Identification Of Novel Receptors For Subgroup J Avian Leukosis Virus

Avian leukosis viruses are members of the Alpharetrovirus genus of the family Retroviridae which can induce some types of tumors. According to antigenicity of the envelope glycoprotein, ALV was classified into 10 distinct subgroups including subgroup A to J, and subgroup A, B, C, D, E and J can infect chickens and induce tumors. In the late 1980s, avian leukosis viruse subgroup J (ALV-J) was first isolated and identified as a new subgroup from broiler chickens by Payne and colleagues.Retroviral envelope glycoprotein contains two subunits, one is surface protein (SU) that is encoded by gp85 gene, and another is transmembrane protein (TM). SU protein consists of variable region and determinants of virus-receptor interaction, and SU has the ability to mediate the virus binding to cellular receptor, it is the key factor for determining the host range. Once virus particle bound to its receptor on the host cell, virion-cell fusion was activated through a two-step mechanism. First, the envelope glycoprotein undergoes a conformational change at the cell surface that mediates viral uptake and endosomal trafficking.A large number of retroviral receptors have to date been identified, for instance, CD4 and CD134 act as primary receptors for feline immunodeficiency virus (FIV). CD4, CCR5 and CXCR4 are primary receptor and co-receptors for human immunofeficiency virus (HIV) in infection of human CD4 T cells, likewise, CD4, CCR5 and CXCR4 also act as receptors for Simian immunofeficiency virus (SIV) involved in SIV infection, these receptors have played significant role in further research on infection mechanism and disease control.So far, four distinct cellular receptors have been identified that mediate ALV entry. The Tva, Tvc, and chNHEl genes confer susceptibility to ALV of subgroup A, C and J respectively, while Tvb encodes the host cell receptor for subgroup B, D and E. The avian leukosis virus that mainly can infect chicken has been intensively used as a model for analysis of viral entry into target cells. However, ALV-J has been an infection confined to meat-type stock generally, but in 2002 it had spread in China to egg-type chickens and gained a stronger foothold in China where the disease spectrum showed differences from western countries, and not only widespread ALV-J-induced disease in layer-type chickens from many parts of China was observed, but also ALV-J infections have also been reported in several Chinese local breeds of chickens, indicating that the pathogenicity of ALV-J might have been enhanced and the host range gradually expanded, and ALV-J infection changed from eliciting hemangioma or myeloma alone to occurrence of hemangioma, myeloma and liomyoma simultaneously. In recent years, report in succession indicated that ALV-J co-infection with other immunosuppression virus in vivo, for instance MDV and REV.This phenomeno was partially due to the antigenic variation which was resulted from mutation in sequence changes within the variable regions of envelope (env) gene, nevertheless, a more important reason is that there probably also exist one or more receptors or attachment factors on host cells for ALV-J infection except for chNHE1.For this reason, in this study, ALV-J was taken as the object to isolate and identify novel receptor for ALV-J on DF1 cell through immunoprecipitation, mass spectrum analysis, real-time PCR and other approaches. GRP78 and ANXA2 were isolated from DF1 cells and identified as two novel receptors for ALV-J that specially binding to ALV-J SU and/or envelope glycoprotein and their function to mediate the ALV-J entry into DF-1 cells.1. Fusion expression of ALV-J gp85 gene and  fragment of rabbit IgG in Adenovirus expression systemFirst, we cloned SUJ-rIgGFc into pShuttle-CMV from pcDNA-SUJ-rIgGFc and constructed the pShuttle-CMV-SUJ-rIgGFc, and it was linearized and co-transformed into BJ5183-AD-1 competent cells with pAdEasy-1 to produce recombinant plasmid pAd-SUJ-rIgGFc, which was subsequently transfected into 293 T cells to produce recombinant adenovirus rAd-SUJ-rlgGFc. As expected, a recombinant adenovirus plasmid pAD-SUJ-rlgGFc containing a 1.8-kilobase (kb) fragment which fused ALV-J SU gene and the Fc region of rabbit IgG was constructed, and was transfected into human 293T cell to generate fusion protein SUJ-rIgGFc for subsequent co-immunoprecipitation. The purification and western blot of SUJ-rlgGFc showed that the molecular weight of this fusion protein is approximately 90-95kd, and the IFA results indicated that fusion protein is expressed on the membrane of 293 T cells and MDCK cells. So we have succeeded in preparation of the fusion protein for isolating receptor in next immunoprecipitation assay.2. Isolation and mass spectrometry analysis of putative receptor for ALVJIn order to search for novel receptor for ALV-J, we used DF1 cells and pcDNA-env DF1 cell line as donor cells to isolate receptor by co-immunoprecipitation use two different methods. Firstly, DF1 cell surface proteins that were capable of interacting with ALV-J envelope glycoprotein were precipitated with SUJ-rIgGFc and Protein A Agarose, and rabbit IgGFc bond to Protein A Agarose immunoprecipitation with DF1 membrane proteins were used as negative control. Secondly, the membrane protein complexes extracted from pcDNA-env DF-1 cells were co-immunoprecipitated with the monoclonal antibody JE9 specific to ALV-J