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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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Sensitivity of glycosylation mutants to CXCR4 antagonist. Infection of MYA-1 and CLL-CD134 cells with HIV (FIV) pseudotypes bearing WT, T271I or N342Y mutant GL8 and CPG41 Envs was quantified in the presence of increasing concentrations of CXCR4 antagonist AMD3100. Infection is expressed as luciferase activity (CPM) and percent infection relative to control (no antagonist). Each point represents the mean +/- SE of triplicate samples.
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Figure 4: Sensitivity of glycosylation mutants to CXCR4 antagonist. Infection of MYA-1 and CLL-CD134 cells with HIV (FIV) pseudotypes bearing WT, T271I or N342Y mutant GL8 and CPG41 Envs was quantified in the presence of increasing concentrations of CXCR4 antagonist AMD3100. Infection is expressed as luciferase activity (CPM) and percent infection relative to control (no antagonist). Each point represents the mean +/- SE of triplicate samples.

Mentions: The enhanced usage of the CD134-CRD1 chimaera by the T271 mutants may have arisen due to a less stringent interaction between FIV Env and CD134, the N-linked glycosylation site at 269 contributing to the receptor binding surface. Alternatively, removal of the N-linked glycosylation may have enhanced the ability of the Env to interact with CXCR4, either facilitating partial CD134-independent entry or by enhancing the affinity for CXCR4. To address the latter possibility, we examined the sensitivity of viral pseudotypes bearing the WT, T271I or N342Y Envs to the CXCR4 antagonist AMD3100 (Fig. 4). Sensitivity to AMD3100 was assessed on MYA-1 T cells (expressing low levels of CXCR4 [82]) and CLL-CD134 (expressing high levels of CXCR4 (Willett et al., unpublished data). Where CXCR4 was limiting (MYA-1 cells), infection with both GL8 and CPG41 was inhibited efficiently by AMD3100. Neither the T271 nor the N342Y mutation enhanced or reduced sensitivity to AMD3100 although it was notable that GL8 was significantly more sensitive to antagonism by AMD3100 than CPG41 (at 0.1 μg/ml AMD3100 there was a 100-fold reduction in GL8 entry compared with 5-fold for CPG41), suggesting that CPG41 has a higher affinity for CXCR4 per se. Where CXCR4 was abundant (CLL-CD134), inhibition of infection required significantly higher concentrations of AMD3100 and even at 10 μg/ml antagonist, the degree of inhibition never exceeded 80%. The data suggest that the T271I and N342Y mutations do not alter the affinity of the CXCR4 interaction significantly.


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)

Sensitivity of glycosylation mutants to CXCR4 antagonist. Infection of MYA-1 and CLL-CD134 cells with HIV (FIV) pseudotypes bearing WT, T271I or N342Y mutant GL8 and CPG41 Envs was quantified in the presence of increasing concentrations of CXCR4 antagonist AMD3100. Infection is expressed as luciferase activity (CPM) and percent infection relative to control (no antagonist). Each point represents the mean +/- SE of triplicate samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Sensitivity of glycosylation mutants to CXCR4 antagonist. Infection of MYA-1 and CLL-CD134 cells with HIV (FIV) pseudotypes bearing WT, T271I or N342Y mutant GL8 and CPG41 Envs was quantified in the presence of increasing concentrations of CXCR4 antagonist AMD3100. Infection is expressed as luciferase activity (CPM) and percent infection relative to control (no antagonist). Each point represents the mean +/- SE of triplicate samples.
Mentions: The enhanced usage of the CD134-CRD1 chimaera by the T271 mutants may have arisen due to a less stringent interaction between FIV Env and CD134, the N-linked glycosylation site at 269 contributing to the receptor binding surface. Alternatively, removal of the N-linked glycosylation may have enhanced the ability of the Env to interact with CXCR4, either facilitating partial CD134-independent entry or by enhancing the affinity for CXCR4. To address the latter possibility, we examined the sensitivity of viral pseudotypes bearing the WT, T271I or N342Y Envs to the CXCR4 antagonist AMD3100 (Fig. 4). Sensitivity to AMD3100 was assessed on MYA-1 T cells (expressing low levels of CXCR4 [82]) and CLL-CD134 (expressing high levels of CXCR4 (Willett et al., unpublished data). Where CXCR4 was limiting (MYA-1 cells), infection with both GL8 and CPG41 was inhibited efficiently by AMD3100. Neither the T271 nor the N342Y mutation enhanced or reduced sensitivity to AMD3100 although it was notable that GL8 was significantly more sensitive to antagonism by AMD3100 than CPG41 (at 0.1 μg/ml AMD3100 there was a 100-fold reduction in GL8 entry compared with 5-fold for CPG41), suggesting that CPG41 has a higher affinity for CXCR4 per se. Where CXCR4 was abundant (CLL-CD134), inhibition of infection required significantly higher concentrations of AMD3100 and even at 10 μg/ml antagonist, the degree of inhibition never exceeded 80%. The data suggest that the T271I and N342Y mutations do not alter the affinity of the CXCR4 interaction significantly.

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