key: cord-0295327-8nwymd6m
authors: Georgi, Fanny; Andriasyan, Vardan; Witte, Robert; Murer, Luca; Hemmi, Silvio; Yu, Lisa; Grove, Melanie; Meili, Nicole; Kuttler, Fabien; Yakimovich, Artur; Turcatti, Gerardo; Greber, Urs F
title: The FDA-approved drug Nelfinavir inhibits lytic cell-free transmission of human adenoviruses
date: 2020-05-15
journal: bioRxiv
DOI: 10.1101/2020.05.15.098061
sha: 6548e447e66883e09efc0aca4c39f3bbfdb50853
doc_id: 295327
cord_uid: 8nwymd6m

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.

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

Viruses 134

HAdV-C2-dE3B-GFP was previously described (Yakimovich et al., 2012) (GenBank accession  135 number MT277585). The virus was generated by exchange of the viral E3b genome region with 136 a reporter cassette harbouring the enhanced green fluorescent protein (GFP) under a consti-137 tutively active cytomegalovirus (CMV) promoter. It was grown in A549 cells and purified by double 138

CsCl gradient centrifugation (Greber et al., 1993) . Aliquots supplemented with 10% (v / v) glycerol 139

were stored at -80°C. HAdV-C2-dE3B-GFP was found to be homogeneous by SDS-PAGE and 140

negative-stain analyses in transmission electron microscopy (EM). Recombinant HAdV-C2-dE3B-141

GFP-dADP was generated using homologous recombination according to the Warming 142 recombineering protocols (Sirena et al., 2004; Warming et al., 2005) . For a detailed protocol, see 143

Supplementary methods. HAdV-C2-dE3B-GFP-dADP was plaque-purified and amplified, 144

followed by two rounds of CsCl purification (Hemmi et al., 1998 Impedance-based assays were performed using the xCELLigence system (Roche Applied 195

Science and ACEA Biosciences) as described previously (Prasad et al., 2014 (Prasad et al., , 2020a to the manufacturer's instructions (Spiegel, 2009) Netherlands) at 100 kV. Images were acquired using a CCD camera (Orius SC1000 with 4,000 x 298 2,600 pixels, Gatan, Pleasanton, USA). 299 300

Double CsCl-purified grown in presence / absence of Nelfinavir (HAdV-C5 ±Nelfinavir ) stocks and size 302 standard (PageRuler plus, Thermo Fisher Scientific, Waltham, USA) were size-separated on 12% 303

acrylamide gel under reducing conditions and transferred to a PVDF membrane. HAdV proteins 304

were detected using the following primary antibodies: 1:10,000 R72 rabbit α-fiber ( Waltham, USA). Max projections of confocal z-stacks (25 z steps spaced 1 µm) were acquired on 339 a SP5 resonant APD (Leica, Wetzlar, Germany) at 1.7x zoom using a 63x glycerol objective 340

(numerical aperture 1.4). 341 342

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. 

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.

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

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

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

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 is expressed at high levels late in infection and enhances cell lysis (Tollefson et al., 1992, 548 1996b). To test if ADP was required for Nelfinavir inhibition of lytic spread, we generated an ADP-549

depleted HAdV-C2-dE3B-GFP mutant, HAdV-C2-dE3B-GFP-dADP. The mutant completely lacks 550

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

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

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

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., 1996a Tollefson 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., 1996b Tollefson 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.

Conflict of interest 743

The authors declare no conflict of interest.

