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. 2022 Nov 11;12(1):19303.
doi: 10.1038/s41598-022-23317-3.

Functional and molecular dissection of HCMV long non-coding RNAs

Sungwon Lee #  1   2 , Hyewon Kim #  1   2 , Ari Hong #  2   3 , Jaewon Song #  1   2 , Sungyul Lee  1   2 , Myeonghwan Kim  1   2 , Sung-Yeon Hwang  1   2 , Dongjoon Jeong  1   2 , Jeesoo Kim  1   2 , Ahyeon Son  1   2 , Young-Suk Lee  4 , V Narry Kim  1   2 , Jong-Seo Kim  1   2 , Hyeshik Chang  5   6   7 , Kwangseog Ahn  8   9
Affiliations

Functional and molecular dissection of HCMV long non-coding RNAs

Sungwon Lee et al. Sci Rep. .

Abstract

Small, compact genomes confer a selective advantage to viruses, yet human cytomegalovirus (HCMV) expresses the long non-coding RNAs (lncRNAs); RNA1.2, RNA2.7, RNA4.9, and RNA5.0. Little is known about the function of these lncRNAs in the virus life cycle. Here, we dissected the functional and molecular landscape of HCMV lncRNAs. We found that HCMV lncRNAs occupy ~ 30% and 50-60% of total and poly(A)+viral transcriptome, respectively, throughout virus life cycle. RNA1.2, RNA2.7, and RNA4.9, the three abundantly expressed lncRNAs, appear to be essential in all infection states. Among these three lncRNAs, depletion of RNA2.7 and RNA4.9 results in the greatest defect in maintaining latent reservoir and promoting lytic replication, respectively. Moreover, we delineated the global post-transcriptional nature of HCMV lncRNAs by nanopore direct RNA sequencing and interactome analysis. We revealed that the lncRNAs are modified with N6-methyladenosine (m6A) and interact with m6A readers in all infection states. In-depth analysis demonstrated that m6A machineries stabilize HCMV lncRNAs, which could account for the overwhelming abundance of viral lncRNAs. Our study lays the groundwork for understanding the viral lncRNA-mediated regulation of host-virus interaction throughout the HCMV life cycle.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Profiling of HCMV lncRNAs by Illumina sequencing and nanopore DRS (a) Schematic of the sequencing strategies for profiling HCMV lncRNAs in various infections state. (b) Coverage of HCMV genome mapped reads (left) and statistics of read counts (right) from RNA-seq in latency and reactivation. (c) Coverage of HCMV genome mapped reads (left) and statistics of read counts (right) from nanopore DRS in latency, reactivation, and lytic infection stages.
Figure 2
Figure 2
HCMV lncRNAs other than RNA5.0 are required for lytic replication in HFFs. (a) Schematic representation for generation of HCMV lncRNA-Mut virus. (b) Schematic representation of lytic infection analysis in HFFs using WT, lncRNA-Mut, and lncRNA-Rev viruses. (c) Immunoblot assay of viral proteins, which indicate the lytic replication phase (IE1/2, Immediate early; UL44, early; pp28, late). GAPDH served as a loading control. Images are representative of two independent experiments. (d) Cell-free viral particles from supernatant medium were titrated via limiting dilution assay. Data represents mean ± SEM, and the statistical significance was calculated by two-way ANOVA with Tukey’s multiple comparisons test (n = 3).
Figure 3
Figure 3
HCMV lncRNAs other than RNA5.0 are necessary for latent reservoir maintenance and efficient reactivation in Kasumi-3 cells. (a) Schematic representation of latency and reactivation analysis in Kasumi-3 cells using WT and lncRNA-Mut viruses. (b) The levels of viral DNA at the indicated time points were measured by qPCR of the UL123 region, and normalized against GAPDH. (c, d) The RNA levels of LUNA (c), and UL138 (d) at the indicated time points were quantified using qRT-PCR. (e) WT or lncRNA-Mut HCMV-infected Kasumi-3 cells were treated with DMSO or 20 nM PMA and co-cultured with naïve HFF cells. Frequency of infectious centers was calculated using ELDA. (f) Representative images of HFF cells co-cultured with 1600 PMA-treated Kasumi-3 cells for 14 days. Each image is obtained from each well of HFFs. IE1/2-positive images are presented on the left. Merged images are shown for IE1/2 (green), and DAPI (blue) staining. (b–e) Data represent mean ± SEM. All experiments were performed independently (n = 3), and the statistical significance was calculated by two-tailed unpaired t-test.
