The lysine-rich intracellular loop and cell type-specific co-factors are required for IFITM3 antiviral immunity in hematopoietic stem cells

The interferon-induced transmembrane protein 3 (IFITM3) inhibits lentiviral gene therapy vector entry into hematopoietic stem cells and can be overcome by Cyclosporine H (CsH), but underlying mechanisms remain unclear. Here, we show that mutating the evolutionarily conserved lysines of the IFITM3 intracellular loop abolishes its antiviral activity without affecting either its localization or its degradation by CsH through non-canonical lysosomal pathways. When confined to the plasma membrane, the lysine-competent IFITM3 lost restriction against VSV-G pseudotyped viral vectors but gained antiviral activity against vectors that fuse directly at the plasma membrane. Interestingly, altering the lysines did not alter IFITM3 homodimerization but impacted higher-order protein complex formation, suggesting loss of interaction with cellular co-factors. In agreement, IFITM3 expression was not sufficient to restrict viral vectors in myeloid K562 cells as opposed to promonocytic THP1 or primary HSC. We exclude the involvement of previously identified factors affecting IFITM3 biology and propose a novel model for IFITM3 restriction that depends on the presence of cellular co-factor(s) that may interact with IFITM3 through the intracellular loop lysine residues. Overall, our work provides significant insight into the mechanisms of action of IFITM3 and CsH that can be exploited for improved gene therapies and broadly acting antiviral strategies.


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Here, we have investigated the molecular determinants of IFITM3-mediated 96 inhibition of viral gene therapy vectors in human hematopoietic cell lines as well as in 97 primary HSPC and addressed how CsH induces its degradation. Our work uncovers 98 the requirement of co-factors and a critical region for IFITM3 antiviral activity and 99 provides insight into the proteolytic pathways involved in CsH-mediated counteraction 100 of this antiviral protein.

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104 CsH degrades IFITM3 through lysosomal pathways 105 We have previously shown that CsH increases LV transduction through transient 106 degradation of IFITM3 in HSPC but the mechanisms remain unclear (6). As 107 Cyclosporins are known to bind cyclophilins (Cyp) (Quesniaux et al, 1987), we firstly 108 assessed whether knock-out of the known targets Cyp A, D or F could affect IFITM3 109 restriction or CsH-mediated inhibition of IFITM3 in THP1 (Fig. 1A-B). However, knock-110 out of Cyclophilins did not affect LV transduction sensitivity to IFNα or CsH ( Fig. 1A-111 B). These data suggest that IFITM3 and CsH-mediated effects in THP1 are 112 independent from Cyclophilins.

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IFITM3 has been reported to impede viral egress from the endo-lysosomal 114 compartments (12). In agreement, we observed that LV pseudotyped with mutant 115 VSV-G glycoproteins that are less efficient in fusing with the endo-lysosomal 116 membranes were severely impaired in THP1 over-expressing IFITM3 and HSPC ( Fig.   117 1C-D). Moreover, these vectors were the ones to benefit the most from CsH that 118 restored their transduction to the levels of those of a LV pseudotyped with wild-type 119 VSV-G envelope. However, altering lipid membrane stability through Amphotericin B 120 did not rescue IFITM3 restriction (Fig.1E) as opposed to previous observations for IAV 121 in the lung epithelial A549 cell line (29, 30), suggesting distinct mechanisms of action 122 in this context. In addition, IFITM3 did not inhibit vectors pseudotyped with 123 glycoproteins fusing directly at the plasma membrane such as BaEV-TR, RD114 or 124 Measles envelopes that remained also insensitive to CsH-mediated enhancement of 125 transduction (Fig. 1E-F-G). Of note, we also confirmed that the non-enveloped vector 7 126 AAV6 remains insensitive to IFITM3 restriction and CsH in HSPC despite entering 127 through endocytosis ( Fig. 1H-I) (6).

