key: cord-265211-a7whyds8
authors: Hussein, Hosni A. M.; Akula, Shaw M.
title: miRNA-36 inhibits KSHV, EBV, HSV-2 infection of cells via stifling expression of interferon induced transmembrane protein 1 (IFITM1)
date: 2017-12-21
journal: Sci Rep
DOI: 10.1038/s41598-017-18225-w
sha: 
doc_id: 265211
cord_uid: a7whyds8

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.

recently concluded study, we described a significant increase in the expression of host cell encoded miR-36 as early as 15 min PI of cells 20 . In the present study, we monitored expression of this miR-36 at early time points during KSHV infection of human B and endothelial cells. We employed human B (BJAB) and endothelial (HMVEC-d) cells as they are physiologically relevant cells to KSHV biology. Expression of miR-36 gradually increased from 5 min PI and peaked at 30 min PI in KSHV infected BJAB (Fig. 1A) and HMVEC-d cells (Fig. 1B) . Uninfected BJAB and HMVEC-d cells did not express miR-36 (Fig. 1A,B) . Expression of known miRNAs, hsa-let-7c and hsa-miR-3130-5p, were not significantly altered when compared to miR-36 during early stages of KSHV infection of cells (Fig. 1A,B) . Treatment of cells with 10 units/ml of heparinase I/III for 2 h at 37 °C prior to KSHV infection of cells resulted in a significant drop in the expression of miR-36 in BJAB (Fig. 1C) and HMVEC-d cells (Fig. 1D) . Also, infection of both BJAB and HMVEC-d cells with UV.KSHV could induce the expression of miR-36 to comparable levels as the wild-type KSHV (Fig. 1C,D) . These results demonstrate KSHV to induce host cell miR-36 very early upon infection. Key features of the novel miR-36 including the secondary structure are provided in Table 1 and supplemental Fig. 1 , respectively.

To evaluate the biological effects of miR-36 in target cells, we analyzed the effects of miR-36 mimic and inhibitor on KSHV infection. The range of doses tested in this study is comparable to those reported in the earlier studies [21] [22] [23] [24] [25] . The doses of the mimic In panels 'C and D' , Student t test was performed to study the effect of HS and compare infection of cells with KSHV versus UV.KSHV on miR-36 expression at 5, 10, 15, and 30 min PI. Two-tailed P value of 0.05 or less was considered statistically significant. *p < 0.05; **p,0.01; ***p < 0.001; NS-not significant. and inhibitor used in the study did not significantly induce cell death in BJAB and HMVEC-d cells ( Fig. 2A,B) . Transfection of BJAB (Fig. 2C ) and HMVEC-d (Fig. 2D ) cells with miR-36 mimic significantly reduced KSHV infection of cells as monitored by the expression of ORF50 gene as early as 30 min PI. KSHV entry is a quick process and the IE gene, ORF50, is expressed immediately upon infection 26 . KSHV infection was not significantly altered in BJAB (Fig. 2C ) and HMVEC-d ( Fig. 2D ) cells that were either transfected with scrambled miRNA control (miR-NC) or mock transfected. Interestingly, the effect of miR-36 mimic on KSHV infection of BJAB and HMVEC-d cells could be significantly reversed by co-transfecting cells with 10 nM of miR-36 inhibitor (Fig. 2E ). Co-transfection of cells with miR-NS did not alter the effects of miR-36 mimic (Fig. 2E ). To ascertain that the effect of miR-36 mimic was at a post-binding stage of infection, we performed a binding assay. The binding assay performed on BJAB and HMVEC-d cells demonstrated that miR-36 mimic and the miR-36 inhibitor did not block KSHV from binding the target cells (Fig. 2F ). Incubating KSHV with heparin but not CSA significantly blocked KSHV from binding cells (Fig. 2F ). Our results clearly implicate miR-36 to inhibit KSHV infection of cells. To extend our understanding of the role of miR-36 on other related viruses, we tested the effect of miR-36 mimic and the miR-36 inhibitor on EBV and HSV-2 infection of BJAB cells. Interestingly, miR-36 mimic could significantly block EBV and HSV-2 infection of BJAB cells and this inhibition could be specifically reversed by the miR-inhibitor (Fig. 3 ). miR-36 targets IFITM1. By using the DIANA and MiRmap tool algorithms, we identified a putative miR-36 binding site located in the 3′-UTR of IFITM1 mRNA (Supplemental Fig. 2 ). To confirm the ability of miR-36 to specifically inhibit IFITM1 expression, we monitored the expression of IFITM1 in target cells that were untransfected, transfected with miR-36 mimic, or miR-NC prior to infection. Transfection of BJAB and HMVEC-d cells with miR-36 mimic significantly lowered the expression of IFITM1 at 15 min PI compared to untransfected cells and cells transfected with miR-NC (Fig. 4A) . Transfection of cells with miR-36 mimic could specifically inhibit IFITM1 expression from 5 min till 48 h PI (data not shown). These results authenticate the fact that IFITM1 expression may well be regulated by miR- 36. In order to determine the bona fide target of miR-36, a luciferase reporter assay was performed. In this assay, two quantifiable genes encoding luciferase proteins were put on a vector. The IFITM1 3′ UTR with the target region was placed downstream GLuc to regulate its translation, and SEAP was placed under no regulation for normalization. 293 cells were co-transfected with the IFITM1 3′ UTR vector plasmid and miR-36 mimic. miR-36 mimic significantly decreased the relative luciferase activity compared to the cells that were transfected with miR-NC (Fig. 4B ). In contrast, transfection of cells with miR-inihibitor reversed the ability of miR-36 mimic from lowering the luciferase activity (Fig. 4B ). There was an inverse correlation observed in the expression of miR-36 and IFITM1 during the course of KSHV infection of BJAB (Fig. 4C ) and HMVEC-d (Fig. 4D ) cells. These results suggest that miR-36 directly targets IFITM1 and thereby downregulates its expression.

