized in cytoplasm and imported into nucleus via its own nucleus localization signal (NLS) to participate in viral replication and assembly, it was predicted that PCV1 ORF2 protein contains several potential monopartite or bipartite nuclear localization signals in its N-terminus. Contribution of these partially overlapping motifs to nuclear importing was identified by expression of mutated PCV1 Cap versions fused to enhanced green fluorescent protein (eGFP). The C-terminus truncated PCV1 Cap-EGFP was localized in nuclei of PK-15 cells similar to the wild-type PCV1 Cap-EGFP, whereas truncation of the N-terminus rendered the fusion protein distributed throughout cytoplasm, indicating that the nuclear import of PCV1 Cap was efficiently mediated by its N-terminal region. Substitutions of basic residues in stretches 9RRRR12 or the right part of 25RRPYLAHPAFRNRYRWRRK43 resulted in a diffused distribution of the fusion protein in both nuclei and cytoplasm, indicating that the two NLSs were responsible for restricted nuclear targeting of PCV1 Cap, which was different from that of PCV2 Cap.We constructd a DNA clone that contains a PCV2 genome and partial duplication in pUC-18. Also, a chimeric PCV12 DNA clone was consucted, in which PCV1 ORF2 was replaced by PCV2 ORP2 in PCV1 genome. Other two chemeric DNA clones were constucted based on PCV2 and PCV12 DNA clones, in which the nuclear localization signal sequence of PCV2 ORF2 was replaced by that of PCV1 ORF2, resulting in PCV2-2NLS1 and PCV12-2NLS1 infectious clones. The transfection of PK-15 cells with each of the four DNA clones led to the expression of a functional virus genome, indicating that they are all infectious. To confirm the viability of the progeny viruses after initial transfection and to compare the in vitro replication levels, the four recombinant viruses were subjected to 14 passages in PK-15 cells and the virus titers reached 105.5 and 105 50% tissue culture infective doses (TCID50/ml) for progeny virues PCV2 (mPCV2) and PCV12. However, PCV2-2NLS1 and PCV12-2NLS1 had lower titers of only 103.5 TCID50/ml. To further determine the growth characteristics of the four viruses, a one-step growth curve was performed simultaneously for each virus. By 96 h postinfection, mPCV2 and PCV12 had a titer of 104.5 and 104 TCID50/ml respectively, while PCV2-2NLS1 and PCV12-2NLS1 titers were only 103 TCID50/ml, indicating that mPCV2 and PCV12 showed higher replication levels in vitro.To evaluate the immunogenicity and pathogenicity of the recombinant viruses, ninety-six 8 weeks-old BALB/c mice were randomly assigned into six groups,16 per group, and inoculated with the viruses or MEM as control. The results showed that they had similar level of antibody to PCV2 ORF2 although the mice inoculated with mPCV2 developed more viremic than those inoculated with PCV12. However, only a few mice inoculated PCV2-2NLS1 or PCV12-2NLS1 had low level of viremia and low PCV antibody response. Almost none of the mice in the study had lung lesions. Nevertheless, mild to moderate microscopic lesions in spleens of mice inoculated with wild type PCV2 and mPCV2 were observed, which were significantly different from those of mice in other groups (p<0.05). However, there were no statistical differences of spleen lesions between mice inoculated with three chimeric viruses and those in control group. Consistently, mice inoculated with wild type PCV2 and mPCV2 showed significant downshifts of the CD4+ and CD8+ T-cell subsets of peripheral blood lymphocytes compared with the control mice (p<0.05), while the proportions of the CD4+ and CD8+ T-cells in PCV 12 inoculated mice were similar to that of control mice (p>0.05). Thus, PCV 12 could serve as a vaccine candidate as it showed high replication level both in vitro and in vivo, and induced specific antibody response similar to that of wild PCV2 but was somehow attenuated in vivo. However, PCV2-2NLS1 and PCV12-2NLS1 showed only low level of replication in vitro or in vivo, indicating that replacement of N-terimus of PCV2 ORF2 containing NLS of PCV1 may result in the disruption of the structure or function of the Cap, and lead to low level of viral replication.In conclusion, PCV2 infections were spreading widely in southeastern China by an epidemiology study and the PCV2 isolates in this area were significantly different from those of other regions especially on its epitopes in ORF2 protein. And we first reported that the nuclear localization of PCV1 ORF2 protein was directed by two motifs (9RRRR12 and 25RRPYLAHPAFRNRYRWRRK43) in its N-terminus. The progeny virus of PCV12 displayed a low pathogenesis in mice copared with that of PCV2 SH04 and mPCV2, however, they all showed similar replicating characteristic and immunogenicity. The construction and application of infectious clones of PCV based on pUC-18 plasmid and infection of viruses in mice are useful for further study which may focus on the mutation of the key amino acids of PCV2 NLS rather than the whole N-terminal sequence, which can ensure the integrality and compatibility of ORF2 protein.