miRNA-36 inhibits KSHV, EBV, HSV-2 infection of cells via stifling expression of interferon induced transmembrane protein 1 (IFITM1)

Kaposi’s sarcoma-associated herpesvirus (KSHV) is etiologically associated with all forms of Kaposi’s sarcoma worldwide. Little is currently known about the role of microRNAs (miRNAs) in KSHV entry. We recently demonstrated that KSHV induces a plethora of host cell miRNAs during the early stages of infection. In this study, we show the ability of host cell novel miR-36 to specifically inhibit KSHV-induced expression of interferon induced transmembrane protein 1 (IFITM1) to limit virus infection of cells. Transfecting cells with miR-36 mimic specifically lowered IFITM1 expression and thereby significantly dampening KSHV infection. In contrast, inhibition of miR-36 using miR-36 inhibitor had the direct opposite effect on KSHV infection of cells, allowing enhanced viral infection of cells. The effect of miR-36 on KSHV infection of cells was at a post-binding stage of virus entry. The highlight of this work was in deciphering a common theme in the ability of miR-36 to regulate infection of closely related DNA viruses: KSHV, Epstein-Barr virus (EBV), and herpes simplexvirus-2 (HSV-2). Taken together, we report for the first time the ability of host cell miRNA to regulate internalization of KSHV, EBV, and HSV-2 in hematopoietic and endothelial cells.


Cytotoxicity assay
Target cells were treated with different concentrations of miR-36 mimic and inhibitor at 37°C in a V-bottom 96-well plate. After a 24h incubation, the cells were analyzed for the expression of LDH, as an indicator of cell death. The LDH assay was performed using the CytoTox 96 nonradioactive kit (Promega) as per earlier studies 33 . G418 (Sigma-Aldridge, St. Louis, MO) and cytochalasin D (Sigma-Aldridge) were used as known cell death inducers.

Virus infection of cells, RNA extraction, and monitoring virus infection
BJAB, HFF, and HMVEC-d cells were infected with 10 multiplicity of infection (MOI) 42,79 of KSHV, EBV, and HSV-2. The cells were left uninfected or infected for 5, 10, 15, and 30min prior to washing the cells twice in PBS and processed appropriately for RNA extraction. Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA). The RNA concentration was measured with a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA), and then verified for quality using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Only the RNA samples with 260/280 ratios of 1.8 to 2.0 were used in the study.
Extracted RNA was used to synthesize cDNA and the expression of ORF50 was monitored by qRT-PCR using specific primers as per earlier studies 33 . Expression of ORF50 was used as a scale to measure KSHV infection of cells. As reported earlier 42 , the lowest limit of detection in the standard samples was 6-60 copies for the ORF50 gene. The results from the use of ORF50 primers were consistently confirmed by monitoring the expression of another viral immediate early (IE) gene, vGPCR (data not shown). EBV and HSV-2 infection was monitored using specific primers to BRLF1 (homolog of KSHV ORF50) 81 and HSV-2 IE gene, US1 82 .

Flow cytometry
Flow cytometry was used to monitor expression of IFITM1 in the cells. Briefly, target cells were fixed in 10ml of ice-cold acetone for 20min, washed thrice in PBS prior to incubating cells in 25µg/ml of rabbit polyclonal antibody to IFITM1 (EMD Millipore, Billerica, MA) for 60min at 4°C. The cells were washed thrice in PBS and further incubated with FITC conjugated appropriate secondary IgG at 4°C for 30 min, washed and analyzed in a FACScan flow cytometer (Becton Dickinson) with appropriate gating parameters.

Real-time qRT-PCR analysis of the expression of miRNAs
The quality of RNA was tested using a spectrophotometer. Only the RNA samples with 260/280 ratios of 1.8 to 2.0 were used in the study. Approximately 500ng of RNA was reverse transcribed in a 25µl reaction volume using the All-in-one TM miRNA qRT-PCR detection kit (GeneCopoeia, Rockville, MD). Briefly, the cDNA was synthesized in a 25μl reaction mix containing 5μl of 5x reaction buffer, 2.5U/μl Poly A Polymerase, 10ng/μl MS2 RNA, and 1µl RTase Mix. The reaction was performed at 37°C for 60 min and terminated at 85°C for 5 min. cDNA that was produced in the RT reaction was diluted ten-fold and was used as the template for the PCR reaction in an Applied Biosystems ViiA 7 Real-Time PCR System (Life Technologies, USA). In this system, MS2 RNA was used as an external reference for the quality of the extracted miRNAs, and RNU6B, RNU44, RNU48, and RNU49 were used for normalization. The expression levels of miRNAs were measured employing qRT-PCR with the SYBR green detection and specific forward primer for the mature miRNA sequence and the universal adaptor reverse primer (GeneCopoeia, USA).

Silencing IFITM1 using siRNA
Expression of IFITM1 was inhibited by the transfection of double-stranded (ds) RNA oligos as per standard protocols 80 . IFITM1 siRNA was purchased from Dharmacon RNA Technologies (Lafayette, CO). Briefly, 1 x 10 6 cells were washed twice in RPMI and incubated in phenol redfree RPMI supplemented with 5% FBS at 37°C. After 24 hours incubation (considered as 0h for experiments in Fig. 6A), the target cells were transfected with either ds short interfering RNAs (siRNAs) or the nonspecific (NS) controls using Fugene HD as per manufacturer's recommendations (Promega). At 0, 12, 24, and 48 hours after transfection, total RNA was isolated from the cells and subjected to Northern blotting to monitor the expression of IFITM1