CG dinucleotide suppression enables antiviral defence targeting non-self RNA

Abstract

Vertebrate genomes exhibit marked CG suppression-that is, lower than expected numbers of 5'-CG-3' dinucleotides. This feature is likely to be due to C-to-T mutations that have accumulated over hundreds of millions of years, driven by CG-specific DNA methyl transferases and spontaneous methyl-cytosine deamination. Many RNA viruses of vertebrates that are not substrates for DNA methyl transferases mimic the CG suppression of their hosts. This property of viral genomes is unexplained. Here we show, using synonymous mutagenesis, that CG suppression is essential for HIV-1 replication. The deleterious effect of CG dinucleotides on HIV-1 replication was cumulative, associated with cytoplasmic RNA depletion, and was exerted by CG dinucleotides in both translated and non-translated exonic RNA sequences. A focused screen using small inhibitory RNAs revealed that zinc-finger antiviral protein (ZAP) inhibited virion production by cells infected with CG-enriched HIV-1. Crucially, HIV-1 mutants containing segments whose CG content mimicked random nucleotide sequence were defective in unmanipulated cells, but replicated normally in ZAP-deficient cells. Crosslinking-immunoprecipitation-sequencing assays demonstrated that ZAP binds directly and selectively to RNA sequences containing CG dinucleotides. These findings suggest that ZAP exploits host CG suppression to identify non-self RNA. The dinucleotide composition of HIV-1, and perhaps other RNA viruses, appears to have adapted to evade this host defence.

Extended data Figure 1. CG-enriched HIV-1 clones yield near WT levels of virus from transfected 293T cells but are attenuated in replication in primary lymphocytes

a, Yield of infectious virus from proviral plasmid transfected 293T cells, as measured by infection of MT4 cells (mean ± sem, n=3, 4 or 5 independent experiments) b, c, Spreading replication of HIV-1 mutants in primary lymphocytes from two additional donors as measured by reverse transcriptase activity in the supernatant of infected cells over time.

Extended data Figure 2. Effects of CG dinucleotides on HIV-1 infectious virion yield, RNA and protein levels in single-cycle replication assays

a, Yield of infectious virus, in a single-cycle of replication following infection of MT4 cells with equal titers of HIV-1_WT and pol mutants (mean ± sem n=3 independent experiments). b, expression of gfp in MT4 cells, as measured by flow cytometry, 48h after infection with equal titers of the indicated viruses. Numerical values are mean fluorescent intensity (MFI) of infected cells (indicated by the dotted box). c, Western blot analysis (anti-Gag, anti-Env, anti-GFP and anti-HSP90) of viral, reporter and cellular protein expression, 48h after a single-cycle infection of MT4 cells with WT and synonymous pol mutant HIV-1. Representative of 3 experiments. d, Q-RT-PCR quantification of unspliced RNA in MT4 cells in a single-cycle infection assay with WT and synonymous pol mutant HIV-1 (mean ± sem n=2 or 3 independent experiments). e. Quantification of RNA molecules (fluorescent spots) by smFISH in cytoplasm using a probe targeting all spliced and unspliced HIV-1 RNA species after infection of HOS/CD4-CXCR4 cells. Each symbol represents an individual cell. Horizontal lines represent mean values, p-values were determined using Mann-Whitney test (n=10).

Extended data Figure 3. smFISH quantification of unspliced HIV-1 RNA in infected cells

Examples of smFISH analysis of WT and synonymous mutant HIV-1 infected cells (red=smFISH gag probe (see Fig 2c), green=GFP, blue=Hoescht dye). The boxed areas indicate regions selected for expanded views in Fig. 2f. Clusters of RNA molecules in the nuclei of some infected cells may represent sites of proviral integration. Representative of 3 independent experiments. Scale bar = 5μm.

Extended data Figure 4. smFISH quantification of total HIV-1 RNA in infected cells

Examples of smFISH analysis of WT and synonymous mutant HIV-1 infected cells (red=smFISH probe targeting all viral mRNA species (see Fig 2c), green=GFP, blue=Hoescht dye) Clusters of RNA molecules in the nuclei of some infected cells may represent sites of proviral integration. Representative of 3 independent experiments. Scale bar = 5μm.

