Faculty Bibliography

HIV-1-infected individuals are treated with lifelong antiretroviral drugs to control the infection. A means to strengthen the antiviral T cell response might allow them to control viral loads without antiretroviral drugs. We report the development of a lentiviral vector-based dendritic cell (DC) vaccine in which HIV-1 antigen is co-expressed with CD40 ligand (CD40L) and a soluble, high-affinity programmed cell death 1 (PD-1) dimer. CD40L activates the DCs, whereas PD-1 binds programmed death ligand 1 (PD-L1) to prevent checkpoint activation and strengthen the cytotoxic T lymphocyte (CTL) response. The injection of humanized mice with DCs transduced with vector expressing CD40L and the HIV-1 SL9 epitope induced antigen-specific T cell proliferation and memory differentiation. Upon HIV-1 challenge of vaccinated mice, viral load was suppressed by 2 logs for 6 weeks. Introduction of the soluble PD-1 dimer into a vector that expressed full-length HIV-1 proteins accelerated the antiviral response. The results support development of this approach as a therapeutic vaccine that might allow HIV-1-infected individuals to control virus replication without antiretroviral therapy.

Zika virus (ZIKV) is an emerging pathogen linked to neurological disorders for which there is currently no targeted therapy. To identify host innate immune response proteins that restrict ZIKV replication, we treated monocytes and macrophages with toll-like receptor (TLR) agonists. Of those tested, the TLR7/8 agonist R848 (resiquimod) was the most potent inhibitor of ZIKV replication. RNA-seq analysis identified several genes strongly induced by R848 in monocytes. Testing of several of these for their ability to restrict ZIKV replication identified viperin, an interferon-induced gene active against several viruses. Transduction of microglial CHME3 cells with a viperin lentiviral expression vector rendered them resistant to ZIKV infection, preventing the synthesis of viral RNA and protein. CRISPR/Cas9 knock-out of viperin in macrophages relieved the block to infection, demonstrating that viperin is a major innate immune response protein able to block ZIKV replication. TLR agonists may be useful for the prophylactic or therapeutic treatment for ZIKV.

Monocyte-derived macrophages (MDMs) are an important target for HIV-1 despite SAMHD1, a myeloid restriction factor for which HIV-1 lacks a counteracting accessory protein. The antiviral activity of SAMHD1 is modulated by phosphorylation of T592 by cyclin-dependent kinases (CDK). We show that treatment of MDMs with neocarzinostatin, a compound that introduces double strand breaks (DBS) in genomic DNA, results in the decrease of phosphorylated SAMHD1, activating its antiviral activity and blocking HIV-1 infection. The effect was specific for DSB as DNA damage induced by UV light irradiation did not affect SAMHD1 phosphorylation and did not block infection. The block to infection was at reverse transcription and was counteracted by Vpx, demonstrating that it was caused by SAMHD1. Neocarzinostatin treatment also activated an innate immune response that induced interferon-stimulated genes but this was not involved in the block to HIV-1 infection, as it was not relieved by an interferon-blocking antibody. In response to Neocarzinostatin-induced DNA damage, the level of the CDK inhibitor p21^cip1increased which could account for the decrease of phosphorylated SAMHD1. The results show that the susceptibility of MDMs to HIV-1 infection can be affected by stimuli that alter the phosphorylation state of SAMHD1, one of which is the DNA damage response.

The triphosphohydrolase SAMHD1 restricts HIV-1 replication in nondividing myeloid cells by depleting the dNTP pool, preventing reverse transcription. SAMHD1 is also reported to have ribonuclease activity that degrades the virus genomic RNA. Human SAMHD1 is regulated by phosphorylation of its carboxy-terminus at T592, which abrogates its antiviral function yet has only a small effect on its phosphohydrolase activity. In the mouse, SAMHD1 is expressed as two isoforms (ISF1 and ISF2) that differ at the carboxy-terminus due to alternative splicing of the last coding exon. In this study, we characterized the biochemical and antiviral properties of the two mouse isoforms of SAMHD1. Both are antiviral in nondividing cells. Mass spectrometry analysis showed that SAMHD1 is phosphorylated at several amino acid residues, one of which (T634) is homologous to T592. Phosphomimetic mutation at T634 of ISF1 ablates its antiviral activity yet has little effect on phosphohydrolase activity in vitro. dGTP caused ISF1 to tetramerize, activating its catalytic activity. In contrast, ISF2, which lacks the phosphorylation site, was significantly more active, tetramerized and was active without added dGTP. Neither isoform, nor human SAMHD1 had detectable RNase activity in vitro or affected HIV-1 genomic RNA stability in newly infected cells. These data support a model in which SAMHD1 catalytic activity is regulated through tetramer stabilization by the carboxy-terminal tail, phosphorylation destabilizing the complexes and inactivating the enzyme. ISF2 may serve to reduce the dNTP pool to very low levels as a means of restricting virus replication.

