Abstract: BACKGROUND: DSB, the 3-O-(3',3'dimethylsuccinyl) derivative of betulinic acid, blocks the last step of protease-mediated processing of HIV-1 Gag precursor (Pr55Gag), which leads to immature, noninfectious virions. When administered to Pr55Gag-expressing insect cells (Sf9), DSB inhibits the assembly and budding of membrane-enveloped virus-like particles (VLP). In order to explore the possibility that viral factors could modulate the susceptibility to DSB of the VLP assembly process, several viral proteins were coexpressed individually with Pr55Gag in DSB-treated cells, and VLP yields assayed in the extracellular medium. RESULTS: Wild-type Vif (Vifwt) restored the VLP production in DSB-treated cells to levels observed in control, untreated cells. DSB-counteracting effect was also observed with Vif mutants defective in encapsidation into VLP, suggesting that packaging and anti-DSB effect were separate functions in Vif. The anti-DSB effect was abolished for VifC133S and VifS116V, two mutants which lacked the zinc binding domain (ZBD) formed by the four H(108)C(114)C(133)H(139) coordinates with a Zn atom. Electron microscopic analysis of cells coexpressing Pr55Gag and Vifwt showed that a large proportion of VLP budded into cytoplasmic vesicles and were released from Sf9 cells by exocytosis. However, in the presence of mutant VifC133S or VifS116V, most of the VLP assembled and budded at the plasma membrane, as in control cells expressing Pr55Gag alone. CONCLUSION: The function of HIV-1 Vif protein which negated the DSB inhibition of VLP assembly was independent of its packaging capability, but depended on the integrity of ZBD. In the presence of Vifwt, but not with ZBD mutants VifC133S and VifS116V, VLP were redirected to a vesicular compartment and egressed via the exocytic pathway.

Abstract: Intranuclear crystalline inclusions have been observed in the nucleus of epithelial cells infected with Adenovirus serotype 5 (Ad5) at late steps of the virus life cycle. Using immuno-electron microscopy and confocal microscopy of cells infected with various Ad5 recombinants modified in their penton base or fiber domains, we found that these inclusions represented crystals of penton capsomers, the heteromeric capsid protein formed of penton base and fiber subunits. The occurrence of protein crystals within the nucleus of infected cells required the integrity of the fiber knob and part of the shaft domain. In the knob domain, the region overlapping residues 489-492 in the FG loop was found to be essential for crystal formation. In the shaft, a large deletion of repeats 4 to 16 had no detrimental effect on crystal inclusions, whereas deletion of repeats 8 to 21 abolished crystal formation without altering the level of fiber protein expression. This suggested a crucial role of the five penultimate repeats in the crystallisation process. Chimeric pentons made of Ad5 penton base and fiber domains from different serotypes were analyzed with respect to crystal formation. No crystal was found when fiber consisted of shaft (S) from Ad5 and knob (K) from Ad3 (heterotypic S5-K3 fiber), but occurred with homotypic S3K3 fiber. However, less regular crystals were observed with homotypic S35-K35 fiber. TB5, a monoclonal antibody directed against the Ad5 fiber knob was found by immunofluorescence microscopy to react with high efficiency with the intranuclear protein crystals in situ. Data obtained with Ad fiber mutants indicated that the absence of crystalline inclusions correlated with a lower infectivity and/or lower yields of virus progeny, suggesting that the protein crystals might be involved in virion assembly. Thus, we propose that TB5 staining of Ad-infected 293 cells can be used as a prognostic assay for the viability and productivity of fiber-modified Ad5 vectors.

