Constitutive TRIM22 expression within the respiratory tract identifies tissue-specific and cell-type dependent intrinsic immune barriers to influenza A virus infection

We hypothesized that increased expression of antiviral host factors at portals of viral entry may protect exposed tissues from the constant threat of invading pathogens. Comparative transcriptomic analysis identified the broad-acting restriction factor TRIM22 (TRIpartite Motif 22) to be among the most abundantly expressed antiviral host factors in the lung, a major portal of entry for many respiratory pathogens. This was surprising, as TRIM22 is currently considered to be an interferon stimulated gene (ISG) product that confers protection following the activation of pathogen-induced cytokine-mediated innate immune defences. Using human respiratory cell lines and the airways of rhesus macaques, we experimentally confirmed high levels of constitutive TRIM22 expression in the lung. In contrast, TRIM22 expression in many widely used transformed cell lines could only be observed following immune stimulation. Endogenous levels of TRIM22 in non-transformed cells were sufficient to restrict human and avian influenza A virus (IAV) infection by inhibiting the onset of viral transcription independently of cytokine-mediated innate immune defences. Thus, TRIM22 confers a pre-existing (intrinsic) tissue-specific immune barrier to IAV infection in the respiratory tract. We investigated whether the constitutive expression of TRIM22 was a characteristic shared by other ISGs in human lung tissue. Transcriptomic analysis identified a large group of ISGs and IAV immuno-regulatory host factors that were similarly enriched in the lung relative to other mucosal tissues, but whose expression was downregulated in transformed cell-lines. We identify common networks of immune gene downregulation which correlated with enhanced permissivity of transformed cells to initiate IAV replication. Our data highlight the importance of tissue-specific and cell-type dependent patterns of pre-existing immune gene expression in the intrinsic intracellular restriction of IAV; findings highly relevant to the immune regulation of many clinically important respiratory pathogens. Author Summary The respiratory tract is a major portal of virus entry for many clinically important viruses, including seasonal and pandemic influenza A virus (IAV). We reasoned that cells within the respiratory tract might differentially express antiviral host factors to protect against the constant challenge of viral infection. We found the broad-acting antiviral protein TRIM22, conventionally regarded as an interferon stimulated gene (ISG) product upregulated in response to virus infection, to be constitutively expressed to high levels in the lung. We found that constitutive expression of TRIM22 restricted the initiation of human and avian IAV infection independently of cytokine-mediated innate immune defences. We identified pre-existing tissue-specific and cell-type dependent patterns of constitutive immune gene expression that strongly correlated with enhanced resistance to IAV replication from the outset of infection. Importantly, we show that these constitutive patterns of immune gene expression are lost or downregulated in many transformed cell lines widely used for respiratory virus research. Our data highlight the importance of pre-existing tissue-specific and cell-type dependent patterns of constitutive antiviral gene expression in the intracellular restriction of respiratory viral pathogens not captured in conventional cell culture model systems of infection.

151 As the respiratory tract is a major portal for virus entry, we hypothesized that cells in the 152 respiratory mucosa might express antiviral proteins to higher levels than cells in less exposed 153 locations, thereby creating localized pre-existing (intrinsic) immune barriers to virus 154 infection. We initially explored this hypothesis using RNA-seq data and protein expression  Table). Analysis of RNA-seq data 169 obtained by the GTEx project independently confirmed TRIM8, 22, and 28 to be the most 170 abundantly expressed TRIMs in human lung tissue (S1 Fig, S1A Table), with the highest 171 levels of TRIM22 expression observed in the spleen and lung (S1 Fig, S1C Table). Together, 172 these data demonstrate that TRIM22 transcript levels show tissue-specific patterns of gene 173 expression and to be enriched within the lung relative to other tissues or TRIM family 174 members. Analysis of HPA immunohistochemistry (IHC) expression records demonstrated 175 the nasopharynx and bronchus to be among tissues with the highest levels of TRIM22 176 expression (Fig 1C, D). These relatively high levels of tissue-specific protein expression 177 suggest that TRIM22 could make a substantial contribution to a pre-existing and localized 178 intrinsic immune barrier to respiratory airway infection. 179 However, the above data contrast with many previous studies of transformed cultured 184 detectable in unstimulated cells by immunofluorescence (Fig 1E), showing the same 185 predominantly nuclear localisation observed by IHC in respiratory epithelia ( Fig 1C). The 186 addition of IFN-β caused an intense upregulation of the ISG Mx1 at both the transcript and 187 protein level. In contrast, TRIM22 expression in the same cells was only increased at the 188 transcript level and not detectably increased at the protein level (Fig 1F-H 290 Comparing the effects of Ruxo (4 M) or DMSO (carrier control) treatment on the relative . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted June 21, 2019. ; https://doi.org/10.1101/679159 doi: bioRxiv preprint 291 plaque titre of IAV in TRIM22 depleted or control MRC5t cells showed that TRIM22 292 depletion caused the same increase in relative plaque titre regardless of the inhibition of JAK-293 STAT signalling ( Fig 4B). Thus, constitutively expressed TRIM22 restricts IAV replication 294 independently of pathogen-induced cytokine-mediated innate immune defences.

