Activation of the complement system generates potent chemoattractants and opsonizes 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 depends on dimerization of C3 with C3b of the convertase. SCIN blocked the formation of a productive enzyme-substrate complex. Irreversible dissociation of C3bBb is crucial to complement regulation and was determined by slow binding kinetics of the Mg2+-adhesion site in Bb. Understanding the mechanistic basis of the central complement activation step and microbial immune evasion strategies targeting this step will aid the development of complement therapeutics.

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.

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 Leu439, 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 Leu439 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.

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.

Human cytomegalovirus (HCMV) is the most frequent viral cause of congenital defects and HCMV infection in immunocompromised patients may trigger devastating disease. Cytotoxic lymphocytes control HCMV by releasing granzymes towards virus-infected cells. In mice, granzyme M (GrM) has a physiological role in controlling murine CMV infection. However, the underlying mechanism remains poorly understood. In this study, we showed that human GrM was expressed by HCMV-specific CD8 þ T cells both in latently infected healthy individuals and in transplant patients during primary HCMV infection. We identified host cell heterogeneous nuclear ribonucleoprotein K (hnRNP K) as a physiological GrM substrate. GrM most efficiently cleaved hnRNP K in the presence of RNA at multiple sites, thereby likely destroying hnRNP K function. Host cell hnRNP K was essential for HCMV replication not only by promoting viability of HCMV-infected cells but predominantly by regulating viral immediate-early 2 (IE2) protein levels. Furthermore, hnRNP K interacted with IE2 mRNA. Finally, GrM decreased IE2 protein expression in HCMV-infected cells. Our data suggest that targeting of hnRNP K by GrM contributes to the mechanism by which cytotoxic lymphocytes inhibit HCMV replication. This is the first evidence that cytotoxic lymphocytes target host cell proteins to control HCMV infections.

Background: Granzymes have distinct substrate specificities and trigger diverse cell death pathways. Results: All human granzymes can target hnRNP K, which is essential for tumor cell viability. Conclusion: Granzyme-mediated cleavage of hnRNP K contributes to the elimination of tumor cells by cytotoxic lymphocytes. Significance: Elucidating granzyme function provides insights into the role of cytotoxic lymphocytes in tumor immunology.

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.

Immune checkpoints are several co-stimulatory and inhibitory pathways that regulate T cell immune responses. Most of the discoveries about immune checkpoints were made in cancer research where inhibitory immune checkpoints cause immune exhaustion and down-regulate anti-tumor responses. In addition to cancer, immune checkpoints are exploited in chronic infectious diseases. In human immunodeficiency virus (HIV) infection, the immune checkpoint molecule called programmed cell death protein 1 (PD-1) has been determined as being a major regulatory factor for T cell exhaustion. Recent studies with antibodies blocking either PD-1 ligand 1 (PD-L1) or PD-1 show not only promising results in the enhancement of HIV-specific immune responses but even in reducing the latent HIV reservoir. Apart from the therapeutic target for a functional cure of HIV-1, immune checkpoint molecules might be used as biomarkers for monitoring disease progression and therapeutic response. In this review, we will summarize and discuss the inhibitory immune checkpoint molecules PD-1, cytotoxic T-lymphocyte-associated protein 4 (CTLA4), lymphocyte-activation gene 3 (LAG3), and T cell immunoglobulin and mucin-domain-containing-3 (TIM3) as well as the co-stimulatory molecules CD40L and CD70, including their role in immunity, with a particular focus on HIV infection, and being potential targets for a functional HIV cure.