key: cord-0297815-e7hzh94e
authors: Hörnich, Bojan F.; Großkopf, Anna K.; Dcosta, Candice J.; Schlagowski, Sarah; Hahn, Alexander S.
title: Interferon-Induced Transmembrane Proteins Inhibit Infection by the Kaposi’s Sarcoma-Associated Herpesvirus and the Related Rhesus Monkey Rhadinovirus in a Cell Type-Specific Manner
date: 2021-07-17
journal: bioRxiv
DOI: 10.1101/2021.07.16.452764
sha: 76a779ecf76cb4bf8e76c46bcc2d28350d757c75
doc_id: 297815
cord_uid: e7hzh94e

The interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral proteins that inhibit the entry of enveloped viruses. We analyzed the effect of IFITMs on the gamma2-herpesviruses Kaposi’s sarcoma-associated herpesvirus (KSHV) and the closely related rhesus monkey rhadinovirus (RRV). We used CRISPR/Cas9-mediated gene knockout to generate A549, human foreskin fibroblast (HFF) and human umbilical vein endothelial cells (HUVEC) with combined IFITM1/2/3 knockout and identified IFITMs as cell type-dependent inhibitors of KSHV and RRV infection in A549 and HFF but not HUVEC. IFITM overexpression revealed IFITM1 as the relevant IFITM that inhibits KSHV and RRV infection. Fluorescent KSHV particles did not pronouncedly colocalize with IFITM-positive compartments. However, we found that KSHV and RRV glycoprotein-mediated cell-cell fusion is enhanced upon IFITM1/2/3 knockout. Taken together, we identified IFITM1 as a cell type-dependent restriction factor of KSHV and RRV that acts at the level of membrane fusion. Strikingly, we observed that the endotheliotropic KSHV circumvents IFITM-mediated restriction in HUVEC despite high IFITM expression, while influenza A virus (IAV) glycoprotein-driven entry into HUVEC is potently restricted by IFITMs even in the absence of interferon. IMPORTANCE IFITM proteins are the first line of defense against infection by many pathogens, which may also have therapeutic importance, as they, among other effectors, mediate the antiviral effect of interferons. Neither their function against herpesviruses nor their mechanism of action are well understood. We report here that in some cells, but not in, for example, primary umbilical vein endothelial cells, IFITM1 restricts KSHV and RRV, and that, mechanistically, this is likely effected by reducing the fusogenicity of the cell membrane. Further, we demonstrate potent inhibition of IAV glycoprotein-driven infection of cells of extrapulmonary origin by high constitutive IFITM expression.

and RRV infection in A549 and HFF but not HUVEC. IFITM overexpression revealed IFITM1 as the 22 relevant IFITM that inhibits KSHV and RRV infection. Fluorescent KSHV particles did not 23 pronouncedly colocalize with IFITM-positive compartments. However, we found that KSHV and 24 RRV glycoprotein-mediated cell-cell fusion is enhanced upon IFITM1/2/3 knockout. Taken 25 together, we identified IFITM1 as a cell type-dependent restriction factor of KSHV and RRV that 26 acts at the level of membrane fusion. Strikingly, we observed that the endotheliotropic KSHV 27 circumvents IFITM-mediated restriction in HUVEC despite high IFITM expression, while influenza 28 A virus (IAV) glycoprotein-driven entry into HUVEC is potently restricted by IFITMs even in the 29 absence of interferon. 30 31 IMPORTANCE 32 IFITM proteins are the first line of defense against infection by many pathogens, which may also 33 have therapeutic importance, as they, among other effectors, mediate the antiviral effect of 34 interferons. Neither their function against herpesviruses nor their mechanism of action are well 35 understood. We report here that in some cells, but not in, for example, primary umbilical vein 36 endothelial cells, IFITM1 restricts KSHV and RRV, and that, mechanistically, this is likely effected 37 by reducing the fusogenicity of the cell membrane. Further, we demonstrate potent inhibition 38 of IAV glycoprotein-driven infection of cells of extrapulmonary origin by high constitutive IFITM 39 expression. 40 41 INTRODUCTION 43 plasma membrane, while IFITM2 and IFITM3 localize to endosomes/lysosomes (5, 8) . 48

