Human MAIT cells respond to and suppress HIV-1

Human MAIT cells sit at the interface between innate and adaptive immunity, are polyfunctional and are capable of killing pathogen infected cells via recognition of the Class IB molecule MR1. MAIT cells have recently been shown to possess an antiviral protective role in vivo and we therefore sought to explore this in relation to HIV-1 infection. There was marked activation of MAIT cells in vivo in HIV-1 infected individuals, which decreased following ART. Stimulation of THP1 monocytes with R5 tropic HIVBAL potently activated MAIT cells in vitro. This activation was dependent on IL-12 and IL-18 but was independent of the TCR. Upon activation, MAIT cells were able to up-regulate granzyme B, IFNg and HIV-1 restriction factors CCL3, 4 and 5. Restriction factors produced by MAIT cells inhibited HIV-1 infection of primary PBMCs and immortalized target cells in vitro. These data reveal MAIT cells to be an additional T cell population responding to HIV-1, with a potentially important role in controlling viral replication at mucosal sites.

A Conserved uORF Regulates APOBEC3G Translation and Is Targeted by HIV-1 Vif Protein to Repress the Antiviral Factor

The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular restriction factors APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutation during reverse transcription. Vif counteracts A3G at several levels (transcription, translation, and protein degradation) that altogether reduce the levels of A3G in cells and prevent its incorporation into viral particles. How Vif affects A3G translation remains unclear. Here, we uncovered the importance of a short conserved uORF (upstream ORF) located within two critical stem-loop structures of the 5′ untranslated region (5′-UTR) of A3G mRNA for this process. A3G translation occurs through a combination of leaky scanning and translation re-initiation and the presence of an intact uORF decreases the extent of global A3G translation under normal conditions. Interestingly, the uORF is also absolutely required for Vif-mediated translation inhibition and redirection of A3G mRNA into stress granules. Overall, we discovered that A3G translation is regulated by a small uORF conserved in the human population and that Vif uses this specific feature to repress its translation.

HPV16 Entry into Epithelial Cells: Running a Gauntlet

During initial infection, human papillomaviruses (HPV) take an unusual trafficking pathway through their host cell. It begins with a long period on the cell surface, during which the capsid is primed and a virus entry platform is formed. A specific type of clathrin-independent endocytosis and subsequent retrograde trafficking to the trans-Golgi network follow this. Cellular reorganization processes, which take place during mitosis, enable further virus transport and the establishment of infection while evading intrinsic cellular immune defenses. First, the fragmentation of the Golgi allows the release of membrane-encased virions, which are partially protected from cytoplasmic restriction factors. Second, the nuclear envelope breakdown opens the gate for these virus–vesicles to the cell nucleus. Third, the dis- and re-assembly of the PML nuclear bodies leads to the formation of modified virus-associated PML subnuclear structures, enabling viral transcription and replication. While remnants of the major capsid protein L1 and the viral DNA remain in a transport vesicle, the viral capsid protein L2 plays a crucial role during virus entry, as it adopts a membrane-spanning conformation for interaction with various cellular proteins to establish a successful infection. In this review, we follow the oncogenic HPV type 16 during its long journey into the nucleus, and contrast pro- and antiviral processes.

Amphotericin b effect on the sensitivity to influenza infection of WI-38 VA-13 cells with IFITM3 gene knockout

BACKGROUND: The application of CRISPR/Cas9 is one of the most rapidly developing areas in biotechnology. This method was used to obtain clones of а human origin cell line with knockout of one or more genes of the IFITM family, representing host restriction factors for influenza infection. Amphotericin B has previously been shown to promote influenza infection by blocking IFITM3 function. AIM: The aim of this study was to evaluate the effect of amphotericin B on the sensitivity of IFITM knockout cells to influenza A virus infection. MATERIALS AND METHODS: WI-38 VA-13 cells and mutant clones with IFITM3 knockout (F3 clone) or IFITM1, IFITM3 knockout (clone E12) were infected with influenza virus A/PR/8/34 (H1N1) in the presence or absence of amphotericin B. Forty-four hours after infection, the culture medium was taken to determine the infectious activity of the virus by titration in the MDCK cell culture, as well as the hemagglutinating activity of the virus. The infected cells were stained with fluorescently labeled antibodies against the viral NP protein, and the number of NP-positive cells was determined by flow cytometry. RESULTS: The addition of amphotericin B increased the hemagglutinating and infectious activity of the virus in WI-38 VA-13cells, while the difference was insignificant for clones with IFITM gene knockout. A similar dependency was obtained for the percent of infected cells. CONCLUSIONS: Mutant cells with a knockout of one or several genes of the IFITM family were equally susceptible to influenza infection regardless of the addition of amphotericin B, which confirms the crucial importance of a defect in the IFITM3 protein in increasing the permissiveness of cells to influenza A virus.

