Li Wu, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Human immunodeficiency virus (HIV) infection is the leading killer worldwide among infectious diseases, incurring 2-3 million deaths annually. Defining the mechanisms of HIV transmission and understanding the role of host cells that participate in the process are essential in developing effective strategies to combat HIV infection. Dendritic cells (DC) perform an important role in HIV infection and dissemination. DC-SIGN, a C- type lectin predominately expressed on myeloid DC, has been identified as a key mediator of DC-mediated HIV transmission. However, the precise mechanisms of DC-enhanced HIV infection and the role of DC-SIGN in the viral transmission process remain elusive. Our long-term objective is to elucidate the mechanisms of DC-mediated HIV dissemination and understand their role in HIV pathogenesis. The proposed studies will facilitate understanding of the mechanisms and the development of more effective interventions against HIV transmission, and potentially aid in development of novel HIV vaccine strategies. We hypothesize that cell- type-dependent HIV trafficking determines efficiency of DC-SIGN- or DC-mediated HIV transmission, and HIV Nef protein regulates DC-mediated HIV transmission through modulation of CD4 and DC-SIGN expression. This hypothesis is based on the observations that 1) DC-SIGN-mediated HIV transmission is cell-type dependent and requires cell-cell contact. The ability of DC-SIGN to promote HIV transfer correlates with the localization of the viral particles on the DC-SIGN-expressing cell; 2) CD4 coexpression in DC-SIGN transfectants abolishes HIV transmission to T cell targets. HIV Nef protein downregulates CD4 expression, but upregulates DC-SIGN expression and promotes HIV spread; 3) HIV transmission efficiency is significantly enhanced by maturation of DC, and the enhanced viral transmission is independent of DC- SIGN. Localization of HIV in immature DC is distinct from that in mature DC. The specific aims of this proposal are to: 1. Characterize cell-type restriction of DC-SIGN-mediated HIV transmission. 2. Examine the role of CD4 and Nef proteins in modulation of DC-SIGN-mediated HIV transmission. 3. Identify mechanisms of maturation of DC enhancing HIV transmission. These experimental designs will yield a better molecular and cellular description of HIV interactions with DC, which have relevance to control of HIV mucosal transmission and understanding of viral pathogenesis. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Cellular restriction of HIV infection in host cells reflects innate antiviral immunity. Monocytes are important immune sentinels and precursors of antigen presenting cells. Undifferentiated human monocytes are resistant to HIV infection despite the expression of HIV receptors. The mechanisms of postentry HIV restriction in monocytes are not fully understood. Given that monocyte-differentiated macrophages are permissive for productive HIV infection, it has been speculated that differentiation-dependent cellular cofactors in monocytes might be responsible for the anti-HIV activity. However, specific HIV restriction factors in monocytes remain to be identified. Our long-term goal is to elucidate the molecular mechanisms underlying the postentry HIV restriction in monocytes and to identify host factors that contribute to the restriction. Our preliminary studies indicate that i) HIV infection is profoundly blocked in monocytes freshly isolated from healthy donors despite efficient viral reverse transcription, while the nuclear import of HIV DNA is significantly impaired in HIV-infected monocytes;ii) The postentry HIV restriction does not correlate with the expression levels of a few HIV restriction factors reported in monocytes. Thus, we hypothesize that unidentified cellular factors in monocytes may account for the postentry restriction of HIV infection. Two specific aims are proposed to test this hypothesis. 1). To explore the HIV restriction phenotype by cell fusion between primary monocytes and HIV-permissive CD4+ T cells. 2). To perform a genetic screen for potential HIV restriction factors from a cDNA library of undifferentiated monocytes. The research design and methods include cell-fusion-based HIV infection assays and a genetic analysis for potential HIV restriction factors in monocytes. The proposed studies will provide new insights into understanding of HIV interactions with monocytes. PUBLIC HEALTH RELEVANCE: HIV infection is the leading killer worldwide among infectious diseases, incurring 2-3 million AIDS deaths annually. This proposal attempts to explore the mechanisms of HIV restriction in monocytes, which will help to understand host factor-HIV interactions and potentially aid in developing effective strategies to combat HIV infection.