was significantly elevated with KSHV infection of BJAB and HMVEC-d cells (Fig. 5A ). The expression of IFITM1 increased in KSHV infected cells as early as 5 min PI which was elevated by 15 min and 10 min PI in BJAB and HMVEC-d cells, respectively, but declined sharply by 30 min PI (Fig. 5A ). To confirm a role for IFITM1 in KSHV infection of target cells, we transiently transfected BJAB and HMVEC-d cells with pQCXIP/IFITM1 and the expression of IFITM1 was confirmed by flow cytometry (Fig. 5B ). KSHV infection of the above IFITM1 expressing BJAB and HMVEC-d cells was a measure of the expression of ORF50 at 30 min PI. The idea was to strictly understand the effects of IFITM1 expression on early stages of KSHV infection. BJAB (Fig. 5C ) and HMVEC-d ( Fig. 5D ) cells expressing IFITM1 supported a significantly enhanced KSHV infection compared to those cells that were left untransfected, mock transfected, or transfected with the empty vector. Surprisingly, IFITM1 expression also enhanced HSV-2 and EBV infection of BJAB and HMVEC-d cells (Fig. 5C,D) . Interestingly, co-transfection of the above cells with miR-36 mimic could significantly drop KSHV infection of cells compared to miR-NC (Fig. 5C,D) .

To further confirm the role of IFITM1 in KSHV infection of cells, we first transfected cells with siRNA specific for IFITM1. Northern blotting was performed at 0, 12, 24, and 48 hours after transfection as per the standard protocols to monitor IFITM1 mRNA expression (Fig. 6A) . The levels of IFITM1 mRNA was significantly suppressed in BJAB and HMVEC-d cells by siRNA when compared with a (NS)siRNA control (Fig. 6A) inhibition of 82% ± 7%, 74% ± 9%, and 45% ± 6% was observed in BJAB cells at 12, 24, and 48 hours, respectively, after siRNA transfection (Fig. 6A) . A IFITM1 mRNA inhibition of 78% ± 9%, 65% ± 7%, and 34% ± 7% was observed in HMVEC-d cells at 12, 24, and 48 hours, respectively, after siRNA transfection. IFITM1 expression levels were not significantly altered by the (NS)siRNA controls in both the cells tested, demonstrating the specificity of the siRNA used (Fig. 6A ). IFITM1 expression in target cells transfected with siRNA specific to IFITM1 was significantly lowered at 15 min post KSHV infection (Fig. 6B ). In contrast, KSHV induced IFITM1 expression in untransfected or cells transfected with (NS)siRNA were not altered (Fig. 6B ). On the same lines, KSHV infection in cells silenced for the expression of IFITM1 was significantly lower compared to cells that were untransfected or transfected with (NS)siRNA (Fig. 6C ). Silencing the expression of IFITM1 also decreased EBV and HSV-2 infection of the above cells (Fig. 6C) . The above viral infections were monitored by performing qRT-PCR. Taken together, the results clearly implicate a role for IFITM1 in enhancing KSHV, EBV, and HSV-2 infection of cells.

