Martijn J. W. E. Rabelink

Baculovirus Infection of Nondividing Mammalian Cells: Mechanisms of Entry and Nuclear Transport of Capsids

ABSTRACT We have studied the infection pathway of Autographa californica multinuclear polyhedrosis virus (baculovirus) in mammalian cells. By titration with a baculovirus containing a green fluorescent protein cassette, we found that several, but not all, mammalian cell types can be infected efficiently. In contrast to previous suggestions, our data show that the asialoglycoprotein receptor is not required for efficient infection. We demonstrate for the first time that this baculovirus can infect nondividing mammalian cells, which implies that the baculovirus is able to transport its genome across the nuclear membrane of mammalian cells. Our data further show that the virus enters via endocytosis, followed by an acid-induced fusion event, which releases the nucleocapsid into the cytoplasm. Cytochalasin D strongly reduces the infection efficiency but not the delivery of nucleocapsids to the cytoplasm, suggesting involvement of actin filaments in cytoplasmic transport of the capsids. Electron microscopic analysis shows the cigar-shaped nucleocapsids located at nuclear pores of nondividing cells. Under these conditions, we observed the viral genome, major capsid protein, and electron-dense capsids inside the nucleus. This suggests that the nucleocapsid is transported through the nuclear pore. This mode of transport seems different from viruses with large spherical capsids, such as herpes simplex virus and adenovirus, which are disassembled before nuclear transport of the genome. The implications for the application of baculovirus or its capsid proteins in gene therapy are discussed.

Spacers increase the accessibility of peptide ligands linked to the carboxyl terminus of adenovirus minor capsid protein IX.

ABSTRACT The efficiency and specificity of gene transfer with human adenovirus (hAd)-derived gene transfer vectors would be improved if the native viral tropism could be modified. Here, we demonstrate that the minor capsid protein IX (pIX), which is present in 240 copies in the Ad capsid, can be exploited as an anchor for heterologous polypeptides. Protein IX-deleted hAd5 vectors were propagated in hAd5 helper cells expressing pIX variants, with heterologous carboxyl-terminal extensions of up to 113 amino acids in length. The extensions evaluated consist of alpha-helical spacers up to 75 Å in length and to which peptide ligands were fused. The pIX variants were efficiently incorporated into the capsids of Ad particles. On intact particles, the MYC-tagged-pIX molecules were readily accessible to anti-MYC antibodies, as demonstrated by electron microscopic analyses of immunogold-labeled virus particles. The labeling efficiency improved with increasing spacer length, suggesting that the spacers lift and expose the ligand at the capsid surface. Furthermore, we found that the addition of an integrin-binding RGD motif to the pIX markedly stimulated the transduction of coxsackievirus group B and hAd receptor-deficient endothelioma cells, demonstrating the utility of pIX modification in gene transfer. Our data demonstrate that the minor capsid protein IX can be used as an anchor for the addition of polypeptide ligands to Ad particles.

Ube2j2 ubiquitinates hydroxylated amino acids on ER-associated degradation substrates.

An E2–E3 complex can ubiquitinate substrates via either an isopeptide bond (to a lysine) or an ester bond (to a serine or threonine) and preferentially uses the latter to induce ERAD.

The Coiled-Coil Domain of the Adenovirus Type 5 Protein IX Is Dispensable for Capsid Incorporation and Thermostability

ABSTRACT The 14.4-kDa hexon-associated protein IX (pIX) acts as a cement in the capsids of primate adenoviruses and confers a thermostable phenotype. Here we show that deletion of amino acids 100 to 114 of adenovirus type 5 pIX, which eliminates the conserved coiled-coil domain, impairs its capacity to self-associate. However, pIXΔ100-114 is efficiently incorporated into the viral capsid, and the resulting virions are thermostable. Deletion of the central alanine-rich domain, as in pIXΔ60-72, does not impair self-association, incorporation into the capsid, or the thermostable phenotype. These data demonstrate, first, that the self-association of pIX is dispensable for its incorporation into the capsid and generation of the thermostability phenotype and, second, that the increased thermostability results from pIX monomers binding to different hexon capsomers rather than capsid stabilization by pIX multimers.

