Welcome to the
IVANOV LABORATORY
Research
The main topic of our research is the molecular basis of innate antiviral defenses. We investigate how distinctive molecular patterns associated with infection are recognized by the innate immune system, and what defense mechanisms block viral replication within cells. We are particularly interested in restriction factors—host proteins that disrupt specific steps in the viral life cycle and form the first line of defense against viruses. Evolutionary antagonism between viruses and their hosts’ restriction factors has shaped what organisms are currently susceptible or resistant to what viruses. Understanding how these defenses function and how viruses evade them may open novel avenues for treatment of viral infections, autoimmune diseases and cancer.
TRIM5α
TRIM5α restriction factors bind retroviral capsids after cell entry and impair reverse transcription and nuclear import of the viral genetic material. Species-specific sequence variations within TRIM5α proteins contribute to host tropism of primate immunodeficiency viruses. Most notably, the human TRIM5α has poor affinity for the HIV capsid making humans susceptible to AIDS. We are interested in how primate TRIM5α proteins recognize retroviral capsids and how they impair viral infectivity. Our studies revealed that TRIM5α of the rhesus monkey, a powerful restrictor of HIV replication, uses long flexible loops of its SPRY domain to recognize multiple epitopes on the surface of the assembled viral capsid. The unusual features of this protein-protein interaction make it remarkably resistant to viral escape by mutagenesis. We also investigate how E3 ubiquitin ligase activity of TRIM5α is enhanced by the interaction with the capsid. For example, we determined that the E3 activity depends on the transient dimerization of the RING domains, which occurs upon binding of multiple TRIM5α molecules to the capsid. The findings revealed how E3 ubiquitin ligase activity is controlled by ligand binding in the TRIM E3 family.
SAMHD1
SAMHD1 is an innate immune factor with roles in antiretroviral defenses and in interferon signaling. SAMHD1 catalyzes hydrolysis of deoxynucleotides (dNTPs) to triphosphate and unphosphorylated nucleosides and acts as a key regulator of dNTP supply in the cell. Retroviral restriction is thought to arise from the SAMHD1-catalyzed depletion of cellular dNTPs, which directly impairs the ability of the virus to complete reverse transcription of its genome. Controlled depletion of dNTPs is likely an ancient antiviral defense strategy that dates back to prokaryotes. By studying biochemical and structural basis of SAMHD1 activity we seek to elucidate the tantalizing functional link between antiviral immunity and dNTP metabolism. Most recently we have focused on the role of nucleic acid binding by SAMHD1, which remains poorly understood. We have discovered that SAMHD1 recognizes phosphorothioate bonds in the Rp configuration located 3’ to G nucleotides (GpsN), the modification pattern that occurs in the enzymatic DNA phosphorothioation in prokaryotes. The findings shed light on the immunomodulatory effects of oligonucleotide phosphorothioation and raise questions about the role of phosphorothioated nucleic acids in human innate immunity.
TOOLS
In our research we use many different experimental tools of modern biochemistry and structural biology. For example, we use NMR spectroscopy to map and characterize interactions of restriction factors with other proteins, nucleic acids and small-molecule modulators. NMR is a powerful and versatile method for studying biomolecular interactions in solution. We also use NMR to investigate kinetics of chemical reactions catalyzed by SAMHD1 and other enzymes. Once the interacting domain boundaries and locations of binding sites are determined, we use X-ray crystallography to reveal atomic details of macromolecular interactions. The high resolution of X-ray structures helps us develop mechanistic models, which we then test by mutagenesis and other approaches joining forces with our collaborators. Fluorescent labeling and analytical ultracentrifugation are other biophysical tools that we commonly use to characterize how individual interactions contribute to the assembly and function of complex macromolecular machines. Our studies are facilitated by advanced biochemical techniques for protein expression and purification
ENVIRONMENT
We are part of the Biochemistry and Structural Biology Department at the UT Health San Antonio. Our department hosts several institutional core facilities that offer access to state-of-the-art instrumentations for NMR spectroscopy, X-ray crystallography, Mass Spectrometry, Analytical Ultracentrifugation, Calorimetry and Surface Plasmon Resonance. Students from the Biochemical Mechanisms in Medicine (BMM), Molecular Immunology & Microbiology (MIM) and Cancer Biology (CB) Disciplines of the Integrated Biomedical Science (IBMS) Ph.D. program are encouraged to join the lab for their Ph.D. projects, and everyone is welcome for research rotations.
