Fadila Bouamr, Ph.D.
Investigator
Dr. Fadila Bouamr received her Ph.D. from Victor Segalen Bordeaux University in 1997. She performed her postdoctoral research with Dr. Carol Carter at SUNY at Stony Brook and with Dr. Steve Goff at Columbia University. She joined the Laboratory of Molecular Microbiology (LMM) in December, 2004.
Description of Research Program
The work in my laboratory aims to understand various aspects of HIV-1 and HTLV-I assembly, budding, and release. The main areas of research are summarized below.
1. Comparative studies of the structure of HTLV-I and HIV-1 capsids.
The main component of the retroviral core is the capsid (CA) protein. After processing, CA protein separates from the Gag polyprotein and undergoes rearrangements that change the spherical core to a central conical core in HIV-1; a spherical core in HTLV-I; or an irregularly polyhedral core in MoMLV. The progression of these events and the mechanisms involved are not well understood. However, an increasing body of data suggests that retroviral assembly is a highly dynamic process that includes many, and probably different, molecular interactions of CA proteins. While the structures of several retroviral capsid proteins have been solved, many questions remain about how the individual CA proteins are arranged in the mature infectious virion.
To gain insight into molecular mechanisms that govern capsid assembly, we studied the relationships between the HTLV-I capsid structure and its function. We previously solved the structure of the N-terminal domain (NTD) of CA by NMR and determined that the protein shares with HIV-1 CA a highly conserved, predominantly helical structure composed of six alpha-helices with an N-terminal beta-hairpin (Cornilescu, et al., 2001). However, comparison of both structures showed differences in the orientation of the N-terminal beta-hairpin and a central loop located in the helical core. These findings suggested that the orientation and perhaps the flexibility of the beta-hairpin, rather than formation of the structure per se, might influence the exposure and function of CA-CA interfaces during particle assembly (Cornilescu, et al., 2003). In addition the result suggested that the backbone flexibility of CA might play a role during the highly dynamic process of core assembly.
Since studies with HIV-1 CA suggested that the beta-hairpin is critical for formation of a CA-CA interface, we disrupted the salt bridge holding the beta-hairpin in the NTD of the HTLV-I and examined the structure of the mutant protein by NMR. This caused structural alterations localized in the region near the mutation in the beta-hairpin structure (Bouamr, et al., 2005). In contrast, biochemical and electron microscopic analyses showed no detectable effect on assembly, size, or morphology of HTLV-I viral particles (Bouamr, et al., 2005). Mutations introduced throughout the HTLV-I NTD prevented particle assembly and release (Rayne, et al., 2001). These results indicate that the integrity of the HTLV-I NTD is essential for capsid assembly, yet distinguish it from its HIV-1 counterpart, which seems to play a minor role in assembly of viral particle, and is rather required for virion infectivity.
2. Host factors involved in retroviral budding.
A yeast two-hybrid screen has identified the tumor-suppressor-gene 101 protein (Tsg101) as the binding partner of the HIV-1 p6 domain (VerPlank, et al., 2001). This work was first to show that Tsg101 binds the Late domain (L domain) PTAP motif in p6 to facilitate HIV-1 release. A search for host factors that bind the PPPY Late domain motif was conducted and identified members of the Nedd4 E3 ligases family as the cellular partner that binds the PPPY motif and mediates particle budding and release of retroviruses, such as Rous sarcoma virus, which carries a PPPY motif in its Gag polyprotein (Kikonyogo, et al., 2001). The HTLV-I Gag polyprotein contains a proline-rich region in the C-terminal end of matrix (MA) protein that carries a PPPY and a PTAP motif. Mutational analysis of this region showed that both the PPPY and the PTAP motifs are involved in late stages of HTLV-I particle budding and release with the PPPY motif playing a dominant role in these events (Bouamr, et al., 2003). The results also showed that the PPPY and the PTAP motifs bind members of Nedd4 E 3 ligases family members and Tsg101, respectively. These bindings are both required for HTLV-I particle production. Interestingly, the use of interfering dominant-negative fragments of the Nedd4 protein arrested the HTLV-I particle in an early stage of particle morphogenesis and before the membrane curvature necessary to viral particle budding was complete (Bouamr, et al., 2003). These results hint to a possible role of Nedd4 E3 ligases in events that lead to membrane curvature and bending.
