The soluble form of EIAV receptor encoded by an alternative splicing variant inhibits EIAV infection on target cells

Abstract

Equine lentivirus receptor 1 (ELR1) has been identified as the sole receptor for equine infectious anemia virus (EIAV) and is a member of the tumor necrosis factor receptor (TNFR) superfamily. In addition to the previously described membraneassociated form of ELR1, two other major alternative splicing variant mRNAs were identified in equine monocyte-derived macrophages (eMDMs). One major spliced species (ELR1-IN) contained an insertion of 153 nt, which resulted in a premature stop codon situated 561 nt upstream of the predicted membrane spanning domain. The other major species (ELR1-DE) has a deletion of 109 nt that causes a shift of the open reading frame and generates a stop codon 312 nt downstream. Because ELR1-DE presumably encodes a peptide of a mere 23 residues, only ELR1-IN was further analyzed. The expression of a soluble form of ELR1 (sELR1) by ELR1-IN was confirmed by Western blot and immunofluorescence analyses. Similar to ELR1, the transcription level of ELR1-IN varied among individual horses and at different time points in the same individuals. The ratio of ELR1-IN mRNA species to ELR1 mRNA was approximately 1:2.5. Pre-incubation of the recombinant sELR1 with EIAV significantly inhibited EIAV infection in equine macrophages, the primary in vivo target cell of the virus. Fetal equine dermal (FED) cells are susceptible to EIAV in vitro, and the replication of EIAV in FED cells transiently transfected with ELR1-IN was markedly reduced when compared with replication in cells transfected with the empty vector. Finally, the expression levels of both forms of the EIAV receptor were significantly regulated by infection with this virus. Taken together, our data indicate that sELR1 acts as a secreted cellular factor that inhibits EIAV infection in host cells.

Structural insight into equine lentivirus receptor 1

“…The genes encoding full‐length ELR1 and gp90 were provided by Jianhua Zhou at the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Heilongjiang, China) . The ectodomains of ELR1 (residues 39–206) and gp90 (residues 30–448) were amplified and cloned into the pMT/BiP/TEV‐His expression vector, which was modified from the pMT/BiP/V5‐His plasmid (Invitrogen, USA).…”

“…The genes encoding full-length ELR1 and gp90 were provided by Jianhua Zhou at the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Heilongjiang, China). 7,8,35 The ectodomains of ELR1 (residues 39-206) and gp90 (residues 30-448) were amplified and cloned into the pMT/BiP/TEV-His expression vector, which was modified from the pMT/ BiP/V5-His plasmid (Invitrogen, USA). To construct plasmids for the expression of ELR1 containing the mutations Y61A, L70A, G72A and L70AG72A, the pMT-ELR1 expression plasmid was mutated using the QuickChange site-directed mutagenesis kit (Stratagene, USA) according to manufacturer's instructions.…”

Current and Future Therapeutic Strategies for Lentiviral Eradication from Macrophage Reservoirs

“…Clinical studies reported that the EIAVFDDV13 attenuated vaccine is approximately 80% effective at inducing immune protection against EIAV. The vaccine confers protection by blocking the ELR1 receptors with an EIAV SU protein, upregulating secretion of an ELR1-transcript variant identified as soluble ELR1 (sELR1) and increasing expression of IFN-β by toll-like receptor 3 (TLR3) (Lin et al, 2013;Ma et al, 2014). Upregulation of secretion of sELR1 is shown to exert antiviral effects and contribute to viral resistance induced by the vaccine (Lin et al, 2013).…”

“…The vaccine confers protection by blocking the ELR1 receptors with an EIAV SU protein, upregulating secretion of an ELR1-transcript variant identified as soluble ELR1 (sELR1) and increasing expression of IFN-β by toll-like receptor 3 (TLR3) (Lin et al, 2013;Ma et al, 2014). Upregulation of secretion of sELR1 is shown to exert antiviral effects and contribute to viral resistance induced by the vaccine (Lin et al, 2013). Unfortunately, soluble viral receptors were constructed and tested for the HIV-1 receptor (CD4) in the mid to late '90s; sCD4+ treatment Phase I and II clinical trials were unable to show effective antiviral properties in vivo (Norkin, 1995).…”