Abstract: BACKGROUND: Human parainfluenza viruses (hPIV) are respiratory pathogens responsible for upper and lower respiratory tract infections. In most labs, the clinical diagnosis of hPIV is routinely done using techniques based on the detection of viral antigens such as immunofluorescence assay or/and viral isolation. STUDY DESIGN: Five hPIV-2 isolated from respiratory samples exhibited unusual phenotypic and antigenic characteristics. These isolates showed important syncytial cytopathic effect and failed to react with one specific monoclonal antibody. These variant strains were subsequently compared with hPIV-2 prototype strain by cellular and molecular techniques. RESULTS: Both variant and prototype strains showed similar growth kinetics. Observation of plaque formation and syncytia assay indicated a more important fusogenic activity for the variant strains. Sequencing of fusion (F) and hemagglutinin-neuraminidase (HN) genes showed differences between the "atypical" hPIV-2 isolates and the Greer hPIV-2 prototype strain. These differences were analyzed with molecular modelling and structure prediction soft wares. A potential new glycosylation site in HN, in addition to minor changes observed in the predicted structure for the variant strains could explain their antigenic variation. Genetic changes in the fusion peptide and the cleavage site of F could also explain the difference observed in the fusion activity. CONCLUSIONS: Continuous global viral surveillance is essential to monitor antigenic changes that may occur in nature particularly with regards to the implementation of diagnostic assays. The differences observed in F and HN between the prototype strain and clinical hPIV-2 variants could also provide new data for the analysis of Paramyxovirus fusion mechanisms and their pathogenesis.

Abstract: BACKGROUND: The human EED protein, a member of the superfamily of Polycomb group proteins, is involved in multiple cellular protein complexes. Its C-terminal domain, which is common to the four EED isoforms, contains seven repeats of a canonical WD-40 motif. EED is an interactor of three HIV-1 proteins, matrix (MA), integrase (IN) and Nef. An antiviral activity has been found to be associated with isoforms EED3 and EED4 at the late stage of HIV-1 replication, due to a negative effect on virus assembly and genomic RNA packaging. The aim of the present study was to determine the regions of the EED C-terminal core domain which were accessible and available to protein interactions, using three-dimensional (3D) protein homology modelling with a WD-40 protein of known structure, and epitope mapping of anti-EED antibodies. RESULTS: Our data suggested that the C-terminal domain of EED was folded as a seven-bladed beta-propeller protein. During the completion of our work, crystallographic data of EED became available from co-crystals of the EED C-terminal core with the N-terminal domain of its cellular partner EZH2. Our 3D-model was in good congruence with the refined structural model determined from crystallographic data, except for a unique alpha-helix in the fourth beta-blade. More importantly, the position of flexible loops and accessible beta-strands on the beta-propeller was consistent with our mapping of immunogenic epitopes and sites of interaction with HIV-1 MA and IN. Certain immunoreactive regions were found to overlap with the EZH2, MA and IN binding sites, confirming their accessibility and reactivity at the surface of EED. Crystal structure of EED showed that the two discrete regions of interaction with MA and IN did not overlap with each other, nor with the EZH2 binding pocket, but were contiguous, and formed a continuous binding groove running along the lateral face of the beta-propeller. CONCLUSION: Identification of antibody-, MA-, IN- and EZH2-binding sites at the surface of the EED isoform 3 provided a global picture of the immunogenic and protein-protein interacting regions in the EED C-terminal domain, organized as a seven-bladed beta-propeller protein. Mapping of the HIV-1 MA and IN binding sites on the 3D-model of EED core predicted that EED-bound MA and IN ligands would be in close vicinity at the surface of the beta-propeller, and that the occurrence of a ternary complex MA-EED-IN would be possible.

Abstract: Two echovirus 6 (EV6) strains were isolated from a clinical sample after successive sub-cultures in PLC (human hepatocellular carcinoma) and HeLa (human cervical adenocarcinoma) cells. The first strain retained its haemagglutinating capacity (HAEV6) while the second became non-haemagglutinating (NHAEV6). Virus binding assay showed that HAEV6 was capable of binding to DAF-expressing cells but not NHAEV6 confirming the role of DAF in EV6 haemagglutination. The lack of competition between the two viral strains during coinfections suggested that each strain used a different cell entry pathway. We provide evidence showing that HAEV6 used preferentially the lipid raft-dependent caveolae pathway, whereas NHAEV6 followed the clathrin-mediated pathway. Comparison of the sequences of HAEV6 and NHAEV6 revealed five amino acid changes in the VP1, VP2 and VP3 capsid proteins distributed in domains which are known to be highly immunogenic or suggested to be involved in receptor binding, virion stability and pathogenicity.

