Influence of Self-MHC Class I Recognition on the Dynamics of NK Cell Responses to Cytomegalovirus Infection.

Although interactions between inhibitory Ly49 receptors and their self-MHC class I ligands in C57BL/6 mice are known to limit NK cell proliferation during mouse CMV (MCMV) infection, we created a 36-marker mass cytometry (CyTOF) panel to investigate how these inhibitory receptors impact the NK cell response to MCMV in other phenotypically measurable ways. More than two thirds of licensed NK cells (i.e., those expressing Ly49C, Ly49I, or both) in uninfected mice had already differentiated into NK cells with phenotypes indicative of Ag encounter (KLRG1+Ly6C-) or memory-like status (KLRG1+Ly6C+). These pre-existing KLRG1+Ly6C+ NK cells resembled known Ag-specific memory NK cell populations in being less responsive to IL-18 and IFN-α stimulation in vitro and by selecting for NK cell clones with elevated expression of a Ly49 receptor. During MCMV infection, the significant differences between licensed and unlicensed (Ly49C-Ly49I-) NK cells disappeared within both CMV-specific (Ly49H+) and nonspecific (Ly49H-) responses. This lack of heterogeneity carried into the memory phase, with only a difference in CD16 expression manifesting between licensed and unlicensed MCMV-specific memory NK cell populations. Our results suggest that restricting proliferation is the predominant effect licensing has on the NK cell population during MCMV infection, but the inhibitory Ly49-MHC interactions that take place ahead of infection contribute to their limited expansion by shrinking the pool of licensed NK cells capable of robustly responding to new challenges.

Caspase-8 restricts antiviral CD8 T cell hyperaccumulation.

The magnitude of CD8 T cell responses against viruses is checked by the balance of proliferation and death. Caspase-8 (CASP8) has the potential to influence response characteristics through initiation of apoptosis, suppression of necroptosis, and modulation of cell death-independent signal transduction. Mice deficient in CASP8 and RIPK3 (Casp8-/-Ripk3-/- ) mount enhanced peak CD8 T cell levels against the natural mouse pathogen murine cytomegalovirus (MCMV) or the human pathogen herpes simplex virus-1 compared with littermate control RIPK3-deficient or WT C57BL/6 mice, suggesting an impact of CASP8 on the magnitude of antiviral CD8 T cell expansion and not on contraction. The higher peak response to MCMV in Casp8-/-Ripk3-/- mice resulted from accumulation of greater numbers of terminally differentiated KLRG1hi effector CD8 T cell subsets. Antiviral Casp8-/-Ripk3-/- T cells exhibited enhanced proliferation when splenocytes were transferred into WT recipient mice. Thus, cell-autonomous CASP8 normally restricts CD8 T cell proliferation following T cell receptor activation in response to foreign antigen. Memory inflation is a hallmark quality of the T cell response to cytomegalovirus infection. Surprisingly, MCMV-specific memory inflation was not sustained long-term in Casp8-/-Ripk3-/- mice even though these mice retained immunity to secondary challenge. In addition, the accumulation of abnormal B220+CD3+ T cells in these viable CASP8-deficient mice was reduced by chronic MCMV infection. Combined, these data brings to light the cell death-independent role of CASP8 during CD8 T cell expansion in mice lacking the confounding impact of RIPK3-mediated necroptosis.

Caspase-8 restricts natural killer cell accumulation during MCMV Infection.

Natural killer (NK) cells provide important host defense against herpesvirus infections and influence subsequent T cell control of replication and maintenance of latency. NK cells exhibit phases of expansion, contraction and memory formation in response to the natural mouse pathogen murine cytomegalovirus (MCMV). Innate and adaptive immune responses are tightly regulated in mammals to avoid excess tissue damage while preventing acute and chronic viral disease and assuring resistance to reinfection. Caspase (CASP)8 is an autoactivating aspartate-specific cysteine protease that initiates extrinsic apoptosis and prevents receptor interacting protein (RIP) kinase (RIPK)1-RIPK3-driven necroptosis. CASP8 also promotes death-independent signal transduction. All of these activities make contributions to inflammation. Here, we demonstrate that CASP8 restricts NK cell expansion during MCMV infection but does not influence NK memory. Casp8-/-Ripk3-/- mice mount higher NK response levels than Casp8+/-Ripk3-/- littermate controls or WT C57BL/6 J mice, indicating that RIPK3 deficiency alone does not contribute to NK response patterns. MCMV m157-responsive Ly49H+ NK cells support increased expansion of both Ly49H- NK cells and CD8 T cells in Casp8-/-Ripk3-/- mice. Surprisingly, hyperaccumulation of NK cells depends on the pronecrotic kinase RIPK1. Ripk1-/-Casp8-/-Ripk3-/- mice fail to show the enhanced expansion of lymphocytes observed in Casp8-/-Ripk3-/- mice even though development and homeostasis are preserved in uninfected Ripk1-/-Casp8-/-Ripk3-/- mice. Thus, CASP8 naturally regulates the magnitude of NK cell responses in response to infection where strong activation signals depend on another key regulator of death signaling, RIPK1. In addition, the strong NK cell response promotes survival of effector CD8 T cells during their expansion. Thus, hyperaccumulation of NK cells and crosstalk with T cells becomes amplified in the absence of extrinsic cell death machinery.

