A doubly responsive probe for the detection of Cys4-tagged proteins

International audienceRecombinant proteins bearing a tag are crucial tools for assessing protein location or function. Small tags such as Cys4 tag (tetracysteine; Cys–Cys–X–X–Cys–Cys) are less likely disrupt protein function in the living cell than green fluorescent protein. Herein we report the first example of the design and synthesis of a dual fluorescence and hyperpolarized 129Xe NMR-based sensor of Cys4-tagged proteins. This sensor becomes fluorescent when bound to such Cys4-tagged peptides, and the 129Xe NMR spectrum exhibits a specific signal, characteristic of the biosensor-peptide association

Masters of manipulation: viral modulation of the immunological synapse

In order to thrive, viruses have evolved to manipulate host cell machinery for their own benefit. One major obstacle faced by pathogens is the immunological synapse. To enable efficient replication and latency in immune cells, viruses have developed a range of strategies to manipulate cellular processes involved in immunological synapse formation to evade immune detection and control T‐cell activation. In vitro, viruses such as human immunodeficiency virus 1 and human T‐lymphotropic virus type 1 utilise structures known as virological synapses to aid transmission of viral particles from cell to cell in a process termed trans‐infection. The formation of the virological synapse provides a gateway for virus to be transferred between cells avoiding the extracellular space, preventing antibody neutralisation or recognition by complement. This review looks at how viruses are able to subvert intracellular signalling to modulate immune function to their advantage and explores the role synapse formation has in viral persistence and cell‐to‐cell transmission

Revisiting HIV-1 uncoating

HIV uncoating is defined as the loss of viral capsid that occurs within the cytoplasm of infected cells before entry of the viral genome into the nucleus. It is an obligatory step of HIV-1 early infection and accompanies the transition between reverse transcription complexes (RTCs), in which reverse transcription occurs, and pre-integration complexes (PICs), which are competent to integrate into the host genome. The study of the nature and timing of HIV-1 uncoating has been paved with difficulties, particularly as a result of the vulnerability of the capsid assembly to experimental manipulation. Nevertheless, recent studies of capsid structure, retroviral restriction and mechanisms of nuclear import, as well as the recent expansion of technical advances in genome-wide studies and cell imagery approaches, have substantially changed our understanding of HIV uncoating. Although early work suggested that uncoating occurs immediately following viral entry in the cell, thus attributing a trivial role for the capsid in infected cells, recent data suggest that uncoating occurs several hours later and that capsid has an all-important role in the cell that it infects: for transport towards the nucleus, reverse transcription and nuclear import. Knowing that uncoating occurs at a later stage suggests that the viral capsid interacts extensively with the cytoskeleton and other cytoplasmic components during its transport to the nucleus, which leads to a considerable reassessment of our efforts to identify potential therapeutic targets for HIV therapy. This review discusses our current understanding of HIV uncoating, the functional interplay between infectivity and timely uncoating, as well as exposing the appropriate methods to study uncoating and addressing the many questions that remain unanswered

The Level of DING Proteins Is Increased in HIV-Infected Patients: In Vitro and In Vivo Studies

DING proteins constitute an interesting family, owing to their intriguing and important activities. However, after a decade of research, little is known about these proteins. In humans, at least five different DING proteins have been identified, which were implicated in important biological processes and diseases, including HIV. Indeed, recent data from different research groups have highlighted the anti-HIV activity of some DING representatives. These proteins share the ability to inhibit the transcriptional step of HIV-1, a key step of the viral cycle that is not yet targeted by the current therapies. Since such proteins have been isolated from humans, we undertook a comprehensive study that focuses on the relationship between these proteins and HIV-infection in an infectious context. Hence, we developed a home-made ELISA for the quantification of the concentration of DING proteins in human serum. Using this method, we were able to determine the concentration of DING proteins in healthy and HIV-infected patients. Interestingly, we observed a significant increase of the concentration of DING proteins in non treated and treated HIV-infected patients compared to controls. In addition, cell cultures infected with HIV also show an increased expression of DING proteins, ruling out the possible role of antiretroviral treatment in the increase of the expression of DING proteins. In conclusion, results from this study show that the organism reacts to HIV-infection by an overexpression of DING proteins

Counteraction of Tetherin Antiviral Activity by Two Closely Related SIVs Differing by the Presence of a Vpu Gene

In different primate lentiviruses, three proteins (Vpu, Env and Nef) have been shown to have anti-tetherin activities. SIVden is a primate lentivirus harbored by a Cercopithecus denti (C. denti) whose genome code for a Vpu gene. We have compared the activity of HIV-1 Vpu and of SIVden Vpu on tetherin proteins from humans, from C. denti and from Cercopithecus neglectus (C. neglectus), a monkey species that is naturally infected by SIVdeb, a virus closely related to SIVden but which does not encode a Vpu protein. Here, we demonstrate that SIVden Vpu, is active against C. denti tetherin, but not against human tetherin. Interestingly, C. neglectus tetherin was more sensitive to SIVden Vpu than to HIV-1 Vpu. We also identify residues in the tetherin transmembrane domains that are responsible for the species-specific Vpu effect. Simultaneous mutation (P40L and T45I) of human tetherin conferred sensitivity to SIVden Vpu, while abolishing its sensitivity to HIV-1 Vpu. We next analyzed the anti-tetherin activity of the Nef proteins from HIV-1, SIVden and SIVdeb. All three Nef proteins were unable to rescue virus release in the presence of human or C. denti tetherin. Conversely, SIVdeb Nef enhanced virus release in the presence of C. neglectus tetherin, suggesting that SIVdeb relies on Nef in its natural host. Finally, while HIV-1 Vpu not only removed human tetherin from the cell surface but also directed it for degradation, SIVden Vpu only induced the redistribution of both C. denti and C. neglectus tetherins, resulting in a predominantly perinuclear localization