The exact mechanism of IFITM-mediated restriction of viral replication is not completely 49 understood. It is, however, clear that restriction mainly occurs at the viral entry stage (3, 9, 10) . 50

According to some reports, IFITMs modify the overall membrane fusogenicity, by modification 51 of the membrane lipid composition and/or the membrane rigidity and thus prevent virus-host 52 membrane fusion (11) (12) (13) (14) , probably causing arrest of the fusion pore opening following 53 hemifusion (11, 12, 15) . Other modes of action such as e.g. recruitment of additional antiviral 54 factors, altered endocytic trafficking or interference with vacuolar ATPase have been postulated 55 as well (reviewed in (16)). 56

The majority of IFITM-restricted viruses are RNA viruses. The interplay of IFITMs with DNA-57 viruses has been studied less extensively and with more ambiguous results. While vaccinia virus 58 and herpes simplex virus (HSV-1) are restricted by overexpression of individual IFITM proteins 59 (17, 18) , human papillomavirus 16 (HPV16) and the non-enveloped adenovirus type 5 are not 60 (19) . Interestingly, for the human cytomegalovirus (HCMV), small interfering RNA (siRNA)-61 mediated IFITM knockdown resulted in reduced infection and disturbed virus assembly (20) . 62

Varying results were obtained for Epstein-Barr virus (EBV), a gammaherpesvirus. While the 63 initial entry of EBV was enhanced by overexpression of IFITM1 (21, 22) , incorporation of 64 IFITM2/3 into viral particles reduced the infectivity of progeny virus, whereas IFITM1 65 incorporation had no effect (23). Together, the literature on IFITM-mediated effects on the 66 158

We next investigated the effect of individual IFITMs through directed expression by retroviral 160 transduction ( Fig. 4 A We excluded HUVEC here, as IFITM1/2/3 knockout was without effect in these cells, despite 164 high IFITM expression, which makes it unlikely that additional overexpression yields meaningful 165 results. 166

Overexpression of IFITM1 in A549 reduced KSHV and RRV infection by over 50%, whereas 167 overexpression of IFITM2 and IFITM3 only resulted in a non-significant reduction (Figure 4 A we observed different subcellular localizations of IFITM1, IFITM2, and IFITM3 in IFN-a-treated 197 A549 (Fig. 5 A) . IFITM2 and IFITM3 partially colocalized with the early endosome marker EEA1 198 and to a larger extent with the late endosome/lysosome marker LAMP1, while IFITM1 localized 199 to the plasma membrane and was distributed more toward the perimeter of the cell. IFITM1 200 was also found colocalized with EEA1 and LAMP1, but not as pronounced as IFITM2/IFITM3. 201

Next, we analyzed colocalization of KSHV particles with IFITMs. We utilized a KSHV_mNeon-202 orf65, which is tagged with mNeonGreen at the capsid protein orf65, to visualize virions in IFN-203 a-treated cells at different timepoints (Fig. 5 B) . KSHV_mNeon-orf65 particles were detectable 204 at the perimeter at the 0-min timepoint and were detected inside the cells from the 30-min 205 timepoint on. Some particles reached the nucleus at the 240-min timepoint. As IFITMs are 206 widely distributed throughout the cell, partial overlap with KSHV_mNeon-orf65 particles was 207 observed for all IFITMs, most prominently at later time points and for IFITM1. While some 208 particles localized to areas of high intensity in the IFITM staining, KSHV_mNeon-orf65 particles 209 were also frequently found in regions with overall lower IFITM signal. These areas were often 210 adjacent to IFITM-positive areas, which might be compatible with the luminal spaces of large 211 vesicles. 212

IFITMs were reported to modulate overall membrane fusogenicity and thereby entry of viral 215 particles (11, 12, 64) . We therefore utilized a cell-cell fusion assay to determine whether KSHV 216 and RRV glycoprotein-mediated fusion activity is modulated by IFITMs. 293T effector cells were 217 transfected with KSHV gH/gL or RRV gH/gL together with RRV gB and a plasmid encoding a 218 VP16-Gal4 transactivator fusion protein. RRV gB was used because KSHV gB does not allow for 219 efficient cell-cell fusion (46). Transfected effector cells were added to IFN-a-treated A549 220 IFITM1/2/3 knockout cells transduced with a lentiviral Gal4-driven TurboGFP-luciferase reporter 221 construct or 293T cells transfected to express IFITM1, IFITM2, or IFITM3, and a Gal4-driven 222