HIV-1 Protease Uses Bi-Specific S2/S2' Subsites to Optimize Cleavage of Two Classes of Target Sites.

Retroviral proteases (PRs) have a unique specificity that allows cleavage of sites with or without a P1' proline. A P1' proline is required at the MA/CA cleavage site due to its role in a post-cleavage conformational change in the capsid protein. However, the HIV-1 PR prefers to have large hydrophobic amino acids flanking the scissile bond, suggesting that PR recognizes two different classes of substrate sequences. We analyzed the cleavage rate of over 150 combinations of six different HIV-1 cleavage sites to explore rate determinants of cleavage. We found that cleavage rates are strongly influenced by the two amino acids flanking the amino acids at the scissile bond (P2-P1/P1'-P2'), with two complementary sets of rules. When P1' is proline, the P2 side chain interacts with a polar region in the S2 subsite of the PR, while the P2' amino acid interacts with a hydrophobic region of the S2' subsite. When P1' is not proline, the orientations of the P2 and P2' side chains with respect to the scissile bond are reversed; P2 residues interact with a hydrophobic face of the S2 subsite, while the P2' amino acid usually engages hydrophilic amino acids in the S2' subsite. These results reveal that the HIV-1 PR has evolved bi-functional S2 and S2' subsites to accommodate the steric effects imposed by a P1' proline on the orientation of P2 and P2' substrate side chains. These results also suggest a new strategy for inhibitor design to engage the multiple specificities in these subsites.

Using Primer-ID Deep Sequencing to Detect Recent Human Immunodeficiency Virus Type 1 Infection.

Intrahost viral sequence diversity can be evaluated over multiple genomic regions using next-generation sequencing (NGS) and scaled to population-level diversity to identify recent human immunodeficiency virus type 1 infection. Using Primer-ID NGS, we sequenced the reverse transcriptase (RT) and env V1-V3 regions from persons with known infection dates, and assessed the mean (π) and first quintile of pairwise diversity distributions over time. The receiver operating characteristic area under the curve (AUC) of RT and V1-V3 combined showed excellent discrimination of recent infection <9 months: using π (only single transmitted variants: AUC, 0.98; threshold <1.03%; sensitivity, 97%; specificity, 89%) and the first quintile (including all variants: AUC, 0.90; threshold <0.60%; sensitivity, 91%; specificity, 92%).

A human anti-IL-2 antibody that potentiates regulatory T cells by a structure-based mechanism.

Interleukin-2 (IL-2) has been shown to suppress immune pathologies by preferentially expanding regulatory T cells (Tregs). However, this therapy has been limited by off-target complications due to pathogenic cell expansion. Recent efforts have been focused on developing a more selective IL-2. It is well documented that certain anti-mouse IL-2 antibodies induce conformational changes that result in selective targeting of Tregs. We report the generation of a fully human anti-IL-2 antibody, F5111.2, that stabilizes IL-2 in a conformation that results in the preferential STAT5 phosphorylation of Tregs in vitro and selective expansion of Tregs in vivo. When complexed with human IL-2, F5111.2 induced remission of type 1 diabetes in the NOD mouse model, reduced disease severity in a model of experimental autoimmune encephalomyelitis and protected mice against xenogeneic graft-versus-host disease. These results suggest that IL-2-F5111.2 may provide an immunotherapy to treat autoimmune diseases and graft-versus-host disease.

Cutting Edge: IL-2-Induced Expression of the Amino Acid Transporters SLC1A5 and CD98 Is a Prerequisite for NKG2D-Mediated Activation of Human NK Cells.