SAMHD1-Deficient CD14+ Cells from Individuals with Aicardi-Goutières 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

Labeling of Multiple HIV-1 Proteins with the Biarsenical-Tetracysteine System

Due to its small size and versatility, the biarsenical-tetracysteine system is an attractive way to label viral proteins for live cell imaging. This study describes the genetic labeling of the human immunodeficiency virus type 1 (HIV-1) structural proteins (matrix, capsid and nucleocapsid), enzymes (protease, reverse transcriptase, RNAse H and integrase) and envelope glycoprotein 120 with a tetracysteine tag in the context of a full-length virus. We measure the impact of these modifications on the natural virus infection and, most importantly, present the first infectious HIV-1 construct containing a fluorescently-labeled nucleocapsid protein. Furthermore, due to the high background levels normally associated with the labeling of tetracysteine-tagged proteins we have also optimized a metabolic labeling system that produces infectious virus containing the natural envelope glycoproteins and specifically labeled tetracysteine-tagged proteins that can easily be detected after virus infection of T-lymphocytes. This approach can be adapted to other viral systems for the visualization of the interplay between virus and host cell during infection

HIV-1 Pre-Integration Complexes Selectively Target Decondensed Chromatin in the Nuclear Periphery

Integration of the double-stranded DNA copy of the HIV-1 genome into host chromosomal DNA is a requirement for efficient viral replication. Integration preferentially occurs within active transcription units, however chromosomal site specificity does not correlate with any strong primary sequence. To investigate whether the nuclear architecture may affect viral integration we have developed an experimental system where HIV-1 viral particles can be visualized within the nuclear compartment. Fluorescently labeled HIV-1 virions were engineered by fusing integrase, the viral protein that catalyzes the integration reaction, to fluorescent proteins. Viral tests demonstrate that the infectivity of fluorescent virions, including the integration step, is not altered as compared to wild-type virus. 3-D confocal microscopy allowed a detailed analysis of the spatial and temporal distribution of the pre-integration complexes (PICs) within the nucleus at different moments following infection; the fluorescently labeled PICs preferentially distribute in decondensed areas of the chromatin with a striking positioning in the nuclear periphery, while heterochromatin regions are largely disfavored. These observations provide a first indication of how the nuclear architecture may initially orient the selection of retroviral integration sites

Alteration in Superoxide Dismutase 1 Causes Oxidative Stress and p38 MAPK Activation Following RVFV Infection

Rift Valley fever (RVF) is a zoonotic disease caused by Rift Valley fever virus (RVFV). RVFV is a category A pathogen that belongs to the genus Phlebovirus, family Bunyaviridae. Understanding early host events to an infectious exposure to RVFV will be of significant use in the development of effective therapeutics that not only control pathogen multiplication, but also contribute to cell survival. In this study, we have carried out infections of human cells with a vaccine strain (MP12) and virulent strain (ZH501) of RVFV and determined host responses to viral infection. We demonstrate that the cellular antioxidant enzyme superoxide dismutase 1 (SOD1) displays altered abundances at early time points following exposure to the virus. We show that the enzyme is down regulated in cases of both a virulent (ZH501) and a vaccine strain (MP12) exposure. Our data demonstrates that the down regulation of SOD1 is likely to be due to post transcriptional processes and may be related to up regulation of TNFα following infection. We also provide evidence for extensive oxidative stress in the MP12 infected cells. Concomitantly, there is an increase in the activation of the p38 MAPK stress response, which our earlier published study demonstrated to be an essential cell survival strategy. Our data suggests that the viral anti-apoptotic protein NSm may play a role in the regulation of the cellular p38 MAPK response. Alterations in the host protein SOD1 following RVFV infection appears to be an early event that occurs in multiple cell types. Activation of the cellular stress response p38 MAPK pathway can be observed in all cell types tested. Our data implies that maintaining oxidative homeostasis in the infected cells may play an important role in improving survival of infected cells

HIV-1 Protease and Reverse Transcriptase Control the Architecture of Their Nucleocapsid Partner

The HIV-1 nucleocapsid is formed during protease (PR)-directed viral maturation, and is transformed into pre-integration complexes following reverse transcription in the cytoplasm of the infected cell. Here, we report a detailed transmission electron microscopy analysis of the impact of HIV-1 PR and reverse transcriptase (RT) on nucleocapsid plasticity, using in vitro reconstitutions. After binding to nucleic acids, NCp15, a proteolytic intermediate of nucleocapsid protein (NC), was processed at its C-terminus by PR, yielding premature NC (NCp9) followed by mature NC (NCp7), through the consecutive removal of p6 and p1. This allowed NC co-aggregation with its single-stranded nucleic-acid substrate. Examination of these co-aggregates for the ability of RT to catalyse reverse transcription showed an effective synthesis of double-stranded DNA that, remarkably, escaped from the aggregates more efficiently with NCp7 than with NCp9. These data offer a compelling explanation for results from previous virological studies that focused on i) Gag processing leading to nucleocapsid condensation, and ii) the disappearance of NCp7 from the HIV-1 pre-integration complexes. We propose that HIV-1 PR and RT, by controlling the nucleocapsid architecture during the steps of condensation and dismantling, engage in a successive nucleoprotein-remodelling process that spatiotemporally coordinates the pre-integration steps of HIV-1. Finally we suggest that nucleoprotein remodelling mechanisms are common features developed by mobile genetic elements to ensure successful replication