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, 106 (33), 14040-5

The Brain-Specific Factor FEZ1 Is a Determinant of Neuronal Susceptibility to HIV-1 Infection

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The Brain-Specific Factor FEZ1 Is a Determinant of Neuronal Susceptibility to HIV-1 Infection

Juliane Haedicke et al. Proc Natl Acad Sci U S A.

Abstract

Neurons are one of the few cell types in the human body that do not support HIV type-1 (HIV-1) replication. Although the lack of key receptors is a major obstacle to infection, studies suggest that additional functions inhibit virus replication to explain the exquisite resistance of neurons to HIV-1. However, specific neuronal factors that may explain this resistance remain to be discovered. In a screen for antiviral factors using a fibroblast line chemically mutagenized and selected for resistance to retroviral infection, we recently identified induction of rat FEZ1 (fasciculation and elongation protein zeta-1), a brain-specific protein, as the cause of this resistance. When exogenously expressed in nonneuronal cell lines rat FEZ1 blocked nuclear entry of retroviral DNA. Here, we demonstrate that among human brain cells, neurons naturally express high levels of FEZ1 compared to astrocytes or microglia cells and are correspondingly less susceptible to infection with pseudotyped HIV-1 that bypasses receptor-mediated viral entry. Demonstrating that endogenous FEZ1 was functionally important in the resistance of neurons to HIV-1 infection, siRNA-mediated knockdown of endogenous FEZ1 increased the infectivity of neurons while sensitive brain cell types like microglia became more resistant upon FEZ1 overexpression. In addition, FEZ1 expression was not induced in response to IFN treatment. As such, in contrast to other widely expressed, IFN-inducible antiviral factors, FEZ1 appears to represent a unique neuron-specific determinant of cellular susceptibility to infection in a cell type that is naturally resistant to HIV-1.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
High levels of FEZ1 expression and reduced HIV-1 infectivity in neurons. (A) qPCR showing the level of endogenous FEZ1 expression in human lines and rat primary cells of brain origin. Cytoplasmic RNA prepared from human brain cell lines; neuronblastoma line (SH-SY5Y), glioblastoma/astrocytoma (1321N1, U87MG, and U138MG), and microglia (CHME3) or from primary rat hippocampus neurons (Rat N) and primary rat cortex astrocytes (Rat A) was reverse transcribed and used as template for qPCR. Data are median copy numbers normalized to cypA (for human cells) or GAPDH (for rat cells) obtained in triplicates from 2 independent RNA preparations. (B) Western blotting showing the levels of FEZ1 protein in representative human brain cell lines. Upper, endogenous FEZ1 expression was detected using anti-FEZ1 antibodies. Lower, loading of equal amounts of protein was confirmed using NCBP1 antibody. (C and D) Susceptibility of brain cell lines to infection with pseudotyped HIV-1. The 3 representative lines as in B were incubated with various amounts of HIV-1-puro (C) or 2 different dilutions (1:2 and 1:10) of HIV-1-luc (D) viruses. Noninfected (NI) cells were included as negative control. Cells were either selected in medium containing puromycin and the number of HIV-1-puro transduced colonies was counted after 8–12 days or were lysed and assayed for luciferase activity 48 h postinfection. Similar results were obtained in at least 3 independent experiments. (E and F) Analysis of viral DNA synthesis using qPCR. The indicated cell lines were infected with different dilutions (1:2, 1:5, and 1:10) of HIV-1-puro pseudotyped with VSV-G envelope. Low molecular hirt DNA was isolated at 24 h after infection and the amount of total viral DNA (E) and circular DNA (F) synthesized was measured by qPCR using primers specific to puromycin or 2-LTR DNA, respectively. Each sample was assayed in triplicate at a minimum of 3 different time points.
Fig. 2.
Fig. 2.