Since miRNAs discovery over 20 years ago, miRNAs have been established as key players in the molecular mechanisms of mammalian biology including the maintenance of normal homeostasis and the regulation of disease pathogenesis. Host miRNAs also play a crucial role in mounting an immune response against microbial infections including those caused by viruses 27 . Several viruses belonging to herpesvirus, polyomavirus, hepadnavirus, and HMVEC-d that were untransfected, mock transfected, transiently transfected with miR-36 mimic, cotransfected with miR-36 mimic and miR-36 inhibitor(miR-mimic + mir-inhib), co-transfected with miR-36 mimic and nonspecific inhibitor (miR-mimic + mir-NS), or untransfected and treated with Heparin or CSA. Data was plotted to represent the percentage of KSHV binding to target cells treated differently compared to the untransfected cells. Bars (panels A-F) represent average ± s.d. of five individual experiments. Student t test was performed to compare groups. Two-tailed P value of 0.05 or less was considered statistically significant. *p < 0.05; **p,0.01; ***p < 0.001; NS-not significant.

SCIENtIfIC RepoRts | (2017) 7:17972 | DOI:10.1038/s41598-017-18225-w adenovirus, and retrovirus encode miRNAs 14, 15 . Virus-encoded miRNAs (vmiRNA) identified in virus-infected cells significantly influence viral replication and disease progression by modulating viral as well as host cellular mRNA. Many of the cellular miRNAs affect viral replication either directly by binding to the viral genome or indirectly by targeting host factors related to replication 28 . There are several published works that describe virus and cellular encoded miRNAs in KSHV pathogenesis 10, 18 . However, to date, there is no report on the role of cellular miRNAs during early stages of KSHV infection. Recent studies from our laboratory using a combination of deep sequencing and qRT-PCR identified cellular miR-36 to activate as early as 15 min PI 20 . In the current studies, we employed physiologically relevant human B cells (BJAB) and endothelial cells (HMVEC-d) to make the study more meaningful to KSHV biology. Accordingly, using specific primers we analyzed the expression profile of cellular miR-36 during the first 30 min of KSHV infection of cells. A successful viral entry was a measure of KSHV to enter cells and express immediate early gene, ORF50. The expression of ORF50 was monitored by qRT-PCR 26, 29 . KSHV infection of BJAB and HMVEC-d cells rapidly triggered the expression of miR-36 as early as 5 min PI that peaked at 30 min PI (Fig. 1) . Expression of miR-36 in KSHV infected BJAB is higher than those in HMVEC cells. This could be attributed to the inherited variations observed between different cells in the manner by which they respond to virus infection 30 . Moreover, the expression level of miRNAs has been shown to vary among tissues, cell types, and even between cells of the same lineage 31, 32 .

Cellular miR-36 is triggered by UV.KSHV infection of cells while treating target cells with heparinase I/III prior to infecting cells with the wild-type KSHV failed to induce expression of miR-36. Treatment of cells with heparinase I/III cleave heparan sulfate (HS) at different sites and liberate them from the cell surface 33 . KSHV binds to a target cell via interacting with HS expressed on the cell surface 34 . Taken together, our results indicate the following: (i) it is the interactions between the virus envelope proteins and the host cells that trigger miR-36 response; and (ii) binding of KSHV to cells is critical to miR-36 expression in cells. miR-36 mimic specifically inhibited KSHV infection of BJAB and HMVEC-d cells (Fig. 2C,D) . Transfection of cells with miR-36 inhibitor reversed the effects of miR-36 mimic on KSHV infection of cells (Fig. 2E) . The effects of miR-36 mimic and inhibitor was specific as the scramble negative control (miR-NC) and non-specific inhibitor (miR-NS) did not significantly alter KSHV infection of cells (Fig. 2C,D,E) . It was concluded that the effects of miR-36 mimic and inhibitor on KSHV infection was at a post attachment stage of internalization as they did not adversely affect virus binding to cells (Fig. 2F) .

