Lubbertus C. F. Mulder

DENV Inhibits Type I IFN Production in Infected Cells by Cleaving Human STING

Dengue virus (DENV) is a pathogen with a high impact on human health. It replicates in a wide range of cells involved in the immune response. To efficiently infect humans, DENV must evade or inhibit fundamental elements of the innate immune system, namely the type I interferon response. DENV circumvents the host immune response by expressing proteins that antagonize the cellular innate immunity. We have recently documented the inhibition of type I IFN production by the proteolytic activity of DENV NS2B3 protease complex in human monocyte derived dendritic cells (MDDCs). In the present report we identify the human adaptor molecule STING as a target of the NS2B3 protease complex. We characterize the mechanism of inhibition of type I IFN production in primary human MDDCs by this viral factor. Using different human and mouse primary cells lacking STING, we show enhanced DENV replication. Conversely, mutated versions of STING that cannot be cleaved by the DENV NS2B3 protease induced higher levels of type I IFN after infection with DENV. Additionally, we show that DENV NS2B3 is not able to degrade the mouse version of STING, a phenomenon that severely restricts the replication of DENV in mouse cells, suggesting that STING plays a key role in the inhibition of DENV infection and spread in mice.

A Family of Mammalian E3 Ubiquitin Ligases That Contain the UBR Box Motif and Recognize N-Degrons

ABSTRACT A subset of proteins targeted by the N-end rule pathway bear degradation signals called N-degrons, whose determinants include destabilizing N-terminal residues. Our previous work identified mouse UBR1 and UBR2 as E3 ubiquitin ligases that recognize N-degrons. Such E3s are called N-recognins. We report here that while double-mutant UBR1−/− UBR2 −/− mice die as early embryos, the rescued UBR1 −/− UBR2 −/− fibroblasts still retain the N-end rule pathway, albeit of lower activity than that of wild-type fibroblasts. An affinity assay for proteins that bind to destabilizing N-terminal residues has identified, in addition to UBR1 and UBR2, a huge (570 kDa) mouse protein, termed UBR4, and also the 300-kDa UBR5, a previously characterized mammalian E3 known as EDD/hHYD. UBR1, UBR2, UBR4, and UBR5 shared a ∼70-amino-acid zinc finger-like domain termed the UBR box. The mammalian genome encodes at least seven UBR box-containing proteins, which we propose to call UBR1 to UBR7. UBR1 −/− UBR2 −/− fibroblasts that have been made deficient in UBR4 as well (through RNA interference) were significantly impaired in the degradation of N-end rule substrates such as the Sindbis virus RNA polymerase nsP4 (bearing N-terminal Tyr) and the human immunodeficiency virus type 1 integrase (bearing N-terminal Phe). Our results establish the UBR box family as a unique class of E3 proteins that recognize N-degrons or structurally related determinants for ubiquitin-dependent proteolysis and perhaps other processes as well.

SAMHD1-Deficient CD14+ Cells from Individuals with Aicardi-Goutieres Syndrome Are Highly Susceptible to HIV-1 Infection

Myeloid blood cells are largely resistant to infection with human immunodeficiency virus type 1 (HIV-1). Recently, it was reported that Vpx from HIV-2/SIVsm facilitates infection of these cells by counteracting the host restriction factor SAMHD1. Here, we independently confirmed that Vpx interacts with SAMHD1 and targets it for ubiquitin-mediated degradation. We found that Vpx-mediated SAMHD1 degradation rendered primary monocytes highly susceptible to HIV-1 infection; Vpx with a T17A mutation, defective for SAMHD1 binding and degradation, did not show this activity. Several single nucleotide polymorphisms in the SAMHD1 gene have been associated with Aicardi-Goutières syndrome (AGS), a very rare and severe autoimmune disease. Primary peripheral blood mononuclear cells (PBMC) from AGS patients homozygous for a nonsense mutation in SAMHD1 (R164X) lacked endogenous SAMHD1 expression and support HIV-1 replication in the absence of exogenous activation. Our results indicate that within PBMC from AGS patients, CD14+ cells were the subpopulation susceptible to HIV-1 infection, whereas cells from healthy donors did not support infection. The monocytic lineage of the infected SAMHD1 -/- cells, in conjunction with mostly undetectable levels of cytokines, chemokines and type I interferon measured prior to infection, indicate that aberrant cellular activation is not the cause for the observed phenotype. Taken together, we propose that SAMHD1 protects primary CD14+ monocytes from HIV-1 infection confirming SAMHD1 as a potent lentiviral restriction factor.

