The FDA-approved drug Nelfinavir inhibits lytic cell-free transmission of human adenoviruses

Adenoviruses (AdVs) are prevalent and give rise to chronic and recurrent disease. The human AdV (HAdV) species B and C, such as HAdV-C2, C5 and B14, cause respiratory disease, and constitute a health threat for immuno-compromised individuals. HAdV-Cs are well known for lysing cells, owing to the E3 CR1-β-encoded adenovirus death protein (ADP). We previously reported a high-throughput image-based screening framework and identified an inhibitor of HAdV-C2 multi-round infection, Nelfinavir Mesylate. Nelfinavir is the active ingredient of Viracept, an FDA-approved inhibitor of the human immuno-deficiency virus (HIV) aspartyl protease, and used to treat acquired immunodeficiency syndrome (AIDS). It is not effective against single round HAdV infections. Here, we show that Nelfinavir inhibits the lytic cell-free transmission of HAdV, indicated by the suppression of comet-shaped infection foci in cell culture. Comet-shaped foci occur upon convection-based transmission of cell-free viral particles from an infected cell to neighbouring uninfected cells. HAdV lacking ADP was insensitive to Nelfinavir, but gave rise to comet-shaped foci indicating that ADP enhances but is not required for cell lysis. This was supported by the notion that HAdV-B14 and B14p1 lacking ADP were highly sensitive to Nelfinavir, although HAdV-A31, B3, B7, B11, B16, B21, D8, D30 or D37 were less sensitive. Conspicuously, Nelfinavir uncovered slow-growing round-shaped HAdV-C2 foci, independent of neutralizing antibodies in the medium, indicative of non-lytic cell-to-cell transmission. Our study demonstrates the repurposing potential of Nelfinavir with post-exposure efficacy against different HAdVs, and describes an alternative non-lytic cell-to-cell transmission mode of HAdV. Graphical Abstract Figure 1.


Introduction 41 42
Adenovirus (AdV) was first described in 1953 by Rowe and co-workers as a cytopathologic agent 43 isolated from human adenoids (Rowe et al., 1953). More than 100 human AdV (HAdV) genotypes 44 have since been characterized by molecular genetics or serology and grouped into seven species 45 (Harrach et  . The N-terminus of ADP is lumenal and the C-terminus protrudes into the cytosol 114 . Following post-translational modifications, ADP is transported to the inner 115 nuclear membrane, where the N-terminus is intruding into the nucleus (Georgi and Greber,116 submitted). At late stages, when capsid assembly in the nucleus has commenced ADP expression 117 is boosted Wold et al., 1984 Here, we report that Nelfinavir is an effective inhibitor of HAdV lytic egress. The identification 122 process of Nelfinavir is described in an accompanying paper using an imaging-based, high 123 content screen of the Prestwick Chemical Library (PCL) comprising 1,280 mostly clinical or 124 preclinical compounds (Georgi et al., 2020;Yakimovich et al., 2015). Nelfinavir is the off-patent 125 active pharmaceutical ingredient of Viracept, FDA-approved, which inhibits the human immuno-126 deficiency virus (HIV) protease (Kaldor et al., 1997). The work here documents the repurposing 127 potential of Nelfinavir, which is effective against a spectrum of HAdV types in a post exposure 128 manner. Nelfinavir is partly, but not exclusively, active against ADP-encoding HAdV types, and 129 uncovers the appearance of round-shaped plaques, which arise upon non-lytic cell-to-cell viral 130 transmission. 131

Assessment of HAdV infectivity of HAdV-C5 ±Nelfinavir 343
Fifteen thousand A549 cells were seeded per 96-well in full DMEM and allowed to attach over 344 night at standard cell culture conditions. The next day, the medium was replaced by double CsCl-345 purified HAdV-C5 ±Nelfinavir virus stocks at 50 to 0.001 pg / well of BCA-based viral protein 346 concentration and incubated at standard cell culture conditions. Cells were fixed at 52 hpi, stained 347 for HAdV hexon expression and imaged following the procedure described under Image-based 348 plaque assay. Images were quantified using Plaque2.0 (Yakimovich et al., 2015). Nuclei were 349 segmented based on Hoechst signal. Infected cells were segmented based on hexon 350 immunofluorescence staining signal.

Quantification of infectious progeny production 367
Four hundred and eighty thousand A549 cells were seeded per 6-well dish and inoculated with 368 1,100 pfu HAdV-C2-dE3B-GFP / well for 1 h at 37°C, washed with PBS and detached by trypsin 369 digestion. Infected cells were centrifuged and resuspended in fresh medium to remove any 370 unbound input virus. Cells were seeded at 180,000 cells / 12-well in medium supplemented with 371 1.25, 3 or 10 µM Nelfinavir or the respective DMSO solvent control. Viral progeny in the cell 372 monolayer and supernatant was harvested at the indicated time pi by three freeze / thaw cycles.

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The lysates were cleared by centrifugation and stored at 4°C until titration on naive A549 cells.