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 

Conditionally 1073 replicating adenoviruses kill tumor cells via a basic apoptotic machinery-independent mechanism that 1074 resembles necrosis-like programmed cell death

DNA restriction analysis of adenovirus 1076 prototypes 1 to 41

Components of adenovirus genome packaging

Rapid and efficient purification of Cowpea chlorotic mottle virus by 1079 sucrose cushion ultracentrifugation

Interaction of adenovirus with antibodies, complement, and 1081 coagulation factors

Deep learning of virus infections reveals mechanics of lytic cells

Innate immunity to adenovirus: lessons from mice

A mini review of the zoonotic threat 1087 potential of influenza viruses, coronaviruses, adenoviruses, and enteroviruses. Front Public Health 6

Diversity within the adenovirus fiber knob hypervariable loops influences 1090 primary receptor interactions

The e3 ubiquitin ligase mind bomb 1 controls adenovirus genome release at the 1093 nuclear pore complex

Studies of the mechanism of enhancement of human 1095 adenovirus infection in monkey cells by simian virus 40

Vaccinia virus hijacks EGFR signalling to enhance virus spread through rapid and 1098 directed infected cell motility

Adenovirus composition, proteolysis, and disassembly studied by in-depth qualitative and 1101 quantitative proteomics

Do nonhuman primate or bat adenoviruses pose a risk 1103 for human health?

Recent lessons in gene expression, cell cycle control, and cell biology from adenovirus

Repositioning HIV protease inhibitors as cancer therapeutics

Adenovirus mutants with splice-enhancing mutations in the E3 complex 1109 transcription unit are also defective in E3A RNA 3'-end formation

Development of Adenovirus Reporter Viruses and Studies on Mouse Adenovirus-2 and -3

Receptor Candidates. Doctoral dissertation. Department of Molecular Life Sciences

What does S-palmitoylation do to membrane 1113 proteins?

Adenovirus transport 1115 via direct interaction of cytoplasmic dynein with the viral capsid hexon subunit

New prospects for nelfinavir in non-HIV-1118 related diseases

Virus movements on the plasma membrane support infection 1120 and transmission between cells

Drifting motions of the adenovirus receptor CAR and immobile integrins initiate virus uncoating and 1123 membrane lytic protein exposure

Some HIV protease inhibitors 1125 alter lamin A/C maturation and stability, SREBP-1 nuclear localization and adipocyte differentiation

CellProfiler: image analysis software for identifying and 1129 quantifying cell phenotypes

Deaths associated with human adenovirus-14p1 infections

Acute respiratory disease associated with 1134 adenovirus serotype 14--four states

Viral and cellular interactions during adenovirus 1136 DNA replication

1138 (2011a). Species D adenoviruses as oncolytics against B-cell cancers

Cross-species transmission of a novel adenovirus associated with 1141 a fulminant pneumonia outbreak in a new world monkey colony

Nelfinavir induces liposarcoma apoptosis and cell cycle 1143 arrest by upregulating sterol regulatory element binding protein-1

Anti-HIV drugs for cancer therapeutics: back to the future?

MxB is an interferon-induced restriction factor of human herpesviruses

Viruses and the diversity of cell death

Homology between the human 1152 cytomegalovirus RL11 gene family and human adenovirus E3 genes

Genetic content and evolution of adenoviruses

Prospects for oral replicating adenovirus-vectored vaccines

Novel deletion mutants 1158 that enhance a distant upstream 5' splice in the E3 transcription unit of adenovirus 2

Molecular evolution of human adenovirus (hadv) species C

Repulsion of superinfecting virions: 1164 a mechanism for rapid virus spread

Overexpression of the ADP (E3-11.6K) protein increases cell lysis and spread of adenovirus

Molecular epidemiology of adenovirus type 7 in the United States

A pathogenic picornavirus acquires an envelope by hijacking cellular membranes

Epigenetic 1175 reprogramming by adenovirus e1a

Viral mechanisms for docking and delivering at nuclear pore 1177 complexes

Structure-activity analysis of niclosamide reveals potential 1180 role for cytoplasmic pH in control of mammalian target of rapamycin complex 1 (mTORC1) signaling

The HIV protease inhibitor nelfinavir inhibits Kaposi's sarcoma-associated 1184 herpesvirus replication in vitro

