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. 2002 Dec 19:2:31.
doi: 10.1186/1471-2334-2-31. Epub 2002 Dec 19.

Infection of cells expressing CXCR4 mutants lacking N-glycosylation at the N-terminal extracellular domain is enhanced for R5X4-dualtropic human immunodeficiency virus type-1

Ingo Thordsen  1 , Svenja Polzer, Michael Schreiber
Affiliations

Infection of cells expressing CXCR4 mutants lacking N-glycosylation at the N-terminal extracellular domain is enhanced for R5X4-dualtropic human immunodeficiency virus type-1

Ingo Thordsen et al. BMC Infect Dis. .

Abstract

Background: Infection with human immunodeficiency virus type-1 (HIV-1) requires binding of the viral envelope gp120 to CD4 and to the CXCR4 coreceptor. Both, gp120 and CXCR4 are subject to N-glycosylation. Here we investigated the influence of the N-linked glycans g1 and g2 present on CXCR4 for HIV-1 infection.

Methods: The two CXCR4 N-glycosylation sites g1 (NYT) and g2 (NVS) were mutated by changing the first or third amino acids N or T/S to Q and A respectively (g1; N11Q or T13A; g2, N176Q or S178A). Human osteosarcoma cells (GHOST) expressing human CD4 and the various CXCR4 glycosylation mutants were tested for infection using NL4-3-based viruses with X4, R5 or R5X4-tropism differing only in the V3 loop region.

Results: All constructed cell lines expressing the various CXCR4 glycomutants showed similar permissiveness for the X4-monotropic virus and no change in the coreceptor specificity that allows infection of a CCR5-dependent R5-monotropic virus. Interestingly, the removal of glycan g1 significantly enhanced the permissiveness of GHOST cells for the R5X4 dualtropic virus. GHOST cells expressing the CXCR4-g1 or CXCR4-g1/2 mutants were infected at higher rates by the R5X4-dualtropic virus compared to cells expressing CXCR4-wt or CXCR4-g2 coreceptors.

Conclusion: Our present observations underscore a role for glycans present on the CXCR4 coreceptor in the entry process of HIV-1. The data will help to better understand the multifaceted mechanism of HIV infection and the selective forces which drive HIV-1 evolution from mono- to dual-tropism.

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Figures

Figure 1
Figure 1
CXCR4 extracellular domains and CXCR4 mutants generated. A, two recognition sites for N-linked glycosylation, designated g1 (aa (NYT, pos. 11–13) and g2 (aa NVS, pos. 176–178) are located in the extracellular CXCR4 domains. B, by replacing the first amino acid N against Q and the third amino acid S against A a set of five CXCR4 mutants lacking g1, g2 or both glycans were generated. Amino acids: A, alanine; N, asparagine; Q, glutamine; S, serine; T, threonine; V, valine; Y, tyrosine.
Figure 2
Figure 2
CXCR4 cell surface expression. The CXCR4 coding sequence was mutated in the pcDNA3.1 plasmid, cloned into pBABE-puro and finally expressed in GHOST.CD4 cells. Cell surface expression was monitored by flow cytometry using phycoerythrin labeled anti-CXCR4 mAb 12G5. Coreceptor expression is expressed as % of CXCR4 wt expression of GHOST.CD4.CXCR4 cells. Error bars are calculated based on three independent experiments.
Figure 3
Figure 3
Construction of NL4-3 mutants differing in coreceptor usage. A, Amino acid V3 loop sequence of the NL4-3 laboratory strain and of two primary isolates PI-952 (R5X4-dualtropic) and PI-991 (R5-monotropic) of different phenotype. B, V3 loop regions of patient isolates were generated by PCR amplification followed by a second PCR. In the second PCR amplification silent mutations were introduced to generate the BglII and NheI restriction sites. PCR fragments were cloned into pUCenv-deltaV3 and the NL4-3 env – V3 loop chimera was cloned into the pNL4-3Bst retroviral vector as a BstEII/BamHI fragment. C, This V3 cloning procedure was carried out to generate mutants of NL4-3, designated NL-952 and NL-991, containing the V3 loop and the coreceptor phenotype of the of the original primary isolates. NL4-3, X4-monotropic; NL-952, R5X4-dualtropic; NL-991, R5-monotropic.
Figure 4
Figure 4
Virus replication and CXCR4 glycosylation. GHOST cells expressing CXCR4 wt or CXCR4-g1, -g2 or -g1/g2 were infected with equal amounts of (A) the X4 tropic strain NL4-3 (0.05 ng p24 / 3 × 10e3 cells), (B) the R5X4 dual-tropic virus NL-952 (0.5 ng p24 / 3 × 10e3 cells) and (C) the R5 tropic HIV-1 NL-991 (0.5 ng p24 / 3 × 10e3 cells. Virus production was measured by a standardized p24 antigen ELISA [(OD405nm – 0,1) / 0,04 = ng p24, linear range OD405m 0,2–1,5]. Values are calculated based on two independent experiments. White symbols GHOST.CD4 cells expressing CXCR4 lacking g1, black symbols GHOST.CD4 cells expressing CXCR4 containing g1.
Figure 5
Figure 5
Charged amino acids in the extracellular domains of CXCR4 and CCR5. The diagramm shows a schematic representation of the extracellular domains of CXCR4 and CCR5. Indicated are positively and negatively charged amino acids and the glycans attached to asparagine residues. Symbol plus, lysine (K) and arginine (R) amino acid residues; symbol minus, glutamic (E) and aspartic (D) acid amino acid residues.
Figure 6
Figure 6
Comparison of charged amino acids between coreceptors and viral V3 loops. This comparison shows the number of charged amino acids in the extracellular domains of CXCR4 and CCR5 and the number in the V3 loop of the three viruses NL4-3, NL-952 and NL-991. Symbol plus (formula image), lysine (K) and arginine (R) amino acid residues, Symbol minus (formula image), glutamic (E) and aspartic (D) acid amino acid residues. Boxed, amino acids related to coreceptor usage
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