Downregulation Of Cellular Protein Annexin 2 Inhibits Replication Of Classical Swine Fever Virus

Classical swine fever virus (CSFV), is a small, enveloped virus with a non-segmented positive-stranded RNA genome, belonging to the genus Pestivirus within the family Flaviviridae. CSFV is the pathogen of classical swine fever (CSF), a highly contagious swine disease with high morbility and mortality, featuring symptoms of hemorrhagic fever and immune suppression. The disease is a notifiable one of the World organization for animal health (OIE), usually leading to substantial economic losses to the pig industry worldwide. In recent years, CSF with lower morbility and mortality and mild, chronic, atypical symptoms was often observed, even in immune pigs herd.At present, nothing is known about the interaction between CSFV and host cells. To uncover cellular protein responses, Dr. Shi zixue have analyzed the differential gene expression in CSFV-infected white blood cell of swine, screened 1745 expression upwards or downwards gene, in which Annexin 2 upregulates 2.483 times, that the difference is remarkable. Moreover, Dr. Sun Jinfu have conducted a differentially proteomic analysis in CSFV-infected PBMC, and also have discovered that Annexin 2 remarkably upregulates.Annexin 2(AnxA2) is a Ca2+-dependent, anionic phospholipid (aPL)-binding protein that belongs to the ubiquitous multigene annexin family. More than 50 different annexin isoforms have been identified. Like the majority of annexins, AnxA2 consists of a conserved C-terminal core domain and a short variable N-terminal segment. AnxA2 can coordinate Ca2+ ions via conserved a-helical repeats (annexin repeats). They have unique Ca2+-binding sites, which enables them to bind with negatively charged lipids. This protein, which plays a role in membrane organization and dynamics in particular along the endocytic pathway, exists as a heterotetrameric complex, consisting of two Annexin 2 molecules and a dimer of p11/S100A10. Interestingly, Annexin 2 is a cellular protein that promotes the formation of lipid microdomains, which are necessary for the budding of virus from infected host cells. The viral envelope of influenza viruses is derived from the host cell plasma membrane, and thus it is likely that enveloped viruses incorporate annexin, which is present in the plasma membrane of the host cell from which the viruses bud. The presence of Annexin 2 in the virus particles provides the virus a means to convert PLG into plasmin, which could act as an alternative protease for the cleavage of the HA precursor molecule into the HA1 and HA2 subunits. For the infection of a target cell by IAV, the cleavage of the precursor HA molecule into the HA1 and HA2 subunits by trypsin-like proteases is required Annexin 2, which is a member of the annexin protein family of Ca2+-dependent membrane binding, also was associated with other enveloped viruses, like cytomegalovirus and human immunodeficiency virus type 1. This suggests that the acquisition of Annexin 2 from the host cell membrane is a property shared by many enveloped viruses.Therefore, we studied the change of Annexin 2 expression in CSFV-infected PK-15 cells. Western-bloting detected the upregulation of Annexin 2, which has confirmed the earlier period experiment result. In 12, 24, 36, 48, 60 and 72 hours, Annexin 2 upregulated in comparison with that for the control cells, concomitant with the upgrade in CSFV E2 .We successed to knockdown the Annexin 2expression by siRNA treatment in PK-15 cell. When detected SiAnx suppression effect by Western Blotting, we discovered Annexin 2 downregulation after transfection 8 hours, and in 12, 18 and 24 hours Annexin 2 is nothing, but can examine Annexin 2 expression in 96 hour. This indicated this downregulation is a short process. Simultaneously in 24, 48, 72 hour, Annexin 2 is downregulate by above 7 times in comparison with the control cells. These data indicated, although this downregulation is short, but the effect is remarkable.After Annexin 2 downregulation, we studied the change of CSFV duplication and multiplication. Fistly, we examined the CSFV genome duplication by RT-PCR after Annexin 2 downregulation, and then discovered that Annexin 2downregulation suppresses the CSFV genome duplication. The data indicated that, in comparison with No Treat , the copy number was decreased by 2-8 times. Secondly, we studide CSFV infection titer variation by TCID50 when Annexin 2 downregulation. The data suggested Annexin 2 downregulation similarly reduces in CSFV infection titer. Lastly, we detected the CSFV E2 expression by Western blotting, and found E2 was decreased in comparison with that for the control cells, concomitant with the reduction in Anx2.. All data suggested annexin 2 down -regulation influence CSFV duplication and multiplication existence.To examine the relative intracellular locations of Anx2 and E2, we visualized both proteins in CSFV-infected PK-15 cells by using laser scanning confocal microscopy. Although the staining pattern for E2 was somewhat diffuse because of abundant virus expression, clear accumulation of E2 fluorescence was seen at the limiting membrane and interior of perinuclear vesicles ranging from 0.8 to 4μM in diameter; these were also positive for Anx2. To corroborate the interaction between Anx2 and E2, we establishmented the cell lines of stable E2 expression. Then we detected Annexin 2 expression, and found Annexin 2 significantly increased along with the stability of E2 expression, compared to empty vector transfected PK-15 cells. The results suggested that CSFV E2 alone induced Annexin 2 upregulation. But it did not suggest that Annexin 2 and E2 directly correlate, E2 may also indirectly regulate the expression of Annexin 2.To sum up, Annexin 2 may play an important role in CSFV infection. Current studies suggest that the incidence and development of virus disease is a multi-gene, multi-step, multi-stage process of participation, in which Annexin 2need to be further studied.