DESCRIPTION (provided by applicant): The HIV-1 pre-integration complex (PIC) that comprises of HIV-1 full-length DNA, viral proteins, and host proteins is essential for viral integration and replication. Previous studies have identified and characterized several human proteins that interact with HIV-1 integrase and play important roles in viral infection. However, the technical limitations in acquiring sufficient amounts of catalytically active PICs has hindered a comprehensive identification of host proteins associated with the HIV-1 PICs. We recently developed a novel approach and identified 18 new human proteins that are specifically associated with the HIV-1 PICs isolated from infected CD4+ T cells. Our preliminary study suggested that one of these proteins, non-POU domain-containing octamer-binding protein (NonO), is required for efficient HIV-1 infection in CD4+ T cells. However, it is unclear whether the other 17 PIC- associated host proteins that we identified can affect HIV-1 infection. NonO is a multifunctional nuclear protein involved in transcription regulation, mRNA splicing, and DNA repair in the cell, but the mechanism by which NonO affects HIV-1 infection is not known. We seek to address these two significant questions in this R21 proposal. We hypothesize that host proteins (such as NonO) specifically associated with catalytically active PICs in HIV-1-infected cells play a role in viral integration and infection. The following two specific aims are designed o test this hypothesis: Aim 1. To determine the role of novel PIC-associated host proteins in HIV-1 infection. In addition to NonO, we may identify other PIC-associated host proteins among these 18 candidates that can significantly affect HIV-1 infection in Aim 1. We will select 2-3 protein candidates that have the most significant effect on viral infection to further study their mechanisms of action. Aim 2. To investigate the mechanisms by which PIC-associated host proteins affect HIV-1 infection. We will first investigate the mechanism by which NonO affects HIV-1 infection, and we will then perform similar studies to examine other protein candidates identified in Aim 1. The long-term goal of this project is to understand the function and mechanism of PIC-associated host proteins in HIV-1 integration or infection and to develop novel therapeutic strategies. An emerging anti-HIV therapeutic strategy is to block the interactions between HIV-1 integrase and its critical cellular cofactors. Our proposed study has the potential to identify novel drug targets to block HIV-1 integration and infection. PUBLIC HEALTH RELEVANCE: There are over 33 million people living with HIV worldwide and approximately 56,300 new HIV infections each year in the US. Although antiretroviral therapy can control HIV-1 infection in infected individuals, new anti-HIV drug targets are needed to increase efficacy and prevent drug resistance. This project aims to identify and characterize novel host proteins that are essential for the HIV integration process. The findings from the proposed studies will help identify novel drug targets for blocking HIV integration and infection.

DESCRIPTION (provided by applicant): Monocytes are precursors of dendritic cells and macrophages, and these cell types play an important and multifaceted role in HIV-1 infection and pathogenesis. Undifferentiated primary monocytes are resistant to HIV-1 postentry infection, which is likely due to the expression of a potential restriction factor. A challenge in he field is to identify the host factor and understand its mechanisms underlying HIV-1 restriction of primary monocytes. In our preliminary study, we identified a cellular protein named UBE2V1 (ubiquitin-conjugating enzyme E2 variant 1) that can inhibit HIV-1 infection in monocytic cells. The goal of this R21 proposal is to explore the function and mechanisms of UBE2V1 in blocking HIV-1 infection in primary monocytes. We propose to test the hypothesis that post-translationally modified UBE2V1 inhibits HIV-1 replication in primary monocytes. The expected outcome of our proposed studies is to define a novel mechanism by which post-translational modifications of UBE2V1 regulate HIV-1 infection in monocyte-lineage cells. UBE2V1 (also called UEV1, UEV1A or CROC-1) belongs to the subfamily of ubiquitin-conjugating enzyme variant proteins and acts as a regulatory protein in DNA damage and cell differentiation. A recent study indicated that the retroviral restriction factor TRIM5 functions as an innate immune sensor for the retrovirus capsid lattice by interacting with the ubiquitin-conjugating enzyme complex UBC13-UBE2V1. This finding suggests that UBE2V1 is a novel component of the innate immune response. Interestingly, our preliminary studies indicate that post-translational modification of UBE2V1 is associated with HIV-1 restriction in primary monocytes. We propose to explore the role and mechanisms of UBE2V1 in HIV-1 restriction in primary monocytes in two specific aims. Aim 1. Characterize UBE2V1-mediated restriction of the HIV-1 early lifecycle in primary monocytes. Aim 2. Define the critical domains and residues of the modified UBE2V1 responsible for HIV-1 restriction in primary monocytes. The significant impact of our proposed studies will be to open a new area in the study of post- translational modifications of host factor involved in HIV-1 replication. We will define the molecular mechanism of HIV-1 restriction in monocyte-lineage cells by characterizing the function of UBE2V1 in HIV-1 infection. Accomplishing the proposed aims has the potential to advance basic knowledge of HIV-host interactions. Ultimately, the knowledge learned from the proposed studies will enable design of novel approaches to block HIV-1 replication in target cells. PUBLIC HEALTH RELEVANCE: HIV-1 infection is a leading killer worldwide among infectious diseases, causing 2-3 million AIDS deaths annually. HIV-1 persistence is the major barrier to successful AIDS treatment. We propose to explore novel mechanisms by which a cellular protein named UBE2V1 restricts HIV- 1 replication in primary monocytes. The knowledge learned from the proposed studies will enable design of novel approaches to block HIV-1 persistent infection in target cells.