Nelfinavir impairs 1186 glycosylation of herpes simplex virus 1 envelope proteins and blocks virus maturation

Prevalence and quantitation of species C 1189 adenovirus DNA in human mucosal lymphocytes

Infectious adenovirus 1191 type 2 transport through early but not late endosomes

High-content image-based drug screen identifies a clinical compound against cell transmission of 1194 adenovirus

Nelfinavir, A lead HIV protease inhibitor, is a broad-spectrum, 1197 anticancer agent that induces endoplasmic reticulum stress, autophagy, and apoptosis in vitro and in vivo

Gene therapy clinical trials 1200 worldwide to 2017: An update

Inhibition of herpes simplex virus type 1 replication by 1203 adeno-associated virus rep proteins depends on their combined DNA-binding and ATPase/helicase 1204 activities

Genotype prevalence and risk factors for severe clinical 1207 adenovirus infection, United States

Virus assembly and disassembly: the adenovirus cysteine protease as a trigger factor

Signalling in viral entry

Adenovirus entry: from infection to immunity

Stepwise dismantling of adenovirus 2 1214 during entry into cells

The 1216 role of the nuclear pore complex in adenovirus DNA entry

Adenoviruses -from pathogens 1218 to therapeutics: a report on the 10th International Adenovirus Meeting

Nelfinavir 1220 induces liposarcoma apoptosis through inhibition of regulated intramembrane proteolysis of SREBP-1 and 1221 ATF6

Nelfinavir inhibits regulated intramembrane proteolysis of 1223 sterol regulatory element binding protein-1 and activating transcription factor 6 in castration-resistant 1224 prostate cancer

Nelfinavir and nelfinavir analogs block site-2 1226 protease cleavage to inhibit castration-resistant prostate cancer

Structural basis of vesicle formation at the inner nuclear 1229 membrane

Polyinosinic acid enhances delivery of adenovirus vectors in vivo by preventing sequestration in liver 1232 macrophages

Worldwide increased 1234 prevalence of human adenovirus type 3 (HAdV-3) respiratory infections is well correlated with 1235 heterogeneous hypervariable regions (HVRs) of hexon

Adenoviruses across the animal kingdom: a walk in the 1237 zoo

Hyperactive sleeping beauty transposase enables persistent phenotypic 1240 correction in mice and a canine model for hemophilia B

Adenovirus death protein, a 1242 transmembrane protein encoded in the E3 region, is palmitoylated at the cytoplasmic tail

The presence of human 1245 coxsackievirus and adenovirus receptor is associated with efficient adenovirus-mediated transgene 1246 expression in human melanoma cell cultures

Study on Mouse Adenovirus Biology and Development of Their Vectors. Doctoral 1248 dissertation

The biology of the adenovirus E1B 55K 1250 protein

Brincidofovir is highly efficacious in controlling adenoviremia in pediatric 1253 recipients of hematopoietic cell transplant

Management of adenovirus infection in patients after haematopoietic stem cell transplantation: State-of-1256 the-art and real-life current approach: A position statement on behalf of the Infectious Diseases Working 1257 Party of the European Society of Blood and Marrow Transplantation

ESCRTs are everywhere

Outbreak 1260 of febrile respiratory illness caused by adenovirus at a South Korean military training facility: clinical and 1261 radiological characteristics of adenovirus pneumonia

Genetic reconstitution of the 1263 human adenovirus type 2 temperature-sensitive 1 mutant defective in endosomal escape

Genomic foundations of 1265 evolution and ocular pathogenesis in human adenovirus species D

Autophagic cell death of malignant glioma cells induced by a conditionally replicating 1268 adenovirus

Human 1278 adenovirus type 5 induces cell lysis through autophagy and autophagy-triggered caspase activity

Oncolytic adenovirus research evolution: from cell-cycle checkpoints to immune checkpoints

Niclosamide 1284 is a proton carrier and targets acidic endosomes with broad antiviral effects