Extended data Figure 5. ZAP mediates deleterious effects of CG-dinucleotides on HIV-1 replication

a, Western blot analyses, using the indicated antibodies, following transfection of HeLa cells with the corresponding siRNAs, or control siRNAs, in the single-cycle replication assays described in Fig. 3a. Representative of 2 experiments. b, Western blot analysis of ZAP expression in control, CRISPR knockout MT4 cells and doxycycline-inducible ZAP-S reconstituted MT4 cells. Asterisks indicate protein species that appeared in some CRISPR knockout clones, reacted with an anti-ZAP antibody and arose after extended passage. These likely represent truncated forms of ZAP-L whose translation initiated at methionine codons 3′ to the CRISPR target site (that was near the ZAP N-terminus) Representative of 3 experiments. c, Western blot analysis (anti-Gag, anti-Env anti-GFP and anti-Tubulin) of viral, and cellular protein levels in cells and virions, 48h after a single-cycle WT and mutant HIV-1infection of ZAP^−/− MT4 cells that had been reconstituted with a doxycycline-inducible ZAP-S expression construct (ZAP_DI) and left untreated or treated with doxycycline. Representative of 3 experiments.

Extended data Figure 6. CG dinucleotides in 3′UTRs confer sensitivity to inhibition by ZAP

a, Schematic representation of a reporter construct encoding a CG-dinucleotide depleted fluc cDNA into which were inserted the indicated sequences as 3′UTRs. b, Western blot analysis of ZAP expression following CRISPR mutation of ZAP exon 1 in HeLa cells. Representative of 3 experiments. c, Number of CG dinucleotides present in a 200-nucleotide sliding window in the indicated viral cDNA sequences that were left unmanipulated (WT), or recoded with synonymous mutations to contain the maximum number of CG dinucleotides (CG+). d, Luciferase expression following transfection of 293T ZAP−/− cells with CG-dinucleotide depleted fluc reporter plasmids incorporating the indicated VSV or influenza A virus (IAV) RNA sequences as 3′UTRs, in the presence or absence of a cotransfected ZAP-L expression plasmid (mean ± sem n=4 independent experiments).

Extended data Figure 7. Dinucleotide composition of ORFs, 3′UTRs, and preferred ZAP binding sites in cellular mRNAs

a, Expanded views of the portion of the CLIP graphs in Fig4. a corresponding to unmutated portions of the viral genome b, Sources of RNA reads bound to ZAP in a typical CLIP-seq experiment, done using HIV-1 infected cells c-e, Ratio of the observed frequency to the expected frequency (obs/exp, based on mononucleotide composition) for each of the 16 possible dinucleotides, in ORFs (c), 3′ UTR (d) sequences as well as the 100 sites in cellular mRNAs that were most frequently bound by ZAP, based on CLIP read numbers (e). Plotted values are mean ± sd of all ORF (n=35170) and 3′UTRs (n=135557) in the respective libraries (n=?) or the most preferred ZAP binding sites (n=100). f, Frequency distributions of CG dinucleotide observed/expected frequencies in human ORFs, 3′UTRs and top 100, top 1000 and top 10000 ZAP-binding sites in CLIP experiments. The top 100, top 1000 and top 10000 ZAP-binding sites account for 6.7%, 18.9% and 46.7% of total reads. g, Frequency distributions of CG, GC, UA and UG dinucleotide observed/expected frequencies in human ORFs, 3′UTRs and the top 100 APOBEC3G-binding sites in CLIP assays.

Extended data Figure 8. Analysis of CG-suppression in previously reported ZAP-sensitive and ZAP-resistant viruses and ZAP-sensitizing elements

a, CG suppression in RNA and reverse transcribing viruses previously reported to be ZAP sensitive (n=9, open symbols) and ZAP resistant (n=4, filled symbols) ^,–. The viruses included in the analysis and their degrees of CG suppression (CG observed/expected) are: ZAP-sensitive: Sinbis virus (0.90), Semliki forest Virus (0.89), Venezuelan equine encephalitis virus (0.76), Ebolavirus (0.60), Hepatitis B virus (0.52), Moloney Murine Leukemia Virus (0.51), Marburg virus (0.53), Alphavirus M1 (0.89), Ross River Virus (0.82); ZAP-insensitive: HIV-1 (0.21), Yellow fever virus (0.38) Vesicular stomatitis virus (0.48) Poliovirus (0.54). The p value was calculated using the students T-test (2-sided, n=9 ZAP sensitive viruses and n=4 ZAP resistant viruses). Influenza virus (CG obs/exp = 0.44) that has been reported to be ZAP-resistant due to the presence of an antagonist and ZAP-L sensitive via an entirely distinct protein interaction based mechanism was excluded from this analysis. b, Analysis of previous published data on ZAP inhibition of reporter gene expression. Each RNA element derived from the indicated RNA viruses was placed in a 3′UTR of a luciferase reporter plasmid and fold inhibition by coexpressed ZAP is plotted against the product of CG suppression (CG observed/expected) and length for each RNA element. A data point that is a quantitative outlier from the general trend (indicated in red) is from the Sinbis (SINV) genome, but is nevertheless included in the linear regression analysis, P value was calculated using the F-test (2-sided, n=32 data points) Data are from references ^and.