TurboGFP-luciferase reporter construct. Luciferase activity was measured as a readout for 223

Treatment with IFITM-targeting sgRNAs resulted in an increase of KSHV and RRV gH/gL/gB-225 mediated cell-cell fusion compared to non-targeting controls (Fig. 6 A) . Viral glycoprotein 226 expression and IFITM1/2/3 knockout in target cells was confirmed by Western blot (Fig. 6 B) . 227

Under conditions of recombinant overexpression, all three IFITMs were capable of reducing 228 KSHV and RRV gH/gL/gB-mediated cell-cell fusion, with IFITM1 being the most effective ( (18) (19) (20) (21) 23) . Here, we report that human IFITM1 inhibits the entry of 241 KSHV and of the closely related rhesus macaque virus RRV in a cell type-dependent manner. We 242 identified inhibition of membrane fusion as a potential mechanism through which IFITMs can 243 modulate KSHV and RRV infection. 244

Combined knockout of all three IFITMs enabled us to study IFITM-mediated restriction through 245 loss-of-function at expression levels that are induced through IFN signaling and free from 246 potential artefacts through overexpression-induced mislocalization. Our approach revealed that 247 KSHV and RRV infection are enhanced upon IFITM1/2/3 knockout in A549 and HFF but not in 248 HUVEC. This finding may be explained by differences in entry routes that KSHV and RRV utilize 249 to enter these different cell types. KSHV was shown to enter HFF (65) and RRV rhesus fibroblasts 250 (33, 44) via clathrin-mediated endocytosis, whereas KSHV enters HUVEC via macropinocytosis 251 (42). While most viruses that are restricted by IFITMs enter cells via clathrin-or caveolin-252 mediated endocytosis, only Ebola and Marburg viruses are restricted and enter their target cells 253 predominantly via macropinocytosis (reviewed in (66)). However, Ebola and Marburg virus 254 glycoproteins are activated by endosomal cathepsins, which are mainly found in endolysosomal 255 vesicles that also contain IFITMs (reviewed in (67, 68)). In contrast, KSHV might already fuse in 256 acidified IFITM-negative macropinocytotic compartments and thereby avoid IFITM restriction in 257 HUVEC cells. Although IFITM1/2/3 knockout enhanced KSHV and RRV infection in A549 and HFF, 258 the overall contribution to the IFN-mediated block to infection was different. In HFF, the 259 enhancement was mainly observable in IFN-a-treated cells, while in A549 the enhancement was 260 also observable in non-IFN-treated cells. In A549, the IFITM1/2/3 knockout-mediated 261 enhancement practically cancelled out the IFN-a-mediated inhibition of KSHV and RRV 262 infection, similar to what was observed for the highly restricted IAV-LP. Unfortunately, the 263 detailed mechanism of KSHV and RRV entry into A549 cells is presently unknown. Differences in 264 the response to IFITM1/2/3 knockout may very well represent differences in the viral entry 265 routes. Despite minor differences, IFITM1/2/3 knockout similarly impacted KSHV and RRV 266 infection, compatible with a broadly acting mechanism like decreasing membrane fusogenicity. 

glycoprotein-driven entry, and inhibition by IFITM2 and IFITM3 was not observable. This is 283 compatible with our results in colocalization experiments. Several groups reported that IAV 284 colocalized strongly with IFITM3 (15, 53, 73). We were unable to observe a pronounced 285 colocalization of IFITMs with KSHV particles. Rather, KSHV_mNeon-orf65 particles that entered 286 the cell were frequently observed in regions with low IFITM signal. While these findings argue 287 against concentration of IFITMs at viral particles, they would be compatible with indirect 288 mechanisms of action such as rerouting of endocytotic pathways or reduction of membrane 289 fusogenicity. 290