3. Inhibition of HIV-1 particle budding and release using Hrs mutant proteins.
Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) was shown to be the natural partner of Tsg101 (Lu, et al., 2003). Hrs is believed to act as scaffold protein that coordinates the components of the sorting machinery at the surface of the endosome. HIV-1 Gag and Hrs proteins share structural similarities: they both bind membranes and Tsg101 and both carry a PT/SAP motif. For these reasons, it has been proposed that HIV-1 Gag competes Tsg101 away from its natural partner, Hrs, and subverts the machinery that mediate virus release from infected. To test this hypothesis, we generated mutants and fragments of Hrs and tested their ability to interfere with HIV-1 particle production. An N-terminal fragment of Hrs, containing the conserved PSAP motif, had no effect on HIV-1 particle production. In contrast, a C-terminal fragment that contains a coil-coil and proline/glutamic domains, caused a significant decrease in particle production. Similarly, mutants of Hrs, either lacking the membrane-binding domain or carrying a mutated PSAP motif (ASAA), both potently inhibited HIV-1 particle release. The Hrs ASAA mutant interfered with Tsg101 binding to HIV-1 Gag, implying that the ability of mutant Hrs to interfere with Tsg101 binding to HIV-1 Gag, and thereby inhibit HIV-1 release, does not require the PSAP motif. This result suggested that Tsg101 might bind other regions in Hrs. Yeast two hybrid assays showed that Hrs bound Tsg101 in a PSAP independent manner, and two new and independent Tsg101 binding sites were identified in the C-terminal portion of Hrs; they lie within the coil-coil and the P/Q-rich domains. Together these data indicate that mutants of Hrs that contain the new Tsg101 binding sites efficiently captured Tsg101 from the PTAP domain in HIV-1 p6 and caused potent inhibition of HIV-1 particle release. This finding was confirmed by scanning electron microscopy, showing that expression of Hrs ASAA mutant with HIV-1 arrested particles production during the late stages of budding, causing them to concentrate into "lump-like" structures. The finding that Hrs contains redundant binding sites for Tsg101 might have therapeutic implications, because molecules that inhibit the p6-PTAP binding of Tsg101 could disrupt viral budding without completely compromising the cellular function of the Tsg101-Hrs complex.
Research Group Members
Rebecca Casaday, Ph.D., Postdoctoral Fellow, dcasadayr@niaid.nih.go; Swathi Challa, IRTA, challas@niaid.nih.gov.
Selected Publications
(View list in PubMed.)
Medina G, Zhang Y, Tang Y, Gottwein E, Vana ML, Bouamr F, Leis J, Carter CA. The functionally exchangeable L domains in RSV and HIV-1 Gag direct particle release through pathways linked by Tsg101. Traffic. 2005. 6(10): 880-894.
Bouamr F, Cornilescu CC, Goff SP, Tjandra N, Carter CA. Structural and dynamics studies of the D54A mutant of human T cell leukemia virus-1 capsid protein. J Biol Chem. 2005. 280(8): 6792-6801.
Bouamr F, Melillo JA, Wang MQ, Nagashima K, de Los Santos M, Rein A, Goff SP. PPPYVEPTAP motif is the late domain of human T-cell leukemia virus type 1 Gag and mediates its functional interaction with cellular proteins Nedd4 and Tsg101 [corrected]. J Virol. 2003. 77(22): 11882-11895. Erratum in: J Virol. 2004. 78(8): 4383.
Bouamr F, Scarlata S, Carter C. Role of myristylation in HIV-1 Gag assembly. Biochemistry. 2003. 42(21): 6408-6417.
Cornilescu CC, Bouamr F, Carter C, Tjandra N. Backbone (15)N relaxation analysis of the N-terminal domain of the HTLV-I capsid protein and comparison with the capsid protein of HIV-1. Protein Sci. 2003. 12(5): 973-981.
Kikonyogo A, Bouamr F, Vana ML, Xiang Y, Aiyar A, Carter C, Leis J. Proteins related to the Nedd4 family of ubiquitin protein ligases interact with the L domain of Rous sarcoma virus and are required for gag budding from cells. Proc Natl Acad Sci U S A. 2001. 98(20): 11199-11204.
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