Figure 1. Synonymous mutagenesis reveals inhibitory effects of CG dinucleotides on HIV-1 replication

a, Representation of HIV-1_NHG GFP provirus, indicating synonymous mutant blocks, and corresponding phenotypes (see text). b–e, Replication of HIV-1 mutants in MT4 cells, as measured by FACS enumeration of infected cells. f, Number of CG dinucleotides in a 200 nucleotide sliding window in viral and random sequences. g, Replication of HIV-1 mutants in MT4 cells, measured as in b. h, Replication of HIV-1 mutants in primary lymphocytes, measured by supernatant reverse transcriptase activity.

Figure 2. CG dinucleotides cause depletion of cytoplasmic RNA

a, Single-cycle infectious virus yield, following infection of MT4 cells with equal titers of HIV-1_WT and mutants (mean ± sem n=3 independent experiments). b, Western blot analysis 48h after a single-cycle infection of MT4 cells with WT and mutant HIV-1, representative of 3 experiments. c, Location of salient exons (black lines), mutated segments (red shading) and smFISH probes (green shading) in HIV-1 mRNAs. d, Q-RT-PCR quantification of unspliced RNA in MT4 cells in a single-cycle infection assay (mean ± sem n= 2 or 4 independent experiments). e, Quantification of unspliced RNA (fluorescent spots) by smFISH in cytoplasm and nucleus of infected HOS/CD4-CXCR4 cells. Each symbol represents an individual cell nucleus or cytoplasm. Horizontal lines = mean. P-values determined using Mann-Whitney test, n=6, 8 or 9 individual cells. NS = not significant. f, Examples of smFISH analysis of an HIV-1_WT and mutant infected cell (red=smFISH gag probe, green=GFP, blue=Hoescht dye). Blue line indicates nucleus/cytoplasm boundary. Representative of 3 independent experiments.

Figure 3. ZAP specifically inhibits CG-enriched HIV-1 replication

a, Single-cycle infectious HIV-1_WT and L_CG-HI yield from siRNA transfected Hela cells (mean ± sem, n=3 independent experiments). b, Western blot analysis of MT4 cells following CRISPR mutation of ZAP exon 1. Representative of 3 experiments. c, Replication of HIV-1 mutants in ZAP+/+, ZAP−/− and doxycycline-inducible ZAP (ZAP_DI) reconstituted ZAP−/− MT4 cells, as measured by FACS enumeration of infected cells. d, Western blot analysis of cells and virions 48h after a single-cycle infection of ZAP+/+ and ZAP−/− MT4 cells with WT and mutant HIV-1. e, Q-RT-PCR quantification of unspliced RNA in MT4 cells in a single-cycle infection assay (mean ± sem n=3 independent experiments). f, Luciferase expression following transfection of Hela or HeLa ZAP−/− cells with reporter plasmids incorporating HIV-1 RNA segments as 3′UTRs (mean ± sem n=3 independent experiments).

Figure 4. ZAP binds directly and preferentially to CG dinucleotide-containing RNA

a, CLIP analysis of the frequency with which L mutant and HIV-1_WT RNA sequences are bound to ZAP in infected cells, versus their position in the viral genome. CG dinucelotides are indicated as blue lines. The L-mutant segment occupies positions 6307 to 6805. b, Expanded views of the ‘L’ portion of the CLIP graphs in a. c, Frequency distributions of CG, GC, UA and UG dinucleotide observed/expected frequencies in human ORFs, 3′UTRs and the top 100 ZAP-binding sites. P-values for ZAP binding sites (n=100) versus ORFs (n=35170) or 3′UTRs (n=135557) calculated using Welch’s unequal variance t-tests.