Abstract: In this study, a prototype Adenovirus type 5 (Ad5) vector deleted of the fiber knob domain and carrying an Affibody molecule as the targeting ligand showed decreased susceptibility to human pre-existing antibodies. This vector, Ad5/R7-Z(taq)Z(taq), has short fibers carrying seven shaft repeats, a non-native trimerization signal and an affibody molecule (Z(taq)) reactive to Taq polymerase. Ad5/R7-Z(taq)Z(taq) could be specifically targeted to 293 cells stably expressing membrane-bound anti-Z(taq) idiotypic affibody called Z(ztaq) (293Z(ztaq)). Sera from 50 blood donors were analyzed for neutralization activity (NA) against the parental Ad5/Fiwt vector and knobless Ad5/R7-Z(taq)Z(taq) on 293Z(ztaq) cells. Twenty-three sera had NA titers (> or =1:64) against Ad5/Fiwt (46%) and only two against Ad5/R7-Z(taq)Z(taq) (4%). Characterization of sera with NA titers showed that the knob domain is one of the targets of the antibodies. Neutralization assays using sera pre-adsorbed on knob and hexon proteins showed that the NA of the sera was carried mainly by anti-knob and anti-hexon antibodies, but in certain sera the anti-hexon antibodies represent the major population of the neutralizing antibodies (NAbs). Our results suggested that a combination of knob deletion and hexon switching could be an effective strategy for Ad vectors to better evade the anti-Ad NAbs.

Abstract: In order to use adenovirus (Ad) type 5 (Ad5) for cancer gene therapy, Ad needs to be de-targeted from its native receptors and re-targeted to a tumor antigen. A limiting factor for this has been to find a ligand that (i) binds a relevant target, (ii) is able to fold correctly in the reducing environment of the cytoplasm and (iii) when incorporated at an optimal position on the virion results in a virus with a low physical particle to plaque-forming units ratio to diminish the viral load to be administered to a future patient. Here, we present a solution to these problems by producing a genetically re-targeted Ad with a tandem repeat of the HER2/neu reactive Affibody molecule (ZH) in the HI-loop of a Coxsackie B virus and Ad receptor (CAR) binding ablated fiber genetically modified to contain sequences for flexible linkers between the ZH and the knob sequences. ZH is an Affibody molecule specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) that is overexpressed in inter alia breast and ovarian carcinomas. The virus presented here exhibits near wild-type growth characteristics, infects cells via HER2/neu instead of CAR and represents an important step toward the development of genetically re-targeted adenoviruses with clinical relevance.

Abstract: BACKGROUND: The 3-O-(3',3'-dimethylsuccinyl) derivative of betulinic acid (DSB) blocks HIV-1 maturation by interfering with viral protease (PR) at the capsid (CA)-SP1 cleavage site, a crucial region in HIV-1 morphogenesis. METHODS: We analysed the effect of DSB on the assembly of HIV-1 Gag precursor (Pr55Gag(HIV)) into membrane-enveloped virus-like particles (VLP) in baculovirus-infected cells expressing Pr55Gag(HIV), in a cellular context devoid of viral PR. RESULTS: DSB showed a dose-dependent negative effect on VLP assembly, with an IC50 approximately 10 microM. The DSB inhibitory effect was p6-independent and was also observed for intracellular assembly of non-N-myristoylated Gag core-like particles. HIV-1 VLP assembled in the presence of DSB exhibited a lower stability of their inner cores upon membrane delipidation compared with control VLP, suggesting weaker Gag-Gag interactions. DSB also inhibited the assembly of simian immunodeficiency virus SLVmac251 VLP, although with a twofold lower efficacy (IC50 approximately 20 microM). No detectable inhibitory activity was observed for murine leukaemia virus (MLV) VLP; however, fusion of the SP1-NC-p6 domains from HIV-1 to the matrix (MA)-CA domains from MLV conferred DSB sensitivity to the chimaeric Gag precursor Pr72Gag(MLV-HIV) (IC50 = 30 microM). This observation suggested that the main DSB target on Pr55Gag was the SP1 domain, but the higher degree of DSB resistance for Pr72Gag(MLV-HIV) compared with Pr55Gag(HIV) implied that other upstream Gag region(s) might contribute to DSB reactivity. CONCLUSIONS: Sequence alignment and three-dimensional modelling by homology of the CA-SP1-NC junction in HIV-1, SLVmac251 and Pr72Gag(MLV-HIV) suggested that a higher hydrophilic character of the CA region immediately upstream to the HIV-1 CA-SP1 junction, as occurred in Pr72Gag(MLV-HIV), correlated with a lower DSB sensitivity.

