Mäntylä et al., 2018 and 2020 [
14] neutralized impβ with antibodies by microinjection through canine parvovirus (CPV) entry nucleus experiment The statistical result of CPV-infected cells showed that the number of infected cells was few, which verified that impβ plays an important role in the process of the virus entering and crossing the nuclear membrane. Subsequently, co-immunoprecipitation and in situ proximity ligation assay (PLA) experiments were carried out. The results of PLA experiments revealed that the number and density of signals close to the nuclear envelope(NE) were the highest in the nucleus. A small part of the signals was also located deeper, 0.75 ~ 6.0 m away from the NE. Quantitative analysis showed that the capsid enters the nucleus before interacting with the cytoplasmic impβ. Subsequently the capsid enters the nucleus, unlike classical nucleophiles, where the capsid still interacts with Impβ. Based on the phenomenon of nuclear permeability of viral capsids as it enters the nucleus observed in other viruses. Figure
5 illustrates a theory that the viral capsid’s structure change due to internal acidification after entering the cell, resulting in exposure of the VP1 N-terminal domain, which contains phospholipase A2 and NLS on the capsid surface [
42,
43]. The phospholipase activity disassembles the endosomal membrane, while the NLS interacts with impα and β after the endosome opens. Alternatively after capsid release from endosomes during capsid transport using MTs [
25,
44‐
46]. Impβ binds to the NPC and passes through the pore. The process is also observed during CPV nucleation. The portion of the capsid that impβ does not mask interacts with Nup358, Nup62, and Nup153. In anticipation of the interaction of impβ with Nup153 to terminate translocation in the nuclear basket, RanGTP dissociates and imports into the complex [
47,
48], leading to the recycling of Impβ into the cytoplasm. At the same time, NPC-bound capsids disintegrate the NPC/NE, possibly because impβ regulates cell mitosis [
49] and penetrates the nuclear membrane, triggering Ca
2+ release, a known initiator of NE degradation in mitosis. Subsequently, the cytoplasmic capsid-imp complex can then passively enter the nucleus thanks to the pore in the NE. This is a novel hypothesis about the nuclear pathway of the parvovirus genome (CPV), classical nuclear import through the nuclear pore complex, accompanied by instantaneous rupture of the nuclear envelope, which also allows passive entry of the protein capsid complex into the nucleus. The low transduction/infection efficiency could not be fully explained by the restriction of virus-receptor (transferrin receptor, a transferrin receptor on the cell membrane surface of canine parvovirus) [
50] interaction or endocytic entry, indicating a bottleneck occurs after viral entry into the cytoplasm. Therefore, this hypothesis can be a relatively accurate and complete elaboration of the parvovirus nucleation process.