Acute inflammatory response and remodeling of airway 1286 epithelium after subspecies B1 human adenovirus infection of the mouse lower respiratory tract

Molecular Epidemiology of Adenovirus Type 21 Respiratory Strains 1290

Isolated From US Military Trainees (1996-2014)

Viracept (nelfinavir mesylate, AG1343): a potent, orally 1293 bioavailable inhibitor of HIV-1 protease

Nelfinavir 1295 inhibits maturation and export of herpes simplex virus 1

Hacking the cell: network 1297 intrusion and exploitation by adenovirus E1A

Biology of the adenovirus E4orf4 protein: from virus infection to cancer cell death

Nelfinavir and other protease inhibitors in cancer: mechanisms involved in anticancer 1301 activity

Persistence and reactivation of human adenoviruses in the gastrointestinal 1304 tract

Adenovirus infections in the immunocompromised host

Effective 1308 apical infection of differentiated human bronchial epithelial cells and induction of proinflammatory 1309 chemokines by the highly pneumotropic human adenovirus type 14p1

Oncolytic viruses in cancer treatment: 1311 A review

Dynein-and microtubule-mediated translocation of adenovirus serotype 5 occurs after 1314 endosomal lysis

Adenovirus persistence, reactivation, and clinical management

Mediated Human Immunodeficiency Virus Type 1 Abscission

Atomic structure of 1319 human adenovirus by cryo-EM reveals interactions among protein networks

Preformulation studies of a novel HIV protease 1321 inhibitor, AG1343

Severe pneumonia due to adenovirus serotype 14: a new respiratory threat?

Co-option of Membrane Wounding Enables Virus Penetration into Cells

Adenovirus: epidemiology, global spread of novel serotypes, and 1329 advances in treatment and prevention

Epigenetics and 1331 the dynamics of chromatin during adenovirus infections

Viroporins from RNA viruses induce caspase-dependent 1333 apoptosis

Structure, function and dynamics in adenovirus maturation

Human adenoviruses of subgenera B, C, and E with various 1337 tropisms differ in both binding to and replication in the epithelial A549 and 293 cells

Adenovirus endocytosis

Abrupt 1340 emergence of diverse species B adenoviruses at US military recruit training centers

Virus cell-to-cell transmission

Safety, pharmacokinetics, and antiretroviral activity of the potent, specific human 1346 immunodeficiency virus protease inhibitor nelfinavir: results of a phase I/II trial and extended follow-up in 1347 patients infected with human immunodeficiency virus

Adenovirus death protein (ADP) is required for lytic infection of human 1350 lymphocytes

Lessons learned from adenovirus

First reported cases of human adenovirus serotype 14p1 infection

Immune evasion by adenoviruses: a window into host-virus 1357 adaptation

recombinant adenoviral hexon protein fragments for the production of virus-type specific antibodies

Intrinsic structural disorder 1362 in adenovirus E1A: a viral molecular hub linking multiple diverse processes

Imaging the adenovirus infection cycle

Adenovirus-associated deaths in US 1365 military during postvaccination period

Chemical induction of unfolded protein response enhances cancer cell killing through lytic virus 1368 infection

The UPR sensor IRE1α and the adenovirus E3-19Kglycoprotein sustain persistent and lytic 1371 infections

The UPR sensor IRE1α and the adenovirus E3-19K glycoprotein sustain persistent and lytic 1374 infections

Metabolic reprogramming of the host cell by human 1376 adenovirus infection

R:A Language and Environment for Statistical Computing

Dramatic decline of respiratory illness among US military recruits after the renewed use of adenovirus 1381 vaccines

Low-Level Expression of the E1B 20-Kilodalton Protein by 1383

Adenovirus 14p1 Enhances Viral Immunopathogenesis

The adenovirus 1385 E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and Bcl-2 proteins

