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Human cytomegalovirus long non-coding RNA counteracts nuclear cGAS to facilitate immune evasion

Nature Microbiology volume 10, pages 2275–2290 (2025)Cite this article

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Abstract

Viruses have evolved diverse immune evasion strategies, including the targeting of host pattern recognition receptors. The role of viral non-coding RNAs in modulating pattern recognition receptor activity is unclear. Here we show that human cytomegalovirus (HCMV) produces long non-coding RNA4.9 that counteracts the nuclear cyclic GMP–AMP synthase (cGAS)-mediated immune response to facilitate viral infection in human foreskin fibroblasts. RNA4.9 interacts with host cGAS via its 75-nucleotide RNA region with predicted hairpin loops. Binding of RNA4.9 to cGAS inhibits cGAS enzymatic activity as well as downstream interferon response and facilitates productive viral replication. Sterically blocking the folding of the 75-nucleotide region with antisense oligonucleotides during HCMV infection restores cGAS activity and impairs viral replication. In addition, we found that the specific localization of RNA4.9, which concentrates near HCMV DNA, is correlated with its efficient binding to cGAS and subsequent immune suppression. Our findings identify viral non-coding RNAs as key regulators of cGAS and highlight their potential as therapeutic targets.

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Fig. 1: HCMV RNA4.9 represses host innate immunity.
Fig. 2: RNA4.9 targets cGAS to accelerate the productive viral lytic replication.
Fig. 3: RNA4.9 interacts with cGAS through a prominent cGAS-binding domain with RNA hairpin loops.
Fig. 4: The 75-nt region of cGAS-binding hairpin loops within RNA4.9 is important for HCMV immune evasion.
Fig. 5: The 3.0–4.0-kb region directs the local accumulation of RNA4.9 near the HCMV DNA and contributes to cGAS inhibition.
Fig. 6: RNA4.9 localization is correlated with efficient RNA4.9–cGAS interaction.

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Data availability

The raw sequencing data of mRNA-seq and CLIP-seq were deposited in the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) database: GSE268887 and GSE270524, respectively. The whole-genome sequencing data from progeny viruses of the WT and mutant viruses were deposited in the NCBI Sequence Read Archive (SRA) under the BioProject PRJNA1232851. Reads from mRNA sequencing were aligned to the GRCh38 primary assembly (GENCODE V32). Reads from CLIP-seq were aligned to the GRCh38 primary assembly (GENCODE V45) and human herpesvirus 5 strain Toledo genome (GenBank accession GU937742.2). Reads from the whole-genome sequencing were aligned to the human herpesvirus 5 strain Toledo genome (GenBank accession GU937742.2). Source data are provided with this paper.

Code availability

The codes used for analysis of mRNA-seq, CLIP-seq and viral genome sequencing are available via GitHub at https://github.com/dohlee/viral-lncRNA-cGAS, https://github.com/hyejun18/cgas-clip-hcmv and https://github.com/hyejun18/hcmv-wgs-rna49, respectively.

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Acknowledgements

We thank all laboratory members for their productive discussions. We also thank T. Shenk for providing HCMV Toledo-BAC (AC146905.1). This work was supported by Institute for Basic Science IBS-R008-D1 (to K.A.), National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) RS-2020-NR049538 (to K.A.) and NRF-2020R1A2C3011298 to (to K.A.). This work was partially supported by funds from the SNU Institute for Virus Research.

Author information

Authors and Affiliations

  1. Center for RNA Research, Institute for Basic Science, Seoul, Republic of Korea

    Sungwon Lee, Sungchul Kim, Hyejun Kim, Dongjoon Jeong, Hyewon Kim, Junhyun Park, Kiwon Park, V. Narry Kim & Kwangseog Ahn

  2. School of Biological Sciences, Seoul National University, Seoul, Republic of Korea

    Sungwon Lee, Hyejun Kim, Dongjoon Jeong, Hyewon Kim, Junhyun Park, Kiwon Park, V. Narry Kim & Kwangseog Ahn

  3. Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea

    Sungchul Kim

  4. Bioinformatics Institute, Seoul National University, Seoul, Republic of Korea

    Dohoon Lee

  5. The Research Institute of Basic Sciences, Seoul National University, Seoul, Republic of Korea

    Junhyun Park

  6. PNU GRAND Convergence Medical Science Education Research Center, Pusan National University School of Medicine, Yangsan, Republic of Korea

