Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
HIV (human immunodeficiency virus) and EIAV (equine infectious anemia virus) are closely related lentiviruses that both infect immune cells but whose pathogenesis differs. Membrane binding of the (matrix) MA protein of HIV appears to be primarily driven by a cluster of basic residues in the MA domain and possibly assisted by an N-myristoylation signal. Interestingly, the MA protein of EIAV does not contain either of these signals. To understand what factors may promote EIAV assembly we characterized the membrane binding properties of its MA proteins using fluorescence methods and compared them to our previous HIV-MA results. We find that like HIV-MA, EIAV-MA exists as a multimer in solution whose protein-protein interactions are destabilized by membrane binding. Unlike HIV-MA, EIAV-MA binds strongly to electrically neutral membranes (POPC) as well as negatively charged (POPS) ones and our results indicate a different exposure of the EIAV-MA Trp residues when bound to the two types of membranes. Based on these data and the known structures of closely related matrix proteins, we constructed a structural model of EIAV-MA. This model predicts that EIAV-MA binds to POPS similar to HIV-MA, but EIAV-MA has an additional membrane binding region that allows for hydrophobic membrane interactions.
Figure 1: Gene map of Gag and depiction of mature virus assembly
Matrix protein is instrumental in membrane binding of Gag
-Decrease in fluorescence EIAV homotransfer as seen by an increase in anisotropy as the pH is lowered
-Decrease in homotransfer indicates subunit dissociation
-This was verified by SDS-PAGE electrophoresis (data not shown)
Figure 2: pH dependence of oligomerization.
Membrane Binding of EIAV
-Binding to LUVs was followed by the decrease in intrinsic fluorescence of EIAV-MA
-Lipid membrane was added to a solution containing EIAV-MA. This may promote protein-protein interactions so an alternate assay was done (Fig. 4)
Figure 4: Membrane Binding of EIAV-MA to POPS and POPC
- Membranes were labeled with the environmentally-sensitive probe Laurdan. The shift in Laurdan fluorescence as the protein displaces water from the surface was followed and is shown.
Figure 5: Quenching of Trp by KI differs when EIAV is
bound to POPS and POPC
80 uM POPS slope 0.0056
80 uM POPC slope 0.0039
Figure 6: Molecular model for the EIAV-MA structure.
The amino acid sequences of the EIAV and HIV-1 matrix domains were aligned with the multiple sequence alignment program ClustalW (Higgins et al., 1991). The sequence identity of the alignment is 19% (23 identities over 120 residues). Homology models of the EIAV matrix protein in monomeric and trimeric forms were constructed with the model routine of the homology modeling program Modeller (Sali and Blundell, 1993). The ClustalW alignment was used to match the EIAV matrix sequence to the HIV-1 matrix sequence; the NMR structure of HIV-1 matrix protein (PDB code, 1tam) was used as the structural template for the monomer model and the x-ray structure of the HIV-1 matrix protein trimer (PDB code, 1hiw) was used as the template for the trimer model. Residues present in the HIV-1 MA trimer interfaces are only partly conserved in EIAV MA:
HIV-1 MA trimer interface residues:ERFAVNQQQTGS-EE
Corresponding residues in EIAV MA:DLFHDTDLQTLSGEE
Models of the EIAV monomer based on other alignments share common properties with the model described above: 1) electrostatic polarity: the “front” surface is basic, while the “back” surface is slightly acidic with exposed hydrophobic residues that may penetrate the membrane interface; 2) surface Trp residues are more prominent on the hydrophobic face than the basic face; 3) the sole Cys residue is partially buried in the hydrophobic core. Fluorescence studies measuring the accessibility of this Cys support this model.
Figure 7: Theoretical model of Membrane Binding of EIAV-MA to POPS and POPC
From the binding data presented here we theorize that EIAV-MA binds to POPS through electrostatic interactions. The binding then promotes subunit dissociation through the use of the anionic phosphates of the membrane surface. We believe that EIAV-MA binds to POPC and the subunits dissociate through the same mechanisms as POPS. However, the dissociation exposes hydrophobic residues found within the slightly acidic region on the “back” of the protein, and “rolling” of the protein for the hydrophobic residues to penetrate the membrane occurs on POPC.
1. Higgins, D.G., Bleasby, A.J., and Fuchs, R. (1991). ClustalW: improved
software for multiple sequence alignment. CABIOS 8, 189-191.
2. Sali, A. and Blundell, T.J. (1993). Comparative protein modeling by
satisfaction of spatial restraints. J.Molec.Biol. 234, 779-815.
3. Scarlata, S., Ehrlich, L.S., and Carter, C.A. (1998). Membrane-induced
alterations in HIV-1 Gag and matrix protein-protein interactions.
J.Mol.Biol. 277, 161-169.
4. Ehrlich, L.S., Fong, S., Scarlata, S., Zybarth, G., and Carter, C. (1996).
Partitioning of HIV-1 Gag and Gag-related proteins to membranes.
Biochemistry. 35, 3933-3943.
N. Tijandra, I. Jayatilaka
Supported by NIH 05827101