Activin A impairs insulin action in cardiomyocytes via up-regulation of miR-143

AIMS Enhanced activin A release from epicardial adipose tissue (EAT) has been linked to the development of cardiac dysfunction in type 2 diabetes (T2D). This study examined whether the inhibition of insulin action induced by epicardial adipokines in cardiomyocytes can be ascribed to alterations in miRNA expression. METHODS AND RESULTS Expression levels of miRNAs were assessed by real-time PCR in primary adult rat cardiomyocytes (ARC) exposed to conditioned media generated from EAT biopsies (CM-EAT) from patients with and without T2D. CM-EAT-T2D altered the expression of eight miRNAs in ARC vs. CM-EAT from patients without T2D. Of these, only expression of the miR-143/145 cluster was affected by activin A in the same direction as CM-EAT-T2D. Accordingly, activin A neutralizing antibodies prevented the induction of the miR-143/145 cluster by CM-EAT-T2D. Subsequently, the impact of the miR-143/145 cluster on insulin action was investigated. Transfection of HL-1 cells with precursor-miR-143 (pre-miR-143), but not pre-miR-145, blunted the insulin-mediated phosphorylation of Akt and its substrate proline-rich Akt substrate of 40 kDa (PRAS40), and reduced insulin-stimulated glucose uptake. Also lentivirus-mediated expression of pre-miR-143 in ARC reduced insulin-induced Akt phosphorylation. These effects were ascribed to down-regulation of the miR-143 target and regulator of insulin action, the oxysterol-binding protein-related protein 8 (ORP8) in both ARC and HL-1 cells. Finally, LNA-anti-miR-143 protected against the detrimental effects of CM-EAT-T2D on insulin action in ARC. CONCLUSION Activin A released from EAT-T2D inhibits insulin action via the induction of miR-143 in cardiomyocytes. This miRNA inhibits the Akt pathway through down-regulation of the novel regulator of insulin action, ORP8.

A system for efficient generation of adenovirus protein IX-producing helper cell lines

The adenovirus 14.3 kDa hexon‐associated protein IX (pIX) functions in the viral capsid as ‘cement’ and assembles the hexons in stable groups‐of‐nine (GONs). Although viruses lacking pIX do not form GONs, and are less heat‐stable than wild‐type (wt) viruses, they can be propagated with the same kinetics and yields as the wt viruses. To facilitate ‘pseudotyping’ of adenoviral vectors we have set up an efficient system for the generation of pIX‐producing helper cell lines.

New Role of Signal Peptide Peptidase To Liberate C-Terminal Peptides for MHC Class I Presentation

The signal peptide peptidase (SPP) is an intramembrane cleaving aspartyl protease involved in release of leader peptide remnants from the endoplasmic reticulum membrane, hence its name. We now found a new activity of SPP that mediates liberation of C-terminal peptides. In our search for novel proteolytic enzymes involved in MHC class I (MHC-I) presentation, we found that SPP generates the C-terminal peptide-epitope of a ceramide synthase. The display of this immunogenic peptide–MHC-I complex at the cell surface was independent of conventional processing components like proteasome and peptide transporter TAP. Absence of TAP activity even increased the MHC-I presentation of this Ag. Mutagenesis studies revealed the crucial role of the C-terminal location of the epitope and “helix-breaking” residues in the transmembrane region just upstream of the peptide, indicating that SPP directly liberated the minimal 9-mer peptide. Moreover, silencing of SPP and its family member SPPL2a led to a general reduction of surface peptide–MHC-I complexes, underlining the involvement of these enzymes in Ag processing and presentation.

A comparative study between catalase gene therapy and the cardioprotector monohydroxyethylrutoside (MonoHER) in protecting against doxorubicin-induced cardiotoxicity in vitro.