Meta- and Orthogonal Integration of Influenza “OMICs” Data Defines a Role for UBR4 in Virus Budding

Several systems-level datasets designed to dissect host-pathogen interactions during influenza A infection have been reported. However, apparent discordance among these data has hampered their full utility toward advancing mechanistic and therapeutic knowledge. To collectively reconcile these datasets, we performed a meta-analysis of data from eight published RNAi screens and integrated these data with three protein interaction datasets, including one generated within the context of this study. Further integration of these data with global virus-host interaction analyses revealed a functionally validated biochemical landscape of the influenza-host interface, which can be queried through a simplified and customizable web portal (http://www.metascape.org/IAV). Follow-up studies revealed that the putative ubiquitin ligase UBR4 associates with the viral M2 protein and promotes apical transport of viral proteins. Taken together, the integrative analysis of influenza OMICs datasets illuminates a viral-host network of high-confidence human proteins that are essential for influenza A virus replication.

Cytidine deamination induced HIV-1 drug resistance

The HIV-1 Vif protein is essential for overcoming the antiviral activity of DNA-editing apolipoprotein B mRNA editing enzyme, catalytic polypeptide 3 (APOBEC3) cytidine deaminases. We show that naturally occurring HIV-1 Vif point mutants with suboptimal anti-APOBEC3G activity induce the appearance of proviruses with lamivudine (3TC) drug resistance-associated mutations before any drug exposure. These mutations, ensuing from cytidine deamination events, were detected in >40% of proviruses with partially defective Vif mutants. Transfer of drug resistance from hypermutated proviruses via recombination allowed for 3TC escape under culture conditions prohibitive for any WT viral growth. These results demonstrate that defective hypermutated genomes can shape the phenotype of the circulating viral population. Partially active Vif alleles resulting in incomplete neutralization of cytoplasmic APOBEC3 molecules are directly responsible for the generation of a highly diverse, yet G-to-A biased, proviral reservoir, which can be exploited by HIV-1 to generate viable and drug-resistant progenies.

Polymorphisms and Splice Variants Influence the Antiretroviral Activity of Human APOBEC3H

ABSTRACT Human APOBEC3H belongs to the APOBEC3 family of cytidine deaminases that potently inhibit exogenous and endogenous retroviruses. The impact of single nucleotide polymorphisms (SNP) and alternative splicing on the antiretroviral activity of human APOBEC3H is currently unknown. In this study, we show that APOBEC3H transcripts derived from human peripheral blood mononuclear cells are polymorphic in sequence and subject to alternative splicing. We found that APOBEC3H variants encoding a SNP cluster (G105R, K121D and E178D, hapII-RDD) restricted human immunodeficiency virus type 1 (HIV-1) more efficiently than wild-type APOBEC3H (hapI-GKE). All APOBEC3H variants tested were resistant to HIV-1 Vif, the viral protein that efficiently counteracts APOBEC3G/3F activity. Alternative splicing of APOBEC3H was common and resulted in variants with distinct C-terminal regions and variable antiretroviral activities. Splice variants of hapI-GKE displayed a wide range of antiviral activities, whereas similar splicing events in hapII-RDD resulted in proteins that uniformly and efficiently restricted viral infectivity (>20-fold). Site-directed mutagenesis identified G105R in hapI-GKE and D121K in hapII-RDD as critical substitutions leading to an average additional 10-fold increase in antiviral activity. APOBEC3H variants were catalytically active and, similarly to APOBEC3F, favored a GA dinucleotide context. HIV-1 mutagenesis as a mode of action for APOBEC3H is suggested by the decrease of restriction observed with a cytidine deaminase domain mutant and the inverse correlation between G-to-A mutations and infectivity. Thus, the anti-HIV activity of APOBEC3H seems to be regulated by a combination of genomic variation and alternative splicing. Since prevalence of hapII-RDD is high in populations of African descent, these findings raise the possibility that some individuals may harbor effective as well as HIV-1 Vif-resistant intracellular antiviral defense mechanisms.

Degradation of HIV-1 integrase by the N-end rule pathway.

Human immunodeficiency virus type-1 (HIV-1) integrase catalyzes the irreversible insertion of the viral genome into host chromosomal DNA. We have developed a mammalian expression system for the synthesis of authentic HIV-1 integrase in the absence of other viral proteins. Integrase, which bears a N-terminal phenylalanine, was found to be a short-lived protein in human embryo kidney 293T cells. The degradation of integrase could be suppressed by proteasome inhibitors. N-terminal phenylalanine is recognized as a degradation signal by a ubiquitin-proteasome proteolytic system known as the N-end rule pathway. The replacement of N-terminal phenylalanine with methionine, valine, or glycine, which are stabilizing residues in the N-end rule, resulted in metabolically stabilized integrase proteins (half-life of N-terminal Met-integrase was at least 3 h). Conversely, the substitution of N-terminal phenylalanine with other destabilizing residues retained the metabolic instability of integrase. These findings indicate that the HIV-1 integrase is a physiological substrate of the N-end rule. We discuss a possible functional similarity to the better understood turnover of the bacteriophage Mu transposase and functions of integrase instability to the maintenance and integrity of the host cell genome.

