Sexual transmission accounts for most cases of HIV infection worldwide, with semen being the main carrier of viral particles during this process. Recent studies revealed that positively charged amyloid fibrils from human semen can substantially boost HIV infectivity rates. In two separate studies, M?nch et al. and our group identified two distinct HIV-enhancing semen amyloids. More recently, endogenous amyloid fibrils have been observed directly in human semen using a variety of techniques including electron microscopy (EM), atomic force microscopy (AFM), amyloidspecific dyes and antibodies. Thus far, the molecular mechanisms by which semen amyloids enhance HIV infection are not understood. In this proposal, we test two hypotheses. First, we theorize that specific structures or conformations of semen amyloids have superior activity in enhancing HIV infection. Second, we posit that semen contains factors that promote or limit this enhancing activity. In Aim 1, we will isolate, identify, and characterize structure-function relationships of endogenous or synthetic semen amyloids using mass spectrometry (MS), synchrotron Fourier transform infrared (SR-FTIR) spectroscopy, EM, computational tools, and HIV infectivity assays. We will apply an innovative combination of SR-FTIR and MS tools to determine the composition of endogenous semen amyloids. We will then use ion mobility MS approaches coupled with computational methods to characterize the structures and oligomeric conformations of synthetic semen amyloids that best enhance HIV infection. In Aim 2, we will use affinity-based pull-downs to identify the factors that interact with synthetic semen amyloids, and assess whether they modify the HIV-enhancing activity of the amyloids. This information will lead to the development of microbicides that target viral-enhancing factors in semen. The work proposed here will be carried out during a period of one year. The data acquired will be important for my planned application for extramural funding.