Abstract: Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and the cellular trafficking of the CFTR protein is an essential factor that determines its function in cells. The aim of our study was to develop an Ad vector expressing a biologically active green fluorescent protein (GFP)-CFTR chimera that can be tracked by both its localization and chloride channel function. No study thus far has demonstrated a GFP-CFTR construct that displayed both of these functions in the airway epithelia. Tracheal glandular cells, MM39 (CFTRwt) and CF-KM4 (CFTRDeltaF508), as well as human airway epithelial cells from a patient with cystic fibrosis (CF-HAE) and from a healthy donor (HAE) were used for the functional analysis of our Ad vectors, Ad5/GFP-CFTRwt and Ad5/GFP-CFTRDeltaF508. The GFP-CFTRwt protein expressed was efficiently addressed to the plasma membrane of tracheal cells and to the apical surface of polarized CF-HAE cells, while GFP-CFTRDeltaF508 mutant was sequestered intracellularly. The functionality of the GFP-CFTRwt protein was demonstrated by its capacity to correct the chloride channel activity both in CF-KM4 and CF-HAE cells after Ad transduction. A correlation between the proportion of Ad5-transduced CF-KM4 cells and correction of CFTR function showed that 55 to 70% transduction resulted in 70% correction of the Cl- channel function. In reconstituted CF-HAE, GFP-CFTRwt appeared as active as the nontagged CFTRwt protein in correcting the transepithelial Cl- transport. We show for the first time a GFP-CFTR chimera that localized to the apical surface of human airway epithelia and restored epithelial chloride transport to similar levels as nontagged CFTR.

Abstract: BACKGROUND: The human EED protein, a member of the superfamily of Polycomb group (PcG) proteins with WD-40 repeats, has been found to interact with three HIV-1 components, namely the structural Gag matrix protein (MA), the integrase enzyme (IN) and the Nef protein. The aim of the present study was to analyze the possible biological role of EED in HIV-1 replication, using the HIV-1-based vector HIV-Luc and EED protein expressed by DNA transfection of 293T cells. RESULTS: During the early phase of HIV-1 infection, a slight negative effect on virus infectivity occurred in EED-expressing cells, which appeared to be dependent on EED-MA interaction. At late times post infection, EED caused an important reduction of virus production, from 20- to 25-fold as determined by CAp24 immunoassay, to 10- to 80-fold based on genomic RNA levels, and this decrease was not due to a reduction of Gag protein synthesis. Coexpression of WTNef, or the non-N-myristoylated mutant NefG2A, restored virus yields to levels obtained in the absence of exogenous EED protein. This effect was not observed with mutant NefDelta57 mimicking the Nef core, or with the lipid raft-retargeted fusion protein LAT-Nef. LATAA-Nef, a mutant defective in the lipid raft addressing function, had the same anti-EED effect as WTNef. Cell fractionation and confocal imaging showed that, in the absence of Nef, EED mainly localized in membrane domains different from the lipid rafts. Upon co-expression with WTNef, NefG2A or LATAA-Nef, but not with NefDelta57 or LAT-Nef, EED was found to relocate into an insoluble fraction along with Nef protein. Electron microscopy of HIV-Luc producer cells overexpressing EED showed significant less virus budding at the cell surface compared to control cells, and ectopic assembly and clustering of nuclear pore complexes within the cytoplasm. CONCLUSION: Our data suggested that EED exerted an antiviral activity at the late stage of HIV-1 replication, which included genomic RNA packaging and virus assembly, resulting possibly from a mistrafficking of viral genomic RNA (gRNA) or gRNA/Gag complex. Nef reversed the EED negative effect on virus production, a function which required the integrity of the Nef N-terminal domain, but not its N-myristoyl group. The antagonistic effect of Nef correlated with a cellular redistribution of both EED and Nef.

