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Profiling of cellular microRNA responses during the early stages of KSHV infection

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Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) causes a variety of cancers, including Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). Host cellular microRNAs (miRNAs) play important post-transcriptional regulatory roles in gene expression and can greatly influence virus-host cell interactions. This study investigated cellular miRNA expression profiles operating in response to early stages of KSHV infection of human Burkitt lymphoma B cells (BJAB). We employed deep sequencing to analyze miRNA expression in KSHV-infected BJAB cells 15 min post infection (PI) and compared this to uninfected BJAB cells. A total of 32 known miRNAs and 28 novel miRNA candidates were differentially expressed in KSHV-infected compared to uninfected BJAB cells. Interestingly, miRNA expression profiles during the early stages of viral infection yielded comparable results when UV-inactivated KSHV was used. The deep sequencing results were further confirmed by performing real-time reverse transcription PCR. The target genes predicted to be regulated by both the known and novel miRNAs are mainly involved in assisting virus entry, inducing critical cell signaling, initiating transcription of immediate early genes, promoting latent infection, and modulating the host immune response. For the first time, we provide insight into the host cellular miRNA expression profiles in response to early stages of KSHV infection of human B cells. Furthermore, this study offers a valuable basis for further investigation on the roles of cellular miRNAs in the KSHV entry process.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, NC, 27834, USA

    Hosni A. M. Hussein & Shaw M. Akula

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  1. Hosni A. M. Hussein
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Correspondence to Shaw M. Akula.

Ethics declarations

We thank Dr. Blossom Damania (University of North Carolina at Chapel Hill) to have kindly provided us with the BJAB cells.

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The authors declare no conflict of interest.

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This article does not contain any studies with human participants or animals performed by any of the authors.

Electronic supplementary material

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705_2017_3478_MOESM1_ESM.doc

Supplementary Figure 1: UV exposure inactivates KSHV. BJAB cells were infected with 10 MOI of KSHV or UV-KSHV for 30 min prior to monitoring infection of cells by qRT-PCR measuring the expression of ORF50. Bars represent average ± s.d. of three individual experiments. Data was plotted to represent the change in RNA copy numbers of KSHV-ORF50, used as an indicator for successful infection. A student’s t test was performed to compare expression of ORF50 RNA in KSHV-infected cells and UV-KSHV infected cells. All the changes plotted are statistically significant (P value of 0.05 or less) (DOC 26 kb)

705_2017_3478_MOESM2_ESM.doc

Supplementary Figure 2: UV-KSHV modulates expression of miRNAs. The qRT-PCR data was plotted for the 28 novel miRNAs that are differentially expressed in UV-KSHV-infected BJAB cells, when compared to uninfected cells at 15min PI. The x-axis indicates the ID/name of the novel miRNAs and the y axis indicates fold change in expression of these miRNAs. The relative expression of miRNAs was measured in terms of threshold cycle value (Ct) and normalized to snRNA RNU6B. Bars represent average ± s.d. of three individual experiments. A student’s t test was performed to compare expression of a specific miRNA in KSHV-infected cells and uninfected cells. All the changes plotted are statistically significant (P value of 0.05 or less) (DOC 206 kb)

705_2017_3478_MOESM3_ESM.doc

Supplementary Figure 3: Putative targets for the novel miRNAs expressed during early stages of KSHV infection of BJAB cells. 24 validated novel miRNAs that are differentially expressed by KSHV infection were predicted to modulate expression of 84 target genes with confirmed roles during the early stages of virus infection. MIRanda, DIANA, and miRTar tools were used for identifying miRNA-target interactions (DOC 120 kb)

705_2017_3478_MOESM4_ESM.doc

Supplementary Figure 4: Validation of miR-targets by qRT-PCR (A) and Western blotting (B). (A) Expression of IRF5, DLG5, CPLX1 and SPTBN4 was detected by qRT-PCR at 15 minutes post KSHV infection. The qRT-PCR data was plotted using fold changes for the expression of IRF5, DLG5, CPLX1, and SPTBN4 in BJAB cells infected with 10 MOI of KSHV, when compared to uninfected cells. Bars represent average ± s.d. of three individual experiments. A student’s t test was performed to compare expression of specific target genes in KSHV-infected cells and uninfected cells. All the changes plotted are statistically significant (P value of 0.05 or less). (B) Western blotting analysis demonstrated changes in the expression levels of the identified putative targets for the novel miRNAs expressed during the early stages of KSHV infection. BJAB cells were either left uninfected or infected with 10 MOI of KSHV for 30 min. The cells were then lysed and the expression of IRF5, DLG5, CPLX1 and SPTBN4 protein levels was monitored using appropriate antibodies, normalized to β-actin protein levels. The link between the miRNAs and the putative target genes is depicted using a schematic. Red and green lines denote inhibition and enhancement, respectively (DOC 169 kb)

Supplementary Table 1. sRNA candidates in uninfected BJAB cells (XLS 326 kb)

Supplementary Table 2. sRNA candidates in KSHV-infected BJAB cells (XLS 261 kb)

Supplementary Table 3. Mature miRNA in uninfected BJAB cells at 15min PI (DOC 1481 kb)

Supplementary Table 4. Mature miRNA in KSHV-infected BJAB cells at 15min PI (DOC 1306 kb)

Supplementary Table 5. Predicted Novel miRNA in KSHV-infected BJAB cells at 15min PI (DOC 218 kb)

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Hussein, H.A.M., Akula, S.M. Profiling of cellular microRNA responses during the early stages of KSHV infection. Arch Virol 162, 3293–3303 (2017). https://doi.org/10.1007/s00705-017-3478-y

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