DESCRIPTION (provided by applicant): Immune activation is the driving force of HIV-1 replication in vivo, which facilitates viral infection in target cells and fundamentally contribute to AIDS pathogenesis. Myeloid cells, including monocytes, dendritic cells (DCs) and macrophages, play a critical role in innate immunity against viral infection. Our long-term goal is to define th mechanisms regulating immune activation of myeloid cells during HIV-1 infection. SAMHD1 is a recently identified HIV-1 restriction factor in myeloid cells. SAMHD1 a nuclear protein involved in innate immunity and has been proposed to act as a negative regulator of the interferon response. However, the role of SAMHD1 in regulating immune activation of myeloid cells remains unknown. The molecular mechanisms of SAMHD1-mediated HIV-1 restriction in DCs are not fully understood. In this project, we aim to study the mechanisms of SAMHD1-mediated HIV-1 restriction in primary DCs and the role of SAMHD1 in regulating immune activation of DCs. In our preliminary studies, we observed that HIV-1 infection of DCs significantly up-regulated the release of some early pro-inflammatory cytokines. Intriguingly, we found that HIV-1 infection of DCs resulted in the translocation of SAMHD1 from the nucleus to the cytoplasm. Our central hypotheses are: HIV-1 infection of DCs triggers SAMHD1 cytoplasmic translocation, which is important for HIV-1 restriction and immune suppression of DCs. As a result, HIV-1 restriction by SAMHD1 in DCs negatively regulates DC-mediated activation of CD4+ T-cells and HIV-1 transmission, which sets up a less permissive environment for HIV-1 spreading. We propose two specific aims to test these novel hypotheses. Aim 1. To examine the role of SAMHD1 in suppression of immune activation of DCs during HIV-1 infection; and Aim 2. To define the mechanisms of SAMHD1-mediated HIV-1 restriction in DCs. Our proposed studies will reveal the unique role of SAMHD1 in regulating immune activation of myeloid DCs during HIV-1 infection and define the precise mechanisms of SAMHD1-mediated HIV-1 restriction in primary DCs. Accomplishing the proposed studies will also elucidate the mechanisms by which SAMHD1 negatively regulates DC-mediated HIV-1 trans-infection and activation of CD4+ T-cells. Overall, our results will provide new insights into intrinsic immunity against HIV-1 infectin in myeloid DCs, which can help us to develop novel interventions to block HIV-1 infection and transmission.