Priming of human NK cells with IL-2 is necessary to render them functionally competent upon NKG2D engagement. We examined the underlying mechanisms that control NKG2D responsiveness in NK cells and found that IL-2 upregulates expression of the amino acid transporters SLC1A5 and CD98. Using specific inhibitors to block SLC1A5 and CD98 function, we found that production of IFN-γ and degranulation by CD56bright and CD56dim NK cells following NKG2D stimulation were dependent on both transporters. IL-2 priming increased the activity of mTORC1, and inhibition of mTORC1 abrogated the ability of the IL-2-primed NK cells to produce IFN-γ in response to NKG2D-mediated stimulation. This study identifies a series of IL-2-induced cellular changes that regulates the NKG2D responsiveness in human NK cells.

A Direct Interaction with RNA Dramatically Enhances the Catalytic Activity of the HIV-1 Protease In Vitro.

Though the steps of human immunodeficiency virus type 1 (HIV-1) virion maturation are well documented, the mechanisms regulating the proteolysis of the Gag and Gag-Pro-Pol polyproteins by the HIV-1 protease (PR) remain obscure. One proposed mechanism argues that the maturation intermediate p15NC must interact with RNA for efficient cleavage by the PR. We investigated this phenomenon and found that processing of multiple substrates by the HIV-1 PR was enhanced in the presence of RNA. The acceleration of proteolysis occurred independently from the substrate's ability to interact with nucleic acid, indicating that a direct interaction between substrate and RNA is not necessary for enhancement. Gel-shift assays demonstrated the HIV-1 PR is capable of interacting with nucleic acids, suggesting that RNA accelerates processing reactions by interacting with the PR rather than the substrate. All HIV-1 PRs examined have this ability; however, the HIV-2 PR does not interact with RNA and does not exhibit enhanced catalytic activity in the presence of RNA. No specific sequence or structure was required in the RNA for a productive interaction with the HIV-1 PR, which appears to be principally, though not exclusively, driven by electrostatic forces. For a peptide substrate, RNA increased the kinetic efficiency of the HIV-1 PR by an order of magnitude, affecting both turnover rate (k(cat)) and substrate affinity (K(m)). These results suggest that an allosteric binding site exists on the HIV-1 PR and that HIV-1 PR activity during maturation could be regulated in part by the juxtaposition of the enzyme with virion-packaged RNA.

The triple threat of HIV-1 protease inhibitors.

Newly released human immunodeficiency virus type 1 (HIV-1) particles obligatorily undergo a maturation process to become infectious. The HIV-1 protease (PR) initiates this step, catalyzing the cleavage of the Gag and Gag-Pro-Pol structural polyproteins. Proper organization of the mature virus core requires that cleavage of these polyprotein substrates proceeds in a highly regulated, specific series of events. The vital role the HIV-1 PR plays in the viral life cycle has made it an extremely attractive target for inhibition and has accordingly fostered the development of a number of highly potent substrate-analog inhibitors. Though the PR inhibitors (PIs) inhibit only the HIV-1 PR, their effects manifest at multiple different stages in the life cycle due to the critical importance of the PR in preparing the virus for these subsequent events. Effectively, PIs masquerade as entry inhibitors, reverse transcription inhibitors, and potentially even inhibitors of post-reverse transcription steps. In this chapter, we review the triple threat of PIs: the intermolecular cooperativity in the form of a cooperative dose-response for inhibition in which the apparent potency increases with increasing inhibition; the pleiotropic effects of HIV-1 PR inhibition on entry, reverse transcription, and post-reverse transcription steps; and their potency as transition state analogs that have the potential for further improvement that could lead to an inability of the virus to evolve resistance in the context of single drug therapy.

A sensitive assay using a native protein substrate for screening HIV-1 maturation inhibitors targeting the protease cleavage site between the matrix and capsid.

The matrix/capsid processing site in the HIV-1 Gag precursor is likely the most sensitive target to inhibit HIV-1 replication. We have previously shown that modest incomplete processing at the site leads to a complete loss of virion infectivity. In the study presented here, a sensitive assay based on fluorescence polarization that can monitor cleavage at the MA/CA site in the context of the folded protein substrate is described. The substrate, an MA/CA fusion protein, was labeled with the fluorescein-based FlAsH (fluorescein arsenical hairpin) reagent that binds to a tetracysteine motif (CCGPCC) that was introduced within the N-terminal domain of CA. By limiting the size of CA and increasing the size of MA (with an N-terminal GST fusion), we were able to measure significant differences in polarization values as a function of HIV-1 protease cleavage. The sensitivity of the assay was tested in the presence of increasing amounts of an HIV-1 protease inhibitor, which resulted in a gradual decrease in the fluorescence polarization values demonstrating that the assay is sensitive in discerning changes in protease processing. The high-throughput screening assay validation in 384-well plates showed that the assay is reproducible and robust with an average Z' value of 0.79 and average coefficient of variation values of <3%. The robustness and reproducibility of the assay were further validated using the LOPAC(1280) compound library, demonstrating that the assay provides a sensitive high-throughput screening platform that can be used with large compound libraries for identifying novel maturation inhibitors targeting the MA/CA site of the HIV-1 Gag polyprotein.

