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A single site for N-linked glycosylation in the envelope glycoprotein of feline immunodeficiency virus modulates the virus-receptor interaction.

Willett BJ, McMonagle EL, Logan N, Samman A, Hosie MJ - Retrovirology (2008)

Bottom Line: Prototypic FIV vaccines based on the FL4 strain of FIV contain a cell culture-adapted strain of FIV Petaluma, a CD134-independent strain of FIV that interacts directly with CXCR4.In addition to a characteristic increase in charge in the V3 loop homologue of FIVFL4, we identified two mutations in potential sites for N-linked glycosylation in the region of FIV Env analogous to the V1-V2 region of HIV and SIV Env, T271I and N342Y.The T271I mutation did not confer CD134-independent infection upon GL8 or CPG41, nor did it increase the affinity of the CXCR4 interaction, suggesting that the principal effect was targeted at reducing the complexity of the Env-CD134 interaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Retrovirus Research Laboratory, Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow, G61 1QH, UK. b.willett@vet.gla.ac.uk

ABSTRACT
Feline immunodeficiency virus (FIV) targets helper T cells by attachment of the envelope glycoprotein (Env) to CD134, a subsequent interaction with CXCR4 then facilitating the process of viral entry. As the CXCR4 binding site is not exposed until CD134-binding has occurred then the virus is protected from neutralising antibodies targeting the CXCR4-binding site on Env. Prototypic FIV vaccines based on the FL4 strain of FIV contain a cell culture-adapted strain of FIV Petaluma, a CD134-independent strain of FIV that interacts directly with CXCR4. In addition to a characteristic increase in charge in the V3 loop homologue of FIVFL4, we identified two mutations in potential sites for N-linked glycosylation in the region of FIV Env analogous to the V1-V2 region of HIV and SIV Env, T271I and N342Y. When these mutations were introduced into the primary GL8 and CPG41 strains of FIV, the T271I mutation was found to alter the nature of the virus-CD134 interaction; primary viruses carrying the T271I mutation no longer required determinants in cysteine-rich domain (CRD) 2 of CD134 for viral entry. The T271I mutation did not confer CD134-independent infection upon GL8 or CPG41, nor did it increase the affinity of the CXCR4 interaction, suggesting that the principal effect was targeted at reducing the complexity of the Env-CD134 interaction.

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Binding of FIV Fc-SU fusion proteins to CD134-expressing cells. (A) MCC cells stably-transduced with retroviral vectors encoding full-length fCD134 (Feline), CRD1 of fCD134 in the context of hCD134 (CRD1), human CD134 (Human) or vector only control (CON) were incubated with Fc-SU fusion proteins derived from GL8 WT or glycosylation site mutants T271I and N342Y and bound protein detected using PE-anti-human IgG Fc by flow cytometry. CXCR4 and CD134 expression were assayed using anti-CXCR4 (44701) and CD134 (7D6) respectively followed by PE-anti-mouse IgG. (B) Inhibition of residual WT GL8 Fc-SU binding to human CD134 expressing cells by anti-CXCR4. Cells were pre-incubated with AMD3100 and 44701 prior to binding of WT Fc-SU and detection of bound protein by flow cytometry using PE-anti-human IgG Fc. Histograms show 10,000 events and are representative of at least two independent experiments.
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Figure 6: Binding of FIV Fc-SU fusion proteins to CD134-expressing cells. (A) MCC cells stably-transduced with retroviral vectors encoding full-length fCD134 (Feline), CRD1 of fCD134 in the context of hCD134 (CRD1), human CD134 (Human) or vector only control (CON) were incubated with Fc-SU fusion proteins derived from GL8 WT or glycosylation site mutants T271I and N342Y and bound protein detected using PE-anti-human IgG Fc by flow cytometry. CXCR4 and CD134 expression were assayed using anti-CXCR4 (44701) and CD134 (7D6) respectively followed by PE-anti-mouse IgG. (B) Inhibition of residual WT GL8 Fc-SU binding to human CD134 expressing cells by anti-CXCR4. Cells were pre-incubated with AMD3100 and 44701 prior to binding of WT Fc-SU and detection of bound protein by flow cytometry using PE-anti-human IgG Fc. Histograms show 10,000 events and are representative of at least two independent experiments.