In this study, we originally wanted to use two relevant viruses as negative controls to better understand the specificity of miR-36 on KSHV infection of cells. EBV and HSV-2 were selected as controls: EBV, like KSHV, belongs to γ-herpesvirinae while HSV-2 belongs to α-herpesvirinae. Interestingly, we observed a similar effect of miR-36 mimic and inhibitor on EBV and HSV-2 infection of cells (Fig. 2F ). This could be due to the fact that α, β, and γ-herpesviruses exhibit and share a common three-dimensional capsid structure along with the fact that there is quite a bit of homology in the glycoproteins being expressed on the viral envelope 35 .

One miRNA may regulate many genes as its targets, while one gene may also be targeted by many miRNAs 36 . Accordingly, miR-36 can possibly target several genes (Supplemental Fig. 6 ). Using bioinformatics tools, we identified IFITM1 to be the most promising targets to miR-36. IFITM is a member of the interferon-induced 125-133 amino acid protein family including IFITM1, IFITM2, IFITM3, IFITM5 and IFITM10. This family of proteins is located on chromosome 11 of the human genome and originally described as highly inducible genes by αand γ-interferons (IFNs) 37, 38 . The three members of the IFITM proteins (IFITM1, IFITM2, and IFITM3) have gained prominence as novel antiviral IFN-stimulated genes (ISGs) 39 . Hence, we set out to test the effect of transfecting target cells with IFITM1-3 genes on KSHV infection of target cells. Over-expressing IFITM1 significantly enhanced KSHV infection of cells; IFITM3 moderately enhanced KSHV infection while IFITM2 did not alter the viral infection (Supplemental Fig. 7) . Based on these results, we focused our further studies on IFITM1 in terms of miR-36 and early stages of KSHV infection. Using bioinformatic tools it was determined that the miR-36 target IFITM1 expression. Luciferase assay demonstrated the ability of miR-36 to physically interact with IFITM1 (Fig. 4B) . miR-36 mimic specifically inhibited IFITM1 expression that could be reverted by transfecting cells with miR-36 inhibitor (Fig. 4B) . There was an apparent inverse correlation observed between the KSHV-induced IFITM1 expression and miR-36 response (Fig. 4C,D) . We propose the sharp decline in the expression of IFITM1 30 min PI is because of an increase in the expression of miR-36 (Fig. 4C,D) . Taken together, our study established a direct association between virus-induced IFITM1 and endogenous miR-36 expression in the biology of KSHV.

IFITM1 is expressed in many cell types including leukocytes and endothelial cells 37, 40 . IFITM1 modulates cell functions including immunological responses, cell proliferation, cell adhesion, and germ cell maturation 41 . As other IFITM proteins, IFITM1 is significantly upregulated by interferons type I and II and is critical for anti-virus 48 . In general, the IFITM family of proteins affects virus entry of cells. Over-expression of IFITM1 significantly enhanced KSHV infection of target cells (Fig. 5C,D) while silencing expression of IFITM1 had the opposite effect (Fig. 6 ). More interestingly, we observed identical effects of IFITM1 in enhancing EBV and HSV-2 infection of cells (Fig. 6C) .

IFNs are generally considered to be antiviral cytokines that inhibit virus infection of cells by stimulating ISGs 49 . In fact, we observed a significant increase in the expression of IFN-α and -γ during the early stages of KSHV infection of BJAB and HMVEC-d cells (Supplemental Fig. 8 ). If that is the case, how can IFITM1 enhance infection of not just KSHV; but also of EBV and HSV-2? Such a scenario can be possible only if the virus infection is not altered by IFNs and associated proteins 48 . Interestingly enough, herpesviruses as a group (including KSHV) is considered to be relatively insensitive to the antiviral effects of IFNs in a variety of different cell systems 50, 51 . In a way, our results demonstrate for the first time that herpesviruses, KSHV, EBV, and HSV-2, not only hijack IFITM1 to their benefit in facilitating virus entry into cells but also evade the IFN-induced antiviral effects.

