Naturally occurring dipeptide from elite controllers with dual anti-HIV-1 mechanism.

BACKGROUND: Enhanced levels of a dipeptide, WG-am, have been reported among elite controllers - patients who spontaneously control their HIV-1 infection. This study aimed to evaluate anti-HIV-1 activity and mechanism of action of WG-am. METHODS: Drug sensitivity assays in TZM.bl cells, PBMCs and ACH-2 cells using WT and mutated HIV-1 strainswere performed to evaluate the antiviral mechanism of WG-am. Mass spectrometry-based proteomics and Real-time PCR analysis of reverse transcription steps were performed to unravel the second anti-HIV-1 mechanism of WG-am. RESULTS: The data suggest that WG-am binds to the CD4 binding pocket of HIV-1 gp120 and blocks its binding to the host cell receptors. Additionally, the time course assay showed that WG-am also inhibited HIV-1 at 4-6 hours post-infection, suggesting a second antiviral mechanism. Drug sensitivity assays under acidic wash conditions confirmed the ability of WG-am to internalise into the host cell in an HIV independent manner. Proteomic studies showed a clustering of all samples treated with WG-am independent of the number of doses or presence or absence of HIV-1. Differentially expressed proteins due to the WG-am treatment indicated an effect on HIV-1 reverse transcription, which was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). CONCLUSION: Naturally occurring in HIV-1 elite controllers, WG-am stands out as a new kind of antiviral compound with two independent inhibitory mechanisms of action on HIV-1 replication. WG-am halts HIV-1 entry to the host cell by binding to HIV-1 gp120, thereby blocking the binding of HIV-1 to the host cell. WG-am also exerts a post-entry but pre-integration antiviral effect related to RT-activity.

Natural killer cells induce HIV-1 latency reversal after treatment with pan-caspase inhibitors.

The establishment of a latency reservoir is the major obstacle for a cure of HIV-1. The shock-and-kill strategy aims to reactivate HIV-1 replication in HIV -1 latently infected cells, exposing the HIV-1-infected cells to cytotoxic lymphocytes. However, none of the latency reversal agents (LRAs) tested so far have shown the desired effect in people living with HIV-1. We observed that NK cells stimulated with a pan-caspase inhibitor induced latency reversal in co-cultures with HIV-1 latently infected cells. Synergy in HIV-1 reactivation was observed with LRAs prostratin and JQ1. The supernatants of the pan-caspase inhibitor-treated NK cells activated the HIV-1 LTR promoter, indicating that a secreted factor by NK cells was responsible for the HIV-1 reactivation. Assessing changes in the secreted cytokine profile of pan-caspase inhibitor-treated NK cells revealed increased levels of the HIV-1 suppressor chemokines MIP1α (CCL3), MIP1ß (CCL4) and RANTES (CCL5). However, these cytokines individually or together did not induce LTR promoter activation, suggesting that CCL3-5 were not responsible for the observed HIV-1 reactivation. The cytokine profile did indicate that pan-caspase inhibitors induce NK cell activation. Altogether, our approach might be-in combination with other shock-and-kill strategies or LRAs-a strategy for reducing viral latency reservoirs and a step forward towards eradication of functionally active HIV-1 in infected individuals.

Cytotoxic Lymphocytes Target HIV-1 Gag Through Granzyme M-Mediated Cleavage.

