The Function Of Cold Inducible RNA-binding Protein In The Replication Of H1N1 Virus And Screening Of Its Target Proteins

Cold-inducible RNA binding protein(Cirbp) is the first identified protein relating to cold stress. As a RNA binding protein, Cirbp widely expressed in various tissues and cells. And it is highly conserved among vertebrate. Researches show that Cirbp participates in the process of embryonic development, neurodevelopment, tumorigenesis and circadian clock regulation in physical condition. In addition, Cirbp also makes response to various stress conditions. Recent studies showed that Cirbp can also mediate inflammatory response in different pathological conditions, such as LPS stimuli, brain ischemic shock, sepsis and alcohol stimulation. The previous researches in our lab showed that Cirbp transcript increased in chickens infected with Newcastle disease and Cirbp transcripts and protein expression increased in tissues of SPF mice infected with H1N1 virus. What’s more, overexpression of Cirbp can promore H1N1 virus replication through up-regulating NF-Kappa B signal pathway. However, the function of Cirbp in the process of H1N1 virus replication is still unknown. In order to further study the function of Cirbp in H1N1 virus replication process and discuss its molecular mechanism, screen its target protein, we proceed the following study and obtained the following results. 1. Expression pattern and subcellular localization of Cirbp in cells infected with H1N1 virusIn order to study the expression pattern of Cirbp in cells infected with H1N1 virus, BHK-21, MH-S and PUVEC were infected with a MOI of 1. The BHK-21 and MH-S cells were infected with A/Puerto Rico/8/1934 H1N1, PUVEC cell was infected with A/Swine/GD/2/12 H1N1. Collecting samples respectively at 0 h, 4 h, 8 h, 16 h, 24 h post-infection, and detecting the expression of Cirbp using immunohistochemical staining, then analysis the results using IPP6.0 software. The results showed that Cirbp expression significantly increased at 8 h ~ 24 h post-infection in BHK-21 cell; Cirbp expression significantly increased at 4 h~24 h post-infection in MH-S cell; Cirbp expression significantly increased at 8 h~24 h post-infection in PUVEC cell. These results indicate that,after H1N1 virus infection, Cirbp expression increased in all the BHK-21, MH-S and PUVEC cells. And the increasing of Cirbp appears earlier in infected MH-S cell.In order to study the subcellular localization of Cirbp in cells infected with H1N1 virus, BHK-21 cell was infected with a MOI of 1, and staining the samples respectively at 2 h, 4 h, 6 h, 8 h post-infection using indirect immunofluorescence assay, then observed the results under confoeal laser scanning microscopy. Results showed that Cirbp localized in the nuclear and cytoplasm 2 h post-infection; and localized in the nuclear 4 h post-infection; localized in the nuclear and cytoplasm 6 h post-infection; localized in the nuclear 8 h post-infection. In consideration of Cirbp exist in nuclear under physical condition, the results indicate that Cirbp can shuttle between the nuclear and cytoplasm under condition of H1N1 virus infection.The results above indicate that Cirbp expression increases and changes its localization exposure to H1N1 infecion. 2. Knockdown Cirbp suppress H1N1 virus replication through decreasing viral m RNA stabilities.To analysis the effect of Cirbp on H1N1 virus m RNA stability, RNA and protein synthesis, we analysis the half-life of viral M and NP m RNA using real-time PCR in Cirbp knockdown BHK-21(Cirbp-BHK-21) cell and control cell infected with H1N1 virus(MOI=1). Also we analysis the effect of Cirbp knockdown on viral M and NP m RNA, c RNA and v RNA synthesis using real-time PCR at 3 h, 9 h, 15 h, 21 h post-infection(MOI=1). Then we detected the NP protein using IFA assay in Cirbp-BHK-21 and control cells at 3 h and 6 h post-infection(MOI=1). At last, we measured the TCID50 titer in BHK-21 and Cirbp-BHK-21 cells. The real-time PCR results show that the relative half-life of H1N1 viral NP and M m RNA is 7.5 h and 8 h respectively in Cirbp-BHK-21 cell, while they are exceed 12 h respectively in control cell; knockdown Cirbp significantly decreased M m RNA synthesis at 3 h, 9 h, 15 h, 21 h post-infection, the test group was 21.3%,18.6%, 12.4%,7.6% times respectively to the control group; knockdown Cirbp significantly suppressed the synthesis of M and NP c RNA, v RNA1, v RNA2, the test group was 16.9%、5.3%、11.3% and 5.1%、2.2%、2.6% times respectively to the control group. The IFA result showed that knockdown Cirbp suppressed the synthesis of NP protein at 3 h and 6 h post-infection. TCID50 titer detection results showed that knockdown Cirbp increased the TCID50 titer, the test group was 57.54 times to the control group. These results indicate that knockdown Cirbp decreased the viral m RNA stability and suppress the synthesis of viral c RNA, v RNA and protein. Analysis of these results,we conclude that knockdown Cirbp suppress H1N1 virus replication through decreasing viral m RNA stabilities. 