Behavioral Analysis in International Business Negotiations Based on the Bargaining Model of Game Theory

In the process of global digital trade rules negotiation, game and cooperation coexist, and all parties are actively seeking cooperative relationship while negotiating game. Against this background, this paper provides a comprehensive overview of the negotiation process of global digital trade rules and analyzes the core issues of the negotiations and their implied political game. This paper firstly compares the core issues of global digital trade rules negotiations at multilateral and regional levels and quantitatively reflects the weighting relationship between core issues and keywords by means of a dynamic thematic model. In order to judge the potential partnership of each country, this paper constructs a theoretical model of partner selection for global digital trade rules negotiation from the perspective of political game and uses the data of 62 major digital trade countries for empirical testing. Compared with traditional trade agreements, the consensus reached in digital trade negotiations is less influenced by traditional economic geography factors and more influenced by the level of digital trade restrictions and political distance. When signing digital trade terms, a country tends to choose countries with similar levels of digital trade restrictions and high political similarity as partners. Financial restrictions and trade restrictions are the most important digital trade restriction factors that countries pay attention to when negotiating, and political stability is the most important political factor that countries pay attention to when negotiating. Compared with developed countries, developing countries pay more attention to market size, cultural differences, and digital infrastructure when negotiating. The formulation of global digital trade rules may follow a reconstructive path from bilateral to regional to multilateral, promoting regional digital trade rule systems at this stage and gradually expanding the scope of negotiations to the multilateral level.

Host Restriction Factors Modulating HIV Latency and Replication in Myeloid Cells

In addition to CD4+ T lymphocytes, myeloid cells, and, particularly, differentiated macrophages, are targets of the human immunodeficiency virus type-1 (HIV-1) infection via interaction of gp120Env with CD4 and CCR5 or CXCR4. Both T cells and macrophages support virus replication although with substantial differences. In contrast to activated CD4+ T lymphocytes, HIV-1 replication in macrophages occurs in nondividing cells and it is characterized by virtual absence of cytopathicity both in vitro and in vivo. These general features should be considered in evaluating the role of cell-associated restriction factors aiming at preventing of curtailing virus replication in macrophages and T cells particularly in the context of designing strategies to tackle the viral reservoir in infected individuals receiving combination antiretroviral therapy. In this regard, we will here also discuss a model of reversible HIV-1 latency in primary human macrophages and the role of host factor determining restriction or reactivation of virus replication in myeloid cells.

Methamphetamine facilitates HIV infection of primary human monocytes through inhibiting cellular viral restriction factors

Background Methamphetamine (METH), a potent addictive psychostimulant, is highly prevalent in HIV-infected individuals. Clinically, METH use is implicated in alteration of immune system and increase of HIV spread/replication. Therefore, it is of importance to examine whether METH has direct effect on HIV infection of monocytes, the major target and reservoir cells for the virus. Results METH-treated monocytes were more susceptible to HIV infection as evidenced by increased levels of viral proteins (p24 and Pr55Gag) and expression of viral GAG gene. In addition, using HIV Bal with luciferase reporter gene (HIV Bal-eLuc), we showed that METH-treated cells expressed higher luciferase activities than untreated monocytes. Mechanistically, METH inhibited the expression of IFN-λ1, IRF7, STAT1, and the antiviral IFN-stimulated genes (ISGs: OAS2, GBP5, ISG56, Viperin and ISG15). In addition, METH down-regulated the expression of the HIV restriction microRNAs (miR-28, miR-29a, miR-125b, miR-146a, miR-155, miR-223, and miR-382). Conclusions METH compromises the intracellular anti-HIV immunity and facilitates HIV replication in primary human monocytes.

An Energy-Saving Forwarding Mechanism Based on Clustering for Opportunistic Networks

In Opportunistic Networks (OppNets), mobility of and contact between nodes are explored to create communication opportunities and exchange messages and information. A basic premise for a better performance of these networks is a collaboration of the nodes during communication. However, due to energy restriction factors, nodes may eventually fail to collaborate with message exchanges. In this work, we propose a routing mechanism called eGPDMI to improve message probability of delivery while optimizing nodes’ energy consumption. Unlike other algorithms proposed in OppNets literature, eGPDMI groups nodes by energy level and nodes interests using clustering techniques. Our major assumption is that retaining messages in nodes with the highest energy levels can improve network performance, thus overcoming the problem of nodes’ disconnection due to unwillingness to cooperate due to low energy values. Through questionnaire application and factorial design experiments, we characterize the impacts of energy levels in OppNets. Further, we apply performance evaluation of the eGPDMI mechanism in terms of effectiveness using mobility from real-world scenarios. The results show that our mechanism effectively reduces the degradation of the probability of delivery when the minimum energy level used for nodes to cooperate with communication increases.