DESCRIPTION (provided by applicant): Epigenetic mechanisms play an important role in cancer initiation and progression through modulation of gene expression. Cutaneous T cell lymphoma (CTCL) is defined by infiltration of activated and malignant CD4+ T- cells in the skin. The molecular mechanisms underlying the proliferation of neoplastic CD4+ T-cells in CTCL are not fully understood, but involve epigenetic silencing of multiple tumor suppressor genes. Sterile alpha motif (SAM) and HD domain-containing protein 1 (SAMHD1) is the first identified mammalian triphosphohydrolase that hydrolyzes cellular deoxynucleoside triphosphates (dNTPs). By depleting intracellular dNTPs required for retroviral replication, SAMHD1 blocks retroviral infection in resting CD4+ T-cells and myeloid cells. These cells express high levels of SAMHD1 protein and have 6- to 100-fold lower levels of intracellular dNTPs compared to activated CD4+ T-cells, while several leukemia and lymphoma CD4+ T-cell lines lack SAMHD1 protein expression and have increased dNTP levels. The goal of this project is to better understand the mechanisms and pathogenic consequences of epigenetic silencing of SAMHD1 in CTCL, which may aid in developing novel therapeutic approaches to treat CTCL. A key question is whether CD4+ T-cells from CTCL patients down- regulate or lack SAMHD1 expression to maintain high levels of intracellular dNTPs and support efficient DNA replication and uncontrolled cell growth. Our preliminary results suggest that human SAMHD1 may function as a tumor suppressor in CTCL. Interestingly, we observed 3-fold lower SAMHD1 mRNA levels in peripheral blood mononuclear cells from 9 CTCL patients compared to 14 healthy donors (p=0.0013), indicating that SAMHD1 expression is down-regulated in CTCL patients. We also found that epigenetic modification of the SAMHD1 gene is critical for silencing of SAMHD1 expression, which may in turn regulate DNA replication and cell growth of malignant CD4+ T-cells. Thus, we hypothesize that SAMHD1 functions as a tumor suppressor, and that epigenetic silencing of SAMHD1 expression in malignant CD4+ T-cells leads to increased intracellular dNTPs and contributes to uncontrolled T-cell growth in CTCL. To test this hypothesis, we propose two specific aims: Aim 1. To determine the correlation between SAMHD1 expression and dNTP levels in CD4+ T-cells from 50 patients with CTCL; and Aim 2. To investigate the mechanisms of epigenetic silencing of SAMHD1 in malignant CD4+ T-cells and to validate the results using a CTCL mouse model and a SAMHD1 knockout mouse model. Achieving our goals will provide critical knowledge into a novel mechanism that regulates dNTP metabolism in malignant CD4+ T-cells, which will facilitate the development of more effective drugs against CTCL and other T-cell lymphomas. Discovery of the mechanisms leading to loss of SAMHD1 protein expression could support the development of strategies to re-induce SAMHD1 gene expression as a new therapeutic modality in CTCL. SAMHD1's function as a novel tumor suppressor may also be important in other types of cancer and have a broader impact beyond CTCL.

? DESCRIPTION (provided by applicant): The HIV-1 pathogenic factor Nef and its interactions with host proteins are important for viral pathogenesis, but their role in HIV-1 latency is not known. Nef-associated factor 1 (Naf1), also known as A20-binding inhibitor of NF-kB (ABIN1), is a host protein that inhibits NF-kB activation. NF-kB is a well-known regulator HIV-1 gene expression and viral latency. Intriguingly, our preliminary studies suggest that Naf1 and Nef interplay regulates HIV-1 gene transcription and viral latency. We found that (1) Naf1 suppresses NF-kB- dependent HIV-1 gene expression and Nef counteracts the effects; and (2) Naf1 maintains HIV-1 latency by suppressing viral gene transcription, while Nef antagonizes the effects. Therefore, we propose to delineate the mechanisms of Nef-Naf1 interactions in regulating HIV-1 latency and to seek a new approach to overcome viral latency. Our central hypotheses are (1) Naf1 maintains HIV-1 latency by suppressing NF- kB-dependent viral gene transcription; (2) Nef antagonizes the inhibitory effects of Naf1 by inducing Naf1 phosphorylation, which activates PI3K/Akt signaling and leads to HIV-1 reactivation from latency. We propose two specific aims to test these novel hypotheses. Aim 1. To investigate the mechanisms by which the Naf1-Nef interaction regulates HIV-1 gene expression. Aim 2. To determine the function of Naf1 and Nef interactions in modulating HIV-1 latency in primary CD4+ T cells. Accomplishing the proposed studies through our collaborative efforts will provide novel insights into the viral and cellular mechanisms of modulating HIV-1 latency. Our expected results will provide a basis to target Nef-Naf1 interactions and to enhance anti-retroviral therapy toward a functional cure for HIV-1/AIDS.

