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

Human Adenoviruses infect the upper and lower respiratory tracts, the urinary and digestive tracts, lymphoid systems and heart, and give rise to epidemic conjunctivitis. More than 51 human serotypes have been identified to-date, and classified into 6 species A-F. The species C Adenoviruses Ad2 and Ad5 (Ad2/5) cause upper and lower respiratory disease, but how viral structure relates to the selection of particular infectious uptake pathways is not known. An adenovirus mutant, Ad2-ts1 had been isolated upon chemical mutagenesis in the past, and shown to have unprocessed capsid proteins. Ad2-ts1 fails to package the viral protease L3/p23, and Ad2-ts1 virions do not efficiently escape from endosomes. It had been suggested that the C22187T point mutation leading to the substitution of the conserved proline 137 to leucine (P137L) in the L3/p23 protease was at least in part responsible for this phenotype. To clarify if the C22187T mutation is necessary and sufficient for the Ad2-ts1 phenotype, we sequenced the genes encoding the structural proteins of Ad2-ts1, and confirmed that the Ad2-ts1 DNA carries the point mutation C22187T. Introduction of C22187T to the wild-type Ad2 genome in a bacterial artificial chromosome (Ad2-BAC) gave Ad2-BAC46 virions with the full Ad2-ts1 phenotype. Reversion of Ad2-BAC46 gave wild-type Ad2 particles indicating that P137L is necessary and sufficient for the Ad2-ts1 phenotype. The kinetics of Ad2-ts1 uptake into cells were comparable to Ad2 suggesting similar endocytic uptake mechanisms. Surprisingly, infectious Ad2 or Ad5 but not Ad2-ts1 uptake required CALM (clathrin assembly lymphoid myeloid protein), which controls clathrin-mediated endocytosis and membrane transport between endosomes and the trans-Golgi-network. The data show that no other mutations than P137L in the viral protease are necessary to give rise to particles that are defective in capsid processing and endosomal escape. This provides a basis for genetic analyses of distinct host requirements for Ad endocytosis and escape from endosomes

The Dynactin Complex Enhances the Speed of Microtubule-Dependent Motions of Adenovirus Both Towards and Away from the Nucleus

Unlike transport vesicles or organelles, human adenovirus (HAdV) directly binds to the microtubule minus end-directed motor dynein for transport to the nucleus. The dynein cofactor dynactin enhances nuclear transport of HAdV and boosts infection. To determine if dynactin has a specific role in cytoplasmic trafficking of incoming HAdV on microtubules, we used live cell spinning disc confocal microscopy at 25 Hz acquisition frequency and automated tracking of single virus particles at 20–50 nm spatial resolution. Computational dissection by machine-learning algorithms extracted specific motion patterns of viral trajectories. We found that unperturbed cells supported two kinds of microtubule-dependent motions, directed motions (DM) and fast drifts (FD). DM had speeds of 0.2 to 2 μm/s and run lengths of 0.4 up to 7 μm, while FD were slower and less extensive at 0.02 to 0.4 μm/s and 0.05 to 2.5 μm. Dynactin interference by overexpression of p50/dynamitin or a coiled-coil domain of p150/Glued reduced the speeds and amounts of both center- and periphery-directed DM but not FD, and inhibited infection. These results indicate that dynactin enhances adenovirus infection by increasing the speed and efficiency of dynein-mediated virus motion to the nucleus, and, surprisingly, also supports a hereto unknown motor activity for virus transport to the cell periphery

