Xiaojiang Chen - US grants

DESCRIPTION: (Adapted from the Investigator's abstract): Papilloma viruses are DNA tumor viruses that infect higher vertebrates including humans. Human papilloma viruses (HPVs) include a group of "high-risk" viruses that are the major etiologic factor for cervical cancer and genital carcinoma. Thus, a structural analysis of papilloma viruses bears significant medical relevance The long term objectives of this proposal are to 1) determine the atomic structure of papilloma viruses in order to understand molecular mechanisms in capsid assembly and disassembly (uncoating), and 2) understand the structural basis of papilloma virus surface antigenicity, and the mechanisms of viral neutralization by antibodies. The specific aims build upon our recent determination of the atomic structure of the HPV16 L1 major capsid protein assembled m a T=1 structure. Structures of HPV11 and HPV5 L1 will be determined so that surface loop domains, predicted to be different between viral types, can be compared with the HPV16 structure. This comparison will help define the structural basis of surface antigenicity differences between viral subtypes, and may also provide insight into the location of the cell receptor binding sites. The interaction between viral surface epitopes and neutralizing antibodies will be studied by determining the structure of a complex between HPV 16 L1 and an Fab from a neutralizing monoclonal antibody. This structure will define antibody binding sites at high resolution and provide insight into the mechanism of viral neutralization. The structure of bovine papillomavirus will be determined to extend the current HPV16 structure from a T=1 to a complete T=7 particle. The complete virion structure will identify the contacts between "hexavalent" L1 pentamers as well as other bonds important for virion assembly (e.g. disulfide bonds). The structure of the minor capsid protein L2, in complex with L1 will be determined. This structure will define the domain of L2 that interacts with L1 and will hopefully provide initial information on how the L2 protein interacts with the viral genome and contributes to viral infectivity. Thus, these studies will not only provide information relevant to fundamental problems in structural virology but will also provide a basis for designing improved vaccines against papilloma viruses, and facilitating drug design to block viral entry and uncoating.

DESCRIPTION (provided by applicant): Complement receptor type 2 (CR2/CD21) is a cell surface receptor that functions in diverse biological processes that include activating B-cell and T-dependent humoral immune responses, maintaining long-term B-cell memory and mediating the development of B-cell tolerance to self-antigen. CR2 carries out these diverse functions by interacting with different ligands. Additionally, CR2 is also the receptor for a virus ligand: EBVgp35O. One of the fundamental questions is: what is the structural basis for the recognition of CR2 by these different ligands in order to generate different downstream signals for a particular function. Our goal is to provide answers to this fundamental biological question, which also bears significant medical relevance because both CR2 malfunction and EBV infection are associated with important human diseases such as autoimmune diseases, lymphomas and carcinomas. Four specific aims are built upon our recent success in the structural studies of CR2-C3d interactions. Aim 1: The crystal structure of CR2 containing SCR 1-4 will be determined to learn the SCR domain structure and orientation that will provide insight into the ligand-binding specificity and diversity of CR2. Aim 2: The domain(s) of gp350 important in binding to CR2 will be determined using molecular genetic and biochemical methods. Aim 3: The crystal structure of CR2 binding to gp350 will be determined to understand the initial step of EBV infection and to extend our knowledge of diversity of CR2-ligand interactions. The structure will identify the detailed bonding contact between CR2 and gp350, which is known to be non-identical from those in the CR2-C3d complex. Aim 4: The structure-guided mutagenesis of gp350 will be performed to generate mutant gp350 as well as mutant EBV virus to probe the EBV-CR2 interactions in more details. At the same time, phage peptide library will be employed to screen for peptide ligands for gp350 that can specifically inhibit the binding of gp350 with CR2. These specific aims outline an interdisciplinary approach to increase our understanding of the functions of CR2 and its interactions with various ligands. The results will likely provide a basis for designing drugs to treat the diseases associated with CR2 malfunctions and EBV infection.

structural biology; Epstein Barr virus; virus protein; glycoprotein structure; crystallization; biomedical resource;

