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Regulation of host and virus genes by neuronal miR-138 favours herpes simplex virus 1 latency

Abstract

MicroRNA miR-138, which is highly expressed in neurons, represses herpes simplex virus 1 (HSV-1) lytic cycle genes by targeting viral ICP0 messenger RNA, thereby promoting viral latency in mice. We found that overexpressed miR-138 also represses lytic processes independently of ICP0 in murine and human neuronal cells; therefore, we investigated whether miR-138 has targets besides ICP0. Using genome-wide RNA sequencing/photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation followed by short interfering RNA knockdown of candidate targets, we identified the host Oct-1 and Foxc1 messenger mRNAs as miR-138’s targets, whose gene products are transcription factors important for HSV-1 replication in neuronal cells. OCT-1 has a known role in the initiation of HSV transcription. Overexpression of FOXC1, which was not known to affect HSV-1, promoted HSV-1 replication in murine neurons and ganglia. CRISPR–Cas9 knockout of FOXC1 reduced viral replication, lytic gene expression and miR-138 repression in murine neuronal cells. FOXC1 also collaborated with ICP0 to decrease heterochromatin on viral genes and compensated for the defect of an ICP0-null virus. In summary, miR-138 targets ICP0, Oct-1 and Foxc1 to repress HSV-1 lytic cycle genes and promote epigenetic gene silencing, which together enable favourable conditions for latent infection.

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Fig. 1: ICP0-independent suppression of viral replication by miR-138 in Neuro-2a cells.
Fig. 2: miR-138 reduces viral gene expression in acutely infected mouse trigeminal ganglia.
Fig. 3: Host targets of miR-138 identified by PAR-CLIP/RNA-seq/siRNA screening.
Fig. 4: Repression of Oct-1 and Foxc1 expression by miR-138.
Fig. 5: FOXC1 promotes HSV-1 replication in neuronal cells and mouse trigeminal ganglia.
Fig. 6: FOXC1 effects on HSV-1 gene expression and heterochromatin in Neuro-2a cells.

Data availability

Raw high-throughput sequencing data for RNA-seq (Figs. 1g and 3 and Extended Data Figs. 2 and 8b) and PAR-CLIP (Fig. 3 and Extended Data Fig. 5) experiments have been deposited with the Gene Expression Omnibus under accession no. GSE127504. The HSV-1 KOS strain genome sequence and annotation were obtained from GenBank with accession no. JQ673480.1. Human (hg19) and mouse (mm10) genome sequences and annotations were downloaded from the Harvard Medical School Research Computing server (https://rc.hms.harvard.edu). Source data are provided with this paper.

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Acknowledgements

We thank B. Cullen, C. Jones and S.-H. Chen for the generous provision of plasmids, K. Holton from Harvard Medical School Research Computing for help with the sequencing data analysis, and the Core Facility of Zhejiang University School of Medicine and the Biopolymers Facility at Harvard Medical School for expertise and instrument availability. This work was supported by the National Key R & D Program of China (no. 2017YFC1200204 to D.P.), National Natural Science Foundation of China (no. 81671993 to D.P.), Natural Science Foundation of Zhejiang Province, China (no. LR18H190001 to D.P.), National Institutes of Health (no. P01 AI098681 to D.M.C. and D.M.K.), a Harvard Medical School Dean’s Initiative for Innovation Grant (D.M.K. and D.M.C.), a Natural Sciences and Engineering Research Council of Canada Postgraduate Fellowship and a Peter and Carolyn Lynch Foundation Fellowship (to A.H.M.N.), the National Human Genome Research Institute (no. RM1 HG008525 to G.M.C.) and the Blavatnik Biomedical Accelerator at Harvard University (to G.M.C.).

