Studies On The Transportation Mechanism Of Porcine Circovirus Type 2 In Host Cells

Porcine circovirus 2 (PCV2), the smallest virus that autonomously replicates in mammalian cells, is a major causative agent of postweaning multisystemic wasting syndrome (PMWS). Previous studies demonstrated the process of internalization, but the mechanism of PCV2 transport in host cell is unknown, which is important for virus locating in nucleus to proceed a full replication. A lot of evidence implicated that microtubules and microtubule-associated motors play a critical role in intracellular viral transport. The study is aimed at clarifying the function of microtubules and their motors in PCV2 nuclear trafficking.1. Analysis of microtubules function in nuclear trafficking of PCV2In the study, we first analyzed the dynamic location of PCV2 particles in the early infection stage. Viral particles were mostly localized at the peripheral region at 3 hpi, and then accumulated near the nucleus at 12 hpi. Finally, the viral protein was detected in the nucleus at 15 hpi, which suggest that PCV2 has completed nuclear trafficking and start to replicate. Further analysis provided us a set of close-up, which revealed the viral particles were adsorbed on microtubules. Then we found that the efficient of PCV2 transport and replication was severely inhibited, when PK15 cells were treated with nocodazole. The results showed that internalized PCV2 localized within endosome compartments, and transport in a microtubules-dependent manner.We further analyzed the acetylated tubulin levels in normal cells or in rBV-Cap pretreated cells. The result showed that the Cap could promoted the acetylated tubulin levels and microtubule polymerization, however, the effect could be weakened by HDAC6 overexpression. PCV2 infection was promoted by HDAC6 inhibitor, and meanwhile, the acetylated tubulin levels were enhanced, which suggested that acetylated tubulin is involved with PCV2 infection.2. Analysis of cytoplasmic dynein function in PCV2 infectionIt is a universal mechanism for viral nuclear trafficking to hijack the motor dynein. In the present study, we analyzed the effect of dynein-disrupting agents (Na3VO4) on PCV2 infection, and found that PCV2 infection was inhibited in drug pretreated cells, including lower efficient of transport. Furthermore, we analyzed the relationship between subunits of dynein and PCV2 in fixed infected PK15 cells. Higher immunoreactivity of IC1 was observed for PCV2 located at the peripheral region and remained prominent on those that had already reached the nuclear envelope, which suggested that the recruitment of IC1 was directly mediated by Cap.Furthermore, to determine the recruitment of IC1 only exist in PCV2 transport or not, we analyzed the subcelluar localization of Cap and IC1 after nuclear trafficking. It’s amazing that there is a dynamic and abnormal subcellular localization of IC1, which is same to Cap protein. In infected 3D4/31 cells, the abnormal subcellular localization of IC1 is similar to PK15 cells. Besides infection, abnormal subcellular localization of IC1 was also found in the condition of transfection.It has been reported previously that Cap is a necleo-cytoplasmic transport protein, but IC1 is a subunit of dynein, which conservatively locates in cytoplasm. To examine whether the NLS of Cap mediate IC1 locating in nucleus, PK15 cells were transfected truncated form of Cap with a deletion of NLS (dCap), and neither dCap nor IC1 aggregated in nucleus, suggested that the nuclear localization of IC1 is mediated by NLS of Cap.Based on above results, we speculate that Cap could interacts with IC1. By co-IP and GST-pulldown assays, we found the direct interaction between Cap and IC1, which occurs in infection of PCV2 and in vitro. IC1-binding sequence in Cap was localized at the middle domain of Cap protein. When the expression of IC1 in PK15 cells was interfered by specific shRNAs-mediated knockdown, infectivity of PCV2 was largely decreased, particially the low efficiency of transport, which suggested that IC1 plays a critical role in PCV2 infection.The study showed that IC1 is recruited by PCV2 viral particles to process its nuclear trafficking in early infection. However, in the late stage, IC1 may have a potential function to participate viral life cycle, which was lack of research evidence.