Mechanistically, we found that IFITMs modulate KSHV and RRV glycoprotein-induced 291 membrane fusion at IFN-a-induced levels. Overexpression of IFITM1, IFITM2, and IFITM3 292 revealed that all three IFITMs can in principle reduce the KSHV and RRV glycoprotein-induced 293 cell-cell fusion to a different degree. It should be noted that overexpression of IFITMs leads to 294 abnormal localization, thereby potentially broadening activity. This supports the theory that all 295 IFITMs are, in principle, capable of restricting fusion (6, 16, 74), which might be counteracted by 296 avoidance of IFITM-positive compartments. In line with our experiments, IFITM overexpression 297 was reported to reduce the fusion activity of other viral fusion proteins including the IAV-HA 298 (12, 13, 15 ) and severe acute respiratory syndrome coronavirus 2 spike (75) as well as the 299 glycoprotein of the otherwise non-restricted Lassa virus (15). Although cell-cell fusion does not 300 universally mirror virus-cell fusion (76), our findings support a model of IFITM1 rendering the 301 membrane less fusogenic. A general impact of IFITMs on membrane properties is also 302 supported by a report that IFITMs inhibit trophoblast fusion (64). While our approach of a triple 303 knockout was also intended to identify potential synergism between the three IFITMs, it did not 304 do so. In HFF, IFITM1 might even counteract the mild enhancing effect that IFITM2 had on RRV 305 infection (Fig. 4 B) . Overexpression of IFITM1 was sufficient to effect inhibition with a similar 306 magnitude as the enhancement that was observed after knockout. In light of our results and a 307 recent report that IFITM3 blocks the IAV fusion process through increasing membrane stiffness 308 (13), one might speculate that the three IFITMs exert their inhibitory activity through a similar 309 mechanism at different locations. 310

Entry driven by the HA and NA glycoproteins of IAV, a respiratory pathogen, was far more 311 potently restricted by IFITMs in fibroblasts and endothelial cells, particularly at constitutive 312 expression levels, than in A549 lung epithelial cells. KSHV and likely RRV (77) Endothelial Cell Growth Medium 2 (PromoCell), and iSLK cells, which were maintained in D10 328 supplemented with 2.5 µg/ml puromycin (InvivoGen) and 250 µg/ml G418 (Carl Roth). IFN-a 329 treatment was performed by supplementing the respective culture medium with IFN-a 2b 330 (Sigma; 5000 U/ml). For seeding and subculturing of cells, the medium was removed, the cells 331

were washed with phosphate-buffered saline (PBS; PAN-Biotech), and detached with trypsin 332 (PAN-Biotech). All transfections were performed using polyethylenimine (PEI; Polysciences) at a 333 1:3 ratio (mg DNA/mg PEI) mixed in Opti-MEM (Thermo Fisher Scientific). 334 335

Retroviruses, lentiviruses and lentiviral pseudotypes were produced by PEI-mediated 337 transfection of 293T cells (see Table 2 for plasmids). For retrovirus production, plasmids 338 encoding gag/pol, pMD2.G encoding VSV-G, and the respective pQCXIP-contructs were 339 transfected (ratio 1.6:1:1.6). For production of lentiviruses used for transduction, psPAX2 340 encoding gag/pol, pMD2.G encoding VSV-G and the respective lentiviral construct, Gal4-driven 341 EPKans_forward_mNeon_504-523_ov for the insert, followed by Gibson assembly. 361

KSHV_mNeon-orf65 was generated by inserting the mNeonGreen cassette 5ʹ of the first amino 362 acid of orf65 with the addition of a glycine-serine linker according to the protocol described by 363 Tischer et al. (78) . The recombination cassette was generated using primers mNeon-GS-364

KSHVorf65_for 365 plus mNeon-GS-KSHVorf65_rev and Ax185_ pCNSmNeonGreen_Kana as a template. 366

Infectious KSHV and RRV reporter viruses were produced as described previously (36). See Table  367 3 for oligonucleotide sequences. 368 369