Figure 4
Figure 4
Post-transcriptional landscape of HCMV transcripts throughout the viral life cycle. (a, b) Differentially modified regions in viral transcripts. In comparison of test and control groups (test/control), enriched modification score (odds ratio) was analyzed by ELIGOS2. (a) Viral transcripts were divided into 300 nt tiles, and the median log2 odds ratio for each tile is presented as a heatmap. (b) Scatter plot showing the relationship between number of differential modification sites (odds ratio > 1.8) and average odds ratio for each gene. (c) Location and modification levels of RNA1.2, RNA2.7, and RNA4.9 in latency, reactivation, and lytic infection. Putative modification sites (odds ratio > 1.8) with AGACH motifs (red) and the others (black) are marked. (d) Position-specific base frequency of a motif enriched in the putative modified sites. (e) MeRIP-qPCR of latency, reactivation, and lytic infection samples. HPRT1 and GAPDH served as m6A-negative controls. CREBBP and SON served as m6A-positive controls. Data represent mean ± SEM. All experiments were performed independently (n = 3), and the statistical significance was calculated by two-tailed unpaired t-test.
Figure 5
Figure 5
m6A readers interact with and stabilize RNA1.2, RNA2.7, and RNA4.9 (a, b) Significant binding proteins filtered by comparing with no probe and antisense-lncRNA targeting probe samples. Venn diagrams represent the number of significantly binding proteins. Top 10 lists of lncRNA-specific binding proteins in order of adjusted p-value are presented in (a), and common binding proteins of the three lncRNAs are presented in (b). (c) Enrichment of lncRNA in immunoprecipitation of YTHDF2 or IGF2BP3. HPRT1 and GAPDH served as negative controls for m6A modification; CREBBP and SON served as positive controls. (d) RNA1.2, RNA2.7, and RNA4.9 are stabilized by m6A readers. (Left) Protein expression of METTL3, YTHDF2, and IGF2BP3 in each gene targeting siRNA-treated HFFs. GAPDH served as a loading control. (Right) Stability of EU-labeled HCMV lncRNAs. USP42 served as a negative control. Data represents values relative to a 0 h sample. (c, d) Data represent mean ± SEM of independent experiments (n = 3); statistical significance was calculated by two-tailed unpaired t-test.
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References

    1. Zuhair M, et al. Estimation of the worldwide seroprevalence of cytomegalovirus: A systematic review and meta-analysis. Rev. Med. Virol. 2019;29:e2034. doi: 10.1002/rmv.2034. - DOI - PubMed
    1. Griffiths P, Reeves M. Pathogenesis of human cytomegalovirus in the immunocompromised host. Nat. Rev. Microbiol. 2021 doi: 10.1038/s41579-021-00582-z. - DOI - PMC - PubMed
    1. Sinclair J. Human cytomegalovirus: Latency and reactivation in the myeloid lineage. J. Clin. Virol. 2008;41:180–185. doi: 10.1016/j.jcv.2007.11.014. - DOI - PubMed
    1. Sinzger C, Digel M, Jahn G. Cytomegalovirus cell tropism. Curr. Top. Microbiol. Immunol. 2008;325:63–83. doi: 10.1007/978-3-540-77349-8_4. - DOI - PubMed
    1. O'Connor CM, Murphy EA. A myeloid progenitor cell line capable of supporting human cytomegalovirus latency and reactivation, resulting in infectious progeny. J. Virol. 2012;86:9854–9865. doi: 10.1128/JVI.01278-12. - DOI - PMC - PubMed

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