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Principal Investigator: YONG-HUI ZHENG
Organization: UNIVERSITY OF ILLINOIS AT CHICAGO
Fiscal Year: 2023
Award: $399,750
Funding agency: National Institute of Allergy and Infectious Diseases
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 glycosylation and trafficking in the Golgi, but also elucidate a novel antiviral mechanism that can be
broadly applied to several highly pathogenic human viruses including HIV-1, SARS2, EBOV, and IAV.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><AIDS Virus><AIDS test><AIDS/HIV test><Ablation><Acetylgalactosamine><Acquired Immune Deficiency Syndrome Virus><Acquired Immunodeficiency Syndrome Virus><Adopted><Bio-Informatics><Bioinformatics><Bittner Virus><Blood monocyte><CCL7><CCL7 gene><CD4 Cells><CD4 Positive T Lymphocytes><CD4 T cells><CD4 helper T cell><CD4 lymphocyte><CD4+ T-Lymphocyte><CD4-Positive Lymphocytes><CHIKV><COVID-19 virus><COVID19 virus><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cas nuclease technology><Cell Body><Cell Line><Cell membrane><CellLine><Cells><Cellular Membrane><Chikungunya virus><Chimera Protein><Chimeric Proteins><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><CoV-2><CoV2><Complex><Coronaviridae><Coronavirus><Cytoplasmic Membrane><D-Mannose><Defect><E3 Ligase><E3 Ubiquitin Ligase><EBOV><EC 2.4><Ebola virus><Ebola-like Viruses><Endoplasmic Reticulum><Ergastoplasm><Eukaryotic Cell><Family><Fusion Protein><Glycans><Glycohydrolases><Glycoproteins><Glycosidases><Glycoside Hydrolases><Glycoside Transferases><Goals><Golgi><Golgi Apparatus><Golgi Complex><HIV><HIV test><HIV-1><HIV-1 test><HIV-2 test><HIV-I><HIV1><Hemagglutinin><Human><Human Immunodeficiency Virus Type 1><Human Immunodeficiency Viruses><Human immunodeficiency virus 1><Human immunodeficiency virus test><Hybrids><Infection><Influenza A><Influenza A virus><Influenza Virus><Influenza Viruses Type A><Influenzavirus A><Investigation><Knock-out><Knockout><LAV-HTLV-III><LCM Viruses><LCMV><Laboratories><Lymphadenopathy-Associated Virus><Lymphocytic choriomeningitis virus><Lysosomes><MARC><MCP-3><MCP3><MERS><MERS corona virus><MERS coronavirus><MERS coronavirus disease><MERS virus><MERS-CoV><MERS-CoV disease><MMTV><Macrophage><Mammary Cancer Virus><Mannopyranose><Mannopyranoside><Mannose><Marrow monocyte><Mediating><Membrane><Membrane Fusion><Metabolic Glycosylation><Metabolic Protein Degradation><Middle East Respiratory Syndrome><Middle East Respiratory Syndrome CoV disease><Middle East Respiratory Syndrome Corona Virus><Middle East Respiratory Syndrome Coronavirus><Middle East Respiratory Syndrome Virus><Middle East Respiratory Syndrome coronavirus disease><Middle East Respiratory Syndrome-CoV><Middle East Respiratory coronavirus><Middle Eastern Respiratory Syndrome><Middle Eastern Respiratory Syndrome CoV disease><Middle Eastern Respiratory Syndrome Corona virus><Middle Eastern Respiratory Syndrome Coronavirus><Middle Eastern Respiratory Syndrome Virus><Middle Eastern Respiratory Syndrome coronavirus disease><Middle Eastern Respiratory Syndrome-CoV><Modern Man><Molecular><Mouse Mammary Tumor Virus><Mφ><N acetylgalactosamine><NC28><NSF attachment protein receptor><Nucleocapsid><Oligosaccharides><Organelles><Orthomyxovirus Type A><Pathogenicity><Plasma Membrane><Play><Polysaccharides><Polyubiquitination><Post-Transcriptional Gene Silencing><Posttranscriptional Gene Silencing><Process><Protein Biosynthesis><Protein Glycosylation><Protein Trafficking><Protein Turnover><Proteins><Public Health><Quelling><RNA Interference><RNA Silencing><RNAi><Receptor Protein><Regulatory Protein Degradation><Reporting><Ribosomal Peptide Biosynthesis><Ribosomal Protein Biosynthesis><Ribosomal Protein Synthesis><Rod><Role><SARS Virus><SARS corona virus><SARS corona virus 2><SARS coronavirus><SARS-Associated Coronavirus><SARS-CO-V2><SARS-COVID-2><SARS-CoV><SARS-CoV-1><SARS-CoV-2><SARS-CoV2><SARS-Related Coronavirus><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><SCYA7><SIV><SNAP receptor><SNARE><Sequence-Specific Posttranscriptional Gene Silencing><Severe Acute Respiratory Coronavirus><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome Virus><Severe Acute Respiratory Syndrome corona virus><Severe Acute Respiratory Syndrome coronavirus><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome related corona virus 2><Simian Immunodeficiency Viruses><Strains Cell Lines><Structure><Surface><T-Cells><T-Lymphocyte><T4 Cells><T4 Lymphocytes><Testing><Translating><Translational Research><Translational Science><Type A Influenza><Ubiquitin Protein Ligase><Ubiquitin-Protein Ligase Complexes><Ubiquitin-Protein Ligase E3><VSV><Vesicular Stomatitis Virus><Vesicular stomatitis Indiana virus><Viral><Viral Activity><Viral Diseases><Viral Function><Viral Fusion Proteins><Viral Gene Products><Viral Gene Proteins><Viral Physiology><Viral Proteins><Virion><Virus><Virus Diseases><Virus Particle><Virus Replication><Virus-HIV><Wuhan coronavirus><ZIKV><Zika Virus><alpha helix><corona virus><coronavirus disease 2019 virus><coronavirus disease-19 virus><cultured cell line><ebolavirus><experiment><experimental research><experimental study><experiments><glycosylation><glycosyltransferase><gp160><hCoV19><influenzavirus><member><membrane structure><milk agent><monocyte><nCoV2><novel><particle><plasmalemma><polypeptide><porcine epidemic diarrhea virus><protein degradation><protein folding><protein oligomer><protein synthesis><protein transport><receptor><receptor binding><receptor bound><severe acute respiratory syndrome-CoV><social role><soluble N-ethylmaleimide-sensitive-factor attachment protein receptor><thymus derived lymphocyte><trans-Golgi Network><translation research><translational investigation><ubiquitin-protein ligase><viral infection><viral multiplication><viral replication><virology><virus infection><virus multiplication><virus protein><virus-induced disease><zikav><α-helix>