This study provides a new insight to virus infection. KSHV (including EBV and HSV-2) interactions with target cells induce IFITM1 within minutes, which facilitate virus entry into cells. KSHV-induced IFITM1 is part of the innate immune activation system that occurs in an antigen-independent fashion 52 and relies on the ability of the host to recognize virus via specific pattern recognition receptors 53 . To counter the effects of KSHV-induced IFITM1, infected cells respond within a short period of time by inducing expression of miR-36. Cellular miR-36 in turn inhibits expression of IFITM1 and thus limit virus infection of cells. Perhaps, this could be a mechanism of superinfection resistance (SIR) 54 developed by cells towards KSHV and other related viral pathogens. IFITM1 suppression by miR-36 may have direct and indirect effects of KSHV pathobiology: (i) directly limit KSHV infection of cells; (ii) block cell proliferation/division 55 and thereby promote KSHV latency 56 ; and (iii) reduce virus-induced inflammation. Such effects of IFITM1 on the biology of KSHV will be better understood by employing the three-dimensional (3-D) cell culture models as they mimic certain aspects of the tissue environment 57, 58 . For all this time, studies on virus entry have always focused primarily on the roles of virus encoded glycoproteins and their cognate host cell receptors. To our knowledge, this is the first report of a miRNA influencing KSHV infection of cells and this, we hope, will open doors to a further understanding of virus entry; after all, it is the miRNAs that regulate the gene function.

Of late, miRNA-based therapeutics have been used to effectively treat autoimmune diseases 59 , and cancers including prostate cancer, and leukemia 60 in animal models. The fact that miRNAs can influence virus entry is fascinating as miRNA-based therapeutics like the use of miR-mimics can effectively be used to treat virus entry including pathogenesis. There are several questions that need to be answered and they are as follows: (i) How does IFITM1 enhance KSHV infection of cells? Does it physically bind KSHV envelope-associated protein and facilitate endocytosis? (ii) What is the role of host cell receptors in the IFITM1-facilitated virus entry? (iii) What is the dynamics between the expression pattern of IFITM1 and 3 in regulating KSHV infection of cells? And (iv) Can induction of miR-36 by KSHV infection prevent infected cells from being superinfected with KSHV and other related herpesviruses, in vivo? Current studies in our lab are dedicated to answer these questions. fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA), L-glutamine, and antibiotics 61 . HMVEC-d cells were propagated in EGM MV-microvascular endothelial cell medium (Clonetics) as per standard protocols. The passage numbers for HFFs and HMVEC-ds used in this study ranged between 6-10, and 5-9, respectively. All the cells used in this study were negative for mycoplasma as tested by Mycoplasma PCR ELISA, Roche Life Science. For culture conditions, refer to supplemental data.

Virus. The viruses used in this study were wild-type KSHV 26 , herpes simplexvirus-2 (HSV-2) 62 , and Epstein-Barr virus (EBV) 63 . We generated ultraviolet (UV) inactivated KSHV (UV.KSHV) as per early studies 20 .

Cytotoxicity assay. The LDH assay was performed using the CytoTox 96 non-radioactive kit (Promega) as per earlier studies 26 . 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 24 h 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 non-radioactive kit (Promega) as per earlier studies 26 Extracted RNA was used to synthesize cDNA and the expression of ORF50 was monitored by qRT-PCR using specific primers as per earlier studies 26 Expression of ORF50 was used as a scale to measure KSHV infection of cells. As reported earlier 29 , 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) 64 Binding assay. The effect of miR-mimic and inhibitor on KSHV binding to target cells was assessed by PCR detecting the cell-bound KSHV DNA. Briefly, untransfected cells or cells transfected with miR-mimic or inhibitor were infected with 10 MOI of KSHV at +4 °C. After 60 min of incubation with virus, cells were washed three times with PBS to remove the unbound virus. Cells were pelleted, and total DNA including those representing the cell bound KSHV was isolated using DNeasy kit (Qiagen, Valencia, CA) and subjected to qPCR analysis monitoring ORF50 according to recently published work 29 . Incubating KSHV with 100 µg/ml of heparin and chondroitin sulfate A (CSA; Sigma-Aldridge) for 1 h at 37 °C were used as known positive and negative controls. Western blotting. All the buffers used in this project were made with water that was endotoxin and pyrogen free. Western blotting was conducted as per earlier studies 29 using the following primary antibodies: rabbit anti-IFITM1 polyclonal antibody (EMD Millipore) and mouse anti-actin antibodies (Clone AC-74; Sigma-Aldridge).

