Codon-usage-based inhibition of HIV protein synthesis by human schlafen 11

Abstract

In mammals, one of the most pronounced consequences of viral infection is the induction of type I interferons, cytokines with potent antiviral activity. Schlafen (Slfn) genes are a subset of interferon-stimulated early response genes (ISGs) that are also induced directly by pathogens via the interferon regulatory factor 3 (IRF3) pathway. However, many ISGs are of unknown or incompletely understood function. Here we show that human SLFN11 potently and specifically abrogates the production of retroviruses such as human immunodeficiency virus 1 (HIV-1). Our study revealed that SLFN11 has no effect on the early steps of the retroviral infection cycle, including reverse transcription, integration and transcription. Rather, SLFN11 acts at the late stage of virus production by selectively inhibiting the expression of viral proteins in a codon-usage-dependent manner. We further find that SLFN11 binds transfer RNA, and counteracts changes in the tRNA pool elicited by the presence of HIV. Our studies identified a novel antiviral mechanism within the innate immune response, in which SLFN11 selectively inhibits viral protein synthesis in HIV-infected cells by means of codon-bias discrimination.

Figure 1. SLFN11 inhibits retrovirus production without affecting intracellular vRNA levels

a–h, 293T cells were transfected with pNL4-3.Luc.R⁺E⁻/pCMV-VSV-G together with SLFN5, SLFN11, SLFN11-N, SLFN11-C or CAT (a, c, e, g), or 293, 293shRNA^Ctl, 293shRNA^SLFN and 293T cells were transfected with pNL4-3.Luc.R⁺E⁻ and pCMV-VSV-G (b, d, f, h). a, b, VSV-G-pseudotyped HIV production was assayed by titrated infection and luciferase assay. c, d, Viral particle content in supernatants was analysed by p24 ELISA. e, f, extracellular vRNA concentration was analysed by qPCR of p24. g, h, intracellular vRNA was determined by qPCR of p24. (average±s.d.; n = 3).

Figure 2. SLFN11 selectively inhibits viral protein expression on the basis of codon usage

a, 293T cells were transfected with pNL4-3.Luc.R⁺E⁻ and pcDNA5-EGFP together with SLFN5, SLFN11 or CAT, and cell lysates immunoblotted for HIV proteins, EGFP and GAPDH. b, 293, 293shRNA^Ctl and 293shRNA^SLFN cells were transfected with pNL4-3.Luc.R⁺E⁻ and pcDNA5-EGFP, and lysates analysed as in a. c, Top: 293T cells were co-transfected with pNL4-3.Luc.R⁺E⁻ and SLFN5, SLFN11 or CAT (left), or 293, 293shRNA^Ctl and 293shRNA^SLFN cells were transfected with pNL4-3.Luc.R⁺E⁻ (right). Lysates were probed for luciferase and GAPDH. Bottom: as above, except pNL4-3-ΔEnv-EGFP was used instead of pNL4-3.Luc.R⁺E⁻, and lysates were immunoblotted for Nef and GAPDH; d, Cells were transfected with viral codon-usage-based gag (Gag^vir, top) or synonymous human codon usage-optimized gag (Gag^opt, bottom). Expression of Gag in cell lysates was determined by anti-V5 immunoblotting.

Figure 3. SLFN11 binds tRNAs and selectively inhibits protein expression based on codon usage

a, 293shRNA^Ctl and 293shRNA^SLFN cells were transfected with pNL4-3.Luc.R⁺E⁻ or control vector (indicated as +/− HIV). Relative abundances of mature tRNA species were analysed by microarray as described^,. b, Left: increasing amounts of SLFN11-N or GFP were incubated with ³²P-labelled tRNA and subjected to EMSA. Right: 2 × or 10 × unlabelled tRNA or in-vitro-transcribed vRNA corresponding to the gag-pol frame-shifting sequence (120 bases) were added to the binding reaction. c, As in b, except non-specific (NS) or anti-SLFN11 (Anti-S11) monoclonal antibody was added to the binding reaction. d, 293T cells were transfected with V5-tagged GFP, Myc-tagged EGFP and pNL4-3.Luc.R⁺E⁻ together with either CAT, SLFN5 or SLFN11 (left). Lysates were probed for V5–GFP, Myc–EGFP and GAPDH. Similarly, V5-tagged GFP, Myc-tagged EGFP and pNL4-3.Luc.R⁺E⁻ were co-transfected into 293, 293shRNA^Ctl and 293shRNA^SLFN cells (right), and V5–GFP, Myc–EGFP and GAPDH expression determined by immunoblotting. e, V5–GFP and Myc–EGFP protein levels determined from d were quantified and normalized to V5–GFP and Myc–EGFP mRNA levels, respectively (average ± s.d.; n = 4).

Figure 4. SLFN11 inhibits replication of wild-type HIV-1_LAI in CEM cells

a, Human PBMCs were stimulated with 6,000 U ml⁻¹ IFN-β. SLFN11 expression in the derived lysates and in CEM cell lysates was analysed by immunoblotting. b, SLFN11 expression in CEM, CEMshRNA^Ctl and CEMshRNA^SLFN cells as analysed by immunoblotting. c, CEM, CEMshRNA^Ctl and CEMshRNA^SLFN cells were infected with HIV-1_LAI at a multiplicity of infection (m.o.i.) of 0.01, and viral replication was assayed by p24 ELISA of culture supernatants (average ± s.d.; n = 4).