3. Overexpression of Cirbp promotes the replication efficiency of H1N1 virusCirbp expression increased in condition of H1N1 virus infection. In order to study the effect of Cirbp overexpression on viral replication, we detected the absolute expression quantity of viral M m RNA at 3 h, 8 h, 16 h, 24 h post-infection with the MOI of 0.001,0.01 and 2 respectively using real-time PCR in BHK-21 and Cirbp overexpression BHK-21(Cirbp+BHK-21) cells. The real-time PCR showed when infecting the cells with a MOI of 0.001, the test group was 1.28,30.78,79.05,26.89 times respectively to the control group at 3 h, 8 h, 16 h, 24 h post-infection; when infecting the cells with a MOI of 0.01, the test group was 0.85,26.95,83.84,47.88 times respectively to the control group; when infecting the cells with a MOI of 2, the test group was 4.07,6.46,5.01,3.98 times respectively to the control group. This result indicated that overexpression of Cirbp promotes the replication efficiency of H1N1 virus. In addition, this promotion shows more notable when infecting at a lower MOI. 4. Overexpression of Cirbp facilitates v RNP nuclear exportation of H1N1 virusCirbp shuttles between the nuclear and cytoplasm under condition of H1N1 virus infection. In order to study the function of Cirbp on H1N1 virus transportation, we detected the effect of Cirbp overexpression on viral attachment, entry into the host cell, entry of viral RNPs(v RNPs) into the nucleus, export of the v RNPs from the nucleus. The Cirbp+BHK-21 and BHK-21 cells were infected with H1N1 PR-8 virus at a MOI of 50 when study the effect on viral attachment and entry into the host cell. After infection, cells were incubated further for 1 h, and this time point was the optimal time for influenza virus attachment and entry into the host cell. Using IFA assay, the results showed that there was no significantly difference for virus particle in the cytomembrane and cytoplasm of Cirbp+BHK-21 cell and control cells. This result indicates that overexpression of Cirbp have no effect on viral attachment and entry into the host cell.The Cirbp+BHK-21 and BHK-21 cells were infected with H1N1 PR-8 virus at a MOI of 1 when study the effect on entry of viral RNPs(v RNPs) into the nucleus, export of the v RNPs from the nucleus. The time point of 4h~6 h was the optimal time for entry of v RNPs into the nucleus and 6h~8h was the optimal time for export of the v RNPs from the nucleus. IFA assay was performed at 4 h, 6 h, 8 h post infection in BHK-21 and Cirbp+BHK-21 cells. The IFA results showed that a little amount of v RNPs have entry into the nucleus at 4 h post-infection, and no significant difference was found in Cirbp+BHK-21 cell and BHK-21 cell; at 6 h post-infection the v RNPs still exist in the cytoplasm and nucleus of both group with no significant difference found; at 8 h post-infection, the v RNPs was completely transfer into the cytoplasm from the nucleus in the Cirbp+BHK-21 cell, while v RNPs were transferring from the nucleus into cytoplasm in BHK-21 cells. These results indicate that overexpression of Cirbp promotes nucleus exportation of v RNPs rather than nucleus importation.In conclude, overexpression of Cirbp promotes nucleus exportation of v RNPs rather than other process 5. Screen and primary identification of Cirbp target proteins under condition of H1N1 virus infectionTo screen the target proteins of Cirbp under condition of H1N1 virus infection, we screened its target protein using Co-IP combined with mass spectrometry method in BHK-21 cells infected with H1N1 virus. There were twenty-nine potential target proteins screened. Twenty-seven proteins belong to the host proteins. Then we performed the pathway annotation, GO annotation and COG annotation to the host proteins. The pathway annotation results showed that these potential target proteins mainly participated in nucleic acid metabolism and translation related pathway, neuroregulation related pathway and infection and immunity related pathway. The first five host proteins which have higher mass spectrometry scores are Hsp90,hn RNP R, D1Pas1, Ddx17,Cttn with the mass spectrometry score of 1122.72,381.43,230.4,186.49, 185.49 respectively. Hsp90 and Ddx17 are closely relate to influenza virus replication. Hsp90 has the highest score and plays important functions in influenza virus replication. So we further confirmed if Hsp90 colocalized with Cirbp using IFA assay. Doubtless, the IFA results confirms that Hsp90 can colocalized with Cirbp under condition of virus infection. If they combind in a direct physical way need further study to confirm it. Besides, the western-blot result showed that overexpression of Cirbp up-regulated the Hsp90 expression, which indicates that Cirbp have certain regulating effect on Hsp90. Among the two viral potential target proteins, NP has the higher mass spectrometry score which is 108.68. NP plays vital important roles in the process of influenza A virus replication. NP can affect the vrial transcription, replication, viral assembly and transportation. Further IFA assay confirmed that Cirbp can colocalized with Cirbp under condition of virus infection, which indicates that indicated that NP can bind with Cirbp, at least, in a complex manner. In conclusion, these results provide new clue for further study the interaction between Cirbp and H1N1 virus.