Simian immunodeficiency virus gp120

Metastable conformations of the gp and gp41 envelope glycoproteins of human immunodeficiency virus type 1 HIV-1 and simian immunodeficiency virus SIV must be maintained in the unliganded state of the envelope glycoprotein trimer. Binding of gp to the primary receptor, CD4, triggers the transition to an open conformation of the trimer, promoting interaction with the CCR5 chemokine receptor and ultimately leading to gpmediated virus-cell membrane fusion and entry.

Topological layers in the gp inner domain contribute to gptrimer association in the unliganded state and to CD4 binding. Layer 1 of the SIVmac gp inner domain contributes more to trimer association than the corresponding region of HIV-1 gp Activation of SIVmac by soluble CD4 is dependent on tryptophan and on layer 1 residues that determine a tight association of gp with the trimer.

Based on phylogenetic evidence, SIV variants form the primary reservoir, with HIV-1 and HIV-2 resulting from zoonotic cross-species transmissions within the last century or two 7 , 24 , Clear lineage-specific genetic differences are observed between variants of SIV and also between these variants and their more recent human crossovers 4 , 7 , 24 , Entry of HIV-1 and SIV into the host cell is mediated by the viral envelope glycoproteins, which are derived by proteolytic cleavage of a trimeric, glycosylated gp envelope glycoprotein precursor 2 , The resulting mature envelope glycoproteins, the gp exterior envelope glycoprotein SU and the gp41 transmembrane envelope glycoprotein TM , constitute a trimeric complex on the virion surface that is anchored by the gp41 membrane-spanning segments 8 , 18 , 71 , The gp exterior envelope glycoprotein is retained on the trimer via labile, noncovalent interactions with the gp41 ectodomain and perhaps with other gp protomers 21 , 29 , 75 , The gp glycoprotein is the most exposed element on the trimer and mediates binding to the viral receptors on the host cell.

Binding to the initial receptor, CD4 12 , 36 , triggers conformational changes in gp that promote its interaction with one of the chemokine receptors, usually CCR5 1 , 10 , 13 — 15 , 19 , 70 , Further conformational changes lead to the formation of a gp41 six-helix bundle composed of the HR1 and HR2 heptad repeat regions, which provides the energy needed to fuse the viral and target cell membranes 8 , 46 , Premature triggering of the metastable envelope glycoprotein complex to downstream conformations results in functional inactivation 22 , 27 , 28 , 33 , 34 , Because detailed structural information about gp in the unliganded HIV-1 trimer is lacking, only a rudimentary understanding of the sequence of events initiated by CD4 binding exists.

In the CD4-bound state, the gp core consists of a gpinteractive inner domain, an outer domain that faces outward on the assembled envelope glycoprotein trimer, and a minidomain called the bridging sheet. Although the gp inner domain does not contact CD4 41 , 54 , conformational rearrangements within this domain decrease the off rate of CD4 binding and thus increase gpCD4 affinity The interaction of layer 1 and layer 2 within the HIV-1 gp inner domain contributes in an indirect manner to CD4 binding Thus, gp refolds itself upon binding to CD4, with the inner and outer domains and bridging sheet brought into proximity at the interface with CD4.

Some of these cavity-lining residues contribute to an aromatic array that helps stabilize the CD4-bound conformation 21 , 41 , Because changes in some inner domain residues in layer 2 result in shedding of gp from the envelope glycoprotein trimer, it has been suggested that CD4-induced conformational changes involving the inner domain layers might promote partial release of the restraints that keep gp and gp41 in their unliganded conformation 21 , The SIVsmm gp glycoproteins naturally have a tryptophan residue that is predicted to fill the Phe 43 cavity 39 and may contribute to the lower dependence on CD4.

In support of this hypothesis, substitution of tryptophan for serine , which is found in most HIV-1 strains 39 , fills the Phe 43 cavity and allows HIV-1 gp to sample conformations closer to the CD4-bound state spontaneously Intriguingly, the half-life of the sCD4-activated state of HIV-1 was increased by changes in layer 1 of the inner domain Moreover, the impact of alterations in layers 1 and 2 on CD4 binding could be diminished by replacement of serine with tryptophan in HIV-1 gp It is noteworthy that the disulfide loop in layer 1 of the gp inner domain is truncated in SIV relative to that of HIV-1 see below.