    Jin Hur

  7. Department of Computer Science and Engineering, Seoul National University, Seoul, Republic of Korea

    Sun Kim

  8. SNU Institute for Virus Research, Seoul National University, Seoul, Republic of Korea

    Kwangseog Ahn

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Contributions

S.L. and K.A. contributed to the conceptualization and design of overall experiments. S.L. conducted virus infection and cell biological experiments. Sungchul Kim provided support for the construction of the CLIP library and SMI library. Hyejun Kim and V.N.K. contributed to bioinformatics analysis of CLIP-seq. D.L. and Sun Kim conducted mRNA sequencing analysis. J.P. contributed the microscopic image analyses. D.J., Hyewon Kim and K.P. performed biochemical experiments and cell line generation. J.H. provided support for the experiments using primary HUVEC cells. S.L., K.P., J.P. and K.A. wrote the paper.

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Correspondence to Kwangseog Ahn.

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Nature Microbiology thanks Alfredo Castello, Ian Mohr, Søren Paludan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 The 1.9–4.9-kb region of RNA4.9 plays a critical role in suppressing innate immune response.

(a) Graphical abstract describing each recombinant virus. HFFs infected with WT and 1.9–4.9krpsL HCMV (1 MOI, 6 hpi) were analyzed by reverse transcription-qPCR (RT-qPCR) (n = 2). The data represents mean. “n” indicates the number of independent experiments. (b) Top-10 significant GO terms of upregulated DEGs in 1.9–4.9krpsL HCMV infected cells at the indicated time points. The GO term ranking is determined by –log10 (adj.p-value). Adjusted p-values were based on one-sided Fisher’s exact test corrected by Benjamini-Hochberg procedure. Exact adjusted p-values are provided in Supplementray Table 3. (c) IFN-β protein levels in cell-free supernatant of HCMV-infected HFFs (1 MOI) were quantified by enzyme-linked immunosorbent assay (ELISA) at the indicated time points (n = 4). (d) Graphical abstract describing each recombinant virus. (d-e) HFFs infected with WT, 1.9–4.9krpsL, and Δ1.9–4.9k HCMV (1 MOI, 6 hpi) were analyzed by RT-qPCR (n = 3). (c-e) “n” indicates the number of independent experiments. The data are presented as mean ± SEM and P-values are based on two-sided multiple t-tests without correction. Exact P-values are provided in Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 RNA4.9 inhibits innate immunity in multiple HCMV-permissive cell types.

(a-c) U373MG, MRC-5, and HUVEC cells were infected with the WT and the 1.9–4.9krpsL viruses (U373MG and MRC-5; 1 MOI, HUVEC cells; 5 MOI). Viral gDNA levels were measured by qPCR at 3 hpi and RNA expressions of interferon and cytokines were measured by RT-qPCR at the indicated time points (n = 4; a, n = 3; b-c). “n” indicates the number of independent experiments. The data are presented as mean ± SEM and P-values are based on two-sided multiple t-tests without correction. Exact P-values are provided in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 RNA4.9 depletion-mediated immune activation precedes inefficient viral DNA replication and viral gene expression.

(a-d) HFFs infected with WT and 1.9–4.9krpsL HCMV were harvested at the indicated time points and analyzed for viral DNA level (a), viral protein expression (b), viral mRNA level (c), and immune gene expression (d). IE1, UL44, and UL99 represent viral immediate-early, early, and late transcripts, respectively. (b) The representative images of two independent replicates are presented. See Source Data for uncropped data. (a, c-d) Data are derived from four independent experiments. The data are presented as mean ± SEM and P-values are based on two-sided multiple t-tests without correction. Exact P-values are provided in Source Data Extended Data Fig. 3.

Source data

Extended Data Fig. 4 RNA4.9 inhibits cGAS activity to facilitate HCMV lytic replication.

(a and b) HFFs were infected with WT and 1.9–4.9krpsL HCMV (1 MOI) and were fixed at 6 hpi. (a) Phosphorylated STING (S366) and TBK1 (S172) were visualized by immunofluorescence assay (IFA). (b) Phosphorylated IRF3 (S386) and IE1/2 were visualized by IFA. White dotted lines indicate the boundary of nucleus of infected cells. Mean of raw intensity of nuclear p-IRF3 (S386) was quantified by ImageJ software (Fiji) (n = 36; Mock, n = 38; WT, n = 51; 1.9–4.9krpsL). Each “n” indicates the number of cells those were used in this analysis, which is derived from three independent experiments. The dotted lines in the violin plots indicate the median values. (c-d) 2’3’-cGAMP levels in MRC-5 and HUVEC cells infected with the indicated virus (1 MOI; MRC-5, 5 MOI; HUVEC) were measured by ELISA (n = 3). “n” indicates the number of independent experiments. (b-d) The data are presented as mean ± SEM and P-values are based on two-sided multiple t-tests without correction. Exact P-values are provided in Source Data Extended Data Fig. 4. (e) Images representing viral titers at 11 dpi in Fig. 2d. The representative images of three independent experiments are presented.

