Effect of Different Nuclear Localization Signals on the Subcellular Localization and Anti-HIV-1 Function of the MxB Protein

Abstract

Interferon exerts its antiviral activity by stimulating the expression of antiviral proteins. These interferon stimulate genes (ISGs) often target a group of viruses with unique molecular mechanisms. One such ISG is myxovirus resistance B (MxB) that has been reported to inhibit human immunodeficiency virus type 1 (HIV-1) by targeting viral capsid and impairing nuclear import of viral DNA. The antiviral specificity of MxB is determined by its N-terminal 25 amino acids sequence which has the nuclear localization activity, therefore functions as a nuclear localization signal (NLS). In this study, we report that the bipartite NLS, but not the classic NLS, the PY-NLS, nor the arginine-rich NLS, when used to replace the N-terminal sequence of MxB, drastically suppress HIV-1 gene expression and virus production, thus creates a new anti-HIV-1 mechanism. MxB preserves its anti-HIV-1 activity when its N-terminal sequence is replaced by the arginine-rich NLS. Interestingly, the arginine-rich NLS allows MxB to inhibit HIV-1 CA mutants that are otherwise resistant to wild type MxB, which suggests sequence specific targeting of viral capsid. Together, these data implicate that it is not the nuclear import function itself, but rather the sequence and the mechanism of action of the NLS which define the antiviral property of MxB.

Keywords: HIV-1; MxB; antiviral activity; interferon; nuclear localization signal.

FIGURE 1

The bipartite NLS supports MxB localization to the nuclear envelope. (A) Schematic representation of wild type full-length human MxB, N-terminal truncated MxBΔ(1–25), and MxB variants with N-terminal 25 amino acids replaced by heterologous NLS. The amino acid sequences of NLS are shown. NLS, nuclear localization signal. BSE, bundle signaling element. (B) HeLa cells were transfected with either wild type MxB or MxB mutants bearing a C-terminal GFP tag. The subcellular localization of MxB and its mutants was visualized by confocal laser scanning microscopy. Scale bars represent 10 μm.

FIGURE 2

Nuclear localization of BI-NP-NLS-MxBΔ(1–25) depends on importin-β. MxB or MxB mutants bearing a C-terminal GFP tag were expressed in HeLa cells that were transfected with control siRNA (siCtrl) or siRNA targeting importin-β (siImportin-β). Endogenous importin-β was immuno-stained with anti-importin-β antibodies. The subcellular localization of MxB and importin-β was visualized by confocal laser scanning microscopy. Scale bars represent 10 μm.

FIGURE 3

The bipartite NLS-bearing MxB dramatically suppresses HIV-1 gene expression. (A) HEK293T cells were transfected with wild type or mutant MxB DNA bearing a C-terminal Flag tag together with HIV-1 NL4-3 proviral DNA. At 48 h post-transfection, lysates of transfected cells were subjected to Western blotting to examine the levels of Gag/p24, Nef, and MxB. GAPDH was also examined as the internal control. (B) Total RNA was extracted from the above transfected cells. The levels of full-length HIV-1 RNA were determined by RT-qPCR, and the data were normalized to the levels of β-actin mRNA. Data shown are the average from three independent transfection experiments. (C) The levels of viral RT activity in the culture supernatants were determined, the results report the amounts of HIV-1 particles that were produced by the transfected HEK293T cells. Data shown are the average of three independent experiments. (D) The levels of infectious HIV-1 in the culture supernatants were determined by infecting TZM-bl indicator cells using the same volume of supernatants. Data shown are averages from three independent experiments. (E) HeLa cells were transfected with either wild type pQCXIP-MxB or its mutants bearing a C-terminal FLAG tag. The subcellular localization of MxB and its mutants was visualized by immunofluorescence staining and confocal microscopy. Representative images are shown. Scale bars represent 10 μm.

FIGURE 4

The bipartite NLS-bearing MxB suppresses HIV-1 LTR promoter activity. (A) HEK293T cells were co-transfected with HIV-LTR-Luc DNA containing a luciferase reporter gene driven by the HIV-LTR promoter, together with MxB or its mutant DNA (left panel). Same transfections were performed by including HIV-1 Tat DNA (middle panel). HIV-LTR-Luc DNA was also co-transfected with GFP DNA bearing different types of NLS (right panel). At 48 h post-transfection, the transfected cells were collected to measure luciferase activity which is presented in log10 RLU (relative light unit). Expression of MxB or NLS-GFP was examined by Western blotting. Data shown are the average from three independent experiments. (B–D) HEK293T cells were co-transfected with MxB DNA, CMV-Luc DNA (B), or SV40-Luc DNA (C), or PFV-LTR-Luc DNA (D). Each of these reporter DNA construct contains a luciferase reporter gene driven by the CMV, SV40 or PFV-LTR promoter. At 48 h post-transfection, the cells were lysed to measure luciferase activity. Expression of MxB and its mutants in the transfected cells was detected by Western blotting. Data shown are the average from three independent experiments.

FIGURE 5

The search for MxB orthologs carrying bipartite NLS. (A) Alignment of N-terminal sequences of MxB proteins from 65 species. The bipartite NLS consensus sequence is depicted in bold at the top of the aligned sequences. Highlighted in red boxes are the N-terminal sequences of MxB proteins from three species that contains bipartite-like NLS. (B) HEK293T cells were transfected with wild type or mutant MxB DNA together with HIV-1 NL4-3 proviral DNA. At 48 h post-transfection, the transfected cells were subjected to Western blotting to examine the levels of Gag/p24 and MxB. Tubulin was detected as the internal control.

FIGURE 6

MxB and its mutants bearing the arginine-rich NLS differentially inhibit wild type HIV-1 and viral mutants having CA mutations. SupT1 cells were transduced with retroviral particles that express MxB, Tat-NLS-MxBΔ(1–25), or Rev-NLS-MxBΔ(1–25), followed by infection with HIV-1 and its mutants CA-P90T, CA-E187V, or CA-P207S. MxB and its mutants contain a FLAG tag at the C-terminus. (A) Expression of MxB and its mutants was detected by anti-FLAG immunostaining. The percentages of FLAG-positive cells were scored by flow cytometry, and mean fluorescence intensity (MFI) values of FLAG staining were calculated and presented in the bar graphs. Results shown are the average of three independent experiments. *p < 0.05, **p < 0.001, ***p < 0.0001, ns, not significant. (B) HIV-1 infected cells were detected by immunostaining viral p24. The percentages of viral p24 positive cells in the Flag-positive cell population were scored by flow cytometry. Results shown are the average of three independent experiments. Fold of inhibition of HIV-1 infection by MxB or its mutants were calculated with reference to the infection of control cells by wild type HIV-1 or viral mutants. *p < 0.05, **p < 0.001, ***p < 0.0001. ns, not significant.