Abstract: Adenovirus serotype 5 (Ad5) vectors carrying knobless fibers designed to remove their natural tropism were found to have a lower fiber content than recombinant Ad5 with wild-type (WT) capsid, implying a role for the knob-coding sequence or/and the knob domain in fiber encapsidation. Experimental data using a variety of fiber gene constructs showed that the defect did not occur at the fiber mRNA level, but at the protein level. Knobless fiber proteins were found to be synthesized at a significant slower rate compared with knob-carrying fibers, and the trimerization process of knobless fibers paralleled their slow rate of synthesis. A recombinant Ad5 diploid for the fiber gene (referred to as Ad5/R7-ZZ(wt)/E1 : WT-fiber) was constructed to analyse the possible rescue of the knobless low-fiber-content phenotype by co-expression of WT fiber. Ad5/R7-ZZ(wt)/E1 : WT-fiber contained a knobless fiber gene in its natural location (L5) in the viral genome and an additional WT fiber gene in an ectopic position in E1. Knobless fiber was still synthesized at low levels compared with the co-expressed E1 : WT fiber and the recovery of the two fiber species in virus progeny reflected their respective amounts in the infected cells. Our results suggested that deletion of the fiber knob domain had a negative effect on the translation of the fiber mRNA and on the intracellular concentration of fiber protein. They also suggested that the knob control of fiber protein synthesis and encapsidation occurred as a cis effect, which was not modified by WT fiber protein provided in trans by the same Ad5 genome.

Abstract: Recombinant hexons from subgroup C adenoviruses (Ad2 and Ad5) and from a member of subgroup B (Ad3) adenoviruses have been expressed in insect cells. When expressed alone, all three hexons were found to be insoluble and accumulated as inclusion bodies in the cytoplasm. However, co-expression of recombinant Ad2, Ad5 or Ad3 hexon with Ad2 L4-100K protein resulted in the formation of soluble trimeric hexons. EM analysis of hexons revealed that they were indistinguishable from native hexon capsomers isolated from Ad2-infected human cells, or released from partially disrupted adenovirions. This suggests that 100K acts as a chaperone for hexon folding and self-assembly into capsomer in insect cells. Since 100K protein assists in the trimerization of subgroup C hexon, and of subgroup B hexon protein, it implies that it functions in a manner that is both homo- and heterotypic. During the course of recombinant protein expression, the 100K protein was found in association with hexon monomers and trimers within the cytoplasm. In the nucleus, however, 100K was found in complexes with hexon trimers exclusively. EM observation of purified 100K protein samples showed a dumb-bell-shaped molecule compatible with a monomeric protein. EM analysis of hexon-100K protein complexes showed that interaction of hexon with the 100K protein occurred via one of the globular domains of the 100K protein molecule. Our data confirm the role of the 100K protein as a scaffold protein for hexon, and provide evidence suggesting its function in hexon nuclear import in insect cells.

Abstract: Most human carcinoma cell lines lack the high-affinity receptors for adenovirus serotype 5 (Ad5) at their surface and are nonpermissive to Ad5. We therefore tested the efficiency of retargeting Ad5 to alternative cellular receptors via immunoglobulin (Ig)-binding domains inserted at the extremity of short-shafted, knobless fibers. The two recombinant Ad5's constructed, Ad5/R7-Z(wt)-Z(wt) and Ad5/R7-C2-C2, carried tandem Ig-binding domains from Staphylococcal protein A (abbreviated Z(wt)) and from Streptococcal protein G (C2), respectively. Both viruses bound their specific Ig isotypes with the expected affinity. They transduced human carcinoma cells independently of the CAR pathway, via cell surface receptors targeted by specific monoclonal antibodies, that is, EGF-R on A549, HT29 and SW1116, HER-2/neu on SK-OV-3 and SK-BR-3, CA242 (epitope recognized by the monoclonal antibody C242) antigen on HT29 and SW1116, and PSMA (prostate-specific membrane antigen) expressed on HEK-293 cells, respectively. However, Colo201 and Colo205 cells were neither transduced by targeting CA242 or EGF-R nor were LNCaP cells transduced by targeting PSMA. Our results suggested that one given surface receptor could mediate transduction of certain cells but not others, indicating that factors and steps other than cell surface expression and virus-receptor interaction are additional determinants of Ad5-mediated transduction of tumor cells. Using penton base RGD mutants, we found that one of these limiting steps was virus endocytosis.

