Transduction of Nondividing Human Macrophages with Gammaretrovirus-Derived Vectors

Abstract

It is commonly accepted that infection of nondividing cells by gammaretroviruses such as the murine leukemia viruses is inefficient due to their inability to cross the nuclear envelope barrier. Challenging this notion, we now show that human nondividing macrophages display a specific window of susceptibility to transduction with a Friend murine leukemia virus (F-MLV)-derived vector during their differentiation from monocytes. This finding suggests that factors other than the nuclear membrane govern permissiveness to gammaretroviral infection and raises the possibility of using the macrophage tropism of F-MLV in gene therapy.The nuclear envelope poses itself as a barrier between the initial and final phases of the early steps of retroviral infection: that is, between viral genome entry into the cytoplasm and its integration into the host genome in the nucleus. Two basic mechanisms have been proposed for the passage of viral nucleoprotein complexes (called reverse transcription or preintegration complexes [RTCs and PICs, respectively]) (3,7,8,25) through the nuclear membrane: active import through the nuclear pores or passive passage in the nucleus following nuclear membrane disassembly during mitosis. Active nuclear import can occur either through hijacking of the normal cytonucleoplasmic transport machinery or through the induction of distortions that perturb the integrity of the nuclear envelope, as proposed for the human immunodeficiency virus type 1 (HIV-1) Vpr protein (5).Among Retroviridae, lentiviruses such as HIV-1 have developed probably the most efficient way to traverse an intact nuclear membrane, and this results in the broad tropism of HIV-derived lentiviral vectors for most nondividing cells. Although the exact mechanism by which this is accomplished remains to be elucidated, lentiviral PICs appear to be gated through the nuclear pore and a number of viral proteins that compose it have nucleophilic properties, like Vpr, matrix (MA), and integrase (IN). An additional signal for nuclear transport may be provided by a particular single-gap structure determined by the central polypurine tract (cPPT) present on double-stranded proviral DNA (43). However, no consensus has yet been formed as to the importance of each of these elements in driving viral nuclear import. MA, Vpr, and cPPT can be deleted without completely abolishing either nuclear entry or viral infection (6, 9, 30), and the role played by IN nuclear localization signals is still being debated (6).If nuclear localization of certain PIC components is taken as evidence suggesting-but not proving-they may concur in driving the entire viral nucleoprotein complex inside the nucleus, then the ability to infect nondividing cells should hardly be restricted to lentiviruses. Simple retroviruses previously referred to as "oncoretroviruses" should share a similar theoretical ability. For instance, the INs of certain alpharetroviruses are localized in the nucleus (18,19,39), and for gammaretroviruses such as the murine leukemia virus (MLV), clear evidence...

How Samhd1 changes our view of viral restriction

“…Similarly, BST-2 is promiscuous in its ability to counteract enveloped viruses [64,65]. Interestingly, restriction of murine leukemia virus at the reverse transcription step is abrogated by Vpx in monocytes and macrophages [66,67], suggesting that SAMHD1 may exert its restriction activity against viruses other than lentiviruses. Additionally, if the deoxynucleotide triphosphohydrolase activity is found to be involved in SAMHD1-mediated HIV restriction, then SAMHD1 would be expected to act as restriction factor of viruses that require DNA synthesis during their life cycle.…”

Transfer of the Amino-Terminal Nuclear Envelope Targeting Domain of Human MX2 Converts MX1 into an HIV-1 Resistance Factor

“…Human 293T, HeLa, and U87-MG/CD4/ CXCR4 (12) cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum plus L-glutamine and penicillin-streptomycin. The HIV-1 IIIB provirus, HIV-1/Nef-internal ribosome entry signal (IRES)-Renilla reporter virus, the HIV-1-, FIV-, equine infectious anemia virus (EIAV)-, and MLV-derived lentiviral and retroviral vectors (LVs and RVs, respectively) and the human MX1 and MX2 expressing pEasiLV-based vectors have been described (12,(40)(41)(42)(43)(44), as have the HIV-1 P90A capsid mutant (19) and the HIV-1/SIV MAC capsid chimera (45). Murine Mx1 and Mx2 cDNAs (provided by Georg Kochs) were amplified by PCR and cloned into pEasiLV-MCS using the BamHI and XhoI restriction sites.…”

Characterization of the Behavior of Functional Viral Genomes during the Early Steps of Human Immunodeficiency Virus Type 1 Infection

“…Vectors were pseudotyped with the X4-tropic envelope PMA243 (AIDS Reagent and Reference Program of the NIH) or with the vesicular stomatitis virus G envelope protein (VSVg). At 48 h posttransfection, vectors were purified from the supernatant of transfected cells by ultracentrifugation through a 25% sucrose cushion, as previously described (22). Viral particles were then resuspended, and their infectious titer was determined on HeLaP4 cells after flow cytometry analysis.…”