Leukocidin ED (LukED) is a bicomponent pore-forming toxin produced by Staphylococcus aureus that lyses host cells by targeting the chemokine receptors CC chemokine receptor type 5 (CCR5), CXCR1, CXCR2, and DARC. In addition to its role as a receptor for LukED, CCR5 is the major coreceptor for primary isolates of human immunodeficiency virus type 1 (HIV-1) and has been extensively studied. To compare how LukED and HIV-1 target CCR5, we analyzed their respective abilities to use CCR5/CCR2b chimeras to mediate cytotoxicity and virus entry. These analyses showed that the second and third extracellular loops (ECL) of CCR5 are necessary and sufficient for LukED to target the receptor and promote cell lysis. In contrast, the second ECL of CCR5 is necessary but not sufficient for HIV-1 infectivity. The analysis of CCR5 point mutations showed that glycine-163 is critical for HIV-1 infectivity, while arginine-274 and aspartic acid-276 are critical for LukED cytotoxicity. Point mutations in ECL2 diminished both HIV-1 infectivity and LukED cytotoxicity. Treatment of cells with LukED did not interfere with CCR5-tropic HIV-1 infectivity, demonstrating that LukED and the viral envelope glycoprotein use nonoverlapping sites on CCR5. Analysis of point mutations in LukE showed that amino acids 64 to 69 in the rim domain are required for CCR5 targeting and cytotoxicity. Taking the results together, this study identified the molecular basis by which LukED targets CCR5, highlighting the divergent molecular interactions evolved by HIV-1 and LukED to interact with CCR5. IMPORTANCE: The bicomponent pore-forming toxins are thought to play a vital role in the success of Staphylococcus aureus as a mammalian pathogen. One of the leukocidins, LukED, is necessary and sufficient for lethality in mice. At the molecular level, LukED causes cell lysis through binding to specific cellular receptors. CCR5 is one of the receptors targeted by LukED and is the major coreceptor for CCR5-tropic HIV-1. While the molecular interaction of CCR5 and HIV-1 is well characterized, the means by which LukED interacts with CCR5 is less clear. In this study, we demonstrated that receptor specificity is conferred through unique interactions between key domains on CCR5 and LukE. Although HIV-1 and LukED target the same receptor, our data demonstrated that they interact with CCR5 differently, highlighting the molecular complexity of host-pathogen interactions.

IFN-epsilon is a unique type I IFN that is not induced by pattern recognition response elements. IFN-epsilon is constitutively expressed in mucosal tissues, including the female genital mucosa. Although the direct antiviral activity of IFN-epsilon was thought to be weak compared with IFN-alpha, IFN-epsilon controls Chlamydia muridarum and herpes simplex virus 2 in mice, possibly through modulation of immune response. We show here that IFN-epsilon induces an antiviral state in human macrophages that blocks HIV-1 replication. IFN-epsilon had little or no protective effect in activated CD4+ T cells or transformed cell lines unless activated CD4+ T cells were infected with replication-competent HIV-1 at a low MOI. The block to HIV infection of macrophages was maximal after 24 hours of treatment and was reversible. IFN-epsilon acted on early stages of the HIV life cycle, including viral entry, reverse transcription, and nuclear import. The protection did not appear to operate through known type I IFN-induced HIV host restriction factors, such as APOBEC3A and SAMHD1. IFN-epsilon-stimulated immune mediators and pathways had the signature of type I IFNs but were distinct from IFN-alpha in macrophages. IFN-epsilon induced significant phagocytosis and ROS, which contributed to the block to HIV replication. These findings indicate that IFN-epsilon induces an antiviral state in macrophages that is mediated by different factors than those induced by IFN-alpha. Understanding the mechanism of IFN-epsilon-mediated HIV inhibition through immune modulation has implications for prevention.

BACKGROUND: Monocytes, the primary myeloid cell-type in peripheral blood, are resistant to HIV-1 infection as a result of the lentiviral restriction factor SAMHD1. Toll-like receptors recognize microbial pathogen components, inducing the expression of antiviral host proteins and proinflammatory cytokines. TLR agonists that mimic microbial ligands have been found to have activity against HIV-1 in macrophages. The induction of restriction factors in monocytes by TLR agonist activation has not been well studied. To analyze restriction factor induction by TLR activation in monocytes, we used the imidazoquinoline TLR7/8 agonist R848 and infected with HIV-1 reporter virus that contained packaged viral accessory protein Vpx, which allows the virus to escape SAMHD1-mediated restriction. RESULTS: R848 prevented the replication of Vpx-containing HIV-1 and HIV-2 in peripheral blood mononuclear cells and monocytes. The block was post-entry but prior to reverse transcription of the viral genomic RNA. The restriction was associated with destabilization of the genomic RNA molecules of the in-coming virus particle. R848 treatment of activated T cells did not protect them from infection but treated monocytes produced high levels of proinflammatory cytokines, including type-I IFN that protected bystander activated T cells from infection. CONCLUSION: The activation of TLR7/8 induces two independent restrictions to HIV-1 replication in monocytes: a cell-intrinsic block that acts post-entry to prevent reverse transcription; and a cell-extrinsic block, in which monocytes produce high levels of proinflammatory cytokines (primarily type-I IFN) that protects bystander monocytes and T lymphocytes. The cell-intrinsic block may result from the induction of a novel restriction factor, which can be termed Lv5 and acts by destabilizing the in-coming viral genomic RNA, either by the induction of a host ribonuclease or by disrupting the viral capsid. TLR agonists are being developed for therapeutic use to diminish the size of the latent provirus reservoir in HIV-1 infected individuals. Such drugs may both induce latent provirus expression and restrict virus replication during treatment.

To replicate in their hosts, viruses have to navigate the complexities of the mammalian cell, co-opting mechanisms of cellular physiology while defeating restriction factors that are dedicated to halting their progression. Primate lentiviruses devote a relatively large portion of their coding capacity to counteracting restriction factors by encoding accessory proteins dedicated to neutralizing the antiviral function of these intracellular inhibitors. Research into the roles of the accessory proteins has revealed the existence of previously undetected intrinsic defenses, provided insight into the evolution of primate lentiviruses as they adapt to new species and uncovered new targets for the development of therapeutics. This Review discusses the biology of the restriction factors APOBEC3, SAMHD1 and tetherin and the viral accessory proteins that counteract them.