Untreated HIV-1 infection leads to a slow decrease in CD4+ T cell lymphocytes over time resulting in increased susceptibility to opportunistic infections (acquired immunodeficiency syndrome, AIDS) and ultimately death of the infected individual. Initially, the host's immune response controls the infection, but cannot eliminate the HIV-1 from the host. Cytotoxic lymphocytes are the key effector cells in this response and can mediate crucial antiviral responses through the release of a set of proteases called granzymes towards HIV-1-infected cells. However, little is known about the immunological molecular mechanisms by which granzymes could control HIV-1. Since we noted that HIV-1 subtype C (HIV-1C) Gag with the tetrapeptide insertion PYKE contains a putative granzyme M (GrM) cleavage site (KEPL) that overlaps with the PYKE insertion, we analyzed the proteolytic activity of GrM towards Gag. Immunoblot analysis showed that GrM could cleave Gag proteins from HIV-1B and variants from HIV-1C of which the Gag-PYKE variant was cleaved with extremely high efficiency. The main cleavage site was directly after the insertion after leucine residue 483. GrM-mediated cleavage of Gag was also observed in co-cultures using cytotoxic lymphocytes as effector cells and this cleavage could be inhibited by a GrM inhibitor peptide. Altogether, our data indicate towards a noncytotoxic immunological mechanism by which GrM-positive cytotoxic lymphocytes target the HIV-1 Gag protein within infected cells to potentially control HIV-1 infection. This mechanism could be exploited in new therapeutic strategies to treat HIV-1-infected patients to improve immunological control of the infection.

Utility of Proteomics in Emerging and Re-Emerging Infectious Diseases Caused by RNA Viruses.

Emerging and re-emerging infectious diseases due to RNA viruses cause major negative consequences for the quality of life, public health, and overall economic development. Most of the RNA viruses causing illnesses in humans are of zoonotic origin. Zoonotic viruses can directly be transferred from animals to humans through adaptation, followed by human-to-human transmission, such as in human immunodeficiency virus (HIV), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and, more recently, SARS coronavirus 2 (SARS-CoV-2), or they can be transferred through insects or vectors, as in the case of Crimean-Congo hemorrhagic fever virus (CCHFV), Zika virus (ZIKV), and dengue virus (DENV). At the present, there are no vaccines or antiviral compounds against most of these viruses. Because proteins possess a vast array of functions in all known biological systems, proteomics-based strategies can provide important insights into the investigation of disease pathogenesis and the identification of promising antiviral drug targets during an epidemic or pandemic. Mass spectrometry technology has provided the capacity required for the precise identification and the sensitive and high-throughput analysis of proteins on a large scale and has contributed greatly to unravelling key protein-protein interactions, discovering signaling networks, and understanding disease mechanisms. In this Review, we present an account of quantitative proteomics and its application in some prominent recent examples of emerging and re-emerging RNA virus diseases like HIV-1, CCHFV, ZIKV, and DENV, with more detail with respect to coronaviruses (MERS-CoV and SARS-CoV) as well as the recent SARS-CoV-2 pandemic.

HIV-1 Subtype C with PYxE Insertion Has Enhanced Binding of Gag-p6 to Host Cell Protein ALIX and Increased Replication Fitness.

Human immunodeficiency virus type 1 subtype C (HIV-1C) has a natural deletion of a YPxL motif in its Gag-p6 late domain. This domain mediates the binding of Gag to host cell protein ALIX and subsequently facilitates viral budding. In a subset of HIV-1C-infected individuals, the tetrapeptide insertion PYxE has been identified at the deleted YPxL motif site. Here, we report the consequences of PYxE insertion on the interaction with ALIX and the relevance regarding replication fitness and drug sensitivity. In our three HIV-1C cohorts, PYKE and PYQE were most prevalent among PYxE variants. Through in silico predictions and in vitro experiments, we showed that HIV-1C Gag has an increased binding to ALIX when the PYxE motif is present. To go more into the clinical relevance of the PYxE insertion, we obtained patient-derived gag-pol sequences from HIV-1CPYxEi viruses and inserted them in a reference HIV-1 sequence. Viral growth was increased, and the sensitivity to the protease inhibitor (PI) lopinavir (LPV) and nucleoside reverse transcriptase inhibitor tenofovir alafenamide (TAF) was decreased for some of the HIV-1C PYxE variants compared to that of wild-type variants. Our data suggest that PYxE insertion in Gag restores the ability of Gag to bind ALIX and correlates with enhanced viral fitness in the absence or presence of LPV and TAF. The high prevalence and increased replication fitness of the HIV-1C virus with PYxE insertion indicates the clinical importance of these viral variants.IMPORTANCE Genomic differences within HIV-1 subtypes is associated with various degrees of viral spread, disease progression, and clinical outcome. Viral budding is essential in the HIV-1 life cycle and mainly mediated through the interaction of Gag with host proteins. Two motifs within Gag-p6 mediate binding of host cell proteins and facilitate budding. HIV-1C has a natural deletion of one of these two motifs, resulting in an inability to bind to host cell protein ALIX. Previously, we have identified a tetrapeptide (PYxE) insertion at this deleted motif site in a subset of HIV-1C patients. Here, we report the incidence of PYxE insertions in three different HIV-1C cohorts, and the insertion restores the binding of Gag to ALIX. It also increases viral growth even in the presence of the antiretroviral drugs lopinavir and tenofovir alafenamide. Hence, PYxE insertion in HIV-1C might be biologically relevant for viruses and clinically significant among patients.

