IFITM1 expression is crucial to gammaherpesvirus infection, in vivo

The oncogenic gammaherpesviruses, Epstein–Barr virus (EBV) and Kaposi’s sarcoma herpesvirus (KSHV), are etiologically associated with a variety of human cancers, including Burkitt’s lymphoma (BL), Hodgkin lymphoma (HL), Kaposi’s sarcoma (KS), and primary effusion lymphoma (PEL). Recently, we demonstrated KSHV infection of B- and endothelial cells to significantly upregulate the expression of interferon induced transmembrane protein 1 (IFITM1) which in turn enhances virus entry. This is an extension of the above study. In here, we determined EBV infection of cells to trigger IFITM1 expression, in vitro. Silencing IFITM1 expression using siRNA specifically lowered gammaherpesvirus infection of cells at a post binding stage of entry. A natural model system to explore the effect of IFITM1 on gammaherpesvirus infection in vivo is infection of BALB/c mice with murine gammaherpesvirus 68 (MHV-68). Priming mice with siRNA specific to IFITM1 significantly lowered MHV-68 titers in the lung specimens compared to priming with (NS)siRNA or PBS. MHV-68 titers were monitored by plaque assay and qPCR. Taken together, for the first time, this study provides insight into the critical role of IFITM1 to promoting in vivo gammaherpesvirus infections.


Results
Infection of BJAB cells wit γ-herpesviruses induce expression of IFITM1. In a recently concluded study, we demonstrated the ability of KSHV to induce IFITM1 expression during early stages of infection 10 . In the present study, we analyzed the effect of another closely related γ-herpesvirus, EBV, on IFITM1 expression. IFITM1 transcript (Fig. 1A) and protein expression (Fig. 1B) levels were significantly elevated with EBV and KSHV infection of BJAB cells. The expression of IFITM1 increased in virus infected cells as early as 5 min post infection (PI) which was elevated by 10 min and 15 min PI in BJAB, but significantly declined by 30 min PI (Fig. 1B). We observed a similar IFITM1 expression profile during early stages of viral infection in human microvascular dermal endothelial cells (HMVEC-d) cells (Supplemental Fig. 1). We also performed binding assays to decipher if just binding of KSHV and EBV is enough to induce IFITM1 expression in BJAB cells. Our results (Fig. 1C) demonstrate binding of the wild-type or the UV.inactivated virus is not enough to induce IFITM1 expression. The results implicate the ability of γ-herpesviruses to induce the expression of IFITM1 during early stages of infection.

IFITM1 expression is a necessity for γ-herpesvirus infection of cells. In a recently concluded study,
we demonstrated a crucial role for IFITM1 expression in KSHV infection of cells. This was possible by monitoring the expression of ORF50 transcript as a measure of infection. In the current study, we analyzed internalization of the γ-herpesviruses by monitoring the internalized viral DNA ( Fig. 2A) compared to the expression of ORF50 and BRLF-1 transcripts (Fig. 2B). BJAB cells expressing IFITM1 supported a significantly enhanced KSHV and EBV infection compared to those cells that were left untransfected, mock transfected, or transfected with the empty vector. To authenticate the role for IFITM1 in enhancing infection of γ-herpesviruses, we tested the effect of silencing IFITM1 on the internalization of the viruses. Briefly, we first transfected cells with siRNA specific for IFITM1. Northern blotting and Western blotting were performed at 0, 12, 24, and 48 hours after transfection as per the standard protocols to monitor IFITM1 mRNA and protein expression levels, respectively. A maximum IFITM1 mRNA (Fig. 2C) and protein (Fig. 2D) inhibition were observed in cells transfected with siRNA specific to IFITM1 at 12 h post transfection. On the same lines, internalized γ-herpesviruses in cells silenced for the expression of IFITM1 was significantly lower compared to cells that were untransfected or transfected with (NS) siRNA (Fig. 2E). Taken together, the results clearly implicate a role for IFITM1 in enhancing KSHV, and EBV infection of cells.