Source data

Extended Data Fig. 5 RNA4.9 inhibits dsDNA-triggered immune response independently of other viral factors.

(a) Graphical abstract describing each recombinant virus. (b) HFFs infected with the indicated HCMV (1 MOI) were harvested at 3 and 6 hpi, and subjected to RT-qPCR to measure the interferon and cytokine expressions (n = 3). (c) HeLa cells were transfected with pDEST12.2-RNA4.9 and stimulated by 0.5 μg/ml of Poly (dA:dT) or Poly (I:C). After 24 h, the cells were harvested and subjected to RT-qPCR to measure the interferon RNA level (n = 3). (d) HeLa cells were transfected with pDEST12.2-RNA4.9 and stimulated by 0.5 μg/ml of Poly (dA:dT) or 5 μg/ml of 2’3’-cGAMP. After 24 h, the cells were harvested and subjected to RT-qPCR to measure the interferon and cytokine expressions (n = 3). (b-d) “n” indicates the number of independent experiments. The data are presented as mean ± SEM and P-values are based on two-sided multiple t-tests without correction. Exact P-values are provided in Source Data Extended Data Fig. 5.

Source data

Extended Data Fig. 6 RNA4.9-mediated cGAS inhibition occurs in the nucleus.

(a) RNA levels in the nuclear and cytoplasmic fractions of WT HCMV-infected HFFs (1 MOI, 6 hpi) were quantified by RT-qPCR (n = 3). NEAT1 and GAPDH served as controls for nuclear RNA and cytoplasmic RNA, respectively. (b and c) HFFs were infected with WT and 1.9–4.9krpsL HCMV (1 MOI) and subjected to nucleus/cytoplasm fractionation. (b) At 6 hpi, the level of viral DNA was analyzed by qPCR using IE1 and UL44 targeting primers (n = 3). MDM2 served as controls for host DNA. (c) At 6 hpi, the cGAS level was analyzed by immunoblotting. GAPDH and Histone H3 served as controls for cytoplasmic protein and nuclear protein, respectively. The representative images of two independent experiments are presented. See Source Data for uncropped data. (d) HFFs were infected with WT and 1.9–4.9krpsL HCMV (1 MOI). At 6 and 24 hpi, the 23´-cGAMP level in nuclear fraction was analyzed by ELISA (n = 5). (a, b, and d) “n” indicates the number of independent experiments. The data are presented as mean ± SEM and P-values are based on two-sided multiple t-tests without correction. Exact P-values are provided in Source Data Extended Data Fig. 6.

Source data

Extended Data Fig. 7 Library construction for CLIP-seq.

(a) Immunoblot images representing the IP efficiency of the endogenous cGAS in Fig. 3a (E9G9G; Cell Signaling). (b) Immunoblot images representing the IP efficiency of the cGAS variants in Fig. 3c (F3165; Sigma). (a-b) The representative images of two independent experiments are presented. (c-e) Images were obtained during CLIP-seq library construction. (c) Immunoblot images representing the IP efficiency of cGAS antibodies (D1D3G and E9G9G; Cell signaling). (d) Autoradiograph images of 32P-labeled RNA-cGAS complexes and adaptor-ligated RNAs enriched by cGAS-CLIP. PCR amplicons of CLIP-libraries were visualized by staining with EtBR. (e) Autoradiograph images of 32P-labeled fragmented RNAs from non-crosslinked cells and adaptor-ligated RNAs. PCR amplicons of SMI-libraries were visualized by staining with EtBR. (a-e) See Source Data for uncropped data. (c-e) Two independent experiments are indicated as rep #1 and #2.

Source data

Extended Data Fig. 8 The 75-nt of cGAS-binding region of RNA4.9 is critical for accelerating HCMV lytic replication.