Transcriptomics and Targeted Proteomics Analysis to Gain Insights Into the Immune-control Mechanisms of HIV-1 Infected Elite Controllers.

A small subset of HIV-1 infected individuals, the "Elite Controllers" (EC), can control viral replication and restrain progression to immunodeficiency without antiretroviral therapy (ART). In this study, a cross-sectional transcriptomics and targeted proteomics analysis were performed in a well-defined Swedish cohort of untreated EC (n=19), treatment naïve patients with viremia (VP, n=32) and HIV-1-negative healthy controls (HC, n=23). The blood transcriptome identified 151 protein-coding genes that were differentially expressed (DE) in VP compared to EC. Genes like CXCR6 and SIGLEC1 were downregulated in EC compared to VP. A definite distinction in gene expression between males and females among all patient-groups were observed. The gene expression profile between female EC and the healthy females was similar but did differ between male EC and healthy males. At targeted proteomics analysis, 90% (29/32) of VPs clustered together while EC and HC clustered separately from VP. Among the soluble factors, 33 were distinctive to be statistically significant (False discovery rate=0.02). Cell surface receptor signaling pathway, programmed cell death, response to cytokine and cytokine-mediated signaling seem to synergistically play an essential role in HIV-1 control in EC.

Corrigendum: p120-catenin prevents multinucleation through control of MKLP1-dependent RhoA activity during cytokinesis.

[This corrects the article DOI: 10.1038/ncomms13874.].

p120-catenin prevents multinucleation through control of MKLP1-dependent RhoA activity during cytokinesis.

Spatiotemporal activation of RhoA and actomyosin contraction underpins cellular adhesion and division. Loss of cell-cell adhesion and chromosomal instability are cardinal events that drive tumour progression. Here, we show that p120-catenin (p120) not only controls cell-cell adhesion, but also acts as a critical regulator of cytokinesis. We find that p120 regulates actomyosin contractility through concomitant binding to RhoA and the centralspindlin component MKLP1, independent of cadherin association. In anaphase, p120 is enriched at the cleavage furrow where it binds MKLP1 to spatially control RhoA GTPase cycling. Binding of p120 to MKLP1 during cytokinesis depends on the N-terminal coiled-coil domain of p120 isoform 1A. Importantly, clinical data show that loss of p120 expression is a common event in breast cancer that strongly correlates with multinucleation and adverse patient survival. In summary, our study identifies p120 loss as a driver event of chromosomal instability in cancer.

DNA promoter hypermethylation in nipple fluid: a potential tool for early breast cancer detection.