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In order to dissect where CsH-mediated degradation of IFITM3 takes place in the 129 cell, we blocked the two best-characterized pathways involved in protein degradation, 130 namely the proteasomal and lysosomal degradation (31). Neither increased 131 transduction nor IFITM3 degradation were affected by addition of the proteasome 132 inhibitor MG132 in THP1 or HSPC ( Fig. 2A-C; 2H). Moreover, in agreement with 133 previous reports (32), proteasomal inhibition enhanced LV transduction in HSPC and 134 the effect was additive to that of CsH (Fig. 2B), likely indicating that MG132 may act 135 on a different restriction block that we predict is independent from IFITM3. In contrast, 136 inhibition of lysosomal degradation by Bafilomycin was sufficient to block both CsH 137 enhancement of transduction as well as IFITM3 degradation ( Fig. 2D-F; 2H-I).
138 Consistent with a block of IFITM3 turnover in the endo-lysosomes we observed a 139 significant increase in IFITM3 protein expression in THP1 and HSPC treated with 140 Bafilomycin (Fig. 2G-I). This effect, in combination with the capacity of Bafilomycin to 141 inhibit acidification of endo-lysosomes, may explain the strong inhibition of LV 142 transduction observed after Bafilomycin exposure ( Fig. 2D-E). Because Bafilomycin 143 inhibits also early and late autophagy we tested whether autophagy inhibition through 144 different compounds may lead to the same observations. However, neither 3MA nor 145 Spautin-1 treatment altered CsH-mediated rescue of transduction in THP1 or HSPC 146 ( Fig. S1 A-D), indicating that lysosomal-specific degradation pathways are involved in 147 the CsH-mediated degradation of IFITM3.

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Noteworthy, we observed that the plasma-membrane confined IFITM3 Y20 149 phosphomutants that we have previously shown to have lost antiviral activity against 150 VSV-G LV in HSPC (6) inhibited plasma-membrane (PM) fusing vectors such as 8 151 BaEV, RD114 and Measles pseudotyped LV (Fig. 3A). However, the Y20 mutant was 152 resistant to CsH degradation (Fig. 3B), which we suggest explains why CsH could not 153 rescue its antiviral activity against PM-fusing LV in THP1 (Fig. 3A). The Y20 mutant 154 insensitivity to CsH could be ascribed to its altered cellular localization (Fig. 3B) (Fig. 4C).

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Ubiquitination of the lysine residues is central for protein degradation (37). In

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As the infected cells can up-regulate the histone demethylase LSD1 to remove 193 Set7 methylation and promote IFITM3 antiviral activity (40) we also tested a LSD1-194 specific inhibitor and noted that it did not affect IFITM3 antiviral activity in THP1 cells 195 over-expressing IFITM3 or primary HSPC ( Fig. S3 A-B). In agreement with specific 196 inhibition, LSD1 protein down-regulation by LSD1 inhibitor lead to increased 197 methylation of the histone H3K9 but did not affect IFITM3 protein expression ( Fig. S3 198 C). Taken together, these data invoke a mechanism of degradation of IFITM3 by CsH 199 that does not depend on any single lysine of IFITM3 nor Set7-mediated methylation. 229 likely excluding a lack of dimerization as the mechanism behind loss of antiviral activity 230 of the lysine-less IFITM3 (Fig.7B). However, we observed a difference in higher order 231 protein complex formation with increased ratio of IFITM3 dimers over higher molecular 232 weight complexes (Fig. 7B), suggesting potential loss of co-factor interactions 233 necessary for IFITM3 antiviral activity. In agreement, over-expression of IFITM3 was 234 not sufficient to restrict LV transduction in the myeloid cell line K562 (Fig.7C). Of note, 235 also IFNα failed to inhibit transduction in these cells despite readily inducing 236 expression of IFN-stimulated genes, including endogenous IFITM3 (Fig. 7D). Defects 237 in IFITM3 localization were excluded by IF analysis (Fig. 7E). Restoring the lysine in 238 position 24 did not re-establish IFITM3 restriction in THP1 cells (Fig. 7F) indicating

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In agreement with a block at the level of the VSV-G hemi-fusion with the endo-267 lysosomes, IFITM3 Y20 phosphomutants that were retained at the plasma-membrane 268 showed loss of antiviral activity against VSV-G LV. Conversely, these mutants gained 269 antiviral activity against LV entering through direct fusion at the cell surface.