24 research outputs found

    Primate Lentiviral Vpx Commandeers DDB1 to Counteract a Macrophage Restriction

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    Primate lentiviruses encode four “accessory proteins” including Vif, Vpu, Nef, and Vpr/Vpx. Vif and Vpu counteract the antiviral effects of cellular restrictions to early and late steps in the viral replication cycle. We present evidence that the Vpx proteins of HIV-2/SIVSM promote virus infection by antagonizing an antiviral restriction in macrophages. Fusion of macrophages in which Vpx was essential for virus infection, with COS cells in which Vpx was dispensable for virus infection, generated heterokaryons that supported infection by wild-type SIV but not Vpx-deleted SIV. The restriction potently antagonized infection of macrophages by HIV-1, and expression of Vpx in macrophages in trans overcame the restriction to HIV-1 and SIV infection. Vpx was ubiquitylated and both ubiquitylation and the proteasome regulated the activity of Vpx. The ability of Vpx to counteract the restriction to HIV-1 and SIV infection was dependent upon the HIV-1 Vpr interacting protein, damaged DNA binding protein 1 (DDB1), and DDB1 partially substituted for Vpx when fused to Vpr. Our results indicate that macrophage harbor a potent antiviral restriction and that primate lentiviruses have evolved Vpx to counteract this restriction

    Antiviral drug resistance of herpes simplex virus

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    Infections with herpes simplex virus (HSV) usually have an asymptomatic or benign course. However, severe infections do occur, particularly in HIV/AIDS patients or transplant recipients, and may be life-threatening unless adequate antiviral therapy is given. Since its introduction in the early 1980s, aciclovir (ACV) is the drug of choice for treatment of severe HSV infections and it has been increasingly used also for suppressive therapy and for prophylaxis in patients at risk for frequent and/or severe HSV infections. Nowadays, ACV use is even more widespread due to its 'over the counter' availability. Soon after its introduction, emergence of resistance of HSV to ACV was reported. In the past decades it has been shown that ACV-resistant HSV occur relatively frequently especially in immunocompromised patients and can be associated with serious disease. In contrast, ACV-resistant HSV isolates have been reported infrequently in immunocompetent subjects. However, the prevalence in immunocompetent population is less clear. Due to the effective control of HSV infections by host immune system the extensive spread of resistant virus in the population could occur before becoming apparent. In this thesis, antiviral drug resistance of HSV was investigated from a virological and clinical perspective. Focus was directed at the detection, prevalence and characterization of drug-resistant HSV. A real-time PCR was developed and validated for the detection of HSV-1 or HSV-2 infections. The assay was also applied as a sensitive and objective read-out system for HSV phenotypic antiviral susceptibility assay. A phenotypic assay (ELVIRA HSV) was developed for rapid screening of HSV clinical specimens for decreased susceptibility to ACV and it was successfully used in a large-scale survey of the prevalence of ACV-resistant HSV. A survey of resistance to ACV was performed on HSV strains isolated in The Netherlands between 1999 and 2002. A total of 542 isolates from 496 patients were examined; of these, 128 patients were immunocompromised. The prevalence of ACV resistance in the population at large was 0.27%, and thus did not differ from the level of naturally occurring resistance as observed in the era before introduction of ACV. In contrast, the prevalence of ACV-resistant HSV in immunocompromised patients was 7.0%. Our results indicate that 20 years of ACV usage, including over the counter sales, has not resulted in a measurable increase of HSV antiviral resistance in the Dutch population. However, the prevalence of 7.0% in immunocompromised patients indicates that antiviral resistance has become a considerable problem in this patient group. The ACV-resistant HSV strains were examined to determine the molecular changes underlying the emergence of antiviral resistance. A total of 26 HSV isolates were studied, and yielded 10 new mutations conferring resistance against ACV. This information is of crucial importance for our understanding of the emergence of antiviral resistance, for the development of rapid, sensitive genotypic methods for determination of antiviral resistance directly in patients' specimens and for the development of new antiviral drugs

    Cross Talk between Inhibitory Immunoreceptor Tyrosine-Based Activation Motif-Signaling and Toll-Like Receptor Pathways in Macrophages and Dendritic Cells