(a) Graphical summary of ΔRNA4.9Hairpins virus generation. RNA levels in HFFs infected with the indicated HCMV (1 MOI, 6 hpi) were analyzed by RT-qPCR (n = 3). “n” indicates the number of independent experiments. The data are presented as mean ± SEM. Exact data values are provided in Source Data Extended Data Fig 8. (b) Images representing viral titers at 7 dpi in Fig. 4d. (c) Coverage of CLIP-reads and SMI-reads mapped to the 1.9–4.9-kb region of RNA4.9. Two non-cGAS-binding regions (gray box) and a predominant cGAS-binding region (red box) were selected as ASO targets. (d) Images representing viral titers at 7 dpi in Fig. 4h. (b and d) The representative images of three independent experiments are presented.

Source data

Extended Data Fig. 9 RNA4.9-cGAS interaction is uniquely involved in cGAS inhibition.

(a) Coverage of CLIP-reads and SMI-reads mapped to HCMV transcripts. Predominant cGAS-binding regions of the HCMV transcripts (red box) were selected as ASO targets. (b) HFFs were transfected with the ASOs (250 nM) and infected with WT HCMV (1 MOI). At 24 hpi, 2´3´-cGAMP levels were measured by ELISA (n = 3). N.I indicates non-infection. “n” indicates the number of independent experiments. The data are presented as mean ± SEM and P-values are based on two-sided multiple t-tests without correction. Exact P-values are provided in Source Data Extended Data Fig 9.

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Extended Data Fig. 10 Characterization of RNA4.9 localization of the mutant viruses.

(a) Linescan profile from the normalized intensity of HCMV DNA and IE1/2 foci signals (related to Fig. 5a; n = 11). Signal distributions of HCMV DNAs are identical with those in Fig. 5b. “n” indicates the number of nuclear HCMV DNA or IE1/2 signals which were observed in the cells from three independent experiments. The cell numbers are identical with Extended Data Fig. 10b. The bold line and the shaded region indicate mean and 95% confidence interval (CI), respectively (b) The ratio of IE1/2 or RNA4.9 signals associated with EdU signals to total EdU signals in the nucleus was quantified (related to Fig. 5a). For each channel, the signals with an intensity in the upper 0.05 to 0.1% in the nucleus participated in quantification to exclude background noise signal. Each dot represents the individual cell (n = 6). “n” indicates the number of cells those were used in this analysis, which was obtained from three independent experiments. (c and f) Graphical abstract describing each recombinant virus. RNA levels in HFFs infected with the indicated HCMV (1 MOI, 6 hpi) were analyzed by RT-qPCR (n = 3). (d and g) Viral gDNA levels in HFFs infected with the indicated HCMV (1 MOI, 3 hpi) were analyzed by qPCR (n = 3). (e) Viral productions from the HFFs infected with the indicated HCMV (0.1 MOI) were titrated by limiting dilution analysis (n = 3). (c-g) “n” indicates the number of independent experiments. (b-g) The data are presented as mean ± SEM and P-values are based on two-sided multiple t-tests without correction. Exact P-values are provided in Source Data Extended Data Fig 10.

Source data

Supplementary information

Supplementary Information (download PDF )

Supplementary Figs. 1–8 and Source Data for Supplementary Fig. 7.

Reporting Summary (download PDF )

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Supplementary Data (download XLSX )

Statistical source data for Supplementary Figs. 1–3 and 5–6.

Supplementary Table 1 (download XLSX )

Primers for BAC mutagenesis.

Supplementary Table 2 (download XLSX )

Primers for qPCR.

Supplementary Table 3 (download XLSX )

mRNA sequencing data.

Supplementary Table 4 (download XLSX )

Modified ASOs.

Supplementary Table 5 (download XLSX )

smFISH probes.

Supplementary Table 6 (download XLSX )

PCR primers for construction of CLIP and SMI libraries.

Supplementary Table 7 (download XLSX )

TPM for the HCMV transcripts from CLIP-seq.

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Statistical source data for Fig. 6.

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Statistical source data for Extended Data Fig. 1.

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Source Data Figs. 1 and 2, and Extended Data Figs. 3, 6 and 7 (download PDF )

Uncropped immune blots and gels of Figs. 1 and 2, and Extended Data Figs. 3, 6 and 7.

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Lee, S., Kim, S., Kim, H. et al. Human cytomegalovirus long non-coding RNA counteracts nuclear cGAS to facilitate immune evasion. Nat Microbiol 10, 2275–2290 (2025). https://doi.org/10.1038/s41564-025-02078-5

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