Faculty Bibliography

Background: The deoxynucleoside triphosphohydrolase SAMHD1 restricts retroviral replication in myeloid cells. It is thought to work by depleting the pool of intracellular deoxynucleotide triphosphates but has also been reported to have exonuclease activity that could allow it to degrade the viral genomic RNA or viral reverse transcribed DNA. HIV-2 and SIVmac, but not HIV-1, encode Vpx, a virion-packaged accessory protein that counteracts SAMHD1 by inducinits degradation in the nucleus of a newly infected cell. To induce the degradation of SAMHD1, Vpx co-opts the cullin4a-based E3 ubiquitin ligase, CRL4. E3 ubiquitin ligase complexes are regulated by neddylation, the covalent attachment of the small ubiquitin-like protein, Nedd8, to the cullin subunit. The small molecule MLN4924 prevents cullin neddylation. In this study MLN4924 was used to study the mechanism by which SAMHD1 restricts HIV-1. Methodology: Monocyte derived dendritic cells (MDDC) were incubated with MLN4924 prior to infection with the Vpx-containing HIV-1.GFP reporter virusThe degradation of SAMHD1 was then determined by immunoblot analysis and infectivity was analyzed by flow cytometry. The effect of the neddylation inhibitor was also tested on a cell line that expressed a GFP-SAMHD1 fusion protein that contained only the Vpx-interacting domain of SAMHD1. The cell line allowed measurement of the kinetics and cellular requirements for Vpx-mediated degradation of SAMHD1. Results: MLN4924 inhibited the neddylation of CRL4, blocking the Vpx-induced degradation of SAMHD1. Incubation of cells expressing the GFP-SAMHD1 fusion with Vpx-containing HIV-1 resulted in a rapid reduction in GFP fluorescence and the reduction was blocked by MLN4924. The neddylation inhibitor maintained SAMHD1-mediated restriction in cells that expressed SAMHD1 when infected with Vpx-containing HIV-1. In cells that did not express SAMHD1 the drug had no effect on infectivity. Removal of the drug several hours post-infection released the block. Similarly, Vpx-con!

Abstract Dendritic cells are professional antigen-presenting cells of the immune system and are major producers of type-I interferon. Their role in HIV-1 infection is not well understood. They express CD4 and CCR5 yet appear to be resistant to infection. In culture, infection of the cells with HIV-1 is inhibited by the host cell restriction factor SAMHD1. Lentiviruses such as HIV-2/SIVmac counteract the restriction by encoding Vpx, a virion-packaged accessory protein that induces the proteasomal degradation of SAMHD1. In this study we investigated SAMHD1-mediated restriction in the two major dendritic cell subsets: plasmacytoid dendritic cells (pDC) and myeloid dendritic cells (mDC). The cells were highly resistant to HIV-1 and expressed high levels of SAMHD1. SAMHD1 amino acid residue T592, a target of CDK1 phosphorylation, was unphosphorylated, corresponding to the antiviral form of the enzyme. The resistance to infection was not counteracted by Vpx and SAMHD1 was not degraded in these cells. Treatment of pDCs with a cocktail of antibodies that blocked type-I interferon signaling partially restored the ability of Vpx to induce SAMHD1 degradation and caused the cells to become partially permissive to infection. pDCs and mDCs responded to HIV-1 virions by inducing an innate immune response but did not appear to sense newly produced Gag protein. The findings suggest that in vivo, dendritic cells serve as sentinels to alert the immune system to the virus but do not themselves become infected by virtue of high levels of SAMHD1.

The deoxynucleoside triphosphohydrolase SAMHD1 restricts retroviral replication in myeloid cells. Human immunodeficiency virus type 2 (HIV-2) and a simian immunodeficiency virus from rhesus macaques (SIVmac) encode Vpx, a virion-packaged accessory protein that counteracts SAMHD1 by inducing its degradation. SAMHD1 is thought to work by depleting the pool of intracellular deoxynucleoside triphosphates but has also been reported to have exonuclease activity that could allow it to degrade the viral genomic RNA or viral reverse-transcribed DNA. To induce the degradation of SAMHD1, Vpx co-opts the cullin4a-based E3 ubiquitin ligase, CRL4. E3 ubiquitin ligases are regulated by the covalent attachment of the ubiquitin-like protein Nedd8 to the cullin subunit. Neddylation can be prevented by MLN4924, a drug that inhibits the nedd8-activating enzyme. We report that MLN4924 inhibits the neddylation of CRL4, blocking Vpx-induced degradation of SAMHD1 and maintaining the restriction. Removal of the drug several hours postinfection released the block. Similarly, Vpx-containing virus-like particles and deoxynucleosides added to the cells more than 24 h postinfection released the SAMHD1-mediated block. Taken together, these findings support deoxynucleoside triphosphate pool depletion as the primary mechanism of SAMHD1 restriction and argue against a nucleolytic mechanism, which would not be reversible.

Vanishing bile duct syndrome (VBDS) is a group of rare disorders characterized by ductopenia, the progressive destruction and disappearance of intrahepatic bile ducts leading to cholestasis. Described in association with medications, autoimmune disorders, cancer, transplantation, and infections, the specific mechanisms of disease are not known. To date, only 4 cases of VBDS have been reported in human immunodeficiency virus (HIV) infected patients. We report 2 additional cases of HIV-associated VBDS and review the features common to the HIV-associated cases. Presentation includes hyperbilirubinemia, normal liver imaging, and negative viral and autoimmune hepatitis studies. In HIV-infected subjects, VBDS occurred at a range of CD4+ T-cell counts, in some cases following initiation or change in antiretroviral therapy. Lymphoma was associated with two cases; nevirapine, antibiotics, and viral co-infection were suggested as etiologies in the other cases. In HIV-positive patients with progressive cholestasis, early identification of VBDS and referral for transplantation may improve outcomes.

OBJECTIVE: HIV infection is associated with coagulation abnormalities and significantly increased risk of venous thrombosis. It has been shown that higher plasma levels of coagulation and inflammatory biomarkers predicted mortality in HIV. We investigated the relationship between venous thrombosis and HIV-related characteristics, traditional risk factors of hypercoagulability, and pre-event levels of biomarkers. DESIGN: A retrospective case-control study of 23 HIV-infected individuals who experienced an incident venous thromboembolic event while enrolled in National Institutes of Health studies from 1995 to 2010 and 69 age-matched and sex-matched HIV-infected individuals without known venous thromboembolism (VTE). METHODS: Biomarkers of inflammation, endothelial dysfunction, coagulation, tissue fibrosis, and cytomegalovirus (CMV) reactivation were assessed by ELISA-based assays and PCR using plasma obtained prior to the event. RESULTS: VTE events were related to nadir CD4 cell count, lifetime history of multiple opportunistic infections, CMV disease, CMV viremia, immunological AIDS, active infection, and provocation (i.e., recent hospitalization, surgery, or trauma). VTE events were independently associated with increased plasma levels of P-selectin (P = 0.002), D-dimer (P = 0.01), and hyaluronic acid (P = 0.009) in a multivariate analysis. No significant differences in antiretroviral or interleukin-2 exposures, plasma HIV viremia, or other traditional risk factors were observed. CONCLUSION: Severe immunodeficiency, active infection, and provocation are associated with venous thromboembolic disease in HIV. Biomarkers of endothelial dysfunction, coagulation, and tissue fibrosis may help identify HIV-infected patients at elevated risk of VTE.