266 HIV-1 Matrix Protein Interaction with Ezrin Containing Structures: Possible Intermediates in the Assembly Process C. Gomez*, T. Hope Univ of Illinois at Chicago
Background: Human immunodeficiency virus assembly is a highly dynamic multi-step process. Several groups have shown that matrix protein is a key player in assembly. Matrix is responsible for targeting the Gag polyprotein to the plasma membrane and for the incorporation of the viral envelope glycoproteins into budding virions. A number of reports implicate lipid rafts and actin in the assembly and budding processes. Last year we reported an interaction between matrix and actin. An investigation of this interaction may provide new insights into the life cycle of HIV-1 and which types of cytoskeletal proteins it exploits in order to assemble.
Methods: In order to define the membrane structures to which matrix localizes, we are using immunofluorescence microscopy in addition to a biochemical approach. Matrix-GFP, matrix-capsid-GFP, as well as Gag-GFP fusion proteins were utilized. In addition, Triton X-100 cell lysates of transfected cells were fractionated on a sucrose density gradient and used in a Western blot to test association with membrane rafts.
Results: In our studies, we found that matrix protein co-localized with actin microfilaments, but not with actin stress fibers. Interestingly, matrix in the context of the Gag polyprotein did not exhibit the same interaction. We also found matrix co-localized with the protein ezrin and that matrix alone and matrix-capsid appeared to be present in ezrin-containing microvilli. We also saw these proteins localized in GM1-containing structures. In addition, matrix was found to be present in lipid rafts, which also contain ezrin.
Conclusions: The observed interaction between matrix and ezrin points to a variety of possible mechanisms for assembly. Ezrin is a member of the ezrin/radixin/moesin family of proteins which link the cell membrane to the underlying actin cytoskeleton. Ezrin is involved in coupling signal transduction pathways to the actin cytoskeleton and is enriched in cell surface structures, such as microvilli and is associated with lipid rafts. HIV matrix protein may exploit any of these processes to facilitate its assembly. Our results showing MACA localizing to the same actin-dependent, ezrin-containing structures as matrix alone is very compelling due to the fact that matrix-capsid cannot assemble without nucleocapsid. Therefore, we believe that our results may suggest an intermediate step in the assembly process.