Abstract: Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) do not express the coxsackie-adenovirus (Ad) receptor and are poorly permissive to Ad serotype 5 (Ad5). Genetically modified, coxsackie-Ad receptor-independent Ad5 vectors were studied for gene delivery in human RA FLS and synovium explants and murine collagen-induced arthritis. Short-fiber Ad5 vectors with seven fiber shaft repeats Ad5GFP-R7-knob, Ad5GFP-R7-arginine-glycine-aspartic acid (RGD) (RGD-liganded), and Ad5GFPDeltaknob (knob-deleted) were compared with Ad5GFP-FiWT, a conventional wild-type (WT) Ad5 vector. Gene transfer by Ad5GFP-R7-knob and Ad5GFP-R7-RGD was 40- to 50-fold and 25-fold higher, respectively, than Ad5GFP-FiWT in FLS. Ad5GFPDeltaknob was more efficacious than its knob-bearing version Ad5GFP-R7-knob in FLS transduction. Virus attachment and entry required RGD- and LDV-binding integrins including alpha(v), alpha(v)beta3, a(v)beta5, and beta1. Ad5GFP-R7-knob infection of FLS was partially neutralized by synovial fluid (SF), but remained 30- to 40-fold higher than Ad5GFP-FiWT in the presence of SF. Ad5GFPDeltaknob was partially neutralized by SF at low virus input, but escaped viral neutralization by SF at higher virus input. Gene transfer to human synovium ex vivo explants and murine collagen-induced arthritis in vivo was also more efficient with short fiber-modified vectors (with and without the knob domain) than Ad5GFPFiWT. Gene transfer by short fiber-modified vectors was enhanced by inflammatory cytokines in vitro and in the presence of inflammation in murine synovium in vivo. Our data indicated that the highly efficient gene delivery RA was mediated by RGD- and non-RGD-binding integrins and enhanced by inflammation. Short fiber modifications with knob ablation may be a strategy to enhance gene delivery, reducing vector dose and vector-induced inflammation and toxicity.

Abstract: Monolayers of cystic fibrosis transmembrane conductance regulator (CFTR)-deficient human tracheal glandular cells (CF-KM4) were subjected to phage biopanning, and cell-internalized phages were isolated and sequenced, in order to identify CF-KM4-specific peptide ligands that would confer upon adenovirus type 5 (Ad5) vector a novel cell target specificity and/or higher efficiency of gene delivery into airway cells of patients with cystic fibrosis (CF). Three different ligands, corresponding to prototypes of the most represented families of phagotopes recovered from intracellular phages, were designed and individually inserted into Ad5-green fluorescent protein (GFP) (AdGFP) vectors at the extremities of short fiber shafts (seven repeats [R7]) terminated by scissile knobs. Only one vector, carrying the decapeptide GHPRQMSHVY (abbreviated as QM10), showed an enhanced gene transduction of CF-KM4 cells compared to control nonliganded vector with fibers of the same length (AdGFP-R7-knob). The enhancement in gene transfer efficiency was not specific to CF-KM4 cells but was observed in other mammalian cell lines tested. The QM10-liganded vector was referred to as AdGFP-QM10-knob in its knobbed version and as AdGFP-QM10 in its proteolytically deknobbed version. AdGFP-QM10 was found to transduce cells with a higher efficiency than its knob-bearing version, AdGFP-QM10-knob. Consistent with this, competition experiments indicated that the presence of knob domains was not an absolute requirement for cell attachment of the QM10-liganded vector and that the knobless AdGFP-QM10 used alternative cell-binding domains on its capsid, including penton base capsomer, via a site(s) different from its RGD motifs. The QM10-mediated effect on gene transduction seemed to take place at the step of endocytosis in both quantitative and qualitative manners. Virions of AdGFP-QM10 were endocytosed in higher numbers than virions of the control vector and were directed to a compartment different from the early endosomes targeted by members of species C Ad. AdGFP-QM10 was found to accumulate in late endosomal and low-pH compartments, suggesting that QM10 acted as an endocytic ligand of the lysosomal pathway. These results validated the concept of detargeting and retargeting Ad vectors via our deknobbing system and redirecting Ad vectors to an alternative endocytic pathway via a peptide ligand inserted in the fiber shaft domain.