[unreadable] DESCRIPTION (provided by applicant): SV40 large T antigen (LT) is a viral oncoprotein with diverse biological functions. It is involved in cellular transformation through regulating the activities of tumor suppressors. It also plays an important role in viral DNA replication by assembling around the origin into a double hexamer that function not only as a helicase to open up the origin and unwind the fork DNA, but also as a platform for recruiting the essential cellular replication proteins, such as RPA. Our goals are to understand the structural basis of LT functions in these diverse biological processes by studying LT structures in their various oligomeric and conformational states. Four specific aims are built upon our recent progress in the biochemistry/protein chemistry of LT and the crystallization of a LT fragment containing the larger C-terminal portion (residues 251-630). (i) Crystal structures of larger LT containing the N-terminal domains will be determined to leaas well as a logical approach with alternatives and potential problems. The reviewers felt that this was a highly improved application that addressed important questions. Success of these aims will not only provide new and exciting information about SV40 T antigen, but also about helicases and DNA replication initiation, in general. Further strengths of this application include the investigator, who is well trained and has been highly productive, the environment, and the excellent collaborators. The only weaknesses in the application include, in some cases, a lack of experimental detail, and a clear description of how the crystallographic data will address mechanism. The reviewers felt that these were minor compared to the strengths of the overall application. In summary, the study section was highly enthusiastic about this application. They felt that based on the preliminary data there was a high likelihood of success, the questions were significant, and the results from this study will have a high impact on both the understanding of SV40 LT and the field of eukaryotic DNA replication. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): SV40 large T antigen (LT) is a potent carcinogen, and plays an essential role for SV40 DNA replication as the replicative helicase and replication initiator protein. SV40 replication serves as a model for eukaryotic DNA replication, as SV40 uses all the essential cellular replication proteins (primase, polymerase, PCNA, Topoisomerases, etc.), except for the helicase and cellular initiator proteins that consist of multiple initiator factors (such as Orc, Ctd1, Cdc6, MCM in eukaryotes, or DnaA/DnaC/DnaB in prokaryotes) to initiate DNA replication, i.e. marking the replication origin, recruiting helicase, melting origin, and activating helicase. For SV40 replication, LT alone fulfills essentially all the initiator functions and is the helicase for replication fork unwinding during elongation phase. The long-term goal of this research is to understand how LT functions as a helicase to coordinate the functions of the other replication proteins for DNA replication, as well as how LT transforms cells. Specific aims are designed to understand how LT hexameric and double hexameric helicase melt the origin DNA and unwinds dsDNA to initiate DNA replication. We plan to use mainly X-ray protein crystallography, assisted with EM and AFM, single molecule assay, computational method, molecular biology and functional biochemistry in vitro and in cells. The results from this research are expected to have potential impact on the field of DNA replication in eukaryotic cells and on cancer biology.

DESCRIPTION (provided by applicant): Minichromosome maintenance protein (MCM) complex functions as a helicase that unwinds DNA to provide template for DNA replication and plays a critical role in regulating cell growth. MCM helicases are considered to be a licensing factor for cellular DNA replication. MCM proteins from eukaryotes and archaea form hexamers ring complex, with MWt approximating 0.5-1.0 Mega Dolton. Despite extensive efforts, gap of knowledge exists in our understanding of how MCM unwinds DNA, how MCM interacts with DNA during unwinding, and how ATP is utilized to drive the conformational changes of MCM needed for unwinding. MCM are conserved in archaea and eukaryotes. Eukaryotic MCM contains six homologs that form hetero-hexamer and possibly higher order complexes. However, the MCM complex in some archaea contains only one MCM protein that can assemble into homo-hexamers or dodecamers. Thus, archaeal MCM homo-oligomers provide a simpler system for understanding the structure/function of MCM complex. The goal of this proposal is to use archaeal MCM complexes as a model system to understand the structure/functions of MCM complex, with particular emphasis on the mechanisms regarding oligomerization, ATP-triggered conformational changes, and DNA binding and remodeling functions of MCM proteins. A combination of structural biology, biophysics, and functional biochemistry will be employed for the study. The resulting data will provide valuable information for understanding the DNA unwinding mechanism at the replication fork. It will also provide structural/functional insights for the homologous eukaryotic MCM complex, which bears high relevance to cell growth regulation and cancer biology. PUBLIC HEALTH RELEVANCE: One essential process for all living organisms is duplicate the genomic DNA (or DNA replication) to ensure correct genetic information passage. To duplicate genomic DNA, helicases is required to unzip the double helix DNA (dsDNA) to provide ssDNA template for daughter strand synthesis in the replication process. MCM complex is such a helicase that unwinds dsDNA and is critical for DNA replication and cell growth. We propose to study the structure/function of this important MCM helicase using a combination of approaches including structural biology, biophysics, and functional biochemistry, in order to understand how this enzyme utilizes the energy of ATP to unwind the double helix DNA for replication.