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 500 ng of RNA was reverse transcribed in a 25 µl reaction volume using the All-in-one TM miRNA qRT-PCR detection kit (GeneCopoeia, Rockville, MD). Briefly, the cDNA was synthesized in a 25 μl reaction mix containing 5 μl of 5x reaction buffer, 2.5 U/μl Poly A Polymerase, 10 ng/μl MS2 RNA, and 1 µl 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). Expression of IFN-α, -β, and -γ by qRT-PCR was conducted as per earlier protocols 26 using appropriate primers [66] [67] [68] .

SCIENtIfIC RepoRts | (2017) 7:17972 | DOI:10.1038/s41598-017-18225-w Dual-luciferase reporter assay. Luciferase reporter plasmids with wild-type IFITM1 3′-UTR were purchased from GeneCopoeia (Rockville, MD). 293 cells were plated onto 6-well plates. At 24 h post-plating, 293 cells were co-transfected with IFITM1 3′-UTR luciferase reporter plasmid and miR-36 mimic or miR-NCNA scramble control (miR-NC) using FuGene HD (Promega). At 12, 24, 48 h post transfection, supernatants were collected from each treatment and the luciferase activity measured using the Secrete-Pair Dual Luminescence Assay Kit (GeneCopoeia) as per the manufacturers' recommendations.

Northern blotting. Northern blotting to monitor IFITM1 and β-actin expression was performed using a DIG Luminescent Detection Kit (Roche, Indianapolis, IN) as per the manufacturer's recommendations 26 . Silencing IFITM1 using siRNA. Expression of IFITM1 was inhibited by the transfection of double-stranded (ds) RNA oligos as per standard protocols 26 . IFITM1 siRNA was purchased from Dharmacon RNA Technologies (Lafayette, CO). Briefly, 1 × 10 6 cells were washed twice in RPMI and incubated in phenol red-free RPMI supplemented with 5% FBS at 37 °C. After 24 hours incubation (considered as 0 h 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 and β-actin mRNA as per the protocol mentioned in the "Materials and methods" section describing Northern blotting. In another set of experiments, untransfected cells and cells transfected with siRNA or (NS) siRNA for 12 h were infected with 10 MOI of KSHV. At the end of 30 min PI, KSHV infection was assessed by monitoring ORF50 expression by qRT-PCR. Data Availability Statement. All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).

Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma

Kaposi's sarcoma-associated herpesvirus-like DNA sequences in multicentric Castleman's disease

Primary Kaposi's sarcoma of the heart in nonimmunodeficient patient: case report and literature review

Cellular origin of Kaposi's sarcoma and Kaposi's sarcoma-associated herpesvirus-induced cell reprogramming

Serodiagnosis for tumor viruses

Chromatinization of the KSHV Genome During the KSHV Life Cycle

Regulation of herpesvirus reactivation by host microRNAs

Noncoding RNPs of viral origin

Kinetics of Kaposi's sarcoma-associated herpesvirus gene expression

MicroRNAs-Based Imaging Techniques in Cancer Diagnosis and Therapy

Small role with big impact: miRNAs as communicators in the cross-talk between cancer-associated fibroblasts and cancer cells

miRBase: integrating microRNA annotation and deep-sequencing data

New perspective of host microRNAs in the control of PRRSV infection

RNA viruses and microRNAs: challenging discoveries for the 21st century

A novel HIV-1-encoded microRNA enhances its viral replication by targeting the TATA box region

KSHV microRNAs: Tricks of the Devil

Kaposi's sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells

Kaposi's sarcoma herpesvirus (KSHV) microRNA K12-1 functions as an oncogene by activating NF-kappaB/IL-6/STAT3 signaling

Cellular MicroRNA Let-7a Suppresses KSHV Replication through Targeting MAP4K4 Signaling Pathways

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

Transfection of microRNA Mimics Should Be Used with Caution

MicroRNA mimicry blocks pulmonary fibrosis

miR-122-SOCS1-JAK2 axis regulates allergic inflammation and allergic inflammation-promoted cellular interactions

MicroRNA-205-5p regulates the chemotherapeutic resistance of hepatocellular carcinoma cells by targeting PTEN/ JNK/ANXA3 pathway

Differential responses of PPARalpha, PPARdelta, and PPARgamma reporter cell lines to selective PPAR synthetic ligands

