For the past 4 years we have been engaged in characterizing the HIV-1/SIV envelope complex (Env) either isolated from virions or recombinant constructs. Env is on the outer surface of the virus and functions to recognize cellular receptors and promote virus membrane-cell membrane fusion. Env is essential for viral infectivity. Env also represents the major target of antibody response to viral infection. Based on the above considerations, it is extremely important to establish the stoichiometry of the Env complex on the virion as well as develop recombinant constructs that can be used to generate high titer antibody response and for structural studies. Last year, in collaboration with Drs. Moss and Center of the Laboratory of Viral Diseases (LVD), we unequivocally established that detergent solubilized Env complex from virions was trimeric. In contrast, soluble recombinant HIV-1 Env (a fusion protein of gp120 and gp41 with transmembrane region of gp41 removed) forms very heterogeneous oligomers. However, recombinant SIV forms trimeric structures. A trilobe structure can be observed using STEM (scanning transmission electron microscopy) comparable to the structures seen for both the HIV and SIV virion Env. Consequently it should be possible to promote trimerization using HIV/SIV chimeras. This year, the following experimental strategy was employed. The LVD generated a variety of constructs that were characterized by sedimentation equilibrium and velocity analysis in conjunction with STEM. The incorporation of the trimerization (helical heptade repeat) portion of SIV (gp41) in place of the HIV-1 protein sequence provides efficient trimerization of the entire Env. This soluble HIV/SIV Env trimeric chimeria should prove useful for assessment of antigenic structure and immunogenicity. Dr. Richard Wyatt of the Vaccine Research Center (VRC) is also pursuing the generation of trimeric Env via a different strategy. We have validated using sedimentation equilibrium and light scattering analysis that the VRC constructs are indeed trimeric. The VRC hopes to use the trimeric structure to determine the crystal structure of the Env and explore antigenic structure and immunogenicity as well. Additional projects involving viral protein interactions were also pursued. Dr. Barney Graham and graduate student Philip Budge have been working on inhibition of the respiratory syncytial virus (RSV). One of the proteins of RSV interacts with the small GTPase RhoA and a peptide derived from the binding region of RhoA shows antiviral activity. Biochemical evidence suggested that the active form of the peptide was a dimer. This was conclusively established using sedimentation equilibrium analysis. Very recently we initiated a characterization of the self-association of the HIV-gag protein that is precursor polyprotein that is cleaved into the major structural proteins of the virus. (This annual report continues work reported in 1Z01 OD 010485-05)