INTRODUCTION: Nipple fluid aspiration provides direct non-invasive sampling of fluid from the mammary ductal system, where the majority of breast cancers originate. DNA promoter hypermethylation ("methylation") occurs early and at high frequency in breast carcinogenesis, bearing the potential as a biomarker for cancer detection at its earliest stages. We assessed methylation in nipple fluid from breasts of healthy women, of women with sporadic breast cancer and of their contralateral breasts. Our goal was to investigate whether nipple fluid can be used as a reliable methylation biomarker source. MATERIALS AND METHODS: Methylation levels of 13 genes were analysed by quantitative multiplex-methylation specific PCR (QM-MSP) in nipple fluid samples from breasts of healthy women, and from the affected and contralateral breasts of breast cancer patients. RESULTS: Methylation analysis of the low-volume nipple fluid samples was feasible. Despite the generally low methylation levels, cancerous and healthy breasts nipple fluid could be discriminated with an area under the receiver operating characteristic curve (AUC) of 0.64 (p<0.01) based on a multivariate model including AKR1B1, ALX1, RASSF1A and TM6SF1. Within-patient differences between cancerous and contralateral nipple fluid samples were less prominent. CONCLUSIONS: Cancerous nipple fluid contains increased levels of methylation of tumor suppressor genes that potentially could serve as a biomarker for early breast cancer detection.

Elevated granzyme M-expressing lymphocytes during cytomegalovirus latency and reactivation after allogeneic stem cell transplantation.

Human cytomegalovirus (HCMV) reactivation can cause serious complications in allogeneic stem cell transplantation (SCT) patients. HCMV is controlled by cytotoxic lymphocytes that release antiviral granzymes. Recently, we have demonstrated that granzyme M (GrM) inhibits HCMV replication in vitro, however the physiological role of GrM and its cellular distribution during HCMV infection remains unknown. Here, we examined GrM expression in lymphocyte populations during HCMV infection. The percentage of GrM-expressing effector-memory CD4(+) T-cells was higher in HCMV latently-infected healthy individuals compared to that of uninfected individuals. SCT recipients had higher percentages of GrM-expressing CD4(+) T, CD8(+) T, γδT, and NKT cells. Despite lower total T-cell numbers, HCMV reactivation in SCT patients specifically associated with higher percentages of GrM-expressing CD4(+) (total and central-memory) T-cells. GrM was elevated in plasma during HCMV reactivation, pointing to extracellular perforin-independent functions of GrM. We conclude that GrM may be important in regulating HCMV latency and reactivation in SCT patients.

All human granzymes target hnRNP K that is essential for tumor cell viability.

Granule exocytosis by cytotoxic lymphocytes is the key mechanism to eliminate virus-infected cells and tumor cells. These lytic granules contain the pore-forming protein perforin and a set of five serine proteases called granzymes. All human granzymes display distinct substrate specificities and induce cell death by cleaving critical intracellular death substrates. In the present study, we show that all human granzymes directly cleaved the DNA/RNA-binding protein heterogeneous nuclear ribonucleoprotein K (hnRNP K), designating hnRNP K as the first known pan-granzyme substrate. Cleavage of hnRNP K was more efficient in the presence of RNA and occurred in two apparent proteolysis-sensitive amino acid regions, thereby dissecting the functional DNA/RNA-binding hnRNP K domains. HnRNP K was cleaved under physiological conditions when purified granzymes were delivered into living tumor cells and during lymphokine-activated killer cell-mediated attack. HnRNP K is essential for tumor cell viability, since knockdown of hnRNP K resulted in spontaneous tumor cell apoptosis with caspase activation and reactive oxygen species production. This apoptosis was more pronounced at low tumor cell density where hnRNP K knockdown also triggered a caspase-independent apoptotic pathway. This suggests that hnRNP K promotes tumor cell survival in the absence of cell-cell contact. Silencing of hnRNP K protein expression rendered tumor cells more susceptible to cellular cytotoxicity. We conclude that hnRNP K is indispensable for tumor cell viability and our data suggest that targeting of hnRNP K by granzymes contributes to or reinforces the cell death mechanisms by which cytotoxic lymphocytes eliminate tumor cells.

Cell death-independent functions of granzymes: hit viruses where it hurts.

