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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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Mutant proviral clones bearing T271I or N342Y yield infectious virus. A. Pelleted virus from MYA-1 cells infected with GL8 (1–4) and CPG41 (5–8) viruses bearing WT (1,5), T271I (2,6), N342Y (3,7) or Δ2N (4,8) were separated by reducing SDS-PAGE on a 12% acrylamide gel, transferred to nitrocellulose and probed with pooled cat anti-FIV. Approx. molecular mass (kDa) are on left, primary reactivities Env, prGag and CA arrowed on right. B. 10–20% gradient gel analysis of GL8 WT (1), T271 (2), N342Y (3) and Δ2N (4) probed with anti-FIV (subtype-specific) SU monoclonal antibody vpg71.2. Approx. molecular mass (kDa) on left, Env is arrowed on right.
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Figure 2: Mutant proviral clones bearing T271I or N342Y yield infectious virus. A. Pelleted virus from MYA-1 cells infected with GL8 (1–4) and CPG41 (5–8) viruses bearing WT (1,5), T271I (2,6), N342Y (3,7) or Δ2N (4,8) were separated by reducing SDS-PAGE on a 12% acrylamide gel, transferred to nitrocellulose and probed with pooled cat anti-FIV. Approx. molecular mass (kDa) are on left, primary reactivities Env, prGag and CA arrowed on right. B. 10–20% gradient gel analysis of GL8 WT (1), T271 (2), N342Y (3) and Δ2N (4) probed with anti-FIV (subtype-specific) SU monoclonal antibody vpg71.2. Approx. molecular mass (kDa) on left, Env is arrowed on right.

Mentions: The T271I and N342Y mutations were reproduced in molecular clones of FIV GL8 and a chimeric molecular clone bearing the CPG41 Env in the GL8 backbone by site-directed mutagenesis. Constructs were transfected into 293T cells and replication competent virus was recovered into IL2-dependent feline T cells (MYA-1). All viruses replicated with similar efficiency in MYA-1 cells (not shown). Bulk supernatants were prepared, virus pelleted by ultracentrifugation and analysed by SDS-PAGE. Immunoblotting using polyclonal anti-FIV (Fig. 2A) confirmed that similar levels of viral proteins were produced by each virus. Although a marginal reduction in the apparent size of Env was suggested in the Δ2N mutants of both GL8 and CPG41 (Fig. 2A), this was visualised more readily when the GL8 viruses were separated on a 10–20% gradient gel (Fig. 2B) and probed with the subtype-specific monoclonal antibody vpg71.2 (vpg71.2 does not recognise CPG41 Env). Under these conditions, a clear downward shift could be discerned in the Δ2N double mutant virus but in neither of the single mutants.


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)

Mutant proviral clones bearing T271I or N342Y yield infectious virus. A. Pelleted virus from MYA-1 cells infected with GL8 (1–4) and CPG41 (5–8) viruses bearing WT (1,5), T271I (2,6), N342Y (3,7) or Δ2N (4,8) were separated by reducing SDS-PAGE on a 12% acrylamide gel, transferred to nitrocellulose and probed with pooled cat anti-FIV. Approx. molecular mass (kDa) are on left, primary reactivities Env, prGag and CA arrowed on right. B. 10–20% gradient gel analysis of GL8 WT (1), T271 (2), N342Y (3) and Δ2N (4) probed with anti-FIV (subtype-specific) SU monoclonal antibody vpg71.2. Approx. molecular mass (kDa) on left, Env is arrowed on right.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2563026&req=5

Figure 2: Mutant proviral clones bearing T271I or N342Y yield infectious virus. A. Pelleted virus from MYA-1 cells infected with GL8 (1–4) and CPG41 (5–8) viruses bearing WT (1,5), T271I (2,6), N342Y (3,7) or Δ2N (4,8) were separated by reducing SDS-PAGE on a 12% acrylamide gel, transferred to nitrocellulose and probed with pooled cat anti-FIV. Approx. molecular mass (kDa) are on left, primary reactivities Env, prGag and CA arrowed on right. B. 10–20% gradient gel analysis of GL8 WT (1), T271 (2), N342Y (3) and Δ2N (4) probed with anti-FIV (subtype-specific) SU monoclonal antibody vpg71.2. Approx. molecular mass (kDa) on left, Env is arrowed on right.
Mentions: The T271I and N342Y mutations were reproduced in molecular clones of FIV GL8 and a chimeric molecular clone bearing the CPG41 Env in the GL8 backbone by site-directed mutagenesis. Constructs were transfected into 293T cells and replication competent virus was recovered into IL2-dependent feline T cells (MYA-1). All viruses replicated with similar efficiency in MYA-1 cells (not shown). Bulk supernatants were prepared, virus pelleted by ultracentrifugation and analysed by SDS-PAGE. Immunoblotting using polyclonal anti-FIV (Fig. 2A) confirmed that similar levels of viral proteins were produced by each virus. Although a marginal reduction in the apparent size of Env was suggested in the Δ2N mutants of both GL8 and CPG41 (Fig. 2A), this was visualised more readily when the GL8 viruses were separated on a 10–20% gradient gel (Fig. 2B) and probed with the subtype-specific monoclonal antibody vpg71.2 (vpg71.2 does not recognise CPG41 Env). Under these conditions, a clear downward shift could be discerned in the Δ2N double mutant virus but in neither of the single mutants.

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