Crystal structure of human 1388 adenovirus at 3.5 A resolution

Glycosylation in health and disease

Vaccinia virus morphogenesis and dissemination

Molecular evolution of human adenoviruses

Novel immunocompetent murine tumor model for evaluation of conditionally replication-competent 1397 (oncolytic) murine adenoviral vectors

Isolation of a 1399 cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture

The E3-11.6K protein of adenovirus is an 1402

Asn-glycosylated integral membrane protein that localizes to the nuclear membrane

Viroporins: structure, function and potential as antiviral targets

Recent advances in drug repositioning for the discovery of new anticancer 1407 drugs

The human membrane cofactor CD46 is a receptor for species B 1410 adenovirus serotype 3

Adenoviral strategies to overcome innate cellular responses to 1412 infection

Real-time and Dynamic Monitoring of Virus-mediated Cytopathogenicity

Overcome the species barrier of productive human adenovirus infection in murine cells

Microtubule-1420 dependent plus-and minus end-directed motilities are competing processes for nuclear targeting of 1421 adenovirus

Virtual computational chemistry laboratory--design and 1424 description

A novel psittacine adenovirus identified during an outbreak of 1427 avian chlamydiosis and human psittacosis: zoonosis associated with virus-bacterium coinfection in birds

The 11,600-MW protein encoded by region 1430 E3 of adenovirus is expressed early but is greatly amplified at late stages of infection

The 1433 adenovirus death protein (E3-11.6K) is required at very late stages of infection for efficient cell lysis and 1434 release of adenovirus from infected cells

The E3-11.6-kDa 1436 adenovirus death protein (ADP) is required for efficient cell death: characterization of cells infected with adp 1437 mutants

Mutations within the ADP (E3-11.6K) protein 1439 alter processing and localization of ADP and the kinetics of cell lysis of adenovirus-infected cells

Permissive Syrian Hamsters after Infection with Species C Human Adenovirus (HAdV-C) Is the Result of 1443

Virus Replication: HAdV-C6 Replicates More and Causes More Pathology than HAdV-C5

Inhibition of intracellular hepatitis C virus replication by nelfinavir and 1446 synergistic effect with interferon-alpha

Molecular mechanisms of chemical mutagenesis: 9-aminoacridine inhibits DNA 1448 replication in vitro by destabilizing the DNA growing point and interacting with the DNA polymerase

Species D human adenovirus type 9 exhibits better virus-1451 spread ability for antitumor efficacy among alternative serotypes

Intricate relationships 1453 between naked viruses and extracellular vesicles in the crosstalk between pathogen and host

Postentry neutralization of adenovirus type 1456 5 by an antihexon antibody

Palmitoylation of virus proteins

Wash and the WASH Regulatory Complex function in Nuclear Envelope budding

The nuclear export 1461 factor CRM1 controls juxta-nuclear microtubule-dependent virus transport

Imaging, Tracking and 1463 Computational Analyses of Virus Entry and Egress with the Cytoskeleton

Simple and highly 1465 efficient BAC recombineering using galK selection

Adenovirus protein VI mediates 1467 membrane disruption following capsid disassembly

Region E3 of adenovirus: a cassette of genes involved in host 1469 immunosurveillance and virus-cell interactions

Mapping a new gene that encodes an 1471 11,600-molecular-weight protein in the E3 transcription unit of adenovirus 2

Evidence that AGAUA 1473 and CCAGA initiate translation in the same mRNA in region E3 of adenovirus

Drug development against 1475 human adenoviruses and its advancement by Syrian hamster models

Adenovirus signalling in entry

Cell-free transmission of human adenovirus by passive mass transfer in cell culture simulated in a 1479 computer model

Plaque2.0-A High-Throughput Analysis Framework to Score Virus-Cell Transmission and Clonal 1482 Cell Expansion

HIV protease inhibitor nelfinavir inhibits replication of SARS-1485 associated coronavirus