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    International audienceThe innate immune cells sense microbial infection and self-ligands by pathogen recognition receptors (PRRs), such as toll-like receptors (TLRs) and regulatory receptors (RRs), associated with immunoreceptor tyrosine-based activation motif (ITAM). Rapid activation and concerted action of PRRs signaling and feedback inhibitory mechanisms must be engaged to ensure the host defense functions and to prevent cytotoxicity associated with excessive activation. ITAM-associated RRs can generate stimulatory or, paradoxically, inhibitory signals. The network of ITAM-associated RR, together with TLR-signaling pathways, are responsible for immunogenic or tolerogenic responses of macrophages and dendritic cells to their microenvironment. In macrophages, TLR4 signaling is inhibited by low-avidity ligation of ITAM-associated receptors, while high-avidity ligation of ITAM-associated receptors results in potentiation of TLR4 signaling together with resistance to extracellular cytokine microenvironment signals. In contrast to macrophages, TLR7/9 signaling in plasmacytoid DCs (pDCs) is inhibited by high-avidity ligation of ITAM-associated RR, while low-avidity ligation does not show any effect. Surprisingly, interference of ITAM-associated receptor signaling with TLR pathways has not been reported in conventional dendritic cells. Here, we present an overview of molecular mechanisms acting at the crossroads of TLR and ITAM-signaling pathways and address the question of how the high-avidity engagement of the ITAM-associated receptors in pDCs inhibits TLR7/9 signaling. Cellular context and spatiotemporal engagement of ITAM- and TLR-signaling pathways are responsible for different outcomes of macrophage versus pDC activation. While the cross-regulation of cytokine and TLR signaling, together with antigen presentation, are the principal functions of ITAM-associated RR in macrophages, the major role of these receptors in pDCs seems to be related to inhibition of cytokine production and reestablishment of a tolerogenic state following pDC activation. Pharmacologic targeting of TLR and ITAM signaling could be an attractive new therapeutic approach for treatment of chronic infections, cancer, and autoimmune and inflammatory diseases related to pDCs

    A cellular restriction dictates the permissivity of nondividing monocytes/macrophages to lentivirus and gammaretrovirus infection

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    Primate lentiviruses, including HIV-1, transduce terminally differentiated, nondividing myeloid cells; however, these cells are refractory to infection by gammaretroviruses such as murine leukemia virus (MLV). Here, we present evidence that a cellular restriction is the obstacle to transduction of macrophages by MLV. Neutralization of the restriction by Vpx, a primate lentiviral protein previously shown to protect primate lentiviruses from a macrophage restriction, rendered macrophages permissive to MLV infection. We further demonstrate that this restriction prevents transduction of quiescent monocytes by HIV-1. Monocyte-HeLa heterokaryons were resistant to HIV-1 infection, while heterokaryons formed between monocytes and HeLa cells expressing Vpx were permissive to HIV-1 infection. Encapsidation of Vpx within HIV-1 virions conferred the ability to infect quiescent monocytes. Collectively, our results indicate that the relative ability of lentiviruses and gammaretroviruses to transduce nondividing myeloid cells is dependent upon their ability to neutralize a cellular restriction

    A Cellular Restriction Dictates the Permissivity of Nondividing Monocytes/Macrophages to Lentivirus and Gammaretrovirus Infection

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    Primate lentiviruses, including HIV-1, transduce terminally differentiated, nondividing myeloid cells; however, these cells are refractory to infection by gammaretroviruses such as murine leukemia virus (MLV). Here, we present evidence that a cellular restriction is the obstacle to transduction of macrophages by MLV. Neutralization of the restriction by Vpx, a primate lentiviral protein previously shown to protect primate lentiviruses from a macrophage restriction, rendered macrophages permissive to MLV infection. We further demonstrate that this restriction prevents transduction of quiescent monocytes by HIV-1. Monocyte-HeLa heterokaryons were resistant to HIV-1 infection, while heterokaryons formed between monocytes and HeLa cells expressing Vpx were permissive to HIV-1 infection. Encapsidation of Vpx within HIV-1 virions conferred the ability to infect quiescent monocytes. Collectively, our results indicate that the relative ability of lentiviruses and gammaretroviruses to transduce nondividing myeloid cells is dependent upon their ability to neutralize a cellular restriction.status: publishe

    Routine use of a highly automated and internally controlled real-time PCR assay for the diagnosis of herpes simplex and varicella-zoster virus infections