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Authors

Contributions

D.P. conceived the study. B.S., X. Yang, Q.W. and D.P. performed molecular cloning and the experiments in cell culture. X. Yang, J.M.-L., F.H. and D.P. generated recombinant viruses. X. Yang, F.H., X. Yu, J.M.P. and S.M.C. contributed to the animal studies. F.H. and E.A.H.V. performed the immunofluorescence assays. F.H. conducted the experiments using mouse primary neurons. P.R., Q.W. and D.P. performed the ChIP experiments. A.H.M.N. and G.M.C. developed the methods of deriving mixed neurons from iPSCs. H.S.O. differentiated the iPSCs into sensory neurons with a separate protocol and performed the experiments using the iPSC-derived neurons. J.M.P. provided statistical advice. D.P. and D.M.C. prepared the manuscript. D.P., D.M.K. and D.M.C. provided supervision. All authors approved the manuscript.

Corresponding author

Correspondence to Dongli Pan.

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Competing interests

A.H.M.N. and G.M.C. are inventors on patent nos. WO2018049382 and WO2018204262 filed by the Presidents and Fellows of Harvard College. Full disclosure for G.M.C. is available on http://arep.med.harvard.edu/gmc/tech.html. A.H.M.N. and G.M.C. are co-founders and have equity in GC Therapeutics. Other authors declare no conflict of interest.

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Peer review information Nature Microbiology thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Additional data about effects of miR-138 on HSV-1 replication.

a, Neuro-2a cells were mock-infected or infected with the indicated virus (MOI = 5) for 16 h before qRT-PCR analysis of miR-138 and let-7a levels. b, In 4 independent experiments performed as in Fig. 1b, the ratios of the average titers from scrambled transfected cells over those from miR-138 transfected cells were calculated separately for WT and M138 viruses and plotted. c, 293 T cells were transfected with 40 nM miRNA mimic for 16 h, and then infected with WT or M138 virus (left graph), or 7134 (ICP0-null) virus (right graph) for 48 h (MOI = 0.1) before viral titer measurements. d, Same as c, except that Vero cells were infected (MOI = 0.01) and no significant difference was detected between miR-138 and either of the two controls. For b, n = 4 independent experiments. For other panels, n = 3 (a, c right), 4 (d) or 8 (c left) biologically independent samples per condition. For all panels, data are presented as mean values ± S.D. and were analyzed by unpaired, two tailed t tests (a, b), two-way (c left, d) or one-way (c right) ANOVA with Bonferroni’s multiple comparisons tests.

Source data

Extended Data Fig. 2 Global effects of miR-138 on viral gene expression.

Same experiment as Fig. 1g, but effects on individual viral transcripts are plotted. Blue and red bars represent data for WT and M138 virus, respectively. n = 3 biologically independent samples per condition. Each bar represents the read count mean value for the indicated transcript from scrambled mimic transfected cells (after being normalized to total reads of the sample) divided by that from miR-138 mimic transfected cells.

Source data

Extended Data Fig. 3 Expression of miR-138, US11 and US12 from recombinant viruses.

a, 293 T cells were infected with M138miR138 or M138nomiR138 virus (MOI = 5). At 8 hpi, the cells were harvested for RNA purification and Northern blot hybridization. RNA from M138miR138 infected cells was run alongside a dilution series of synthetic miR-138 in the gel and hybridized with a miR-138 probe (left panel). RNA from M138nomiR138 infected cells was run alongside a dilution series of synthetic miR-M138b in the gel and hybridized with a miR-M138b probe (right panel). The integrity of RNA from both samples was verified by ethidium bromide staining, which is shown below the Northern blot images for a set of bands ~80 bases. This experiment was performed once. b, Vero cells were infected with the viruses indicated at the bottom at an MOI of 5 and harvested at 8 h post-infection for qRT-PCR analysis of US11 and US12 mRNA levels normalized to ICP27 mRNA levels. c, miR-138 expression from 293 T cells infected by the indicated viruses (MOI = 5, 8 h post-infection) as measured by qRT-PCR. For b and c, n = 3 biologically independent samples and data are presented as mean values ± s.d. Data were analyzed by one-way ANOVA with Bonferroni’s multiple comparisons tests.