295
As WSN is a highly laboratory-adapted strain, we tested whether constitutively 296 expressed TRIM22 was effective against other IAV strains using an immunofluorescent  318 However, we were unable to detect any alteration of NP accumulation caused by TRIM22 in 319 HEK 293T cells using an equivalent assay (Fig 6C, D). Nor were we able to detect any 320 difference in genome stability between TRIM22 depleted or control MRC5t cells within the 342 Having identified TRIM22 to be a constitutively expressed ISG product within the 343 respiratory tract that confers protection to IAV infection (Fig 1, 3 Table) Table), several were downregulated in all three (defined as core) or in two 384 (defined as shared) transformed cell lines (Fig 8B, C). Importantly, the profile of ISG 385 expression in unstimulated HBEC3 cells for this subset of ISGs was similar to that observed not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted June 21, 2019. ; https://doi.org/10.1101/679159 doi: bioRxiv preprint 390 gene set to show pathway enrichment for defence response to virus and IAV infection (Fig   391 8D). Collectively, these data demonstrate that transformed cells display lineage-specific 392 patterns of constitutive ISG expression, with a significant number of ISGs being 393 downregulated relative to HBEC3 cells or lung tissue.

394
Having identified a subset of constitutively expressed ISGs known to restrict IAV to 395 be downregulated in transformed cells (Fig 8), we extended our analysis to determine 396 whether other immune system-related genes were downregulated relative to HBEC3 cells 397 (HBEC3 ≥ 5-fold change; blue circles in Fig 9A; S4A Table).  Table).
402 STRING analysis identified a significant degree of network connectivity between these   Table) or lung tissue. Consistent with both ISG and immune system . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted June 21, 2019. ; https://doi.org/10.1101/679159 doi: bioRxiv preprint 415 profiling (Fig 8C, 9B, respectively), many of these genes were downregulated in two or more 416 transformed cell lines and showed significant network connectivity (29 of 39 genes; Fig   417 10D). The expression profiles of a subset of these proteins were tested in unstimulated cells 418 by western blotting, which confirmed that UBA7, TRIM22 (positive control), IFITM1, 419 GBP1, IFIH1 and TLR3 were expressed to significantly lower levels in A549 cells relative to 420 HBEC3 cells (Fig 10E, F). In order to determine if the disruption of this immune system 421 network influenced IAV replication, we compared the relative plaque titre of IAV in HBEC3 422 and A549 cells to that of MDCK cells. Similar to diploid lung fibroblasts (Fig 2G, H), human 423 bronchial epithelial cells were highly restrictive to the initiation of IAV plaque formation 424 relative to MDCK cells (≥ 70-fold) or A549 cells (≥ 30-fold) (Fig 9G, H). Ruxolitinib 425 inhibition of JAK-STAT signalling did not influence the initiation of IAV plaque formation 426 in any of the cell-types examined (Fig 9I), although a significant increase in plaque diameter 427 could be observed in each cell-type (Fig 9J). Thus, pharmacological inhibition of cytokine-

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Our initial hypothesis that cells in the respiratory mucosa might differentially express 455 antiviral proteins at higher levels than in cells at less exposed locations led to the 456 identification of TRIM22 to be amongst the most abundantly expressed TRIM proteins in 457 lung tissue and non-transformed cells of lung origin (Fig 1, 2A, S1, S6 Fig). This high level 458 of constitutive TRIM22 expression contrasts with many previous studies, which have 459 reported TRIM22 to be strongly upregulated as an effector ISG in primary lymphocytes and 460 transformed cell lines in response to virus infection or immune stimulation (Fig 2, S3 Fig;   461 [54, 55]). We note that TRIM22 (formerly known as Staf50) has been shown to be 462 upregulated by p53 and its expression has been found to correlate with cell differentiation . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted June 21, 2019.

479
We demonstrate that multiple immune regulators, known to influence the replication 480 of a wide variety of viral pathogens, are downregulated in transformed cell lines widely used 481 for respiratory virus research (Fig 8-10). The loss or downregulation of these constitutively 482 expressed host factors correlates strongly with enhanced permissivity of these cell-types to 483 the initiation of IAV replication leading to plaque formation (Fig 2G, 10H). Many of these 484 immune genes, although downregulated in a lineage-specific manner (Fig 9), share common 485 networks of immune-system regulation known to influence IAV replication (Fig 8C, 9C, 486 10D). These observations may account for much of the gene-specific variability, but 487 interrelated pathway connectedness, observed between genome-wide RNA interference . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted June 21, 2019. ; https://doi.org/10.1101/679159 doi: bioRxiv preprint 488 screens that utilized carcinoma model systems to identify host factors that influence IAV 489 replication [79][80][81]. Collectively, our data highlight the importance of utilizing more 490 physiologically relevant cell culture model systems to improve experimental reproducibility 491 between independent groups and research fields.