RNA interference to FEZ1 increases the susceptibility of human neuronal cells to infection. SH-SY5Y cells were transfected with either 3 independent siRNAs (labeled I, II, and III) targeting human FEZ1 or a nonspecific GFP duplex on 2 consecutive days with equal amounts of RNA duplexes and subsequently seeded and infected with various amounts of HIV-1-puro (B) or HIV-1-luc (C) viruses pseudotyped with VSV-G envelope protein as described in the legend to Fig. 1. Similar results were obtained in at least 3 independent experiments. (A and D) qPCR showing the endogenous levels of FEZ1 in cells from B and C, respectively. Cytoplasmic RNA was reverse transcribed and used as template for qPCR as described in the legend to Fig. 1. (E) Western blotting showing the levels of FEZ1 knockdown in human SH-SY5Y cells. Upper, endogenous FEZ1 expression was detected in the same cells as in C using anti-FEZ1 antibodies. Lower, loading of equal amounts of protein was confirmed using NCBP1 antibody. (F) Analysis of the nuclear accumulation of viral DNA using qPCR. SH-SY5Y cells transfected with control or FEZ1-II duplexes as described in D were subsequently seeded and infected with HIV-1-GFP pseudotyped with VSV-G envelope protein. Low molecular hirt DNA was isolated at 24 h after infection and the amount of circular DNA synthesized in the nucleus of infected cells was measured by qPCR using primers specific to 2-LTR DNA. Each DNA sample was assayed in triplicate at a minimum of 3 different time points. (G) qPCR showing the endogenous levels of FEZ1 (as described above) in the same cells as in F.
Fig. 3.
Fig. 3.
Overexpression of FEZ1 reduces the virus susceptibility of human brain macrophages. FEZ1 was overexpressed by either transient transfection of CHME3 cells with mammalian expression vectors containing full length (FEZ1) or C-terminally Flag-tagged FEZ1 (FEZ1-Flag) or by infection of these cells with pseudotyped retroviruses containing N (Flag-FEZ1) or C-terminally Flag-tagged FEZ1 (FEZ1-Flag). Cells transfected with pcDNA3.1 vector (pcDNA3.1) or infected with pQCXIN containing Flag sequences (Flag) were included as negative controls. (A) qPCR showing expression levels of FEZ1. Cytoplasmic RNA prepared from pcDNA3.1 control line, FEZ1, and FEZ1-Flag overexpressing cells was reverse transcribed and used as template for qPCR as described in the legend to Fig. 1. (B and D) Western blotting showing the level of FEZ1 protein in both CHME3 cells transiently transfected with FEZ1, FEZ1-FLAG, or empty vector pcDNA3.1 (B) and CHME3 pools stably overexpressing Flag alone, Flag-FEZ1, and FEZ1-Flag (D). Upper, FEZ1 overexpression was detected using anti-FEZ1 (B) or anti-Flag antibodies (D). The asterisk (*) indicates a nonspecific band detected by the anti-FLAG antibody in all samples. Lower, loading of equal amounts of protein was confirmed using NCBP1 (B) or GAPDH antibodies (D). (C and E) Susceptibility of FEZ1 overexpressing CHME3 cells to infection with pseudotyped HIV-1. The same cells as in A and D were subsequently seeded and infected with different dilutions (1:2 and 1:10) of HIV-1-luc virus pseudotyped with VSV-G envelope protein as described in the legend to Fig. 1. Noninfected (NI) cells were included as negative control. Similar results were obtained in at least 3 independent experiments. (F and G) Analysis of viral DNA synthesis using qPCR. Flag-FEZ1 overexpressing pool and the control Flag pool were infected with different dilutions (1:2 and 1:10) of HIV-1-puro pseudotyped with VSV-G envelope. Low molecular hirt DNA was isolated at 24 h after infection and the amount of total viral DNA (F) and circular DNA (G) synthesized was measured by qPCR as described in the legend to Fig. 1.
Fig. 4.
Fig. 4.
FEZ1 expression in human brain macrophages is not induced by IFN treatment. qPCR showing the endogenous levels of FEZ1 in IFN-α (A) or IFN-γ (C) induced CHME3 cells, along with the levels of the IFN-α inducible gene PKR (B) and the IFN-γ inducible gene IP10 (D). Cells were either not treated (indicated as 0) or treated with increasing amounts of IFN-α or IFN-γ as indicated. Twenty-four hours posttreatment cytoplasmic RNA was reverse transcribed and used as template for qPCR. Specific primers were used to measure the levels of endogenous FEZ1 and IFN inducible transcripts. Median copy numbers for each transcript was normalized to CypA obtained in triplicates from 2 independent RNA preparations.

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