Source data

Extended Data Fig. 4 Additional in vivo data about miR-138 expressing and control recombinant viruses.

a, Viral DNA and RNA levels in TG infected with WTmiR138 and WTnomiR138 at 5 dpi. The DNA or RNA molecules measured are labeled at the top of each graph. Viruses are indicated at the bottom. Each point represents a value from one trigeminal ganglion, and the horizontal lines represent the geometric means. The displayed n numbers represent the numbers of mice used per condition. Data were analyzed by two-tailed, unpaired t tests. b, Same as a, but WTmiR138R and WTnomiR138 are compared. c, Same as a, but M138miR138R and M138nomiR138 are compared.

Source data

Extended Data Fig. 5 PAR-CLIP experiments identified viral and host targets of miR-138.

a, miR-138 expression in 293 Tcontrol and 293T138 cells. b, PAR-CLIP procedure for detecting viral targets of miR-138 with, from left to right, a schematic showing the samples, a cartoon showing the crosslinking procedure, a representative autoradiograph of an SDS polyacrylamide gel showing a band corresponding to the Ago-RNA complex, the subsequent steps, and an agarose gel showing PCR amplification of the cDNA library. The experiment was performed once. c, Read counts of the indicated sequences from the indicated samples. d, 293T cells were co-transfected with 20 nM miRNA mimic and 100 ng/ml plasmid for 48 h before Western blot analysis of FLAG-tagged UL39 using an anti-FLAG antibody and analysis of ICP0 using an ICP0 antibody. This experiment was repeated once with similar results. e, miR-138 levels in Neuro-2a, N2A138 and N2Aanti138 cells. f, Diagram showing the “anti138” sequence expressed in N2Aanti138 cells. The sequence has a “tough decoy” secondary structure. Red and black horizontal lines represent anti138 and miR-138, respectively. Curved lines above and below the main structure represent bulges (extra nucleotides not bound to miR-138) designed to prevent cleavage. g, miR-138 and total read counts in the N2A138 and N2Aanti138 cells in a PAR-CLIP experiment. h, Fraction of 5’ UTR, CDS or 3’ UTR sites in total sites identified by the single PAR-CLIP approach (panels 1 and 2) or the combined PAR-CLIP/RNAseq approach (panels 3 and 4) in 293 T (panels 1 and 3) and Neuro-2a cells (panels 2 and 4). Relative to the single PAR-CLIP approach, the combined approach identified significantly higher fractions of targets with 3’ UTR sites (P = 0.034 and 0.0006 for 293 T and Neuro-2a cells, respectively by Fisher’s exact tests). For a and e, n = 3 biologically independent samples and data are presented as mean values ± s.d.

Source data

Extended Data Fig. 6 Low expression of OCT-1 and FOXC1 in mouse TG.

a, Fixed trigeminal ganglion cryo-sections were stained using an anti-FOXC1 or anti-OCT-1 rabbit primary antibody (red) or without a rabbit primary antibody (control), stained with a mouse anti-Tuj1 antibody (green), and stained with DAPI (blue). Similar results were obtained for OCT-1 using a different OCT-1 antibody (data not shown). This experiment was repeated twice with similar results. b, Neuro-2a cells were mock-transfected or transfected with 200 ng/ml OCT-1 or FOXC1 expressing plasmid for 48 h. The cells were then fixed and stained with an anti-OCT-1 or anti-FOXC1 antibody (green) or without a primary antibody, and stained with DAPI (blue). This experiment was repeated once with similar results.