IFITM1 enhances virus infection at a post-attachment stage.
To enumerate the role of IFITM1 on binding of viruses to target cells, we used untransfected or BJAB cells transfected with siRNA to IFITM1, or (NS) siRNA. These cells were used in binding assays. The binding assay performed on BJAB cells demonstrated that the transfection of cells with (NS)siRNA or siRNA specific to IFITM1 did not block 10 multiplicity of infection (MOI) of KSHV and EBV from binding the target cells (Fig. 3A). Incubating KSHV with heparin but not CSA significantly blocked KSHV and EBV from binding cells (Fig. 3A). We observed identical results when 1 MOI (Supplemental Fig. 6) and 0.1 MOI (data not shown) of KSHV and EBV were used in the above binding assays. This data was confirmed by electron microscopy by monitoring KSHV (Fig. 3B,C) and EBV (Fig. 3B) binding to BJAB cells. Based on these results, we concluded IFITM1 to alter virus infection at a post-attachment stage of entry.
Silencing IFITM1 expression in BALB/c mice lowered MHV-68 infection. MHV-68 is regarded as a substitute for human γ-herpesvirus and has been widely used in in vivo studies 15 . We used mouse siRNA specific to IFITM1 to appreciate the effect of IFITM1 on MHV-68 infection, in vivo, using BALB/c mice. The experimental design is detailed in the schematic (Fig. 4). Priming mice with siRNA specific to IFITM1 significantly lowered expression of IFITM1 in the lungs of the mice compared to (NS)siRNA (Fig. 5A). Our data demonstrates a significant increase in the leukocyte count in mice that were infected with MHV-68 compared to the uninfected mice ( Table 1). The leukocyte count in infected mice that were primed with siRNA specific to IFITM1 was significantly lower compared to those that were primed with PBS or (NS)siRNA. We did not notice atypical lymphoid monocytes in these animals.
MHV-68 infection of mice was monitored by the traditional plaque assay. Plaque assay could detect MHV-68 in lungs of the infected mice (Fig. 5B). MHV-68 infection was significantly inhibited in the mice that were primed with siRNA specific to IFITM1 compared to those that were primed with (NS)siRNA or PBS (Fig. 5B). The results were confirmed by performing qRT-PCR (Fig. 5C). Taken together, silencing the expression of IFITM1 significantly hampered MHV-68 infection of mice.

Discussion
Many different types of cellular sensors can detect viruses and induce the expression of the type I interferons (IFNs)-IFN-α and IFN-β. Type I IFNs bind to the ubiquitously expressed IFNAR (IFN-α/β receptor), activating the JAK/STAT pathway 17,18 . The type I IFN response can induce the expression of hundreds of interferon stimulated genes (ISGs) which primarily serve to limit further virus spread and infection 19 . IFITMs are a family of proteins that have gained popularity as novel antiviral ISGs 20 . IFITMs are a member of the interferon-induced 125-133 aa 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) 21,22 . IFITM proteins are significantly upregulated by type I and II IFNs and are critical for anti-viral innate immune responses 23,24    In the current study, we demonstrated that EBV, another γ-herpesvirus, induces IFITM1 expression during the early stages of infection and it followed an identical pattern as that of KSHV (Fig. 1A,B). Binding of KSHV and EBV to target cells is not sufficient to induce IFITM1 expression (Fig. 1C). We went one step further in demonstrating that over-expressing IFITM1 significantly alters not only the internalization of the γ-herpesviruses ( Fig. 2A) but also the ability to establish infection as monitored by the expression of the respective viral transcripts (Fig. 2B). Also, silencing expression of IFITM1 by specific siRNAs resulted in a significant drop in the number of internalized viral particles (Fig. 2E). IFITM1 promotes γ-herpesvirus infections at a post-binding stage of entry as silencing IFITM1 did not alter the KSHV and EBV binding to the cell surfaces (Fig. 3). KSHV utilize HS as binding receptor 6 . EBV also interacts with HS expressed on epithelial cells via glycoprotein gp150 27 . Generally, speaking, BJAB cells are thought to be resistant to KSHV and EBV primarily because B cells do not express HS 28 . Recent studies determined BJAB cells to express HS 28 and that KSHV and EBV can efficiently infect them 6,29,30 . In our hands, treating EBV with soluble heparin blocked binding to BJAB cells due to one or both of the following reasons: (i) Just like MHV-68 and KSHV, HS may have a role to play in EBV infection of BJAB cells; We are aware of this discrepancy associated with the role of HS in EBV binding and entry. In the current study, soluble heparin was only used as a control and further elaborate studies should be conducted prior to ruling in or out a role for HS in EBV entry into BJAB cells. A natural model system to investigate γ-herpesvirus-host interactions is the infection of mice with MHV-68, a natural pathogen of wild rodents 31,32 . Therefore, to understand the in vivo effects of IFITM1 on γ-herpesviruses, we used MHV-68 as a model virus. Leukocyte counts were increased in response to MHV-68 infection of mice (Table 1). This increase in the leukocyte count was significantly lowered when the mice were primed with siRNA compared to (NS)siRNA and PBS. We did not observe atypical lymphoid monocytes in the mice from all the groups used as it was too early to observe any such changes. Priming mice with siRNA specific to IFITM1 significantly lowered expression of IFITM1 in lungs compared to PBS, (NS)siRNA (Fig. 5A). It was determined by plaque assay and qPCR that a decrease in the expression of IFITM1 resulted in a significant decrease in MHV-68 infection of lungs obtained from the mice (Fig. 5B,C). This study focused primarily in monitoring infection of lungs because intranasal MHV-68 infection of mice results in acute lytic infection of lungs followed by the establishment of lifelong latency 33 . Taken together, we demonstrate for the first time a crucial role for IFITM1 in the in vivo infection of γ-herpesviruses.
The results from this study put forth crucial questions in terms of better understanding the role of IFITM1 in altering entry of γ-herpesviruses. Numerous mechanisms by which IFITM proteins alter virus entry have been proposed and it is not limited to the following: (i) acting as pattern recognition receptors by sensing virus infection and activation of downstream cellular signaling pathways; 23 and (ii) reducing membrane fluidity and curvature, and by possibly disrupting intracellular cholesterol homeostasis 34,35 . Albeit, none of the mechanisms have been confirmed. Future studies will be aimed at delineating that eluding mechanism by which IFITM1 enhance the internalization of the γ-herpesviruses. These studies are crucial because they help us understand the role of ISGs in modulating γ-herpesvirus infection of cells. In addition, the results from this study will benefit in designing IFITM1 targeted therapies to treat cancers 36,37 , and γ-herpesvirus infections including that of HIV 38 and HCV 39 .