Western blotting was performed as described previously (36) using the respective antibodies 371 (Table 4) . 372 373 CRISPR/Cas9-mediated knockout of immune-related IFITMs 374 IFITM1-, IFITM2-, and IFITM3-knockout cell pools were generated by CRISPR/Cas9-mediated 375 knockout following the protocol described by Sanjana et al. (57) , except that PEI transfection 376 was used. In short, the cells intended for knockout were transduced with lentiviruses harboring 377 the CRISPR/Cas9 gene and sgRNAs targeting IFITM1-3 (sgIFITM1/2/3-a, sgIFITM1/2/3-b) or non-378 targeting sgRNAs (sgNT-a, sgNT-b). For detection of CRISPR/Cas9-mediated knockout, the cells 379 were treated with IFN-a (5000 U/ml) for 16 h. Thereafter, the cells were harvested and 380 subjected to Western blot analysis. Table 4 ) incubation was performed 425 in IF buffer overnight at 4°C. Secondary antibody (see Table 4 

Infection of Lymphoblastoid Cell Lines by Kaposi's 583

Critical Role of Cell-Associated Virus

Lack of Heparan Sulfate Expression in B-Cell 586 Lines: Implications for Kaposi's Sarcoma-Associated Herpesvirus and Murine 587 Gammaherpesvirus 68 Infections

Efficient infection 589 of a human B cell line with cell-free Kaposi's sarcoma-associated herpesvirus

Alpha/Beta Interferon (IFN-α/β)-Independent Induction of 592 IFN-λ1 (Interleukin-29) in Response to Hantaan Virus Infection

Mediated Antiviral Cytokine Expression in Human Endothelial and Epithelial Cells by Up-595 Regulating TLR3 Expression

Differences in Type I 597 interferon response in human lung epithelial cells infected by highly pathogenic H5N1 and low 598 pathogenic H11N1 avian influenza viruses

Hang 600 HC. 2019. IFITM3 directly engages and shuttles incoming virus particles to lysosomes. 3

Interferon 603 induction of IFITM proteins promotes infection by human coronavirus OC43. 18. Proceedings of 604 the

606 Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay

Homology-guided identification of a conserved motif linking the antiviral functions of IFITM3 to 610 its oligomeric state

Herpesvirus DNA Is Consistently Detected in 615 Lungs of Patients with Idiopathic Pulmonary Fibrosis

Genome dynamics of the human embryonic kidney 293 lineage in response 620 to cell biology manipulations. 1

Kaposi's sarcoma-derived 622 cell line SLK is not of endothelial origin, but is a contaminant from a known renal carcinoma cell 623 line

Generation of a doxycycline-inducible KSHV producer cell 625 line of endothelial origin: Maintenance of tight latency with efficient reactivation upon induction

Identification of an endocytic signal essential for the antiviral action of IFITM3: 629 Endocytosis of IFITM3 and its antiviral activity

Antiviral Protein Interferon-inducible Transmembrane Protein 3 (IFITM3) Dually Regulates Its 632 Endocytosis and Ubiquitination

Interferon-induced transmembrane proteins inhibit cell fusion mediated by 635 trophoblast syncytins

EphrinA2 Regulates Clathrin Mediated KSHV Endocytosis in Fibroblast 638 Cells by Coordinating Integrin-Associated Signaling and c-Cbl Directed Polyubiquitination

IFITMs Restrict the Replication of 641

Multiple Pathogenic Viruses

Filovirus Entry: A Novelty in the Viral Fusion 643 World. Viruses

Cysteine Cathepsins and their 645 Extracellular Roles: Shaping the Microenvironment

Rhesus macaque IFITM3 gene polymorphisms and SIV infection. 3

Nonhuman Primate IFITM Proteins 649 Are Potent Inhibitors of HIV and SIV

Evolution of vertebrate 651 interferon inducible transmembrane proteins

Evolutionary Dynamics of the Interferon-653 Induced Transmembrane Gene Family in Vertebrates

IFITM3 Clusters on Virus Containing 655

Endosomes and Lysosomes Early in the Influenza A Infection of Human Airway Epithelial Cells. 656 6

Current Progress on Host Antiviral 658 Factor IFITMs

661 Syncytia formation by SARS-CoV-2-infected cells

SARS-CoV-2 and SARS-CoV Spike-Mediated Cell

Rhesus Monkey 667 Rhadinovirus Isolated from Hemangioma Tissue. Microbiol Resour Announc 9. 668 78. Tischer BK, von Einem J, Kaufer B, Osterrieder N. 2006. Two-step red-mediated 669 recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli

