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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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Effect of ectopic CXCR4 expression on infection with glycosylation mutants. AH927 cells expressing low level endogenous CXCR4 (endog.) or enhanced levels of either feline CXCR4 (+feline) or human CXCR4 (+human), in conjunction with feline CD134 (feCD134), CRD1 of fCD134 in the context of hCD134 (CRD1), or vector-only control (Control), were infected with HIV(FIV) pseudotypes bearing the GL8 or CPG41 Envs. 72 hours post-infection luciferase activity was assayed. Each point represents the mean +/- SE of triplicate samples.
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Figure 5: Effect of ectopic CXCR4 expression on infection with glycosylation mutants. AH927 cells expressing low level endogenous CXCR4 (endog.) or enhanced levels of either feline CXCR4 (+feline) or human CXCR4 (+human), in conjunction with feline CD134 (feCD134), CRD1 of fCD134 in the context of hCD134 (CRD1), or vector-only control (Control), were infected with HIV(FIV) pseudotypes bearing the GL8 or CPG41 Envs. 72 hours post-infection luciferase activity was assayed. Each point represents the mean +/- SE of triplicate samples.

Mentions: We next asked whether the T271I or N342Y mutations conferred CD134-independence upon the GL8 and CPG41 Envs. Feline AH927 cells stably transduced with vectors encoding feline CD134 or the CRD1 chimaera, or the empty expression vector (pDONAI) only, were transduced for a second time with vectors encoding feline CXCR4 or human CXCR4, or expression vector (pBabePuro only). Stable lines were selected that expressed either low level endogenous feline CXCR4 (Fig. 5, endog.)), high level feline CXCR4 (+feline), or high level human CXCR4 (+human) in conjunction with either the CRD1 chimaera or native CD134. The cells were then infected with HIV(FIV) luciferase pseudotypes bearing the GL8 and CPG41 Envs (wild type (WT), T271I, N342Y or Δ2N double mutant) and infectivity assayed (Fig. 5). If the T271I or N342Y mutations conferred CD134-independence, enhanced CXCR4 expression alone would be sufficient to promote viral entry. Control cells expressing endogenous CXCR4 were refractory to infection with all viruses (Fig. 5A,B, "endog.") and ectopic expression of CRD1 did not enhance infection (Fig. 5C,D, "endog."). In contrast, ectopic expression of feCD134 (Fig. 5E,F, "endog.") enhanced infection of the control with all viruses (>10-fold) suggesting that sufficient endogenous CXCR4 is present to facilitate viral entry in the presence of native primary receptor. The addition of exogenous feline or human CXCR4 alone did not enhance infection of the control cells mediated by either the GL8 or CPG41 Envs significantly, irrespective of whether WT, T271I, N342Y or Δ2N (Fig. 5A,B, control). In contrast, the addition of exogenous CXCR4 in the presence of either the feline CD134 CRD1 chimera (Fig. 5C,D, CRD1), or native feline CD134 (Fig. 5E,F, CD134) revealed a significantly more marked enhancement of infection with the T271I and Δ2N viruses than with the WT or N342Y viruses. The data are consistent with the T271I mutation removing the requirement of the viral Env for determinants in CRD2 [30,63], and thus improving its ability to utilise the CRD1 chimaera as an effective viral receptor. Accordingly, the data suggest that the N-linked glycosylation site ablated by the T271I mutation either contributes to the binding site on FIV Env for CD134, or constrains the conformation of Env such that it may only interact with native feline CD134.


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)

Effect of ectopic CXCR4 expression on infection with glycosylation mutants. AH927 cells expressing low level endogenous CXCR4 (endog.) or enhanced levels of either feline CXCR4 (+feline) or human CXCR4 (+human), in conjunction with feline CD134 (feCD134), CRD1 of fCD134 in the context of hCD134 (CRD1), or vector-only control (Control), were infected with HIV(FIV) pseudotypes bearing the GL8 or CPG41 Envs. 72 hours post-infection luciferase activity was assayed. 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 5: Effect of ectopic CXCR4 expression on infection with glycosylation mutants. AH927 cells expressing low level endogenous CXCR4 (endog.) or enhanced levels of either feline CXCR4 (+feline) or human CXCR4 (+human), in conjunction with feline CD134 (feCD134), CRD1 of fCD134 in the context of hCD134 (CRD1), or vector-only control (Control), were infected with HIV(FIV) pseudotypes bearing the GL8 or CPG41 Envs. 72 hours post-infection luciferase activity was assayed. Each point represents the mean +/- SE of triplicate samples.
Mentions: We next asked whether the T271I or N342Y mutations conferred CD134-independence upon the GL8 and CPG41 Envs. Feline AH927 cells stably transduced with vectors encoding feline CD134 or the CRD1 chimaera, or the empty expression vector (pDONAI) only, were transduced for a second time with vectors encoding feline CXCR4 or human CXCR4, or expression vector (pBabePuro only). Stable lines were selected that expressed either low level endogenous feline CXCR4 (Fig. 5, endog.)), high level feline CXCR4 (+feline), or high level human CXCR4 (+human) in conjunction with either the CRD1 chimaera or native CD134. The cells were then infected with HIV(FIV) luciferase pseudotypes bearing the GL8 and CPG41 Envs (wild type (WT), T271I, N342Y or Δ2N double mutant) and infectivity assayed (Fig. 5). If the T271I or N342Y mutations conferred CD134-independence, enhanced CXCR4 expression alone would be sufficient to promote viral entry. Control cells expressing endogenous CXCR4 were refractory to infection with all viruses (Fig. 5A,B, "endog.") and ectopic expression of CRD1 did not enhance infection (Fig. 5C,D, "endog."). In contrast, ectopic expression of feCD134 (Fig. 5E,F, "endog.") enhanced infection of the control with all viruses (>10-fold) suggesting that sufficient endogenous CXCR4 is present to facilitate viral entry in the presence of native primary receptor. The addition of exogenous feline or human CXCR4 alone did not enhance infection of the control cells mediated by either the GL8 or CPG41 Envs significantly, irrespective of whether WT, T271I, N342Y or Δ2N (Fig. 5A,B, control). In contrast, the addition of exogenous CXCR4 in the presence of either the feline CD134 CRD1 chimera (Fig. 5C,D, CRD1), or native feline CD134 (Fig. 5E,F, CD134) revealed a significantly more marked enhancement of infection with the T271I and Δ2N viruses than with the WT or N342Y viruses. The data are consistent with the T271I mutation removing the requirement of the viral Env for determinants in CRD2 [30,63], and thus improving its ability to utilise the CRD1 chimaera as an effective viral receptor. Accordingly, the data suggest that the N-linked glycosylation site ablated by the T271I mutation either contributes to the binding site on FIV Env for CD134, or constrains the conformation of Env such that it may only interact with native feline CD134.

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