DESCRIPTION (provided by applicant): The Structural Basis of APOBEC Functions The APOBEC (Apolioprotein B mRNA-editing Enzyme Catalytic polypeptide) family of cytidine deaminases is a family of enzymes that deaminate cytidine residues of DNA/RNA. APOBEC proteins possess significant cellular functions and anti-viral activity, which partially accounts for the intense attention to this the field of research in recent years. APOBEC proteins are found only in vertebrates and APOBEC3 (Apo3) proteins are found only in primates. By binding and deaminating DNA/RNA, APOBEC enzymes achieve remarkably diverse cellular functions. For example, APOBEC1 (Apo1) edits the mRNA of a protein involved in lipid metabolism; AID plays a key role in somatic hypermutation for antibody maturation; APOBEC2 (Apo2) may play a regulatory role for heart muscle development; and Apo3 proteins, especially Apo3G, can restrict important viral pathogens, including Human Immunodeficiency Virus (HIV) and Hepatitis B Virus (HBV), and retro-element mobility. As a result, a novel approach to HIV therapy focuses on utilizing the potent anti-viral activity of Apo3G and Apo3F. Our long-term goals are to understand the structural/functional relationship for APOBEC cellular function and their anti-viral activity. Our specific aims are to extend our prior success in the structural characterization of APOBEC proteins to the studies of the structural basis of APOBEC's functional mechanisms, including their antiviral activity, with particular focuses on Apo3G and Apo3F. The research will provide valuable information for understanding the molecular details of the APOBEC enzyme family and for the potential drug development to provide therapy for HIV, immune diseases and other diseases related to APOBEC function or malfunction. PUBLIC HEALTH RELEVANCE: Understanding The Structural Basis of APOBEC Functions The Apolioprotein B mRNA-editing enzyme catalytic polypeptide (APOBEC) family of cytidine deaminases are found exclusively in verterbrates. APOBEC nucleic acid deaminases modify genes by deaminating cytosines in mRNA coding sequences and in ssDNA. Their critical biological roles include lipid metabolism, humoral immune response, and potential regulations of developmental process of certain human organs and reproductive system. Additionally, these enzymes can inhibit the replication of retroviruses, such as the human immunodeficiency virus (HIV) and hepatitis B virus (HBV), and retrotransposons. The important beneficial mutational ability of APOBEC proteins can become detrimental to the stability of genome if their activity is not tightly regulated. The understanding of the structural basis of the molecular mechanisms of APOBEC function, which is still poorly understood, bears scientific significance and direct health relevance. We propose to study the structure/function of this important APOBEC deaminase family, with focuses on APOBEC3G and 3F (Apo3G and 3F) proteins and their interactions with cellular and viral ligands, using mainly structural biology, assisted by biophysics, molecular biology, and functional biochemistry.