Mentions: Previous studies have demonstrated that soluble IgG-Fc-FIV SU fusion proteins (Fc-SU) recapitulate the receptor binding specificity of the parent virus [29,83-85]. We therefore asked whether GL8 Fc-SU fusion proteins (proteins that exist primarily as dimers) derived from the WT, T271I or N342Y Envs (trimeric on the native virions) would display the receptor binding properties of their respective parent viruses. WT, T271I or N342Y GL8 Env Fc-SUs bound to feline CD134 expressing MCC cells (Fig. 6A, row 1) but not to control MCC cells (Fig. 6A, row 4), consistent with the usage of feline CD134 as a receptor on MCC cells (Fig. 3B). However, a similar level of binding was detected to cells expressing only CRD1 of feline CD134 (Fig. 6A, row 2) suggesting that when the Envs were expressed in soluble form as Fc-SU fusion proteins, the requirement for determinants in CRD2 of CD134 for receptor binding is lost. These data would suggest that the specificity of receptor binding of the GL8 Env may be dependent on intermolecular interactions in the tertiary complex of the native trimeric Env on virions and accordingly that the T271I mutation modulates this interaction. As an additional control for binding specificity, we assessed the binding of Fc-SU proteins to MCC cells expressing human CD134. Neither the WT nor the T271I and N342Y Envs support infection through human CD134 ([30] and data not shown), however, significant (albeit weak) binding of the WT, T271I and N342Y Fc-SU proteins to human CD134-expressing MCC was detected (Fig. 6A, row 3). It was notable that the MCC-derived cell lines transduced with the feline CD134, CRD1 or human CD134 expression vectors showed up-regulated expression of CXCR4 (48.5%, 63.1% and 87.6% respectively) relative to vector-only control cells (14.8%) indicating that the low level binding of the Fc-SU proteins to human CD134 may not have been mediated by human CD134, rather it could have reflected the marked up-regulation of endogenous CXCR4 on these cells. Accordingly, when we repeated the binding of WT Fc-SU to the four cell lines following pre-treatment of the cells with the CXCR4 antagonist AMD3100 and in the presence of anti-CXCR4 monoclonal antibody 44701 (Fig. 6B.), the low level of background binding of Fc-SU to CXCR4 was reduced from 17.2% to 2.6%, consistent with a direct interaction between the soluble SU and CXCR4, as has been noted previously for both FIV and HIV [82,86]. Given that CXCR4 expression alone does not confer susceptibility to infection with WT GL8, and that WT GL8 does not infect cells expressing CRD1 alone, these data suggest that the Fc-SU fusion proteins are in a distinct conformation that fails to mimic that found on native virions.


A single site for N-linked glycosylation in the envelope glycoprotein of feline immunodeficiency virus modulates the virus-receptor interaction.

Willett BJ, McMonagle EL, Logan N, Samman A, Hosie MJ - Retrovirology (2008)