envelope glycoprotein and protein A agarose resin, and isotype control IgG and protein A agarose resin were used as negative control meanwhile. In the respective co-immunoprecipitation assay, two distinct bands which molecular weight was about 80 kd and 38 kd respectively were both precipitated from two kinds of cell membrane proteins, but not in control groups. Analysis by Mass Spectrometry revealed that the two distinct proteins were 78-kd glucose-regulated protein (GRP78) and Annexin A2 (ANXA2), indicating that two proteins which react with ALV-J SU in co-IP may be the receptors for ALV-J, and their isolation lay the foundation for next identification and analysis of ALV-J receptors.3. Identification and functional analysis of chGRP78 and chANXA2 in ALV-J infectionTo investigate the biological relevance of GRP78 or ANXA2 in viral attachment or in subsequent virus entry, we performed an inhibition of infection on DF1 cells. DF-1 cell cultures were pre-treated with different concentrations of an anti-GRP78 antibody (C-20) or anti-ANXA2 antibody (C-16) or isotype control IgG for 2h, respectively. After treatment, cells were infected with ALV-J at a m.o.i. of 5 and incubated for 2h and subsequently followed by treatment with acid glycine (PH3.0) to inactivate un-internalized virus. Then DF1 cells were further incubated in maintenance medium (DMEM containing 1% fetal calf serum and antibiotics) with antibodies for 48h. The treated and untreated cells were used for analysing the effect of inhibition respectively by IF A, real-time PCR, western blotting, and the supernatants were collected for virus titration by an endpoint dilution assay (TCID50) on DF1 cells. The virus yields of untreated cultures at forty-eight hours post-infection were 105.5 p.f.u. ml-1, the cells treated with control IgG did not show significant viral titre decreases with respect to untreated controls, even when they were used at high concentrations. In contrast, anti-GRP78 or anti-ANXA2 antibody-treated cultures significantly decreased ALV-J titres depending on the antibody dilution used. It was observed that 50 μg antibody ml-1 either anti-GRP78 or anti-ANXA2 gave rise to a significant reduction in virus yields of 104’33 p.f.u. ml-1 and 103.64 p.f.u. ml-1, which indicated respectively 93.2% and 98.4% inhibition of viral infection in DF1 cells. Lower antibody concentrations produced smaller reductions in viral titres. Similarly, the results of TCID50 assay were consistent with the results from IF A, real-time PCR and western blot. As virus control, we also did blocking assay for ALV-A infection in DF1 cells, the p27 expression level in groups treated with antibody against chGRP78 or chANXA2 was similar with that from the mock group, indicating antibody against chGRP78 or ANXA2 could not inhibit the ALV-A infection/replication in DF1 cells, These data clearly demonstrate that the interaction between ALV-J viron and cell surface GRP78 or ANXA2 was blocked owing to the antibody blocking on cell surface chGRP78 and chANXA2, the first step of initiating infection was failed, and viral infectivity remarkably declined, suggesting that GRP78 and ANXA2 are involved in early infection events for ALV-J, and acting as two functional co-receptors.4. Reconstruction of chGRP78 and chANXA2 on resistent cellschGRP78 and chANXA2 expressed on DF1 cells and act as viral receptors for ALV-J to mediate virus entry into host cell to initiate infection. But whether ALV-J can infect resistent cells when chGRP78 and chANXA2 were constructed on them, or whether chGRP78 and chANXA2 can mediate ALV-J entry into non-permissive cells. So, we cloned the full-length cDNA encoding chGRP78 and chANXA2 from DF1 cell total RNA by RT-PCR, and chGRP78 or chANXA2 was transiently transfected or co-transfected into GEF and 293 T cells which were resistent to ALV-J and analyzed with polyclonal antibodies against chGRP78 or chANXA2 developed by gene immunization, and subsequently cells were challenged with ALV-J for 48h and followed by analyzing the expression of intracellular ALV-J env by IF A or real-time PCR.GEFs were inoculated with ALV-J at a m.o.i of 5 for 2h at 37℃. Cells were treated with acid glycine pH3.0 to inactivate un-internalized viruses, then allowed to grow under standard conditions for 48h befor harvesting cell lysates to inoculate DF1 cells. After 7 days, IFA was carried out for detecting ALV-J in DF1 cells. IFA results showed that DF1 cells which inoculated with chANXA2 transfected or co-transfected GEFs lysates can produce visible specific fluorescence, whereas cells inoculated with chGRP78 transfected or vector transfected GEFs lysates did not produce specific fluorescence, suggesting that chANXA2 and chGRP78 expressed on GEFs can mediate virion entry into cells and assisted virus to achieve the first step in viral infection, and chANXA2 may play a more important role in infection than chGRP78.For 293T cell, the results of real-time PCR showed that the relative expression of ALV-J envelope gene was increased during 48h post infection, suggesting that chGRP78 and chANXA2 expressed in 293T cells, and the viral mRNA level presented dynamic change process, their expression on 293 T cells can facilitate viral entry into cell. The real-time PCR results of transfected cells confirmed the interaction between ALV-J and chGRP78 and/or chANXA2 in ALV-J entry into host cells, and also implyed that chANXA2 may more important than chGRP78 for ALV-J infection.