HIV-1 protease inhibitor induces growth arrest and apoptosis of human prostate cancer LNCaP 1488 cells in vitro and in vivo in conjunction with blockade of androgen receptor STAT3 and AKT signaling

ADP-overexpressing adenovirus elicits 1491 enhanced cytopathic effect by induction of apoptosis

The adenovirus E1A C terminus suppresses a delayed antiviral 1493 response and modulates RAS signaling

Plasma membrane changes during programmed 1495 cell deaths

E2f/rb family proteins mediate interferon induced 1497 repression of adenovirus immediate early transcription to promote persistent viral infection

Cell-to-cell transmission of viruses

Role of kinesins in directed adenovirus transport and 1502 cytoplasmic exploration

Overexpression of adenovirus 1504 E3-11.6K protein induces cell killing by both caspase-dependent and caspase-independent mechanisms

pKSB2-AdV-C2-LARAzeo, two PCR-generated fragments were first cloned into pBluescript (pBl). 1031The first fragment encompassed 853 bp of the HAdV-C2 left end sequence and was PCR-1032 amplified using the forward primer 5'-ataagaatGCGGCCGCTAGGGATAACAGGGTAAT 1033catcatcataatataccttattttgg-3' inserting the restriction sites NotI and I-SceI, and the reverse primer 1034 5'-CTCTCTACTAGTAATAAGTCAATCCCTTCCTGC -3' inserting the restriction site SpeI. 1035HAdV-C2-dE3B-GFP genomic DNA isolated from infected A549 cells was used as template. The 1036NotI and SpeI restriction sites were used to clone the left arm fragment into pBl. The second 1037 fragment encompassed 853 bp of the HAdV-C2 right end sequence and was PCR-amplified using 1038the forward primer 5'-agagagACTAGTaaaaacatttaaacattagaagcctg-3' adding the restriction sites 1039SpeI and the reverse primer 5'-gcgcaagcttATTACCCTGTTATCCCTAcatcatcataatataccttattttgg-10403' adding the sites I-SceI and HindIII. This fragment was cloned into pBl-AdV2-C2-LA by SpeI and 1041HindIII restriction sites. A SpeI fragment containing the zeocine resistance marker from pcDNA3.1 1042 zeo (Invitrogen) and generated by PCR using the forward 5'-GACTAGTTTTTCG 1043 GATCTGATCAGCACG-3' and reverse primer 5-GACTAGTGGAAAACGATT CCGAAGCCC-3' 1044 was cloned into the SpeI of pBl-AdV-C2-LARA, connecting the two HAdV-C2 arms and resulting 1045in pBl-AdV-C2-LARAzeo. In order to transfer the AdV-C2-LARAzeo cassette to the BACmid 1046 pKSB2, the NotI-HindIII fragment containing this sequence was ligated with the NotI-HindIII-1047 restricted pKSB2 vector. Colonies containing pKSB2-AdV-C2-LARAzeo were selected using 1048 chloramphenicol and zeocin at concentrations of 10 µg / ml and 25 µg / ml, respectively. In order 1049to generate pKSB2-AdV-C2-dE3B_GFP, homologous recombination was performed in SW102 1050 bacteria using AatII and ApaLI-restricted pKSB2-AdV-C2-LARAzeo and HAdV-C2-dE3B-GFP 1051 genomic DNA isolated from infected A549 cells. 1052As a second, HAdV-C2-dE3B-GFP-dADP, was generated using two recombineering steps. In a 1053 first, the galK cassette was introduced into pKSB2-AdV-C2_dE3B_GFP to replace the ADP 1054 sequence. The GalK cassette was amplified using the forward primer 5'- SceI expression in these cells was accomplished following transduction with MLV-ER-I-SceI-1065HA, which encodes a form of the endonuclease that can be translocated to the nucleus upon 1066 treatment with 4-OH-tamoxifen 3 hours post transfection (Courilleau et al., 2012). Cells were 1067