Granule exocytosis by cytotoxic lymphocytes is the key mechanism of our immune response to eliminate virus-infected cells. These lytic granules contain the pore-forming protein perforin and a set of five serine proteases called granzymes (GrA, GrB, GrH, GrK, GrM) that display distinct substrate specificities. Granzymes have mostly been studied for their ability to induce cell death. However, viruses have evolved many inhibitors to effectively block apoptosis. Evidence is emerging that granzymes also use noncytotoxic strategies to inhibit viral replication and potential viral reactivation from latency. Granzymes directly cleave viral or host cell proteins that are required in the viral life cycle. Furthermore, granzymes induce a pro-inflammatory cytokine response to create an antiviral environment. In this review, we summarize and discuss these novel strategies by which the immune system counteracts viral infections, and we will address the potential therapeutic applications that could emerge from this intriguing mechanism.

Noncytotoxic inhibition of cytomegalovirus replication through NK cell protease granzyme M-mediated cleavage of viral phosphoprotein 71.

Granzyme M (GrM) is highly expressed in cytotoxic granules of NK cells, which provide the first line of defense against viral pathogens. GrM knockout mice show increased susceptibility toward murine CMV infection. Although GrM is a potent inducer of cell death, the mechanism by which GrM eliminates viruses remains elusive. In this paper, we show that purified human GrM in combination with the perforin-analog streptolysin O (SLO) strongly inhibited human CMV (HCMV) replication in fibroblasts in the absence of host cell death. In a proteomic approach, GrM was highly specific toward the HCMV proteome and most efficiently cleaved phosphoprotein 71 (pp71), an HCMV tegument protein that is critical for viral replication. Cleavage of pp71 occurred when viral lysates were incubated with purified GrM, when intact cells expressing recombinant pp71 were challenged with living cytotoxic effector cells, and when HCMV-infected fibroblasts were incubated with SLO and purified GrM. GrM directly cleaved pp71 after Leu(439), which coincided with aberrant cellular localization of both pp71 cleavage fragments as determined by confocal immunofluorescence. In a luciferase reporter assay, cleavage of pp71 after Leu(439) by GrM completely abolished the ability of pp71 to transactivate the HCMV major immediate-early promoter, which is indispensable for effective HCMV replication. Finally, GrM decreased immediate-early 1 protein expression in HCMV-infected fibroblasts. These results indicate that the NK cell protease GrM mediates cell death-independent antiviral activity by direct cleavage of a viral substrate.

Proteomic profiling of proteases: tools for granzyme degradomics.

Proteases are a family of proteolytically active enzymes whose dysfunction is implicated in a wide variety of human diseases. Although an estimated 2% of the human genome encodes for proteases, only a small fraction of these enzymes have well-characterized functions. Identification of the specificity and natural substrates of proteases in complex biological samples is challenging, but proteomic screens for proteases are currently experiencing impressive progress. Such proteomic screens include peptide-based libraries, fluorescent 2D difference gel electrophoresis with mass spectrometry, differential isotope labeling in combination with mass spectrometry, quantitative degradomics analysis of proteolytically generated neo-N-termini, and activity-based protein profiling. In the present article, we summarize and discuss the current status of proteomic techniques to identify protease specificity, cleavage sites and natural substrates with a particular focus on the cytotoxic lymphocyte granule serine proteases granzymes.

Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor.

Activation of the complement system generates potent chemoattractants and leads to the opsonization of cells for immune clearance. Short-lived protease complexes cleave complement component C3 into anaphylatoxin C3a and opsonin C3b. Here we report the crystal structure of the C3 convertase formed by C3b and the protease fragment Bb, which was stabilized by the bacterial immune-evasion protein SCIN. The data suggest that the proteolytic specificity and activity depend on the formation of dimers of C3 with C3b of the convertase. SCIN blocked the formation of a productive enzyme-substrate complex. Irreversible dissociation of the complex of C3b and Bb is crucial to complement regulation and was determined by slow binding kinetics of the Mg(2+)-adhesion site in Bb. Understanding the mechanistic basis of the central complement-activation step and microbial immune evasion strategies targeting this step will aid in the development of complement therapeutics.