Alternative Splicing Vairants Of The EIAV Receptor

Equine infectious anemia virus (EIAV) is a member of Lentivirus Genus of Retroviridae andinfects equine macrophages and endothelial cells in vivo. The functional cellular receptor for EIAV isidentified as equine lentivirus receptor1(ELR1), which belongs to the Tumor Necrosis Factor Receptorfamily. ELR1is predicted to be a type I transmembrane protein, with its ectodomain containing fourcysteinerich domains that are designated as CRD1to CRD4. The C-terminal region of the CRD1segment of ELR1is the essential domain for functional binding of ELR1to the EIAV surfaceglycoprotein gp90. The ELR1-mediated entrance of EIAV into target cells is via a low-pH-dependentendocytic pathway.This study firstly confirmed the presence of our previously identified alternative splicing isoformsof ELR1transcripts and analyzed the sequences and compositions of these isoforms. Results revealedthat besides the published sequence of ELR1, there were several other splicing isoforms of ELR1thatcontained inserts or deletions. Among the transcriptional isoforms of ELR1, species that contained partof the Intron6or absented part of the Exon3were the major types of alternative spliced isoforms.Sequence analysis of ELR1transcripts amplified and cloned from macrophages of three horses at threetime points showed that alternative splicing isoforms of the receptor were detected in all individualhorses examined. In addition, the expression levels and radios of these splicing isoforms significantlyvaried among individuals and different time points of sampling. Results of sequence analyzing revealedthat ELR1, which processed65%of the transcript, was the highest component of the isoforms and wasfollowed by ELR1-IN (25%) and ELR1-DE (10%). ELR1-IN contained an insert of153bases betweenthe786-787nt of the published ELR1cDNA sequence. ELR1-DE had a deletion of109bases betweenthe sites of308-415nt. Both of the insertion and the deletion shifted the translational open reading frame(ORF) of ELR1and generated premature stop codons, which terminated translation at the upstreampoints of the transmembrane domain.The truncated receptor encoded by ELR1-IN was predicted as a soluble protein and designated assELR1, which was secreted out of the cells when expressed in eMDM as a recombinant protein fusedwith eGFP or HA at the C-terminus. The cDNA of ELR1-DE contained two ORFs after the deletion,both of which encoded the EIAV receptor lacking function-related CRD1domain. Therefore, these twotruncated polypeptides of ELR1were considered dysfunctional and were not further investigated in thisstudy.To investigate the implications of EIAV infection for the expression of ELR1and its alternativesplicing isoforms, the transcriptional species of ELR1and ELR1-IN were amplified and quantified byreal-time qPCR. Compared with untreated primary equine monocyte-derived macrophages (eMDM),the expression levels and ratios of species of these two types of mRNA were significantly changed bythe infection of EIAV on eMDM. Other experiments of in vitro infection of EIAV on eMDM andprimary equine fibroblasts further indicated that sELR1, the soluble ELR1encoded by ELR1-IN, remained the function to bind with EIAV. Pre-incubation of sELR1with EIAV, or over-expression ofthe recombinant sELR1in these cells remarkably inhibited the virus infection and replication in thetarget cells. To further understand the alternative splicing, isoforms of ELR1affected by EIAV infectionin equine macrophages,ELR1and ELR1-IN expression were detected by real-time PCR. Results showed that the RNAlevels and ratios of ELR1-IN and ELR1significantly changed in macrophages infected with EIAVstrains in different virulence in vitro. The biological significance of ELR1alternative splicingIsoforms, especially the ELR1-IN that encodes sELR1, in the interaction between EIAV and the hosts isstill unclear and needed to be further studied.