ABSTRACT
Lentiviral vectors and lentiviruses are important tools for basic and applied biomedical research. Yet, biosafety regulations from legal authorities have to be fulfilled when transferring BSL-2 to -3 vectors/viruses to facilities with lower biosafety level. Here, we (re-)evaluated different chemical and thermal approaches to inactivate vesicular stomatitis virus G-protein (VSV-G) pseudotyped lentiviral vectors and either wildtype or VSV-G pseudotyped human immunodeficiency viruses (HIV). Aldehydes, detergents and alcohols were as effective as thermal inactivation procedures to efficiently inactivate purified lentiviral vectors and replication-competent HIV. In addition, no residual infectivity was detected when inactivating HIV-infected TZM-bl reporter cells with selected detergents and aldehydes. Thus, our established inactivation protocols can be used by other laboratories working with lentiviral vectors or infectious lentiviruses and provide a template for viruses with similar physicochemical properties.
Subject(s)
Genetic Vectors/drug effects , HIV/drug effects , Lentivirus/drug effects , Virus Inactivation/drug effects , Alcohols/pharmacology , Aldehydes/pharmacology , Detergents/pharmacology , HEK293 Cells , HIV/pathogenicity , Hot Temperature , Humans , Lentivirus/physiologyABSTRACT
Early after entry into monocytes, macrophages, dendritic cells, and resting CD4 T cells, HIV encounters a block, limiting reverse transcription (RT) of the incoming viral RNA genome. In this context, dNTP triphosphohydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) has been identified as a restriction factor, lowering the concentration of dNTP substrates to limit RT. The accessory lentiviral protein X (Vpx) proteins from the major simian immunodeficiency virus of rhesus macaque, sooty mangabey, and HIV-2 (SIVsmm/SIVmac/HIV-2) lineage packaged into virions target SAMHD1 for proteasomal degradation, increase intracellular dNTP pools, and facilitate HIV cDNA synthesis. We find that virion-packaged Vpx proteins from a second SIV lineage, SIV of red-capped mangabeys or mandrills (SIVrcm/mnd-2), increased HIV infection in resting CD4 T cells, but not in macrophages, and, unexpectedly, acted in the absence of SAMHD1 degradation, dNTP pool elevation, or changes in SAMHD1 phosphorylation. Vpx rcm/mnd-2 virion incorporation resulted in a dramatic increase of HIV-1 RT intermediates and viral cDNA in infected resting CD4 T cells. These analyses also revealed a barrier limiting HIV-1 infection of resting CD4 T cells at the level of nuclear import. Single amino acid changes in the SAMHD1-degrading Vpx mac239 allowed it to enhance early postentry steps in a Vpx rcm/mnd-2-like fashion. Moreover, Vpx enhanced HIV-1 infection of SAMHD1-deficient resting CD4 T cells of a patient with Aicardi-Goutières syndrome. These results indicate that Vpx, in addition to SAMHD1, overcomes a previously unappreciated restriction for lentiviruses at the level of RT that acts independently of dNTP concentrations and is specific to resting CD4 T cells.
Subject(s)
HIV Infections/genetics , Reverse Transcription/genetics , SAM Domain and HD Domain-Containing Protein 1/genetics , Viral Regulatory and Accessory Proteins/genetics , Animals , CD4-Positive T-Lymphocytes/virology , Genome, Viral/genetics , HIV Infections/virology , HIV-1/genetics , HIV-1/pathogenicity , HIV-2/genetics , HIV-2/pathogenicity , Host-Pathogen Interactions/genetics , Humans , Macaca mulatta/genetics , Macaca mulatta/virology , Monocytes/virology , Proteolysis , RNA, Viral/genetics , Virion/genetics , Virion/pathogenicity , Virus Replication/geneticsABSTRACT
The nucleoside analog cytarabine (Ara-C) is an essential component of primary and salvage chemotherapy regimens for acute myeloid leukemia (AML). After cellular uptake, Ara-C is converted into its therapeutically active triphosphate metabolite, Ara-CTP, which exerts antileukemic effects, primarily by inhibiting DNA synthesis in proliferating cells. Currently, a substantial fraction of patients with AML fail to respond effectively to Ara-C therapy, and reliable biomarkers for predicting the therapeutic response to Ara-C are lacking. SAMHD1 is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase that cleaves physiological dNTPs into deoxyribonucleosides and inorganic triphosphate. Although it has been postulated that SAMHD1 sensitizes cancer cells to nucleoside-analog derivatives through the depletion of competing dNTPs, we show here that SAMHD1 reduces Ara-C cytotoxicity in AML cells. Mechanistically, dGTP-activated SAMHD1 hydrolyzes Ara-CTP, which results in a drastic reduction of Ara-CTP in leukemic cells. Loss of SAMHD1 activity-through genetic depletion, mutational inactivation of its triphosphohydrolase activity or proteasomal degradation using specialized, virus-like particles-potentiates the cytotoxicity of Ara-C in AML cells. In mouse models of retroviral AML transplantation, as well as in retrospective analyses of adult patients with AML, the response to Ara-C-containing therapy was inversely correlated with SAMHD1 expression. These results identify SAMHD1 as a potential biomarker for the stratification of patients with AML who might best respond to Ara-C-based therapy and as a target for treating Ara-C-refractory AML.