Upregulation of Antiviral Factors That Inhibit HIV-1 Infection in Sickle Cell Disease

Introduction Patients with Sickle cell disease (SCD) have lower risk for HIV-1 infection. We showed that ex vivo HIV-1 replication is blocked in SCD PBMCs in part because of the increased expression of ferroportin (FPN) and activation of SAMHD1, a host antiviral restriction factor. We hypothesized that rupture of sickling red blood cells releases sickle cell hemoglobin (HbS) that is phagocyted by macrophages leading to upregulation of innate antiviral response and inhibition of HIV-1 replication. We accessed changes in antiviral gene expression in PBMCs obtained from SCD patients compared to healthy controls. We also analyzed antiviral gene expression in macrophages treated with HbS compared to the HbA treatment. Methods The study was approved by Howard University review board (IRB) and all subjects consented to sample collection. Whole blood was collected from 9 SCA patients and 9 age and gender- matched healthy controls. PBMCs were activated with PHA (0.5 μg/ml) for 24-48 hrs followed by IL-2 (10 U/ml) for 24 hrs. Human THP-1 cells were differentiated into macrophages with PMA (25nM) for 72 hrs and treated with purified HbS or HbA (5µM). RNA strand-specific libraries were constructed using TruSeq Stranded Total RNA Gold kit (Illumina) and sequenced on an Illumina NextSeq 500 using 75 bp paired-end sequencing on two v2.5 150 cycle High-Output kits, generating 40-50 million paired-end reads per sample. The sequencing data were mapped using Dragen RNA and compared using Dragen differential expression software (Illumina). Ingenuity Pathway analysis (IPA, Qiagen) was used for pathway analysis. Results In activated SCD PBMCs compared to control PBMCs, 40432 genes were detected including 2230 differentially expressed genes (5.5%, 1.5-fold difference, 287 down and 1943 up) at 5% false discovery rate. In non-activated SCD PBMCs compared to control PBMCs, 33119 genes were detected including 5299 differentially expressed genes (16%, 923 down and 4376 up). In THP-1-differentiated macrophages treated with HbS versus HbA, 28362 genes were detected including 322 differentially expressed genes (1.1%, 187 down and 135 up). We focused our analysis on 61 genes including viral restriction factors and iron regulatory genes. In activated SCD PBMCs, four genes had highest upregulation: APOBEC3A (23-fold, p=2 x 10 -5), CH25H (11-fold, p=4 x 10 -5), heme oxygenase-1 (HMOX1, 13-fold, p=1.5 x 10 -12) and FPN (SLC40A1, 5-fold, p = 9 x 10 -8) (Fig.1). Several additional genes were upregulated with 1.5-3-fold increase and high significance including APOBEC3B, BRD4, CD40, CDKN1A (p21), GDR1, IFIT3, IFITM3 and SAMHD1. In non-activated SCD PBMCs, the most upregulated gene was PKR (EIF2AK2, 15-fold, p=1 x 10 -11) and genes with 1.5-3 fold upregulation included APOBEC3B, BST2, CPSF6, IFI16, IFITM3, ISG15, LGALS3BP, PML and RTF1 (Fig.1). Of these genes, only IFITM3 overlapped between activated and non-activated PBMCs. To test whether circulating HbS leads to the upregulation of antiviral response, we analyzed HbS-treated macrophages and found upregulation of several antiviral restriction factors (1.5-2.3 fold): IFIT3, LGALS3BP, MX2 and RTF1 (Fig.1). Unsupervised IPA showed upregulation of IRF-7 signaling pathway and down regulation of viral infection and replication (Fig.2). We validated the CH25 and HO-1 antiviral role in activated SCD PBMCs using small molecule inhibitors. We also confirmed overexpression of CH25H and HO-1 by western blot and ELISA. We observed higher levels of IRF7 in the activated SCD PBMCs confirming that it may play a role in the induction of antiviral response. Conclusion We propose that HbS released by hemolysis and uptaken by macrophages leads to the IRF-7-triggered induction of antiviral state in macrophages that will induced antiviral state in non-activated circulating PBMCs likely though the cytokines and interferons secretion known to be elevated in SCD patients. Upregulation of PKR (EIFAK2) levels in non-activated PBMCs strongly argue toward this possibility. Upon activation of PBMCs, additional factors are expressed including CH25H, HO-1, APOBEC3A and FPN that facilitated stronger and more robust anti-HIV-1 effect and block viral replication. Taken together, our study point to novel mechanism of upregulation of antiviral factors mediated by sickle cell hemoglobin that included induction of antiviral, heme- and iron- regulatory pathways. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Regulation of Viral Restriction by Post-Translational Modifications

Intrinsic immunity is orchestrated by a wide range of host cellular proteins called restriction factors. They have the capacity to interfere with viral replication, and most of them are tightly regulated by interferons (IFNs). In addition, their regulation through post-translational modifications (PTMs) constitutes a major mechanism to shape their action positively or negatively. Following viral infection, restriction factor modification can be decisive. Palmitoylation of IFITM3, SUMOylation of MxA, SAMHD1 and TRIM5α or glycosylation of BST2 are some of those PTMs required for their antiviral activity. Nonetheless, for their benefit and by manipulating the PTMs machinery, viruses have evolved sophisticated mechanisms to counteract restriction factors. Indeed, many viral proteins evade restriction activity by inducing their ubiquitination and subsequent degradation. Studies on PTMs and their substrates are essential for the understanding of the antiviral defense mechanisms and provide a global vision of all possible regulations of the immune response at a given time and under specific infection conditions. Our aim was to provide an overview of current knowledge regarding the role of PTMs on restriction factors with an emphasis on their impact on viral replication.