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

Transport of large cargo through the cytoplasm requires motor proteins and polarized filaments. Viruses that replicate in the nucleus of post-mitotic cells use microtubules and the dynein/dynactin motor to traffic to the nuclear membrane, and deliver their genome through nuclear pore complexes (NPCs) into the nucleus. How virus particles (virions) or cellular cargo are transferred from microtubules to the NPC is unknown. Here, we analyzed trafficking of incoming cytoplasmic adenoviruses by single particle tracking and super-resolution microscopy. We provide evidence for a regulatory role of CRM1/XPO1 (chromosome-region-maintenance-1, exportin-1) in juxta-nuclear microtubule-dependent adenovirus transport. Leptomycin B (LMB) abolishes nuclear targeting of adenovirus. It binds to CRM1, precludes CRM1-cargo binding and blocks signal-dependent nuclear export. LMB-inhibited CRM1 did not compete with adenovirus for binding to the nucleoporin Nup214 at the NPC. Instead, CRM1 inhibition selectively enhanced virion association with microtubules, and boosted virion motions on microtubules less than about 2 µm from the nuclear membrane. The data show that the nucleus provides positional information for incoming virions to detach from microtubules, engage a slower microtubule-independent motility to the NPC and enhance infection

Analysis of adenovirus trans-complementation-mediated gene expression controlled by melanoma-specific TETP promoter in vitro

BACKGROUND: Human adenoviruses (Ads) have substantial potential for clinical applications in cancer patients. Conditionally replicating adenoviruses (CRAds) include oncolytic adenoviruses in which expression of the immediate early viral transactivator protein E1A is controlled by a cancer cell-selective promoter. To enhance efficacy, CRAds are further armed to contain therapeutic genes. Due to size constraints of the capsid geometry, the capacity for packaging transgenes into Ads is, however, limited. To overcome this limitation, the employment of E1A-deleted replication-deficient viruses carrying therapeutic genes in combination with replication-competent CRAd vectors expressing E1A in trans has been proposed. Most trans-complementing studies involved transgene expressions from strong ubiquitous promoters, and thereby relied entirely on the cancer cell specificity of the CRAd vector. RESULTS: Here we tested the trans-complementation of a CRAd and a replication-deficient transgene vector containing the same cancer cell-selective promoter. Hereto, we generated two new vectors expressing IL-2 and CD40L from a bicistronic expression cassette under the control of the melanoma/melanocyte-specific tyrosinase enhancer tyrosinase promoter (TETP), which we previously described for the melanoma-specific CRAd vector AdDeltaEP-TETP. These vectors gave rise to tightly controlled melanoma-specific transgene expression levels, which were only 5 to 40-fold lower than those from vectors controlled by the nonselective CMV promoter. Reporter analyses using Ad-CMV-eGFP in combination with AdDeltaEP-TETP revealed a high level of trans-complementation in melanoma cells (up to about 30-fold), but not in non-melanoma cells, unlike the AdCMV-eGFP/wtAd5 binary vector system, which was equally efficient in melanoma and non-melanoma cells. Similar findings were obtained when replacing the transgene vector AdCMV-eGFP with AdCMV-IL-2 or AdCMV-CD40L. However, the combination of the novel AdTETP-CD40L/IL-2 vector with AdDeltaEP-TETP or wtAd5 gave reproducible moderate 3-fold enhancements of IL-2 by trans-complementation only. CONCLUSIONS: The cancer cell-selective TETP tested here did not give the expected enforceable transgene expression typically achieved in the Ad trans-complementing system. Reasons for this could include virus-mediated down regulation of limiting transcription factors, and/or competition for such factors by different promoters. Whether this finding is unique to the particular promoter system tested here, or also occurs with other promoters warrants further investigations

Cell Cycle-Dependent Kinase Cdk9 Is a Postexposure Drug Target against Human Adenoviruses