Project Summary/Abstract Our goal is to investigate the functions and mechanisms of N6-methyladenosine (m6A) modification of HIV-1 RNA during viral infection. The internal m6A modification of cellular RNAs is a novel mechanism of post-transcriptional control of gene expression, which is coordinately regulated by three groups of host proteins, including methyltransferases (writers), demethylases (erasers), and m6A-selective- binding proteins (readers). Binding of m6A-modified cellular RNA by the readers significantly affects various aspects of RNA functions during translation. Three recent publications, including one from our group, highlighted the critical role of m6A modification of HIV-1 RNA in regulating viral replication. However, these studies reported some different results and suggested distinct mechanisms. To clarify the discrepancy, it is essential to define the mechanisms by which m6A modification of HIV-1 RNA regulates viral replication in primary CD4+ T-cells. We identified multiple regions of m6A modification in HIV-1 RNA bound by the readers in HIV-1- infected primary CD4+ T-cells. We found that the expression levels of the readers significantly modulated HIV-1 infection in target cells. Knockdown of the writers or erasers in virus-producing cells significantly affected HIV-1 Gag protein synthesis, suggesting the importance of m6A modification of HIV-1 RNA in regulating viral gene expression. Interestingly, we observed that HIV-1 infection upregulated the expression of endogenous readers in primary CD4+ T-cells. Our central hypothesize is that reversible m6A modification of HIV-1 RNA regulates viral replication in CD4+ T- cells by affecting the structure, stability, splicing, and/or trafficking of HIV-1 RNA. We will test this hypothesis in three specific aims using interdisciplinary approaches. Aim 1. To identify the m6A residues in HIV-1 RNA and to investigate the effects of m6A modification on HIV-1 RNA structure and interactions with the reader proteins; Aim 2. To investigate the effects of m6A modification on the stability, splicing and trafficking of HIV-1 RNA in infected primary CD4+ T-cells; and Aim 3. To investigate the effects of HIV-1 infection and proinflammatory cytokines on the expression and localization of the readers, writers, and erasers and to define the underlying mechanisms. Overall impact: Accomplishing our proposed studies will define the mechanisms by which HIV-1 RNA m6A modification regulates viral infection in CD4+ T-cells. Investigations of the HIV-1 RNA m6A modification and interactions with host proteins represent a new area of HIV-1 RNA biology, which can facilitate therapeutic development against HIV-1 infection.

Project Summary/Abstract A critical barrier to developing a cure for HIV-1 infection is the long-lived viral reservoir in cells due to viral latency. Many host proteins regulate HIV-1 gene expression and viral latency. By reducing intracellular dNTP levels, the host protein SAMHD1 inhibits HIV-1 replication in myeloid cells and resting CD4+ T-cells, cell types that are important for HIV-1 latency. Patients with homozygous SAMHD1 mutations experience autoimmune diseases, but the functions and mechanisms of SAMHD1 in modulating inflammation and immunity remain unclear. Despite extensive studies of the mechanisms underlying SAMHD1-mediated restriction of virus replication, it is unknown whether and how SAMHD1 regulates antiviral innate immune responses. Here, we aim to address two key questions: (a) What are the mechanisms of SAMHD1 in modulating the innate immune response to HIV-1 infection? (b) How does SAMHD1 affect HIV-1 gene expression? Our new findings demonstrated that SAMHD1 suppresses innate immune responses to viral infections and inflammatory stimuli by inhibiting nuclear factor-kappa B (NF-?B) activation and type I interferon (IFN-I) induction through distinct mechanisms. We discovered that SAMHD1 interacts with key proteins in the NF-?B and IFN-I pathways, allowing it to act as a multifaceted repressor of innate immune signaling. We also found that SAMHD1 impairs HIV-1 gene expression and reactivation of viral latency in primary CD4+ T-cells. NF-?B is critical for HIV-1 gene transcription and transcriptional inhibition of viral gene expression is the main mechanism of HIV-1 latency. Our central hypothesis is that SAMHD1 modulates HIV-1 antiviral immunity and viral gene expression by suppressing NF-?B activation, IFN-I induction, and viral transcription. We designed three specific aims to test this hypothesis. Aim 1. Elucidate the mechanisms by which SAMHD1 suppresses NF-?B activation in HIV-1 infection; Aim 2. Define the mechanisms by which SAMHD1 suppresses IFN-I induction during HIV-1 infection; Aim 3. Investigate the mechanisms by which SAMHD1 impairs reactivation of HIV-1 gene expression. Overall impact. These studies will reveal novel physiological functions of SAMHD1 during HIV-1 infection of primary target cells, which are beyond its known function in restricting virus replication. Furthermore, our interdisciplinary studies will yield new results to fundamentally enhance our mechanistic understanding of SAMHD1 in regulating HIV-1 infection, viral gene expression, and anti-HIV innate immune responses.