Binding of FIV Fc-SU fusion proteins to CD134-expressing cells. (A) MCC cells stably-transduced with retroviral vectors encoding full-length fCD134 (Feline), CRD1 of fCD134 in the context of hCD134 (CRD1), human CD134 (Human) or vector only control (CON) were incubated with Fc-SU fusion proteins derived from GL8 WT or glycosylation site mutants T271I and N342Y and bound protein detected using PE-anti-human IgG Fc by flow cytometry. CXCR4 and CD134 expression were assayed using anti-CXCR4 (44701) and CD134 (7D6) respectively followed by PE-anti-mouse IgG. (B) Inhibition of residual WT GL8 Fc-SU binding to human CD134 expressing cells by anti-CXCR4. Cells were pre-incubated with AMD3100 and 44701 prior to binding of WT Fc-SU and detection of bound protein by flow cytometry using PE-anti-human IgG Fc. Histograms show 10,000 events and are representative of at least two independent experiments.
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Figure 6: Binding of FIV Fc-SU fusion proteins to CD134-expressing cells. (A) MCC cells stably-transduced with retroviral vectors encoding full-length fCD134 (Feline), CRD1 of fCD134 in the context of hCD134 (CRD1), human CD134 (Human) or vector only control (CON) were incubated with Fc-SU fusion proteins derived from GL8 WT or glycosylation site mutants T271I and N342Y and bound protein detected using PE-anti-human IgG Fc by flow cytometry. CXCR4 and CD134 expression were assayed using anti-CXCR4 (44701) and CD134 (7D6) respectively followed by PE-anti-mouse IgG. (B) Inhibition of residual WT GL8 Fc-SU binding to human CD134 expressing cells by anti-CXCR4. Cells were pre-incubated with AMD3100 and 44701 prior to binding of WT Fc-SU and detection of bound protein by flow cytometry using PE-anti-human IgG Fc. Histograms show 10,000 events and are representative of at least two independent experiments.
Mentions: Previous studies have demonstrated that soluble IgG-Fc-FIV SU fusion proteins (Fc-SU) recapitulate the receptor binding specificity of the parent virus [29,83-85]. We therefore asked whether GL8 Fc-SU fusion proteins (proteins that exist primarily as dimers) derived from the WT, T271I or N342Y Envs (trimeric on the native virions) would display the receptor binding properties of their respective parent viruses. WT, T271I or N342Y GL8 Env Fc-SUs bound to feline CD134 expressing MCC cells (Fig. 6A, row 1) but not to control MCC cells (Fig. 6A, row 4), consistent with the usage of feline CD134 as a receptor on MCC cells (Fig. 3B). However, a similar level of binding was detected to cells expressing only CRD1 of feline CD134 (Fig. 6A, row 2) suggesting that when the Envs were expressed in soluble form as Fc-SU fusion proteins, the requirement for determinants in CRD2 of CD134 for receptor binding is lost. These data would suggest that the specificity of receptor binding of the GL8 Env may be dependent on intermolecular interactions in the tertiary complex of the native trimeric Env on virions and accordingly that the T271I mutation modulates this interaction. As an additional control for binding specificity, we assessed the binding of Fc-SU proteins to MCC cells expressing human CD134. Neither the WT nor the T271I and N342Y Envs support infection through human CD134 ([30] and data not shown), however, significant (albeit weak) binding of the WT, T271I and N342Y Fc-SU proteins to human CD134-expressing MCC was detected (Fig. 6A, row 3). It was notable that the MCC-derived cell lines transduced with the feline CD134, CRD1 or human CD134 expression vectors showed up-regulated expression of CXCR4 (48.5%, 63.1% and 87.6% respectively) relative to vector-only control cells (14.8%) indicating that the low level binding of the Fc-SU proteins to human CD134 may not have been mediated by human CD134, rather it could have reflected the marked up-regulation of endogenous CXCR4 on these cells. Accordingly, when we repeated the binding of WT Fc-SU to the four cell lines following pre-treatment of the cells with the CXCR4 antagonist AMD3100 and in the presence of anti-CXCR4 monoclonal antibody 44701 (Fig. 6B.), the low level of background binding of Fc-SU to CXCR4 was reduced from 17.2% to 2.6%, consistent with a direct interaction between the soluble SU and CXCR4, as has been noted previously for both FIV and HIV [82,86]. Given that CXCR4 expression alone does not confer susceptibility to infection with WT GL8, and that WT GL8 does not infect cells expressing CRD1 alone, these data suggest that the Fc-SU fusion proteins are in a distinct conformation that fails to mimic that found on native virions.

Bottom Line: Prototypic FIV vaccines based on the FL4 strain of FIV contain a cell culture-adapted strain of FIV Petaluma, a CD134-independent strain of FIV that interacts directly with CXCR4.In addition to a characteristic increase in charge in the V3 loop homologue of FIVFL4, we identified two mutations in potential sites for N-linked glycosylation in the region of FIV Env analogous to the V1-V2 region of HIV and SIV Env, T271I and N342Y.The T271I mutation did not confer CD134-independent infection upon GL8 or CPG41, nor did it increase the affinity of the CXCR4 interaction, suggesting that the principal effect was targeted at reducing the complexity of the Env-CD134 interaction.

View Article: PubMed Central - HTML - PubMed

Affiliation: Retrovirus Research Laboratory, Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow, G61 1QH, UK. b.willett@vet.gla.ac.uk

ABSTRACT
Feline immunodeficiency virus (FIV) targets helper T cells by attachment of the envelope glycoprotein (Env) to CD134, a subsequent interaction with CXCR4 then facilitating the process of viral entry. As the CXCR4 binding site is not exposed until CD134-binding has occurred then the virus is protected from neutralising antibodies targeting the CXCR4-binding site on Env. Prototypic FIV vaccines based on the FL4 strain of FIV contain a cell culture-adapted strain of FIV Petaluma, a CD134-independent strain of FIV that interacts directly with CXCR4. In addition to a characteristic increase in charge in the V3 loop homologue of FIVFL4, we identified two mutations in potential sites for N-linked glycosylation in the region of FIV Env analogous to the V1-V2 region of HIV and SIV Env, T271I and N342Y. When these mutations were introduced into the primary GL8 and CPG41 strains of FIV, the T271I mutation was found to alter the nature of the virus-CD134 interaction; primary viruses carrying the T271I mutation no longer required determinants in cysteine-rich domain (CRD) 2 of CD134 for viral entry. The T271I mutation did not confer CD134-independent infection upon GL8 or CPG41, nor did it increase the affinity of the CXCR4 interaction, suggesting that the principal effect was targeted at reducing the complexity of the Env-CD134 interaction.

Show MeSH
Related in: MedlinePlus