These observations led us to hypothesize that the interaction of layers 1 and 2 of the HIV-1 gp inner domain and the Phe 43 cavity-filling tryptophan of SIV gp similarly serve to promote the CD4-bound conformation and thus facilitate CD4 binding. A corollary of this hypothesis is that the contribution of layers 1 and 2 to SIV gp interaction with CD4 will be less than that previously documented for HIV-1 gp Here we tested this hypothesis by investigating the contributions of the gp inner-domain layers 1 and 2 and tryptophan to the distinct biological properties of SIV.

For these studies, we chose the molecularly cloned and well-characterized SIVmac 32 , 35 , 52 from the diverse group of SIVs. The SIVmac envelope glycoproteins exhibit a dependence on target cell CD4 levels similar to that of the HIV-1 YU2 envelope glycoproteins 5 , which were studied in the previous mutagenic analysis of the HIV-1 gp inner domain layers 1 and 2 Cf2Th-CCR5 cells were grown in medium supplemented with 0.

Mutations were introduced individually or in combination into the previously described vector expressing the SIVmac envelope glycoproteins pSIVmac 47 , The presence of the desired mutations was determined by automated DNA sequencing. Precipitation of radiolabeled SIVmac envelope glycoproteins from cell lysates or medium was performed with a mixture of sera from SIV-infected macaques.

Cell lysates were immunoprecipitated with a mixture of sera from SIV-infected macaques. For CD4-Ig and CCR5 immunoprecipitations, samples were analyzed under nonreducing conditions as previously described A horseradish peroxidase-conjugated antibody specific for the Fc region of human IgG Pierce was then incubated with the samples for 45 min at room temperature.

Cells were washed 5 times with blocking buffer and 5 times with washing buffer. Recombinant viruses containing the firefly luciferase gene were produced by calcium phosphate transfection of T cells with the HIV-1 proviral vector pNL4. Two days after transfection, the cell supernatants were harvested; the reverse transcriptase activities of all viruses were measured as described previously Luciferase activity in each well was measured as described above.

We wished to investigate the function of the inner domain of SIVmac gp Because a structure of SIV gp with the complete inner domain is not available 9 , we modeled the SIVmac gp envelope glycoprotein based on the X-ray crystal structure of the HIV-1 gp core with intact N and C termini, bound to soluble CD4 and a neutralizing antibody fragment This X-ray crystallographic study revealed the structure of the complete HIV-1 gp inner domain 54 Fig. Changes in layer 2 can decrease the stable association of HIV-1 gp with the envelope glycoprotein trimer and result in shedding of gp The interaction of layers 1 and 2 in the HIV-1 gp inner domain strengthens gpCD4 binding by reducing the off rate The outer domain OD of gp is colored yellow.

The N and C termini are colored cyan. The other two strands of the bridging sheet are derived from the distal portion of layer 2. The location of the Phe 43 cavity is indicated by a red asterisk. The cysteine residues that form a disulfide bond in layer 1 are colored yellow, and some of the layer 1 and layer 2 residues implicated in layer 1-layer 2 interaction in HIV-1 gp 21 are colored blue.

Interestingly, as observed for HIV-1 gp, the conserved residues Trp 69, Asp , and Leu are predicted to be located at the interface of layers 1 and 2 of SIVmac gp compare Fig. However, a fourth residue, His 66, at the layer 1-layer 2 interface of HIV-1 gp, lacks a counterpart in SIV gp as a result of the truncation of layer 1.

Sequence identity is indicated by a solid vertical line, and amino acid residues that are conserved are connected by a dotted vertical line. Gaps in the sequence are indicated by dashes. Evolution of gp changes in PIVs. The phylogenetic relationship of envelope glycoprotein sequences of the PIVs is shown, based on previous studies 4 , 7 , 66 , The evolutionary distances in this tree are approximate.