Cardiotoxicity is the main dose-limiting side effect of doxorubicin in the clinic. Being a free radical producer, doxorubicin affects the heart specifically because of its low antioxidant capacity. Among those antioxidants, catalase is present in very low levels in the heart compared to other organs. Since catalase is an essential enzyme in detoxifying hydrogen peroxide, the aim of the present study was to investigate the protective effect of catalase as delivered by an adenovirus vector against doxorubicin-induced cardiotoxicity in cultured neonatal rat cardiac myocytes (NeRCaMs). 7-Monohydroxyethylrutoside (MonoHER), a potent cardioprotector currently under clinical investigations, was included in the study as a reference. Neonatal rat cardiac myocytes were infected with different multiplicity of infections (MOIs) of adenovirus encoding catalase (AdCat). A control infection with an adenovirus vector encoding a nonrelated protein was included. The activity and content of catalase in infected cells were determined during 3 days postinfection. One group of NeRCaMs was infected with AdCat before treatment with doxorubicin (0–50 μM). The second and third group were treated with doxorubicin (0–50 μM) with and without 1 mM monohydroxyethylrutoside (monoHER), respectively. The LDH release and viability of treated cells were measured 24 and 48 h after doxorubicin treatment. The beating rate was followed in three other groups of cells receiving the same treatments within 3 days after doxorubicin (0–100 μM) treatment. Catalase activity increased in AdCat-infected cells, with different MOIs, starting from the second day after infection as compared to the mock-infected cells (P<0.03). At the third day of infection, an MOI of more than 50 caused cytopathic effects, which hampered the use of higher viral titres. With an MOI of 50, catalase activity increased 3.5-fold in AdCat-infected cells 3 days postinfection (P=0.021) compared to mock-infected cells. The beating rate and survival of NeRCaMs decreased in a concentration and time-dependent manner after doxorubicin treatment (P<0.0005). This cytotoxicity was associated with an increase in the LDH release from the treated cells (P<0.0005). The cells stopped beating 24 h after treatment with >50 μM doxorubicin. A 3.5-fold increase in the activity of catalase did not protect NeRCaMs against any of the cytotoxic effects of doxorubicin on NeRCaMs. In contrast, monoHER (1 mM) significantly protected NeRCaMs against the lethal effects of doxorubicin on the survival, LDH release and the beating rate of NeRCaMs (P<0.004) during 48 h after doxorubicin treatment. This protection resulted in a prolongation of the beating of doxorubicin-treated cells after the end of the experiment (i.e. >72 h). The present study (1) illustrates that the cytotoxicity of high MOI of AdCat (>50) limited the possibility to increase catalase activity more than 3.5-fold, which was not enough to protect infected NeRCaMs against doxorubicin-induced cardiotoxicity and (2) confirms the efficacy of monoHER as a cardioprotector. Thus, the use of monoHER proves more suitable for the prevention of doxorubicin-induced cardiotoxicity than catalase gene transfer employing adenovirus vectors.

Ammonium sulphate precipitation of recombinant adenovirus from culture medium: an easy method to increase the total virus yield

In the majority of the methods for purifying and concentrating recombinant adenoviruses (rAds) the virus that is associated with the helper cells is harvested, while the virus that is present in the cell-culture medium is discarded. During routine propagation of adenovirus type-5 vectors at optimised conditions we noted that, on average, 47% of the total amount of virus is present in the culture medium. To recover and concentrate these rAds from the medium, we devised a method, which is based on ammonium sulphate ((NH4)2SO4) precipitation. At 40% (NH4)2SO4 saturation, 95 ± 6% of the available virus precipitates from the medium, while the majority of the protein (85%) remains in solution. In contrast to adenovirus precipitation with polyethylene glycol, the (NH4)2SO4precipitation technique allows collection of precipitated rAds by filtration. We demonstrate here that (NH4)2SO4 precipitation of rAds from cell-culture medium is a simple and fast technique that can be used in combination with standard virus isolation methods to increase the yields of rAds.

Directed adenovirus evolution using engineered mutator viral polymerases

Adenoviruses (Ads) are the most frequently used viruses for oncolytic and gene therapy purposes. Most Ad-based vectors have been generated through rational design. Although this led to significant vector improvements, it is often hampered by an insufficient understanding of Ad’s intricate functions and interactions. Here, to evade this issue, we adopted a novel, mutator Ad polymerase-based, ‘accelerated-evolution’ approach that can serve as general method to generate or optimize adenoviral vectors. First, we site specifically substituted Ad polymerase residues located in either the nucleotide binding pocket or the exonuclease domain. This yielded several polymerase mutants that, while fully supportive of viral replication, increased Ad’s intrinsic mutation rate. Mutator activities of these mutants were revealed by performing deep sequencing on pools of replicated viruses. The strongest identified mutators carried replacements of residues implicated in ssDNA binding at the exonuclease active site. Next, we exploited these mutators to generate the genetic diversity required for directed Ad evolution. Using this new forward genetics approach, we isolated viral mutants with improved cytolytic activity. These mutants revealed a common mutation in a splice acceptor site preceding the gene for the adenovirus death protein (ADP). Accordingly, the isolated viruses showed high and untimely expression of ADP, correlating with a severe deregulation of E3 transcript splicing.