Dataset Information

Membrane topology analysis of HIV-1 envelope glycoprotein gp41.

ABSTRACT:

Background

The gp41 subunit of the HIV-1 envelope glycoprotein (Env) has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD). An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in both prokaryotic and mammalian systems. We attached topological markers to the C-termini of serially truncated gp41. In the prokaryotic system, we utilized a green fluorescent protein (GFP) that is only active in the cytoplasm. The tag protein (HaloTag) and a membrane-impermeable ligand specific to HaloTag was used in the mammalian system.

Results

In the absence of membrane fusion, both the prokaryotic and mammalian systems (293FT cells) supported the single MSD model. In the presence of membrane fusion in mammalian cells (293CD4 cells), the data obtained seem to support the multiple MSD model. However, the region predicted to be a potential MSD is the highly hydrophilic Kennedy sequence and is least likely to become a MSD based on several algorithms. Further analysis revealed the induction of membrane permeability during membrane fusion, allowing the membrane-impermeable ligand and antibodies to cross the membrane. Therefore, we cannot completely rule out the possible artifacts. Addition of membrane fusion inhibitors or alterations of the MSD sequence decreased the induction of membrane permeability.

Conclusions

It is likely that a single MSD model for HIV-1 gp41 holds true even in the presence of membrane fusion. The degree of the augmentation of membrane permeability we observed was dependent on the membrane fusion and sequence of the MSD.

SUBMITTER: Liu S

PROVIDER: S-EPMC3012657 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Publications

Membrane topology analysis of HIV-1 envelope glycoprotein gp41.

Liu Shujun S Kondo Naoyuki N Long Yufei Y Xiao Dan D Iwamoto Aikichi A Matsuda Zene Z

Retrovirology 20101130

<h4>Background</h4>The gp41 subunit of the HIV-1 envelope glycoprotein (Env) has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD). An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in ...[more]

PMID: 21118523

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Project description:The mature human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer is produced by proteolytic cleavage of a precursor and consists of three gp120 exterior and three gp41 transmembrane subunits. The metastable Env complex is induced to undergo conformational changes required for virus entry by the binding of gp120 to the receptors, CD4 and CCR5/CXCR4. An isoleucine-to-proline change (I559P) in the gp41 ectodomain has been used to stabilize soluble forms of HIV-1 Env trimers for structural characterization and for use as immunogens. In the native membrane-anchored HIV-1BG505 Env, the I559P change modestly decreased proteolytic maturation, increased the non-covalent association of gp120 with the Env trimer, and resulted in an Env conformation distinctly different from that of the wild-type HIV-1BG505 Env. Compared with the wild-type Env, the I559P Env was recognized inefficiently by polyclonal sera from HIV-1-infected individuals, by several gp41-directed antibodies, by some antibodies against the CD4-binding site of gp120, and by antibodies that preferentially recognize the CD4-bound Env. Some of the gp120-associated antigenic differences between the wild-type HIV-1BG505 Env and the I559P mutant were compensated by the SOS disulfide bond between gp120 and gp41, which has been used to stabilize cleaved soluble Env trimers. Nonetheless, regardless of the presence of the SOS changes, Envs with proline 559 were recognized less efficiently than Envs with isoleucine 559 by the VRC01 neutralizing antibody, which binds the CD4-binding site of gp120, and the PGT151 neutralizing antibody, which binds a hybrid gp120-gp41 epitope. The I559P change completely eliminated the ability of the HIV-1BG505 Env to mediate cell-cell fusion and virus entry, and abolished the capacity of the SOS Env to support virus infection in the presence of a reducing agent. These results suggest that differences exist between the quaternary structures of functional Env spikes and I559P Envs.

Project description:Interactions between the gp120 and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer maintain the metastable unliganded form of the viral spike. Binding of gp120 to the receptor, CD4, changes the Env conformation to promote gp120 interaction with the second receptor, CCR5 or CXCR4. CD4 binding also induces the transformation of Env into the prehairpin intermediate, in which the gp41 heptad repeat 1 (HR1) coiled coil is assembled at the trimer axis. In nature, HIV-1 Envs must balance the requirements to maintain the noncovalent association of gp120 with gp41 and to evade the host antibody response with the need to respond to CD4 binding. Here we show that the gp41 HR1 region contributes to gp120 association with the unliganded Env trimer. Changes in particular amino acid residues in the gp41 HR1 region decreased the efficiency with which Env moved from the unliganded state. Thus, these gp41 changes decreased the sensitivity of HIV-1 to cold inactivation and ligands that require Env conformational changes to bind efficiently. Conversely, these gp41 changes increased HIV-1 sensitivity to small-molecule entry inhibitors that block Env conformational changes induced by CD4. Changes in particular gp41 HR1 amino acid residues can apparently affect the relative stability of the unliganded state and CD4-induced conformations. Thus, the gp41 HR1 region contributes to the association with gp120 and regulates Env transitions from the unliganded state to downstream conformations.IMPORTANCE The development of an efficient vaccine able to prevent HIV infection is a worldwide priority. Knowledge of the envelope glycoprotein structure and the conformational changes that occur after receptor engagement will help researchers to develop an immunogen able to elicit antibodies that block HIV-1 transmission. Here we identify residues in the HIV-1 transmembrane envelope glycoprotein that stabilize the unliganded state by modulating the transitions from the unliganded state to the CD4-bound state.

Dataset Information

Membrane topology analysis of HIV-1 envelope glycoprotein gp41.

Background

Results

Conclusions

Publications

Membrane topology analysis of HIV-1 envelope glycoprotein gp41.

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