Human MxB Protein Is a Pan-herpesvirus Restriction Factor

Abstract

Herpesvirus infections are highly prevalent in the human population and persist for life. They are often acquired subclinically but potentially progress to life-threatening diseases in immunocompromised individuals. The interferon system is indispensable for the control of herpesviral replication. However, the responsible antiviral effector mechanisms are not well characterized. The type I interferon-induced, human myxovirus resistance 2 (MX2) gene product MxB, a dynamin-like large GTPase, has recently been identified as a potent inhibitor of HIV-1. We now show that MxB also interferes with an early step of herpesvirus replication, affecting alpha-, beta-, and gammaherpesviruses before or at the time of immediate early gene expression. Defined MxB mutants influencing GTP binding and hydrolysis revealed that the effector mechanism against herpesviruses is thoroughly different from that against HIV-1. Overall, our findings demonstrate that MxB serves as a broadly acting intracellular restriction factor that controls the establishment of not only retrovirus but also herpesvirus infection of all three subfamilies.IMPORTANCE Human herpesviruses pose a constant threat to human health. Reactivation of persisting herpesvirus infections, particularly in immunocompromised individuals and the elderly, can cause severe diseases, such as zoster, pneumonia, encephalitis, or cancer. The interferon system is relevant for the control of herpesvirus replication as exemplified by fatal disease outcomes in patients with primary immunodeficiencies. Here, we describe the interferon-induced, human MX2 gene product MxB as an efficient restriction factor of alpha-, beta-, and gammaherpesviruses. MxB has previously been described as an inhibitor of HIV-1. Importantly, our mutational analyses of MxB reveal an antiviral mechanism of herpesvirus restriction distinct from that against HIV-1. Thus, the dynamin-like MxB GTPase serves as a broadly acting intracellular restriction factor that controls retrovirus as well as herpesvirus infections.

Keywords: HCMV; HSV-1; MHV68; MxB protein; cytomegalovirus; herpes simplex virus; herpesviruses; human MX2 gene; innate immunity; interferons.

FIG 1

Human MxB is a herpesvirus restriction factor. (a) U87MG cells expressing HA-tagged Mx proteins were infected (MOI of 0.5) with GFP-encoding HIV-1 VSV-G pseudotyped lentiviral vector, HSV-1, MCMV, MHV68, Ad5, MVA, VSV, or IAV (H7N7) and subjected to fluorescence-activated cell sorter (FACS) analysis 20 hpi or 6 hpi (only VSV) or 48 hpi (only HIV-1). The percentage of GFP⁺ cells relative to nontransduced control cells (NT) is shown. For Western blot analysis, Mx was detected by anti-HA antibody. Actin served as a control. (b) A549 cells expressing untagged Mx proteins were infected (MOI of 50) with GFP-encoding HCMV. Data represent percentages of GFP⁺ cells relative to cells transduced with empty vector (ctrl) and the relative values of the mean fluorescence intensities (MFI). For Western blot analysis, MxB was detected by anti-MxB antibody. Actin served as a control. Error bars represent the SEM of results from three independent experiments. Statistical analysis was performed via one-way ANOVA with a post hoc Tukey's test. ***, <0.001; **, < 0.01; *, < 0.1.

FIG 2

Herpesvirus infectivity is restricted at multiple time points and can partially be rescued by MxB depletion. (a) Growth kinetics (MOI of 0.0001) of HSV-1 GFP (17⁺) on U87MG cells and HSV-1 (McIntyre) and HSV-2 (MS) on A549 cells. The titers determined by plaque assay are shown as arithmetic means ± standard deviations (SD) of results from three independent experiments. Statistical analysis was performed on log values of the titers via two-way ANOVA with a post hoc Tukey's test. P values were determined by comparing the mean titers of control (ctrl) and MxB(1-715) cells at the depicted time points. (b) MxB- or MxA-expressing A549 cells were treated with siRNA as indicated and subsequently infected with HSV-1 GFP (MOI of 0.5) for 20 h. Infectivity was analyzed as described for Fig. 1. Error bars represent the SEM of results from three independent experiments. Statistical analysis was performed via one-way ANOVA with a post hoc Tukey's test. (c) Nontreated or IFN-α-pretreated T98G cells (100 U/ml for 16 h) were treated and analyzed as described for panel b. (d) T98G cells with specific MX1 and MX2 gene knockouts were left untreated or pretreated with IFN-α (400 U/ml for 16 h) and then infected with HSV-1 strain McIntyre (MOI of 0.001). IFN-α treatment was continued during the whole experiment. Virus titers in the supernatants of the cultures were determined at the indicated time points by plaque assay. The expression of MX1 and MX2 in the different cell lines was detected by Western blotting using MxA- and MxB-specific antibodies. The titers determined by plaque assay are shown as arithmetic means ± SD of results from three independent experiments. Significance is depicted for the MX2 knockout (MX2-ko) strain relative to the control cells and was determined via two-way ANOVA with a post hoc Tukey's test. ***, <0.001; **, <0.01; ns, nonsignificant.

FIG 3

GTPase activity, the N terminus, and oligomerization of MxB determine antiviral function. (a) U87MG cells expressing MxB variants were infected (MOI of 0.5) with GFP-encoding HIV-1 pseudotyped particles, HSV-1, MCMV, MHV68, or Ad5. Cells were subjected to FACS analysis and Western blotting as described for Fig. 1a. Depicted is the percentage of GFP-positive cells relative to cells expressing MxB(26-715). (b) A549 cells expressing MxB variants were infected (MOI of 0.5) with VSV-G pseudotyped HIV-1 vector or HSV-1, both encoding GFP, and subjected to FACS analysis and Western blotting as described for Fig. 1a. Shown are data from four independent experiments. Significance is depicted relative to the empty vector control. (c) U87MG cells expressing MxB variants were infected and analyzed as described for panel b. Shown are data from four independent experiments. The vertical line in the Western blot indicates a cut combining two parts of the same blot. Significance is depicted relative to the empty vector control. ***, <0.001; **, <0.01; *, <0.1. The chimeric MxB(1–85)-MxA(38-662) construct was described in reference . Single point mutations in MxB were determined according to the method previously described by Mitchell et al. (16).

FIG 4

MxB reduces viral gene expression and amplification of the herpesvirus genome. (a) HSV-1 capsid protein (anti-VP5) expression in A549 cells was analyzed by Western blotting at 24 hpi (MOI of 0.1). Shown is a blot representative of two independent experiments. (b) Expression of immediate early genes of HSV-1 (ICP0) and MCMV (ie2) in infected U87MG or A549 cells was measured by RT-qPCR. C_T values were normalized to actin and plotted relative to the ΔC_T values determined for the infected control cell line. Error bars represent geometric means of results from three independent experiments with technical duplicates. (c and d) Viral genome equivalents relative to GAPDH as a host-specific probe of HSV-1-infected (c) or MCMV-infected (d) A549 cells (MOI of 1). Error bars represent the SEM of (c) three independent HSV-1 experiments with technical quadruplicates or (d) two independent MCMV experiments with technical duplicates. Statistical analysis was performed using an unpaired t test for each time point. Significance is depicted relative to the control cells. ***, <0.001; **, <0.01; *, <0.1; ns, nonsignificant.