Human adenoviruses (HAdVs) infect respiratory, gastrointestinal, and urinary tracts and give rise to eye infections and epidemic keratoconjunctivitis (EKC). They persist in lymphoid tissue and cause morbidity and mortality in immunocompromised people. Treatments with significant postexposure efficacy are not available. Here, we report that inhibition of the cell cycle-dependent kinase 9 (Cdk9) by RNA interference, or the compound flavopiridol, blocked infections with HAdV-C2/5, EKC-causing HAdV-D8/37, and progeny formation in human corneal epithelial and cancer cells. Flavopiridol abrogated the production of the immediate early viral transactivating protein E1A without affecting nuclear import of viral DNA. In morphometric plaque assays, the compound exhibited antiviral efficacy in both pre- and postexposure regimens with therapeutic indexes exceeding 10. The study identifies Cdk9 as a postexposure drug target against adenovirus infections in vitro and suggests that the clinically tested anticancer drug flavopiridol is a candidate for treating adenoviral EKC or adenovirus emergence upon immune suppression

An RNA replication-center assay for high content image-based quantifications of human rhinovirus and coxsackievirus infections

Background: Picornaviruses are common human and animal pathogens, including polio and rhinoviruses of the enterovirus family, and hepatits A or food-and-mouth disease viruses. There are no effective countermeasures against the vast majority of picornaviruses, with the exception of polio and hepatitis A vaccines. Human rhinoviruses (HRV) are the most prevalent picornaviruses comprising more than one hundred serotypes. The existing and also emerging HRVs pose severe health risks for patients with asthma or chronic obstructive pulmonary disease. Here, we developed a serotype-independent infection assay using a commercially available mouse monoclonal antibody (mabJ2) detecting double-strand RNA. Results: Immunocytochemical staining for RNA replication centers using mabJ2 identified cells that were infected with either HRV1A, 2, 14, 16, 37 or coxsackievirus (CV) B3, B4 or A21. MabJ2 labeled-cells were immunocytochemically positive for newly synthesized viral capsid proteins from HRV1A, 14, 16, 37 or CVB3, 4. We optimized the procedure for detection of virus replication in settings for high content screening with automated fluorescence microscopy and single cell analysis. Our data show that the infection signal was dependent on multiplicity, time and temperature of infection, and the mabJ2-positive cell numbers correlated with viral titres determined in single step growth curves. The mabJ2 infection assay was adapted to determine the efficacy of anti-viral compounds and small interfering RNAs (siRNAs) blocking enterovirus infections. Conclusions: We report a broadly applicable, rapid protocol to measure infection of cultured cells with enteroviruses at single cell resolution. This assay can be applied to a wide range of plus-sense RNA viruses, and hence allows comparative studies of viral infection biology without dedicated reagents or procedures. This protocol also allows to directly compare results from small compound or siRNA infection screens for different serotypes without the risk of assay specific artifacts

Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake

Adenovirus type 2 (Ad2) binds the coxsackie B virus Ad receptor and is endocytosed upon activation of the αv integrin coreceptors. Here, we demonstrate that expression of dominant negative clathrin hub, eps15, or K44A-dynamin (dyn) inhibited Ad2 uptake into epithelial cells, indicating clathrin-dependent viral endocytosis. Surprisingly, Ad strongly stimulated the endocytic uptake of fluid phase tracers, coincident with virus internalization but without affecting receptor-mediated transferrin uptake. A large amount of the stimulated endocytic activity was macropinocytosis. Macropinocytosis depended on αv integrins, PKC, F-actin, and the amiloride-sensitive Na+/H+ exchanger, which are all required for Ad escape from endosomes and infection. Macropinocytosis stimulation was not a consequence of viral escape, since it occurred in K44A-dyn–expressing cells. Surprisingly, 30–50% of the endosomal contents were released into the cytosol of control and also K44A-dyn–expressing cells, and the number of fluid phase–positive endosomes dropped below the levels of noninfected cells, indicating macropinosomal lysis. The release of macropinosomal contents was Ad dose dependent, but the presence of Ad particles on macropinosomal membranes was not sufficient for contents release. We conclude that Ad signaling from the cell surface controls the induction of macropinosome formation and leakage, and this correlates with viral exit to the cytosol and infection

Adenovirus core protein V reinforces the capsid and enhances genome release from disrupted particles