Molecular Basis for Ebolavirus VP35 Suppression of Human Dendritic Cell Maturation

ABSTRACT Zaire ebolavirus (EBOV) VP35 is a double-stranded RNA (dsRNA)-binding protein that inhibits RIG-I signaling and alpha/beta interferon (IFN-α/β) responses by both dsRNA-binding-dependent and -independent mechanisms. VP35 also suppresses dendritic cell (DC) maturation. Here, we define the pathways and mechanisms through which VP35 impairs DC maturation. Wild-type VP35 (VP35-WT) and two well-characterized VP35 mutants (F239A and R322A) that independently ablate dsRNA binding and RIG-I inhibition were delivered to primary human monocyte-derived DCs (MDDCs) using a lentivirus-based expression system. VP35-WT suppressed not only IFN-α/β but also proinflammatory responses following stimulation of MDDCs with activators of RIG-I-like receptor (RLR) signaling, including RIG-I activators such as Sendai virus (SeV) or 5′-triphosphate RNA, or MDA5 activators such as encephalomyocarditis virus (EMCV) or poly(I·C). The F239A and R322A mutants exhibited greatly reduced suppression of IFN-α/β and proinflammatory cytokine production following treatment of DCs with RLR agonists. VP35-WT also blocked the upregulation of DC maturation markers and the stimulation of allogeneic T cell responses upon SeV infection, whereas the mutants did not. In contrast to the RLR activators, VP35-WT and the VP35 mutants impaired IFN-β production induced by Toll-like receptor 3 (TLR3) or TLR4 agonists but failed to inhibit proinflammatory cytokine production induced by TLR2, TLR3, or TLR4 agonists. Furthermore, VP35 did not prevent lipopolysaccharide (LPS)-induced upregulation of surface markers of MDDC maturation and did not prevent LPS-triggered allogeneic T cell stimulation. Therefore, VP35 is a general antagonist of DC responses to RLR activation. However, TLR agonists can circumvent many of the inhibitory effects of VP35. Therefore, it may be possible to counteract EBOV immune evasion by using treatments that bypass the VP35-imposed block to DC maturation. IMPORTANCE The VP35 protein, which is an inhibitor of RIG-I signaling and alpha/beta interferon (IFN-α/β) responses, has been implicated as an EBOV-encoded factor that contributes to suppression of dendritic cell (DC) function. We used wild-type VP35 and previously characterized VP35 mutants to clarify VP35-DC interactions. Our data demonstrate that VP35 is a general inhibitor of RIG-I-like receptor (RLR) signaling that blocks not only RIG-I- but also MDA5-mediated induction of IFN-α/β responses. Furthermore, in DCs, VP35 also impairs the RLR-mediated induction of proinflammatory cytokine production, upregulation of costimulatory markers, and activation of T cells. These inhibitory activities require VP35 dsRNA-binding activity, an activity previously correlated to VP35 RIG-I inhibitory function. In contrast, while VP35 can inhibit IFN-α/β production induced by TLR3 or TLR4 agonists, this occurs in a dsRNA-independent fashion, and VP35 does not inhibit TLR-mediated expression of proinflammatory cytokines. These data suggest strategies to overcome VP35 inhibition of DC function.

Moderate Influence of Human APOBEC3F on HIV-1 Replication in Primary Lymphocytes

ABSTRACT Multiple APOBEC3 proteins are expressed in HIV-1 target cells, but their individual contributions to viral suppression when expressed at endogenous levels remain largely unknown. We used an HIV NL4-3 mutant that selectively counteracts APOBEC3G (A3G) but not APOBEC3F (A3F) to dissect the relative contribution of A3F to the inhibition of HIV-1 replication in primary human lymphocytes (peripheral blood mononuclear cells [PBMCs]). This HIV Vif mutant replicated similarly to wild-type virus in PBMCs, suggesting that the effect of A3F on HIV restriction in these cells is limited. The different A3F variants found in PMBC donors displayed either comparable activity or less activity than wild-type A3F. Lastly, the endogenous A3F mRNA and protein expression levels in PBMCs were considerably lower than those of A3G. Our results suggest that A3F neutralization is dispensable for HIV-1 replication in primary human T-lymphocytes.

Dengue virus NS2B protein targets cGAS for degradation and prevents mitochondrial DNA sensing during infection

During the last few decades, the global incidence of dengue virus (DENV) has increased dramatically, and it is now endemic in more than 100 countries. To establish a productive infection in humans, DENV uses different strategies to inhibit or avoid the host innate immune system. Several DENV proteins have been shown to strategically target crucial components of the type I interferon system. Here, we report that the DENV NS2B protease cofactor targets the DNA sensor cyclic GMP-AMP synthase (cGAS) for lysosomal degradation to avoid the detection of mitochondrial DNA during infection. Such degradation subsequently results in the inhibition of type I interferon production in the infected cell. Our data demonstrate a mechanism by which cGAS senses cellular damage upon DENV infection.