Selected Publications
Abstract
SAMHD1 impedes infection of myeloid cells and resting T lymphocytes by retroviruses, and the enzymatic activity of the protein-dephosphorylation of deoxynucleotide triphosphates (dNTPs)-implicates enzymatic dNTP depletion in innate antiviral immunity. Here we show that the allosteric binding sites of the enzyme are plastic and can accommodate oligonucleotides in place of the allosteric activators, GTP and dNTP. SAMHD1 displays a preference for oligonucleotides containing phosphorothioate bonds in the Rp configuration located 3′ to G nucleotides (GpsN), the modification pattern that occurs in a mechanism of antiviral defense in prokaryotes. In the presence of GTP and dNTPs, binding of GpsN-containing oligonucleotides promotes formation of a distinct tetramer with mixed occupancy of the allosteric sites. Mutations that impair formation of the mixed-occupancy complex abolish the antiretroviral activity of SAMHD1, but not its ability to deplete dNTPs. The findings link nucleic acid binding to the antiretroviral activity of SAMHD1, shed light on the immunomodulatory effects of synthetic phosphorothioated oligonucleotides and raise questions about the role of nucleic acid phosphorothioation in human innate immunity.
Abstract
SAMHD1 is a dNTP triphosphohydrolase (dNTPase) that impairs retroviral replication in a subset of non-cycling immune cells. Here we show that SAMHD1 is a redox-sensitive enzyme and identify three redox-active cysteines within the protein: C341, C350, and C522. The three cysteines reside near one another and the allosteric nucleotide binding site. Mutations C341S and C522S abolish the ability of SAMHD1 to restrict HIV replication, whereas the C350S mutant remains restriction competent. The C522S mutation makes the protein resistant to inhibition by hydrogen peroxide but has no effect on the tetramerization-dependent dNTPase activity of SAMHD1 in vitro or on the ability of SAMHD1 to deplete cellular dNTPs. Our results reveal that enzymatic activation of SAMHD1 via nucleotide-dependent tetramerization is not sufficient for the establishment of the antiviral state and that retroviral restriction depends on the ability of the protein to undergo redox transformations.
Abstract
SAMHD1 is a dNTP hydrolase, whose activity is required for maintaining low dNTP concentrations in non-cycling T cells, dendritic cells, and macrophages. SAMHD1-dependent dNTP depletion is thought to impair retroviral replication in these cells, but the relationship between the dNTPase activity and retroviral restriction is not fully understood. In this study, we investigate allosteric activation of SAMHD1 by deoxynucleotide-dependent tetramerization and measure how the lifetime of the enzymatically active tetramer is affected by different dNTP ligands bound in the allosteric site. The EC50dNTP values for SAMHD1 activation by dNTPs are in the 2-20 μm range, and the half-life of the assembled tetramer after deoxynucleotide depletion varies from minutes to hours depending on what dNTP is bound in the A2 allosteric site. Comparison of the wild-type SAMHD1 and the T592D mutant reveals that the phosphomimetic mutation affects the rates of tetramer dissociation, but has no effect on the equilibrium of allosteric activation by deoxynucleotides. Collectively, our data suggest that deoxynucleotide-dependent tetramerization contributes to regulation of deoxynucleotide levels in cycling cells, whereas in non-cycling cells restrictive to retroviral replication, SAMHD1 activation is likely to be achieved through a distinct mechanism.
Abstract
SAMHD1, a dNTP triphosphohydrolase, contributes to interferon signaling and restriction of retroviral replication. SAMHD1-mediated retroviral restriction is thought to result from the depletion of cellular dNTP pools, but it remains controversial whether the dNTPase activity of SAMHD1 is sufficient for restriction. The restriction ability of SAMHD1 is regulated in cells by phosphorylation on T592. Phosphomimetic mutations of T592 are not restriction competent, but appear intact in their ability to deplete cellular dNTPs. Here we use analytical ultracentrifugation, fluorescence polarization and NMR-based enzymatic assays to investigate the impact of phosphomimetic mutations on SAMHD1 tetramerization and dNTPase activity in vitro. We find that phosphomimetic mutations affect kinetics of tetramer assembly and disassembly, but their effects on tetramerization equilibrium and dNTPase activity are insignificant. In contrast, the Y146S/Y154S dimerization-defective mutant displays a severe dNTPase defect in vitro, but is indistinguishable from WT in its ability to deplete cellular dNTP pools and to restrict HIV replication. Our data suggest that the effect of T592 phosphorylation on SAMHD1 tetramerization is not likely to explain the retroviral restriction defect, and we hypothesize that enzymatic activity of SAMHD1 is subject to additional cellular regulatory mechanisms that have not yet been recapitulated in vitro.
Abstract
Members of the tripartite motif (TRIM) protein family of RING E3 ubiquitin (Ub) ligases promote innate immune responses by catalyzing synthesis of polyubiquitin chains linked through lysine 63 (K63). Here, we investigate the mechanism by which the TRIM5α retroviral restriction factor activates Ubc13, the K63-linkage-specific E2. Structural, biochemical, and functional characterization of the TRIM5α:Ubc13-Ub interactions reveals that activation of the Ubc13-Ub conjugate requires dimerization of the TRIM5α RING domain. Our data explain how higher-order oligomerization of TRIM5α, which is promoted by the interaction with the retroviral capsid, enhances the E3 Ub ligase activity of TRIM5α and contributes to its antiretroviral function. This E3 mechanism, in which RING dimerization is transient and depends on the interaction of the TRIM protein with the ligand, is likely to be conserved in many members of the TRIM family and may have evolved to facilitate recognition of repetitive epitope patterns associated with infection.