The graph shows individual data points representing averaged values for 709

YFP + cells of either two non-targeting (sgNT-a, sgNT-b) or IFITM1/2/3 knockout 710 (sgIFITM1/2/3-a, sgIFITM1/2/3-b) transduced cells and floating bars representing the mean 711 averaged from four independent experiments for A549 and HFF (A,B) and three independent 712 experiments for HUVEC (C). Infections for each single experiment were performed in triplicates 713 for each condition

Statistical 715 significance was determined by two-way ANOVA, p-values were corrected for all possible 716 multiple comparisons within one family by Tukey's method

Representative Western blots (A-C, right panel) of IFITM-718 knockout (sgIFITM1/2/3-a or sgIFITM1/2/3-b) or control (sgNT-a or sgNT-b) cells treated with 719

U/ml) or H2O. Indicated IFITM expression was detected with antibodies shown in 720

Fig. 1A; GAPDH served as loading control

A) A549, (B) HFF, (C) 293T and (D) SLK cells were transduced with pQCXIP-constructs to express 725

IFITM1-3 or pQCXIP (empty vector). (A-D, left panel) IFITM overexpressing cells were infected 726 with KSHV-GFP, RRV-YFP, IAV lentiviral pseudotype (IAV-LP) or MLV lentiviral pseudotype

Infection was measured using flow cytometry to detect expression of the fluorescent 728 reporter genes. The data shows values normalized to pQCXIP empty vector, which was set to 729

Representative Western blots 733 (A-C, right panel) of IFITM-overexpressing cells. Expression of myc-tagged IFITMs was 734 determined using anti-myc antibody

A) Confocal microscopy images of IFN-⍺-treated (5000 U/ml) A549 cells stained with IFITM1

Co-staining was performed with antibodies to EEA1

LAMP1, or phalloidin conjugate (yellow) and Hoechst (blue). (B) Confocal microscopy images of 741

Staining was 742 performed using IFITM1, IFITM2, or IFITM3 antibody (magenta) and Hoechst (blue). The Scale 743 bars represents 10 µm

Effector cells (293T transfected with either empty vector (eV) or 747 expression plasmids for the indicated viral glycoproteins together with Vp16-Gal4 expression 748 plasmid) were added to target cells

After 48 h, luciferase activity was measured. Values 751 were normalized to the mean of the two non-targeting controls sgNT-a and sgNT-b (sgNT-a/b), 752 which was set to 100, for each experiment. Error bars represent standard error of the mean of 753 four independent experiments, each performed in triplicates. Statistical significance was 754 determined by two-way ANOVA; p-values were corrected for multiple comparisons by Dunnet's 755 method

The expression of proteins in 293T effector and A549 target cells after co-cultivation was 757 analyzed by Western blot from lysates harvested for determination of luciferase activity shown 758 in (A) using the indicated antibodies

After 48 h, luciferase activity was measured. Values were 763 averaged from three independent experiments, each performed in triplicates. The data was 764 normalized to empty vector control pQCXIP, which was set to 100, error bars represent the 765 standard deviation. Statistical significance was determined by two-way ANOVA, p-values were 766 corrected for multiple comparisons by Dunnet's method

D A549 cells were transduced with a lentiviral vector encoding Cas9 and sgRNAs shown in Figure 769 2. IFITM-knockout (sgIFITM1/2/3-a, sgIFITM1/2/3-b) or control cells (sgNT-a and sgNT-b) treated

U/ml) or H2O (control) were stained for cell surface expression of indicated 771 proteins. The graph shows values for the mean fluorescence intensity fold over isotype control 772 averaged from two non-targeting (sgNT-a, sgNT-b) or IFITM1/2/3 knockout

sgIFITM1/2/3-b) transduced cells from one representative experiment performed in triplicates

Error bars represent the standard deviation. Statistical significance was determined by two-way 775

ANOVA, p-values were corrected for multiple comparisons by Tukey's method (p>0.05, ns