Cell membrane-bound Kaposi's sarcoma-associated herpesvirus-encoded glycoprotein B promotes virus latency by regulating expression of cellular Egr-1

The mammalian microRNA response to bacterial infections

Role in Hepatitis C Virus pathogenesis

Disintegrin-like domain of glycoprotein B regulates Kaposi's sarcoma-associated herpesvirus infection of cells

Upregulation of CD40 expression on endothelial cells infected with human cytomegalovirus

Identification of tissue-specific microRNAs from mouse

MicroRNA expression differences in human hematopoietic cell lineages enable regulated transgene expression

Specific binding of the chemokine platelet factor 4 to heparan sulfate

Human herpesvirus 8 interaction with target cells involves heparan sulfate

Comparative virion structures of human herpesviruses

Multiple-to-multiple relationships between microRNAs and target genes in gastric cancer

Evolution of vertebrate interferon inducible transmembrane proteins

A single DNA response element can confer inducibility by both alpha-and gamma-interferons

Interferon-induced cell membrane proteins, IFITM3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms

Molecular analysis of a human interferon-inducible gene family

IFITM-Family Proteins: The Cell's First Line of Antiviral Defense

The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus

IFITM Proteins Restrict HIV-1 Infection by Antagonizing the Envelope Glycoprotein

The Interferon-induced Transmembrane Proteins, IFITM1, IFITM2, and IFITM3 Inhibit Hepatitis C Virus Entry

IFITM proteins restrict viral membrane hemifusion

The antiviral effector IFITM3 disrupts intracellular cholesterol homeostasis to block viral entry

The antiviral restriction factors IFITM1, 2 and 3 do not inhibit infection of human papillomavirus, cytomegalovirus and adenovirus

Interferon induction of IFITM proteins promotes infection by human coronavirus OC43

Antiviral actions of interferons

Sensitivity of the Epstein-Barr virus transformed human lymphoid cell lines to interferon

Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor confers resistance to the antiproliferative effect of interferon-alpha

Interferon-stimulated genes and their antiviral effector functions

Pattern recognition receptors and inflammation

Retroviral superinfection resistance

Silencing of Interferon-Induced Transmembrane Protein 1 (IFITM1) Inhibits Proliferation, Migration, and Invasion in Lung Cancer Cells

KSHV vCyclin counters the senescence/G1 arrest response triggered by NF-kappaB hyperactivation

3-D Microwell Array System for Culturing Virus Infected Tumor Cells

KSHV-initiated notch activation leads to membrane-type-1 matrix metalloproteinase-dependent lymphatic endothelial-to-mesenchymal transition

Inhibition of Autoimmune Diabetes in NOD Mice by miRNA Therapy

& Berindan-Neagoe, I. MicroRNAs and cancer therapy -from bystanders to major players

B-Raf-dependent expression of vascular endothelial growth factor-A in Kaposi sarcoma-associated herpesvirusinfected human B cells

Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) infection of human fibroblast cells occurs through endocytosis

Epstein-Barr virus BRLF1 inhibits transcription of IRF3 and IRF7 and suppresses induction of interferon-beta

Cellular transcription factor Oct-1 interacts with the Epstein-Barr virus BRLF1 protein to promote disruption of viral latency

Discrimination of herpes simplex virus type 2 strains by nucleotide sequence variations

IFN-alpha is constitutively expressed in the human thymus, but not in peripheral lymphoid organs

RIG-I and IL-6 are negative-feedback regulators of STING induced by double-stranded DNA

A long noncoding RNA signature for ulcerative colitis identifies IFNG-AS1 as an enhancer of inflammation

We sincerely thank Ikenna Okafor and Frank Williams to have conducted the double-blinded qRT-PCR experiments. We thank Dr. Michael Farzan (The Scripps Research Institute, Jupiter, USA) for kindly providing the plasmid, pQCXIP encoding IFITM1. Technical help by Dr. Douglas Weidner with the use of flow cytometer is highly appreciated. We thank Dr. Joseph Pagano (UNC-Chapel Hill, NC) to have provided us with the EBV stock virus. We thank Dr. Blossom Damania (University of North Carolina at Chapel Hill) to have kindly provided us with the BJAB cells. We thank Dr. Adrian Reber, Centers for Disease Control and Prevention, to have read and critiqued this report.

Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-017-18225-w.

The authors declare that they have no competing interests.Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.