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PFA-fixed, Hoechst-stained cells were imaged at 44 hpi using a 4x objective on an 375 epifluorescence IXM-XL (Molecular Devices, San Jose, USA). GFP-positive infected cells were 376 classified based on median nuclear GFP intensity using automated image analysis by CellProfiler 377 (Carpenter et al., 2006 trations, comparable to the known HAdV nucleoside analogue inhibitor 3'-deoxy-3'-fluorothy-475 midine (DFT, Figure 1A, 1B). Dequalinium dichloride, Aminacrine and Thonzonium bromide were 476 excluded from further analyses due to toxicity (Georgi et al., 2020), and potential mutagenic 477 effects (Topal, 1984). Long-term incubations of uninfected A549 cells with Nelfinavir up to 115 h 478 showed median toxicity TC50 of 25.7 µM, as determined by cell impedance measurements using 479 xCELLigence ( Figure 1C), consistent with presto-blue assays and counts of cell nuclei 480 (Supplementary Figure 1A,  The therapeutic index 50 (TI50) of Nelfinavir was 27.1 ( Figure 1D Nelfinavir had no effect on GFP or hexon expression at the tested concentrations, while the 492 formation of fluorescent plaques was completely inhibited (Figure 2A, and Figure 1D). This result 493 was in agreement with the notion that Nelfinavir did not affect the replication of the HAdV-C5  Figure 1B). 500

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To test if Nelfinavir affected virion maturation, we analysed purified virions by SDS-PAGE and 502 Western blotting against proteins pVI/VI and pVII/VII using previously characterized antibodies. 503 There was no evidence for increase of precursor VI or VII (pVI or pVII) in HAdV-C5 from Nelfinavir-504 treated cells, in contrast to temperature-sensitive (ts) 1 particles, which lack the L3/p23 protease 505 due to the point mutation P137L in p23 (Imelli et al., 2009) ( Figure 2C). This showed that Nelfinavir 506 did not affect the proteolytic maturation of the virus by the L3/p23 cysteine protease. In 507 accordance, purified HAdV-C5 from Nelfinavir-treated cells attached to naive A549 cells and gave 508 rise to viral gene expression as effectively as control HAdV-C5 particles ( Figures 2D, 2E). 509 Together, these results indicate that Nelfinavir does not affect the production of infectious virions 510 in single round infections. 511 512

Nelfinavir inhibits HAdV-C egress 513
We investigated the kinetics of HAdV-C2-dE3B-GFP production and the release to the We detected strongly reduced numbers of infected nuclei and plaques in cells treated with 3 µM 532 Nelfinavir at infection concentrations up to 100 pfu/well at 95 hpi, ( Figure 3D). Remarkably, HAdV-533 C2-dE3B-GFP formed delayed plaques in presence of Nelfinavir, starting at 4 dpi ( Figures 3E,  534 3F). These late plaques showed a strikingly round morphology, which was calculated to be 535 significantly different from the comet-shaped plaques early in infection of control cells ( Figure 3G). 536 The direction of the comet tail of lytic plaques can be aligned by tilting of the incubation plate 537 (Yakimovich et al., 2012). Thereby, the cell monolayer is positioned non-orthogonally to the vector 538 of thermal convection flux of the liquid cell culture medium. While the direction of the comet-539 shaped plaques could be aligned using this method in the non-treated infections, the late 540 Nelfinavir plaques remained mostly round ( Supplementary Figures 2A-C). Moreover, there was 541 no correlation between the size of the plaques and their roundness irrespective of Nelfinavir up 542 to 7 dpi, demonstrating that the round plaques did not change morphology over time (Supplemen-543 tary Figure 2D). Collectively, the data indicate that virus transmission in presence of Nelfinavir is 544 not driven by the bulk current of cell free medium. 545 546

ADP expression, as indicated by immunofluorescence and Western blot experiments (Supple-551
mentary Figure 3A, 3B). HAdV-C2-dE3B-GFP-dADP formed particles indistinguishable from 552 HAdV-C2-dE3B-GFP, as indicated by negative stain EM (Supplementary Figure 3C). HAdV-C2-553 dE3B-GFP-dADP showed a delayed onset of plaque formation by about 1 day, compared to 554 HAdV-C2-dE3B-GFP ( Figure 4A). These data are in agreement with previous kinetic studies with 555 the ADP deletion mutant HAdV-C dl712 ) (see also Supplementary Figure  556 3A). HAdV-C2-dE3B-GFP-dADP plaques were comet-shaped, albeit their comet-heads appeared 557 bigger and more dense ( Figure 4A). While the parental virus was highly sensitive to Nelfinavir, 558 HAdV-C2-dE3B-GFP-dADP required much higher concentrations of the compound to show 559 inhibition of plaque formation ( Figure 4B, Supplementary Table 2). In accordance, the ADP-560 deleted virus induced cell death independent of Nelfinavir, unlike the ADP-expressing virus, as 561 concluded from cell impedance measurements with xCELLigence ( Figure 4C in (Supplementary Figures 3F to 3H). Together, the results show that the selective antiviral effects 569 of Nelfinavir are more cell-type dependent in case of HAdV lacking ADP than in ADP-expressing 570 viruses, and the effects are comparatively small for viruses lacking ADP. 571 572 Finally, we performed immunofluorescence experiments with HAdV-C2-dE3B-GFP-infected A549 573 cells at 44 hpi ( Figure 4E). Under non-perturbed conditions, ADP accumulated in cytoplasmic foci 574 and the nuclear envelope. Nelfinavir treatment did not affect the overall ADP expression levels 575 nor the amount of ADP in the nuclear periphery, including the nuclear envelope, but completely 576 abolished the cytoplasmic ADP foci as indicated by granularity quantifications ( Figure 4E, right 577 graph). Intriguingly, Tollefson and co-workers observed earlier that ADP lacking lumenal O-578 glycosylation sites did not localize to large cytoplasmic granules and the corresponding HAdV-C 579 mutant pm734.4 was non-lytic . We speculate that the localization of ADP against HAdV-C2 added to the cell culture medium suppressed the comet-shaped plaques of 593 HAdV-C2-dE3B-GFP, and yielded confined, predominantly round-shaped infection foci 4 dpi, akin 594 to Nelfinavir-treated infections ( Figure 5B). 595

