The capsid protein (Cap) is the sole structural protein and the main antigen of porcine circovirus type 2 (PCV2). Structural loops of the Cap play crucial roles in viral genome packaging, capsid assembly, and virus-host interactions. Although the molecular mechanisms are yet unknown, the carboxyl terminus (CT) of the PCV2 Cap is known to play critical roles in the evolution, pathogenesis, and proliferation of this virus. In this study, we investigated functions of CT. Removal of this loop leads to abrogation of the in vitro Cap self-assembly into virus-like particles (VLPs). Likewise, the mutated virus resists rescue from PK15 cell culture. A conserved PXXP motif in the CT is dispensable for VLP assembly and subsequent cell entry. However, its removal leads to the subsequent failure of virus rescued from PK15 cells. Furthermore, substituting either the PCV1 counterpart or an AXXA for the PXXP motif still supports virus rescue from cell culture but results in a dramatic decrease in viral titers compared with wild type. In particular, a strictly conserved residue (227K) in the CT is essential for VLP entry into PK15 cells, and its mutation to alanine greatly attenuates cell entry of the VLPs, supporting a mechanism for the failure to rescue a mutated PCV2 infectious DNA clone (K227A) from PK15 cell culture. These results suggest the CT of the PCV2 Cap plays critical roles in virus assembly, viral-host cell interaction(s), and virus propagation in vitro IMPORTANCE The carboxyl terminus (CT) of porcine circovirus type 2 (PCV2) capsid protein (Cap) was previously reported to be associated with immunorecognition, alterations of viral titer in swine sera, and pathogenicity. However, the molecular mechanisms underlying these effects remain unknown. In this study, roles of the critical residues and motifs of the CT are investigated with respect to virus-like particle (VLP) assembly, cell entry, and viral proliferation. The results revealed that the positively charged 227K of the CT is essential for both cell entry of PCV2 VLPs and virus proliferation. Our findings, therefore, suggest that the CT should be considered one of the key epitopes, recognized by neutralizing antibodies, for vaccine design and a target for drug development to prevent PCV2-associated diseases (PCVADs). Furthermore, it is important to respect the function of 227K for its role in cell entry if using either PCV2 VLPs for nanoscale DNA/drug cell delivery or using PCV2 VLPs to display a variety of foreign epitopes for immunization.

中文翻译：

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猪圆环病毒2型衣壳蛋白的羧基末端对于病毒样颗粒的组装，细胞进入和繁殖至关重要。

衣壳蛋白（Cap）是2型猪圆环病毒（PCV2）的唯一结构蛋白和主要抗原。Cap的结构环在病毒基因组包装，衣壳装配和病毒-宿主相互作用中起关键作用。尽管尚不清楚其分子机制，但已知PCV2 Cap的羧基末端（CT）在该病毒的进化，发病机理和增殖中起关键作用。在这项研究中，我们调查了CT的功能。该环的去除导致体外帽自组装被废除成病毒样颗粒（VLP）。同样，突变的病毒抵抗从PK15细胞培养物中的拯救。CT中保守的PXXP基序对于VLP组装和随后的细胞进入是必不可少的。但是，将其除去会导致随后从PK15细胞中拯救出病毒的失败。此外，用PCV1对应物或AXXA代替PXXP基序仍然支持从细胞培养中拯救病毒，但与野生型相比，病毒滴度显着降低。特别是，CT中严格保守的残基（227K）对于VLP进入PK15细胞至关重要，并且其突变为丙氨酸会大大减弱VLP的细胞进入，从而支持了挽救突变的PCV2感染性DNA克隆失败的机制（ K227A）来自PK15细胞培养。这些结果表明PCV2 Cap的CT在病毒装配，病毒-宿主细胞相互作用和病毒体外繁殖中起关键作用重要2型猪圆环病毒（PCV2）衣壳蛋白（Cap）的羧基末端（CT）以前据报道与免疫识别，猪血清中病毒滴度的改变和致病性有关。但是，这些作用的分子机制仍然未知。在这项研究中，针对病毒样颗粒（VLP）组装，细胞进入和病毒增殖研究了CT的关键残基和基序的作用。结果表明，带正电的CT 227K对于PCV2 VLP的细胞进入和病毒增殖都是必不可少的。因此，我们的发现表明，CT应该被认为是中和抗体识别的关键表位之一，用于疫苗设计，并且是预防PCV2相关疾病（PCVAD）的药物开发目标。此外，如果使用PCV2 VLP进行纳米级DNA /药物细胞递送或使用PCV2 VLP展示各种外源抗原表位进行免疫，则必须尊重227K在细胞进入中的作用。这些作用的分子机制仍然未知。在这项研究中，针对病毒样颗粒（VLP）组装，细胞进入和病毒增殖研究了CT的关键残基和基序的作用。结果表明，带正电的CT 227K对于PCV2 VLP的细胞进入和病毒增殖都是必不可少的。因此，我们的发现表明，CT应该被认为是中和抗体识别的关键表位之一，用于疫苗设计，并且是预防PCV2相关疾病（PCVAD）的药物开发目标。此外，如果使用PCV2 VLP进行纳米级DNA /药物细胞递送或使用PCV2 VLP展示各种外源抗原表位进行免疫，则必须尊重227K在细胞进入中的作用。这些作用的分子机制仍然未知。在这项研究中，针对病毒样颗粒（VLP）组装，细胞进入和病毒增殖研究了CT的关键残基和基序的作用。结果表明，带正电的CT 227K对于PCV2 VLP的细胞进入和病毒增殖都是必不可少的。因此，我们的发现表明，CT应该被认为是中和抗体识别的关键表位之一，用于疫苗设计，并且是预防PCV2相关疾病（PCVAD）的药物开发目标。此外，如果使用PCV2 VLP进行纳米级DNA /药物细胞递送或使用PCV2 VLP展示各种外源抗原表位进行免疫，则必须尊重227K在细胞进入中的作用。