Abstract: We developed a new type of adenovirus type 5 (Ad5)-derived vector with genetically modified fiber proteins whose knob domains could be stripped off due to the insertion of a single Factor Xa cleavage site in the fiber shaft, between a cellular ligand and the knob domain. This Ad vector did not require a specific cell line for propagation and could be grown in HEK-293 cells. Stripping off the knob domains removed the endogenous cell-binding moiety of Ad but retained the new cell ligand for retargeting purposes. As experimental models for cell ligands, we used two peptides with different sequence complexities: (i) the integrin-binding tripeptide RGD and (ii) a 58-residue oligopeptide termed affibody (Zwt). Zwt binds specifically to the human IgG1 Fc domain or to its Fc3(1) homolog. The modified fibers were efficiently encapsidated into virions, and the Factor Xa sites were fully accessible to proteolysis. In vitro binding assays using recombinant Fc3(1) protein and Ad5-mediated gene transduction of Fc3(1)-expressing cells demonstrated that the proteolytically deknobbed Ad5-Zwt vector was functional and specific for receptor targeting.

Abstract: Sera from 17 patients with primary and secondary liver tumors who had been administered oncolytic adenovirus (Ad) mutant Addl1520 were analyzed for anti-Ad neutralization titers and antibodies to the Ad major capsid proteins hexon, penton base (Pb), and fiber. The antibodies recognized mainly conformational epitopes in hexon and both linear and conformational epitopes in Pb and fiber. Pb-specific antibodies were isolated from serum samples that had been obtained prior to and during the course of the treatment of four of these patients. We found that the Pb antibodies had a significant contribution toward anti-Ad neutralization, and this mainly occurred at the step of virus internalization. The Pb antigenic epitopes were determined by phage biopanning and were mapped to 10 discrete regions, which made up three major immunodominant domains within residues 51 to 120, 193 to 230, and 311 to 408, respectively. One of these domains (residues 311 to 408) overlapped the highly conserved, integrin-binding RGD (Arg-Gly-Asp) motif. The contribution of antibodies directed to RGD and other epitopes in Ad neutralization activity was determined indirectly by using a phage-mediated depletion assay. Our results suggested that circulating RGD antibodies were not prevalent and were poorly neutralizing and that other peptide motifs within residues 51 to 60, 216 to 226, and 311 to 408 in Pb sequence represented major target sites for neutralizing antibodies.

Abstract: We investigated the mechanism of adenovirus serotype 5 (Ad5)-mediated maturation of bone marrow-derived murine dendritic cells (DC) using (i) Ad5 vectors with wild-type capsid (AdE1 degrees, AdGFP); (ii) Ad5 vector mutant deleted of the fiber C-terminal knob domain (AdGFPDeltaknob); and (iii) capsid components isolated from Ad5-infected cells or expressed as recombinant proteins, hexon, penton, penton base, full-length fiber, fiber knob, and fiber mutants. We found that penton capsomer (penton base linked to its fiber projection), full-length fiber protein, and its isolated knob domain were all capable of inducing DC maturation, whereas no significant DC maturation was observed for hexon or penton base alone. This capacity was severely reduced for AdGFPDeltaknob and for fiber protein deletion mutants lacking the beta-stranded region F of the knob (residues Leu-485-Thr-486). The DC maturation effect was fully retained in a recombinant fiber protein deleted of the HI loop (FiDeltaHI), a fiber (Fi) deletion mutant that failed to trimerize, suggesting that the fiber knob-mediated DC activation did not depend on the integrity of the HI loop and on the trimeric status of the fiber. Interestingly, peptide-pulsed DC that had been stimulated with Ad5 knob protein induced a potent CD8+ T cell response in vivo.

Abstract: Human EED, a member of the superfamily of WD-40 repeat proteins and of the Polycomb group proteins, has been identified as a cellular partner of the human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein (R. Peytavi et al., J. Biol. Chem. 274:1635-1645, 1999). In the present study, EED was found to interact with HIV-1 integrase (IN) both in vitro and in vivo in yeast. In vitro, data from mutagenesis studies, pull-down assays, and phage biopanning suggested that EED-binding site(s) are located in the C-terminal domain of IN, between residues 212 and 264. In EED, two putative discrete IN-binding sites were mapped to its N-terminal moiety, at a distance from the MA-binding site, but EED-IN interaction also required the integrity of the EED last two WD repeats. EED showed an apparent positive effect on IN-mediated DNA integration reaction in vitro, in a dose-dependent manner. In situ analysis by immunoelectron microscopy (IEM) of cellular distribution of IN and EED in HIV-1-infected cells (HeLa CD4(+) cells or MT4 lymphoid cells) showed that IN and EED colocalized in the nucleus and near nuclear pores, with maximum colocalization events occurring at 6 h postinfection (p.i.). Triple colocalizations of IN, EED, and MA were also observed in the nucleoplasm of infected cells at 6 h p.i., suggesting the ocurrence of multiprotein complexes involving these three proteins at early steps of the HIV-1 virus life cycle. Such IEM patterns were not observed with a noninfectious, envelope deletion mutant of HIV-1.