Project Summary Structural Basis of APOBEC Functions and Interactions with HIV-Vif The APOBEC (Apolioprotein B mRNA-editing Enzyme Catalytic polypeptide) family of cytidine deaminases deaminate are found only in vertebrates and all APOBEC3 subfamily proteins are found only in primates. By deaminating the cytidine to cause mutation to uridine on DNA/RNA, APOBEC enzymes achieve remarkably diverse cellular functions through specific targeting to the intented ssDNA or RNA through a combination of regulations including spatial and temporal and substrate specificity. For example, APOBEC1 (A1) specifically modigy the mRNA of a protein that play a role in cholestoral metabolism; AID, another member of APOBEC family, is required for antibody maturation process including somatic hypermutation and recombination class switch; APOBEC2 (A2) is involved in cardiac and skeletal muscle development; and A3 proteins, a subfamily contains seven members (AA-H), can restrict foreign and internal nucleic acids that poses danger to the genome integrity, which include internal retroelements and transposons as well as external retroviruses and otherinfectious viral pathogens, such as Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV) . For retroviruses like HIV viruses to overcome the anti-HIV activity of APOBEC enyzmes, they encode a protein called Vif (virus virulent factor) that specifically bind to and inactivate APOBEC enzymes through unbuquitination degradation parthway. Even though their deamination activity is the key to excercute their biological functions, APOBEC enzymes can accidental mutations when proper regulations are not in place, which could lead to human deseases such as immune difficiency and cancer. Our long- term goals are to understand the structural/functional relationship for APOBEC cellular function and their anti-viral activity. Our specific aims are to understand the structural basis of APOBEC's functions and and the mechanisms that underlie substrate specificity and anti-HIV and anti-viral activities, with particular focuses on the APOBEC3 subfamily members that have strong anti- retroelements and anti-HIV activities. The outcome of this proposed research will provide valuable information for understanding the molecular details of the APOBEC enzyme family and the mechanisms of substrate specificity, which can be used for the potential drug development to provide therapy for HIV/AIDS, immune disorders, and other diseases such as cancer.

Project Summary Structural Basis of APOBEC Functions and HIV Restriction Apolipoprotein B mRNA-editing Enzyme Catalytic polypeptide (APOBEC) family of cytidine deaminases are capable of deaminating the cytidine to cause mutation to uridine on DNA or RNA. Human APOBEC deaminases have remarkable diverse cellular functions through specific targeting to the intended ssDNA or RNA through a combination of regulations including spatial and temporal distribution and substrate specificity. For example, APOBEC1 (A1) edits certain RNAs with the help of specific cofactors to regulate cholesterol metabolism; AID has important role in acquired immune response, it is required for antibody maturation including somatic hypermutation (SHM) and class switch recombination (CSR); APOBEC2 (A2) is involved in cardiac and skeletal muscle development; and APOBEC3 proteins (A3s, A3A-H) plays an important role in innate immunity for anti-viral activity, they can restrict internal and external nucleic acids (such as RNA and DNA viruses and retroelements) that poses danger to the genome integrity, which include internal retroelements as well as external retroviruses and other infectious viral pathogens, such as Hepatitis B Virus (HBV), Human Papillomavirus (HPV), and Human Immunodeficiency Virus (HIV). Among APOBEC proteins, A3G/A3F/A3D/A3H display strong anti-HIV activity, which are through deaminase-dependent and -independent mechanisms to inhibit viral replication and infection. However, retroviruses like HIV-1 can overcome the anti-HIV activity of APOBEC enzymes by its virus virulent factor (Vif) that specifically recruit cellular Cul5 E3 ligase to target these APOBECs for ubiquitination and degradation, leading to the viral infection. The deamination activity of APOBECs can also cause accidental genomic mutations, which can lead to various human diseases such as immune deficiency and cancer. Our long-term scientific goals are to understand the structural/functional relationship for APOBEC cellular functions and their anti-viral activities. Our specific aims are to understand the structural basis of APOBEC?s functions and the mechanisms that underlie nucleic acid interactions, multimerization, functional regulation, and viral restriction, with particular focuses on the double domain APOBEC3 subfamily members that have strong anti-retroelements and anti-HIV activities. The outcome of this research will provide valuable information for the fundamental molecular mechanisms of APOBEC functions and their anti viral activities, which can be used for the potential drug development to provide therapy for HIV/AIDS, immune disorders, and other diseases such as cancer.