Out of the three core proteins in human adenovirus, protein V is believed to connect the inner capsid surface to the outer genome layer. Here, we explored mechanical properties and in vitro disassembly of particles lacking protein V (Ad5-ΔV). Ad5-ΔV particles were softer and less brittle than the wild-type ones (Ad5-wt), but they were more prone to release pentons under mechanical fatigue. In Ad5-ΔV, core components did not readily diffuse out of partially disrupted capsids, and the core appeared more condensed than in Ad5-wt. These observations suggest that instead of condensing the genome, protein V antagonizes the condensing action of the other core proteins. Protein V provides mechanical reinforcement and facilitates genome release by keeping DNA connected to capsid fragments that detach during disruption. This scenario is in line with the location of protein V in the virion and its role in Ad5 cell entry

Adenovirus core protein V reinforces the capsid and enhances genome release from disrupted particles

Out of the three core proteins in human adenovirus, protein V is believed to connect the inner capsid surface to the outer genome layer. Here, we explored mechanical properties and in vitro disassembly of particles lacking protein V (Ad5-ΔV). Ad5-ΔV particles were softer and less brittle than the wild-type ones (Ad5-wt), but they were more prone to release pentons under mechanical fatigue. In Ad5-ΔV, core components did not readily diffuse out of partially disrupted capsids, and the core appeared more condensed than in Ad5-wt. These observations suggest that instead of condensing the genome, protein V antagonizes the condensing action of the other core proteins. Protein V provides mechanical reinforcement and facilitates genome release by keeping DNA connected to capsid fragments that detach during disruption. This scenario is in line with the location of protein V in the virion and its role in Ad5 cell entryThis work was supported by grants from the Spanish Ministry of Economy, Industry and Competitiveness (FIS2017-89549-R; “Maria de Maeztu” Program for Units of Excellence in R&D MDM-2014-0377 and FIS2017-90701-REDT) and from the Human Frontiers Science Program (HFSPO RGP0012/2018) to P.J.d.P., as well as grant PID2019-104098GB-I00/AEI/10.13039/501100011033, cofunded by the Spanish State Research Agency and the European Regional Development Fund to C.S.M. The CNB-CSIC was further supported by a Spanish State Research Agency Severo Ochoa Excellence grant (SEV 2017-0712). C.S.M. is a member of the CSIC funded consortium LifeHub (CSIC grant number: 202120E47). M.H.-P. was a recipient of a Juan de la Cierva postdoctoral contract and currently holds a Ramón y Cajal position (RyC2021-030929-I) funded by the Spanish State Research Agency. M.H.-P. acknowledges funding from Ayudas a Proyectos de I+D para Jóvenes Doctores de la Universidad Autónoma de Madrid 2021, SI3/PJI/2021-00216 supported by the Community of Madrid and the University Autónoma de Madrid, and TED2021-129937B-I00 from Spanish Ministry of Science and Innovation. Financial support of the Community of Madrid and the European Union through the European Regional Development Fund (ERDF), financed as part of the Union response to Covid-19 pandemic (VIRMAT-REACT), is also acknowledged. Access to the cryo-EM CNB-CSIC facility was carried out via the CRIOMECORR project (ESFRI-2019-01- CSIC-16). Work in the Greber laboratory was supported by a grant to UFG from Schweizerischer Nationalfonds (Swiss National Science Foundation, 31003A_179256/1). Author contributions: N.M.-G. performed AFM experiments and analyzed the data. M.H.-P. and C.S.M. carried out cryoEM analyses. A.G.-G. and M.B. produced the viral samples and carried out thermostability analyses. U.F.G., C.S.M., and P.J.d.P. designed research. N.M.-G. wrote the manuscript, with input from U.F.G., C.S.M., and P.J.d.P. All authors read and edited the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: The Ad5-ΔV cryo-EM map is deposited at the Electron Microscopy Data Bank (EMDB; www.ebi.ac.uk/pdbe/emdb) with accession number EMD-15694. All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Material