Extended Data Fig. 7 Additional results with HSV1FOXC1 and HSV1FOXC1delAD viruses.

a, Mice were infected on the cornea with 2 × 105 PFU per eye of the indicated viruses. Viral titers in eye swabs collected at 1 d post-infection were determined. b, Following infection as in a, TG were harvested at 31 d post-infection and analyzed for viral genome levels normalized to mouse adipsin gene levels by qPCR. c, Following infection as in a, TG were harvested at 5 d post-infection and viral titers in TG were determined. d, Following mock infection (left) or infection of mice at the cornea with 2 × 105 PFU per eye of HSV1FOXC1delAD (middle) or HSV1FOXC1 (right), fixed TG cryo-sections were stained by DAPI (blue) and an anti-FLAG antibody (green) that can detect FLAG-tagged FOXC11 and FOXC1delAD proteins. This experiment was performed once. e, After corneal inoculation with 2 × 105 pfu/eye of the indicated viruses, mouse TG were harvested at 29 d post-infection and analyzed for viral genome levels normalized to mouse adipsin gene levels by qPCR. No significant difference was detected in e. For all panels, the n numbers represent the numbers of mice used per condition. The horizontal lines represent geometrical means. Data were analyzed by two-tailed, unpaired t tests (a, b, c) or one-way ANOVA with Bonferroni’s multiple comparisons tests (e) with the P values indicated.

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Extended Data Fig. 8 Global effects of FOXC1 on viral gene expression.

a, Additional data for Fig. 6b. Neuro-2a cells were transfected with 200 ng/ml plasmid for 40 h. KOS was then added (MOI = 2). The cells were incubated at 4 °C for 1 h to allow attachment, washed by PBS and incubated at 37 °C for 2 h before qRT-PCR analysis for the transcript indicated at the top normalized to host GAPDH levels. n = 6 biologically independent samples. The horizontal lines represent mean values. Data were analyzed by two-tailed, unpaired t tests. b, Neuro-2a cells were transfected with 200 ng/ml pcDNA or pFOXC1human for 40 h and infected with KOS for 5 h at an MOI of 1 before RNA-seq analysis. n = 3 biologically independent samples. Each bar represents the mean read count for the indicated transcript from pFOXC1human transfected cells (after being normalized by total reads from that sample) divided by that from pcDNA transfected cells.

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Extended Data Fig. 9 FOXC1 is important for HSV-1 replication and gene expression.

a, Left, Foxc1 CDS is depicted as a blue box with the DBD in green. Expanded below is the region of deletion in N2AFOXC1 knockout cells, showing sequences in WT and KO cell lines. The PAM sequence required for guide RNA target recognition is shown in green and the target sequence in red. Right, expression of FOXC1 relative to β-tubulin (loading control) in Neuro-2a and N2AFOXC1 knockout cells was analyzed by Western blots. This experiment was repeated twice with similar results. b, Neuro-2a and N2AFOXC1 knockout cells were infected with KOS (MOI = 0.5) for the indicated times. Mean viral titers ± s.d. are shown. c, Additional data for Fig. 6e. N2AFOXC1 knockout or Neuro-2a cells were transfected with 400 ng/ml pcDNA or pFOXC1human. 40 h later, the cells were infected with KOS for 5 h at an MOI of 1 before qRT-PCR analyses of the transcript indicated at the top. Horizontal lines represent geometrical means. Data were analyzed by two-way ANOVA with Bonferroni’s multiple comparisons tests. For b and c, n = 3 biological independent samples per condition.

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Extended Data Fig. 10 Foxc1 reduced heterochromatin associated with viral genes.

Neuro-2a cells were transfected with 500 ng/ml pcDNA or pFoxc1human, as indicated, for 40 h and infected with KOS for 2 h (MOI = 2) before ChIP-qPCR analysis for association of H3K9me3 with the genes indicated at the bottom. n = 3 biologically independent samples. Mean values ± s.d. are shown. Data were analyzed by two-way ANOVA with Bonferroni’s multiple comparisons tests.

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Sun, B., Yang, X., Hou, F. et al. Regulation of host and virus genes by neuronal miR-138 favours herpes simplex virus 1 latency. Nat Microbiol 6, 682–696 (2021). https://doi.org/10.1038/s41564-020-00860-1

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