The disulfide loop DSL length of layer 1 in the gp inner domain is shown in the first column. The identities of residues 66, , and in gp are shown in the other columns. A dash indicates the absence of a corresponding residue in the gp glycoprotein of the indicated PIV. The above analysis predicts that substantial differences exist between the inner domain layer 1 of HIV-1 and SIVmac gp The side chain of residue is oriented toward the Phe 43 cavity, and thus large residues have the potential to occupy the cavity Based on the CD4-binding phenotypes of HIV-1 gp mutants with different substitutions at Ser 76 , the phylogenetic changes in residue accompanying the evolution of the group M HIV-1 envelope glycoproteins would be expected to decrease the tendency to assume the CD4-bound conformation.

The contribution of the gp inner domain to the structure and function of the SIVmac envelope glycoproteins was evaluated by studying the phenotypes of a panel of mutant envelope glycoproteins Table 1.

Proteolytic processing of the envelope glycoprotein precursor and the noncovalent association of the gp exterior envelope glycoprotein with the gp41 transmembrane envelope glycoprotein in the trimer were evaluated. Several changes in the SIVmac gp inner domain decreased the association of gp with the envelope glycoprotein trimer Fig.

These results were noteworthy, since relatively few changes in layer 1 of HIV-1 gp exerted effects on subunit association 21 Fig. Alteration of several layer 2 residues affected the association of the SIVmac gp glycoprotein with the trimer, consistent with the phenotypes of layer 2 mutants of the HIV-1 gp glycoprotein 21 Fig.

However, changes in particular layer 2 residues Gln , Trp , and Ala affected SIVmac gptrimer association differently than alteration of the equivalent residue in HIV-1 gp These observations suggest that layer 1 contributes significantly to the stability of the SIV envelope glycoprotein trimer, in contrast to layer 1 of HIV-1 gp Precursor processing and gptrimer association of selected SIVmac envelope glycoprotein mutants.

Cell lysates and supernatants SN of 35 S-labeled cells transiently expressing the SIVmac wild-type and indicated mutant envelope glycoproteins were precipitated with serum from an SIV-infected macaque.

Residues important for gptrimer association and CD4 binding. Alteration of the interface between layers 1 and 2 of the HIV-1 gp inner domain has been shown to decrease CD4 binding, revealing an important mechanism whereby HIV-1 gp regulates CD4-induced conformational rearrangement and achieves a reduction in the off rate 21 , 34 , Wild-type and mutant SIVmac envelope glycoproteins were transiently expressed in T cells, which were radiolabeled for 16 h.

The amount of radiolabeled gp glycoproteins shed into the cell medium was normalized by immunoprecipitation with polyclonal serum from SIV-infected macaques before assessing the ability to bind CD4-Ig. Author information Article notes Copyright and License information Disclaimer. Correspondence e-mail: ude. Received Oct 10; Accepted Jan This article has been cited by other articles in PMC. PDB reference: simian immunodeficiency virus gp core, 3fus, r3fussf.

Keywords: conformational flexibility, normal-mode analysis, anisotropic thermal parameters, glycoproteins. Introduction Atomic structures of large biomolecular assemblies determined by X-ray crystallography are vitally important to the understanding of their functions. Open in a separate window. Figure 1. Methods 2. Elastic normal-mode analysis and a new branch normal-mode analysis The normal-mode vectors were calculated by an extended version of the elastic network model recently developed in our laboratory Lu et al.

Positional refinement and manual model adjustment The theory and details of the normal-mode-based refinement method have been reported in previous publications Poon et al. Observations 3.

Normal-mode-based refinement of gp core Structural refinement using different refinement programs tends to yield slightly different values for R factors and later versions of the same refinement programs may yield slightly better R factors than earlier versions.

Table 1 Refinement statistics of the original model and the normal-mode model of the SIV gp core. Figure 2. The addition of new sugar rings In addition to structural adjustments of the protein portion of the model, the improved density map also allowed the addition of five new sugar rings Fig. Figure 3. Structural flexibility reflected in the normal-mode model Overall, the B -factor distribution of the final normal-mode model of gp agrees well with its functionally relevant structural flexibility.

Figure 4. Discussion Here, we report a normal-mode-based refinement of the SIV gp structure that was originally determined to 4. Figure 5. Supplementary Material PDB reference: simian immunodeficiency virus gp core, 3fus, r3fussf.

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