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    BACKGROUND: Detection of herpes viruses can be significantly improved by PCR. The development of real-time PCR, which has overcome several limitations of conventional PCR, improved the prospects for implementation of PCR-based assays in diagnostic laboratory. OBJECTIVES: To compare the diagnostic performance of an automated sample extraction procedure in combination with an internally controlled real-time PCR assay for detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) to conventional shell vial culture. STUDY DESIGN: One hundred eighty-two consecutive specimens from patients suspected of HSV or VZV infection were examined by internally controlled PCR and shell vial culture. An internal control consisting of phocine herpes virus was processed along with the specimens during the entire procedure and permitted to monitor extraction and amplification efficiency, including inhibition. RESULTS: A total of 48 (26.4%) specimens were positive for HSV or VZV by culture, and 77 (42.3%) by real-time PCR. Thus, overall sensitivity increased by 60.4%. All culture-positive specimens were detected and typed correctly by PCR, except for a single specimen that contained PCR inhibitors. Specifically, the real-time PCR assay increased the detection rate for HSV-1 and HSV-2 by 43.9% and 62.5%, respectively. In PCR-positive specimens, lower levels of viral DNA were found in culture-negative than in culture-positive specimens. The increase of HSV detection rates by PCR varied with the origin of specimen and was particularly significant for skin specimens (7/14 versus 3/14 detected by culture) and bronchoalveolar lavages (8/8 versus 1/8). In addition, real-time PCR significantly increased the detection rate for VZV. CONCLUSIONS: Compared to shell vial culture, our real-time PCR assay demonstrated a superior sensitivity and an added value of using internal control for checking the quality of examination of each specimen. These results provide a solid basis for implementation of real-time PCR in the routine diagnosis of HSV and VZV infections in various clinical specimens.status: publishe

    Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma

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    Plasma extracellular vesicles (EVs), especially exosome-like vesicles (ELVs), are being increasingly explored as a source of potential noninvasive disease biomarkers. The discovery of blood-based biomarkers associated with ELVs requires methods that isolate high yields of these EVs without significant contamination with highly abundant plasma proteins and lipoproteins. The rising interest in blood-based EV-associated biomarkers has led to the rapid development of novel EV isolation methods. However, the field suffers from a lack of standardization and often, new techniques are used without critical evaluation. Size exclusion chromatography (SEC) has become the method of choice for rapid isolation of relatively pure EVs from plasma, yet it has technical limitations for certain downstream applications. The recently released exoEasy kit (Qiagen) is a new membrane affinity spin column method for the isolation of highly pure EVs from biofluids with the potential to overcome most of the limitations of SEC.status: publishe

    Investigation of tumor-tumor interactions in a double human cervical carcinoma xenograft model in nude mice.

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    Tumor-tumor distant interactions within one organism are of major clinical relevance determining clinical outcome. To investigate this poorly understood phenomenon, a double human cervical xenograft model in nude mice was developed. A first tumor was induced subcutaneously by injection of human papillomavirus positive cervical carcinoma cells into the mouse lower right flank and 3 weeks later, animals were challenged with the same tumor cell line injected subcutaneously into the upper left flank. These tumors had no direct physical contact and we found no systemic changes induced by the primary tumor affecting the growth of a secondary tumor. However, ablation of the primary tumor by local treatment with cidofovir, a nucleotide analogue with known antiviral and antiproliferative properties, resulted not only in a local antitumor effect but also in a temporary far-reaching effect leading to retarded growth of the challenged tumor. Cidofovir far-reaching effects were linked to a reduced tumor-driven inflammation, to increased anti-tumor immune responses, and could not be enhanced by co-administration with immune stimulating adjuvants. Our findings point to the potential use of cidofovir in novel therapeutic strategies aiming to kill tumor cells as well as to influence the immune system to fight cancer.status: Published onlin

    Small-molecule inhibition of HIV-1 Vif

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    The HIV-1 protein Vif, essential for in vivo viral replication, targets the human DNA-editing enzyme, APOBEC3G (A3G), which inhibits replication of retroviruses and hepatitis B virus. As Vif has no known cellular homologs, it is an attractive, yet unrealized, target for antiviral intervention. Although zinc chelation inhibits Vif and enhances viral sensitivity to A3G, this effect is unrelated to the interaction of Vif with A3G. We identify a small molecule, RN-18, that antagonizes Vif function and inhibits HIV-1 replication only in the presence of A3G. RN-18 increases cellular A3G levels in a Vif-dependent manner and increases A3G incorporation into virions without inhibiting general proteasome-mediated protein degradation. RN-18 enhances Vif degradation only in the presence of A3G, reduces viral infectivity by increasing A3G incorporation into virions and enhances cytidine deamination of the viral genome. These results demonstrate that the HIV-1 Vif-A3G axis is a valid target for developing small molecule-based new therapies for HIV infection or for enhancing innate immunity against viruses.status: publishe
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