HIV-1 envelope (Env) glycoprotein (gp) 160 belongs to class I fusion proteins that are also expressed by other highly pathogenic human viruses including influenza A viruses (IAV), Ebola viruses (EBOV), and coronaviruses (CoV) such as SARS-CoV (SARS1), MERS, and SARS-CoV-2 (SARS2). They build spikes on the viral envelope that induce fusion of viral and cellular membranes to allow viruses to enter cells, which is essential to the viral infection. Class I fusion proteins are synthesized as a type I transmembrane (TM) polypeptide precursor in the endoplasmic reticulum (ER) and delivered to the Golgi apparatus for maturation. The Golgi contains glycosidases/glycosyltransferases for glycosylation and conserved oligomeric Golgi (COG) complex and other associated proteins such as soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins for trafficking. Inside the Golgi, high-mannose-type N-glycans are processed into complex- type and hybrid-type N-glycans after extensive mannose-trimming, and O-glycosylation also occurs. These precursors except for SARS1-spike (S) are further subjected to proteolytic cleavage by furin to complete the maturation process. When these steps are disrupted in the Golgi, no infectious particles are produced, leading to complete inhibition of viral infection. Recently, we and others reported that MARCH8, a member of the membrane-associated RING-CH-type E3 ubiquitin ligase family, broadly inhibits viral replication by targeting a wide range of fusion proteins. Importantly, we reported that MARC causes multiple defects in class I H8 fusion maturation in the Golgi via an unknown mechanism. These defects are found not only in furin-cleavage of HIV-1 gp160, IAV-hemagglutinin (HA), EBOV-glycoprotein (GP), MERS-S, and SARS2-S, but also in N- and O-glycosylation of SARS2-S, MERS-S, and EBOV-GP in the Golgi. Although MARCH8 does not trigger the degradation of these fusion proteins, its E3 ligase function is still required for causing these defects. The goal of this project is to elucidate the molecular mechanism of these multiple defects in HIV-1 gp160 maturation by understanding the MARCH8 antiviral mechanism. We hypothesize that MARCH8 targets glycosidases, glycosyltransferases, furin, COG complex, and/or SNARE to block HIV-1 gp160 maturation. We propose two distinct but inter-related Aims to test this hypothesis. In Aim 1, we will characterize how MARCH8 blocks gp160 maturation during HIV-1 infection. Experiments will be performed in primary cells and human T cell lines in combination with RNA silencing and CRISPR/Cas9 knockout to elucidate the MARCH8 anti- HIV activity. In Aim 2, we will identify the MARCH8 targets that play a critical role in HIV-1 gp160 maturation. We will focus on 18 Golgi proteins selected by high confidence bioinformatic analysis to identify the targets. The significance of this project is very high, which will not only fill in gaps in our understanding of class I fusion protein glycosylati...