492
Using a cell culture system that retained the constitutive expression of TRIM22 493 observed at the natural site of infection (Fig 1, 2), we corroborate previous reports that 494 TRIM22 acts as a restriction factor to inhibit IAV replication (Fig 3F-H; [55, 61]).
495 Importantly, we show that constitutive expression of TRIM22 is sufficient to restrict the 496 initiating cycle of both human and avian IAV replication from the outset of infection by 497 inhibiting the efficient onset of viral transcription (Fig 5, 6). We show that pharmacological 498 inhibition of cytokine-mediated JAK-STAT signalling did not reduce the ability of 499 endogenous TRIM22 to restrict IAV infection (Fig 4; [66 , which can be further upregulated in response to cytokine 507 signalling in a manner dependent on the pre-existing basal levels of endogenous expression in 508 a given cell-type (Fig 1F-H, 2A, B, S3 Fig). Secondly, TRIM22 demonstrates that intrinsic 509 immune defences can be upregulated in a tissue-specific manner. Like TRIM32 and TRIM41 510 [21, 22], TRIM22 can restrict the initiation of IAV infection (Fig 5, 6), but unlike these 511 TRIM proteins TRIM22 is upregulated in a tissue-specific manner, being enriched within the 512 lung relative to other tissues (Fig 1A, B, 7A, B). Collectively, these observations point to a . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted June 21, 2019. ; https://doi.org/10.1101/679159 doi: bioRxiv preprint 513 series of distinct ways in which constitutive levels of immune gene expression can influence 514 the outcome of IAV replication independently of pathogen-induced host defences. Further 515 investigation is warranted to determine the accumulative and strain-dependent effects of such 516 intrinsic barriers to IAV infection. 517 We found that constitutively-expressed TRIM22 could restrict the replication of 518 multiple human (H1N1, including WSN) and avian (H3N2 and H7N1) strains of IAV (Fig 5).
519 These data contrast with recent studies in transformed cells [61], which lack constitutive 520 TRIM22 expression (Fig 2A, S3; (Fig 3F-H) independently of detectible NP degradation, either alone or in the context of 526 incoming vRNPs (Fig 6C-E). While we cannot discount a role for ubiquitination in the 527 TRIM22 mediated restriction of IAV, we show endogenous levels of TRIM22 are sufficient 528 to restrict de novo NP expression by inhibiting the onset of viral transcription (Fig 5, 6). not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted June 21, 2019. ; https://doi.org/10.1101/679159 doi: bioRxiv preprint 538 unprecedented, as we have recently shown that PML (TRIM19) plays spatiotemporally 539 distinct roles in the regulation of intrinsic and innate immune defences to HSV-1 infection 540 [19,82]. Further biochemical investigation will be required to determine whether TRIM22 541 has differential modes of substrate-targeting dependent on the kinetics of infection or cellular 542 immune status.

543
A surprising discovery in our study was the identification of enriched levels of 544 constitutive ISG expression in human lung tissue relative to that of other mucosal and non-545 mucosal tissues (Fig 7). Our analysis suggests that human lung tissue could confer 546 heightened levels of pre-existing immune protection against multiple respiratory viruses 547 immediately upon pathogen entry. Importantly, this was not due to the elevated expression of 548 all ISGs, but rather tissue-specific profiles of individual ISG expression (Fig 7A-E, S2A 549 Table). These data demonstrate that human tissues confer distinct profiles of ISG expression 550 in a tissue-dependent manner which may confer enhanced protection at exposed surfaces.  (Fig 7B). Further work is required to determine how such 561 tissue-specific signatures of pre-existing antiviral gene expression influence the initiation and 562 outcome of respiratory virus infection.
. CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted June 21, 2019. ; https://doi.org/10.1101/679159 doi: bioRxiv preprint

563
In conclusion, we identify pre-existing tissue-specific and cell-type dependent 564 patterns of constitutive immune gene expression which confer a significant intracellular 565 immune barrier to IAV replication from the outset of infection and independently of 566 pathogen-induced cytokine-mediated innate immune defences. These intrinsic barrier 567 defences are downregulated in many transformed cell lines currently used for respiratory 568 virus research, which share common networks of immune system disruption relevant to the 569 immune regulation of many respiratory pathogens.

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. CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted June 21, 2019. . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted June 21, 2019. ; https://doi.org/10.1101/679159 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted June 21, 2019. ; https://doi.org/10.1101/679159 doi: bioRxiv preprint . CC-BY 4.0 International license available under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which was this version posted June 21, 2019. ; https://doi.org/10.1101/679159 doi: bioRxiv preprint