Materials and Methods
Cells. Human Burkitt lymphoma B cell line (BJAB) was used in this study. BJAB cells were propagated in phenol red-free RPMI medium (Invitrogen, Carlsbad, CA) containing 10% charcoal-stripped fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA), L-glutamine, and antibiotics 40 . The cells used in this study were negative for mycoplasma as tested by Mycoplasma PCR ELISA (Roche Life Science, Indianapolis, IN).

Virus and mice.
The viruses used in this study were wild-type KSHV 41 , EBV 42 , and MHV-68 43 . Female 6-week-old inbred BALB/c mice supplied by the Faculty of Veterinary Medicine, Brno, Czech Republic were used in this study. We generated ultraviolet (UV) inactivated KSHV (UV.KSHV) and EBV (UV.EBV) as per early studies 10 .

Virus infection of cells, RNA and DNA extraction, and monitoring virus infection. BJAB cells
were infected with 10 multiplicity of infection (MOI) of KSHV and EBV. The cells were left uninfected or infected for 5, 10, 15, and 30 min prior to washing the cells twice in PBS and processed appropriately for RNA and DNA 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. Viral DNA was extracted by isolating total genomic DNA and determining the internalized virus particles by qPCR 44 .  Extracted RNA was used to synthesize cDNA and the expression of ORF50 was monitored by qRT-PCR using specific primers as per earlier studies 41 . Expression of ORF50 was used as a scale to measure KSHV infection of cells. As reported earlier 45 , the lowest limit of detection in the standard samples was 6-60 copies of 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 infection was monitored using specific primers to BRLF1 (homolog of KSHV ORF50) 46  Binding assay. The effect of IFITM1 on KSHV binding to target cells was assessed by PCR detecting the cell-bound KSHV DNA. Briefly, untransfected cells or cells transfected with (NS)siRNA, or siRNA specific to IFITM1 were infected with 10MOI 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 45 . Incubating KSHV with 500 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.
Electron microscopy. EM studies were conducted to appreciate the effect of IFITM1 on virus binding to cells. Briefly, viruses were allowed to bind at 4 °C to BJAB cells that were untransfected, or transiently transfected with IFITM1-specific siRNA, or (NS) siRNA. After 60 min cells were fixed with 2% glutaraldehyde. Thin sections were examined by transmission electron microscopy. The number of virus particles adsorbed on the cell membranes were counted as per procedures outlined in earlier studies 47 . Animal studies. Animal studies using BALB/c mice was performed to understand the effects of IFITM1 on γ-herpesviruses, in vivo. Briefly, five groups of mice (n = 5/group) used in this study were (i) virus free or uninfected; (ii) MHV-68 infected; (iii) IFITM1 siRNA primed + MHV-68 infected; (iv) (NS)siRNA primed + MHV-68 infected; and (v) PBS primed + MHV-68 infected. Mice were primed with PBS, 20 µg/mouse of IFITM1-siRNA, or (NS)siRNA on day 0 by tail vein injection. The uninfected and the MHV-68 infected groups were left untreated. On day 02, only groups (iii), (iv), and (v) were tail vein-injected with IFITM1 siRNA, (NS)siRNA, and PBS, respectively. After this step, animals in groups (ii), (iii), (iv), and (v) were intranasally (i.n) infected with 2 × 10 4 PFU (20 µl) of MHV-68. On day 04, the animals in groups (iii), (iv), and (v) received tail vein injections of IFITM1 siRNA, (NS)siRNA, and PBS, respectively; while animals in groups (i) and (ii) were left undisturbed. On day 06, mice were sacrificed and lungs were collected. Relative IFITM1 gene expression was calculated using the −∆∆ 2 C T method 48 , using β-actin mRNA expression as reference gene and the virus free group as the calibrator. Plaque assay 49 and qPCR 50 was conducted to determine MHV-68 infection of lungs. All animal experiments were performed according to the European Union standards, and fundamental ethical principles including animal welfare requirements were respected. All experiments were done with the approval of State Veterinary and Food Administration of the Slovak Republic (2937/10221).