The choreography of HIV-1 proteolytic processing and virion assembly.

HIV-1 has been the target of intensive research at the molecular and biochemical levels for >25 years. Collectively, this work has led to a detailed understanding of viral replication and the development of 24 approved drugs that have five different targets on various viral proteins and one cellular target (CCR5). Although most drugs target viral enzymatic activities, our detailed knowledge of so much of the viral life cycle is leading us into other types of inhibitors that can block or disrupt protein-protein interactions. Viruses have compact genomes and employ a strategy of using a small number of proteins that can form repeating structures to enclose space (i.e. condensing the viral genome inside of a protein shell), thus minimizing the need for a large protein coding capacity. This creates a relatively small number of critical protein-protein interactions that are essential for viral replication. For HIV-1, the Gag protein has the role of a polyprotein precursor that contains all of the structural proteins of the virion: matrix, capsid, spacer peptide 1, nucleocapsid, spacer peptide 2, and p6 (which contains protein-binding domains that interact with host proteins during budding). Similarly, the Gag-Pro-Pol precursor encodes most of the Gag protein but now includes the viral enzymes: protease, reverse transcriptase (with its associated RNase H activity), and integrase. Gag and Gag-Pro-Pol are the substrates of the viral protease, which is responsible for cleaving these precursors into their mature and fully active forms (see Fig. 1A).

Context surrounding processing sites is crucial in determining cleavage rate of a subset of processing sites in HIV-1 Gag and Gag-Pro-Pol polyprotein precursors by viral protease.

Processing of the human immunodeficiency virus type 1 (HIV-1) Gag and Gag-Pro-Pol polyproteins by the HIV-1 protease (PR) is essential for the production of infectious particles. However, the determinants governing the rates of processing of these substrates are not clearly understood. We studied the effect of substrate context on processing by utilizing a novel protease assay in which a substrate containing HIV-1 matrix (MA) and the N-terminal domain of capsid (CA) is labeled with a FlAsH (fluorescein arsenical hairpin) reagent. When the seven cleavage sites within the Gag and Gag-Pro-Pol polyproteins were placed at the MA/CA site, the rates of cleavage changed dramatically compared with that of the cognate sites in the natural context reported previously. The rate of processing was affected the most for three sites: CA/spacer peptide 1 (SP1) (≈10-fold increase), SP1/nucleocapsid (NC) (≈10-30-fold decrease), and SP2/p6 (≈30-fold decrease). One of two multidrug-resistant (MDR) PR variants altered the pattern of processing rates significantly. Cleavage sites within the Pro-Pol region were cleaved in a context-independent manner, suggesting for these sites that the sequence itself was the determinant of rate. In addition, a chimera consisting of SP1/NC P4-P1 and MA/CA P1'-P4' residues (ATIM↓PIVQ) abolished processing by wild type and MDR proteases, and the reciprocal chimera consisting of MA/CA P4-P1 and SP1/NC P1'-4' (SQNY↓IQKG) was cleaved only by one of the MDR proteases. These results suggest that complex substrate interactions both beyond the active site of the enzyme and across the scissile bond contribute to defining the rate of processing by the HIV-1 PR.

Mechanism of gemini disulfide detergent mediated oxidative refolding of lysozyme in a new artificial chaperone system.

Gemini surfactants are a new class of surfactants that consist of two hydrophilic head groups and two hydrophobic tails separated by a spacer group. As the properties of geminis are different to their monomeric counterparts, a large number of applications have been investigated. Here we report on the use of a new class of gemini detergents containing a disulfide bond in the spacer (Det-SS-Det) for protein refolding. Using lysozyme as a model protein we could demonstrate that the disulfide gemini detergents allow oxidative refolding of the protein in the absence of any external redox system in an "artificial chaperone system". Refolding kinetics using gemini disulfide detergents differing in their hydrophobicity were analysed to determine the folding and aggregation rate constants. The results point to an important role of the transiently formed mixed disulfides between the protein and the detergent (Prot-SS-Det) in the oxidative refolding process of lysozyme.