Abstract
Upon infection of susceptible cells by HIV-1, the conical capsid formed by ∼250 hexamers and 12 pentamers of the CA protein is delivered to the cytoplasm. The capsid shields the RNA genome and proteins required for reverse transcription. In addition, the surface of the capsid mediates numerous host-virus interactions, which either promote infection or enable viral restriction by innate immune responses. In the intact capsid, there is an intermolecular interface between the N-terminal domain (NTD) of one subunit and the C-terminal domain (CTD) of the adjacent subunit within the same hexameric ring. The NTD-CTD interface is critical for capsid assembly, both as an architectural element of the CA hexamer and pentamer and as a mechanistic element for generating lattice curvature. Here we report biochemical experiments showing that PF-3450074 (PF74), a drug that inhibits HIV-1 infection, as well as host proteins cleavage and polyadenylation specific factor 6 (CPSF6) and nucleoporin 153 kDa (NUP153), bind to the CA hexamer with at least 10-fold higher affinities compared with nonassembled CA or isolated CA domains. The crystal structure of PF74 in complex with the CA hexamer reveals that PF74 binds in a preformed pocket encompassing the NTD-CTD interface, suggesting that the principal inhibitory target of PF74 is the assembled capsid. Likewise, CPSF6 binds in the same pocket. Given that the NTD-CTD interface is a specific molecular signature of assembled hexamers in the capsid, binding of NUP153 at this site suggests that key features of capsid architecture remain intact upon delivery of the preintegration complex to the nucleus.
Abstract
The restriction factor TRIM5α binds to the capsid protein of the retroviral core and blocks retroviral replication. The affinity of TRIM5α for the capsid is a major host tropism determinant of HIV and other primate immunodeficiency viruses, but the molecular interface involved in this host-pathogen interaction remains poorly characterized. Here we use NMR spectroscopy to investigate binding of the rhesus TRIM5α SPRY domain to a selection of HIV capsid constructs. The data are consistent with a model in which one SPRY domain interacts with more than one capsid monomer within the assembled retroviral core. The highly mobile SPRY v1 loop appears to span the gap between neighboring capsid hexamers making interhexamer contacts critical for restriction. The interaction interface is extensive, involves mobile loops and multiple epitopes, and lacks interaction hot spots. These properties, which may enhance resistance of TRIM5α to capsid mutations, result in relatively low affinity of the individual SPRY domains for the capsid, and the TRIM5α-mediated restriction depends on the avidity effect arising from the oligomerization of TRIM5α.
Abstract
Tripartite motif protein TRIM5α blocks retroviral replication after cell entry, and species-specific differences in its activity are determined by sequence variations within the C-terminal B30.2/PRYSPRY domain. Here we report a high-resolution structure of a TRIM5α PRYSPRY domain, the PRYSPRY of the rhesus monkey TRIM5α that potently restricts HIV infection, and identify features involved in its interaction with the HIV capsid. The extensive capsid-binding interface maps on the structurally divergent face of the protein formed by hypervariable loop segments, confirming that TRIM5α evolution is largely determined by its binding specificity. Interactions with the capsid are mediated by flexible variable loops via a mechanism that parallels antigen recognition by IgM antibodies, a similarity that may help explain some of the unusual functional properties of TRIM5α. Distinctive features of this pathogen-recognition interface, such as structural plasticity conferred by the mobile v1 segment and interaction with multiple epitopes, may allow restriction of divergent retroviruses and increase resistance to capsid mutations.
People
Kristin Cano
Technical Director of the
NMR Core Facility
CanoK@uthscsa.edu
Vi Dougherty
Research Associate
DoughertyV@uthscsa.edu
Liezl Francisco
Graduate Student
FranciscoL@livemail.uthscsa.edu
Frank Herkules
Graduate Student
Herkules@livemail.uthscsa.edu
Dmitri Ivanov
Associate Professor
ivanov@uthscsa.edu
Zachary Maxwell
Undergraduate Student
maxwell.zachary@yahoo.com
Corey Yu
Postdoctoral Fellow
YuCH@uthscsa.edu
Contact
Our laboratory is located on the 5th floor of the Research Administration Building (RAB) at the Greehey Research and Academic Campus of the UT Health San Antonio. See below for directions and room numbers. The Greehey Campus Shuttle stops right in front of the building. There are several metered visitors parking spots in front of the RAB (Lot 16A) and two “Official Business” spots behind it in Lot 30 (see map).
Contact:
Dmitri Ivanov, PhD
phone: +1-210-567-8781
email: ivanov@uthscsa.edu
Campus Address:
Office: RAB 5.210.3
Lab: RAB 5.308
Mailing Address:
Dept. of Biochemistry
and Structural Biology
7703 Floyd Curl Drive
San Antonio, TX 78229