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To test if round-shaped infection foci (plaques) occurred in regular HAdV-C2-dE3B-GFP 597 infections, we analysed A549 cells infected with less than 1 plaque forming unit(s) (pfu) per well 598 in 160 wells of 96-well formats up to 8 dpi. Thirty three wells developed a single plaque, and 24 599 of them contained fast emerging comet-shaped plaques ( Figure 5C, upper panel), while nine 600 developed delayed round plaques starting 6 dpi ( Figure 5D, lower panel). The originally infected 601 cell (indicated by the pink arrow), which gave rise to the comet-shaped plaque, disappeared 602 between 2 and 3 dpi. In contrast, the infected cell giving rise to the round plaque (orange arrow) 603 remained GFP-positive and apparently viable when the surrounding cells were infected. These 604 data suggest that HAdV-C2 utilizes both lytic and non-lytic transmission, the former involving cell-605 free transmission, and the latter cell-associated transmission. 606 607

Nelfinavir has a broad anti-HAdV spectrum 608
We finally assessed the inhibition breadth of Nelfinavir against various HAdV types from species dishes for classical plaques ( Figure 6B). Viruses that were highly susceptible to Nelfinavir 627 (exhibiting high TI50 values) formed exclusively comet-shaped plaques. Viruses with low TI50 628 values, such as A31, B11 or D37 had a high fraction of round plaques, even when infected with 629 more than 1 pfu / well. This demonstrates that the slowly growing round infection foci observed in 630 fluorescent microscopy gave similarly shaped lesions due to cytotoxicity, akin to the lytic comet-631 shaped foci. We conclude that HAdV types employ lytic cell-free and non-lytic cell-to-cell trans-632 mission modes and give rise to different plaque phenotypes. 633

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A phenotypic screen of the PCL identified Nelfinavir as a potent, post-exposure inhibitor of HAdV-636 C2-dE3B-GFP plaque formation in cell culture (Georgi et al., 2020). Nelfinavir is a non-nucleoside 637 class inhibitor against a range of HAdV types. Surprisingly, we found Nelfinavir to inhibit HAdV 638 infection, although Nelfinavir was previously classified as inactive against HAdV-C based on 639 replication assays (Gantt et al., 2011). It is the off-patent FDA-approved active pharmaceutical 640 ingredient of Viracept. Nelfinavir was originally developed as an inhibitor against the HIV aspartyl 641 protease. It is orally bioavailable, with an inhibitory concentration in the low nanomolar range ADP-deletion mutants show delayed onset of plaque formation (Tollefson et al., 1996a(Tollefson et al., , 1996b. Lysis is enhanced by increased ADP levels and tuned by post-translational ADP processing 671 (Doronin et al., 2003;Tollefson et al., 1996aTollefson et al., , 1996b. ADP has a single signal/anchor sequence, 672 and its lumenal domain is N-and O-glycosylated. The N-terminal segment is cleaved off in the 673 Golgi lumen, and the membrane-anchored ADP localizes to the inner nuclear membrane (Scaria 674 et al., 1992;Tollefson et al., 1996bTollefson et al., , 2003 cell, including membranes. This is akin to another lipophilic drug with pleiotropic effects, the anti-695 viral and anti-helminthic compound Niclosamide, which is a weak acid and acts as a protonophore 696 extracting protons from acidic organelles, and thereby inhibits virus entry and uncouples mito- scientific discussions with Ivo Sbalzarini. We thank the Center for Microscopy and Image Analysis 739 (ZMB) at UZH providing the EM instrumentation. We thank the members of the Greber lab for 740 constructive discussions.

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Conflict of interest 743 The authors declare no conflict of interest.

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Funding 746 We acknowledge the financial support from the Swiss National Science Foundation (UFG, 747 31003A_179256 / 1), and the National Research Program "NCCR chemical biology" supported 748 by the Swiss National Science Foundation (GT, UFG