Abstract: The capacity of recombinant adenoviruses (rAd) to induce immunization against their transgene products has been well documented. In the present study, we evaluated the vaccinal adjuvant role of rAd independently of its vector function. BALB/c mice received one subcutaneous injection of a mixture of six lipopeptides (LP6) used as a model immunogen, along with AdE1 degrees (10(9) particles), a first-generation rAd empty vector. Although coinjected with a suboptimal dose of lipopeptides, AdE1 degrees significantly improved the effectiveness of the vaccination, even in the absence of booster immunization. In contrast to mice that received LP6 alone or LP6 plus a mock adjuvant, mice injected with AdE1 degrees plus LP6 developed both a polyspecific T-helper type 1 response and an effector CD8 T-cell response specific to at least two class I-restricted epitopes. The helper response was still observed when immunization was performed using LP6 plus a mixture of soluble capsid components released from detergent-disrupted virions. When mice were immunized with LP6 and each individual capsid component, i.e., hexon, penton base, or fiber, the results obtained suggested that hexon protein was responsible for the adjuvant effect exerted by disrupted Ad particles on the helper response to the immunogen. Our results thus have some important implications not only in vaccinology but also for gene therapy using rAd vectors.

Abstract: The use of adenovirus (Ad) as an efficient and versatile vector for in vivo tumor therapy requires the modulation of its cellular tropism. We previously developed a method to genetically alter the tropism of Ad5 fibers by replacing the fiber knob domain by an extrinsic trimerization motif and a new cellular ligand. However, fibers carrying complex ligands such as single-chain antibody fragments did not assemble into functional pentons in vitro in the presence of penton base, and failed to be rescued into infectious virions because of their inability to fold correctly within the cytoplasm of Ad-infected cells. Here we show that the coding sequence for a disulfide bond-independent three-helix bundle scaffold Z, derived from domain B of Staphylococcal protein A and capable of binding to the Fc portion of immunoglobulin (Ig) G1, could be incorporated into modified knobless Ad fiber gene constructs with seven shaft repeats. These fiber gene constructs could be rescued into viable virions that were demonstrated to enter 293 cells engineered for IgG Fc surface expression but not unmodified 293 cells, via a mechanism that could be specifically blocked with soluble Fc target protein. However, the tropism modified viruses showed a slightly impaired cellular entry and a lower infectivity than wildtype (WT) virus. In addition, we generated recombinant fibers containing an IgA binding Affibody ligand, derived from combinatorial specificity-engineering of the Z domain scaffold. Such fiber constructs also showed the expected target specific binding, indicating that the affibody protein class is ideally suited for genetic engineering of Ad tropism.

Abstract: CF-KM4 (cystic fibrosis transmebrane conductance regulator-deficient) and MM-39 (healthy) cells, two serous cell lines from submucosal tracheal glands, were found to be poorly susceptible to adenovirus (Ad)5 infection and Ad5-mediated gene transduction. The major limiting steps apparently resided in the primary events of Ad5 interaction, i.e., cell attachment and entry. Both CF-KM4 and MM-39 cells failed to express the Coxsackie-Ad receptor (CAR), and experimental data suggested that alpha[2-->6]-linked sialic acid residues of sialoglycoproteins (SAGP) in CF-KM4 cells, and heparan sulfate glycosaminoglycans (HS-GAG) in MM-39, were used as receptors by Ad5 virions. Ad5 attached to SAGP and HS-GAG receptors via its fiber knob domain, but entered the cells via a penton base- and Arg-Gly-Asp (RGD)-integrin-independent pathway. The block to Ad5-mediated gene transfer in MM-39 and KM4 cells could be overcome by conferring to the vector a novel cell-binding specificity. Thus, Ad5 vectors carrying a stretch of 7-lysine residues genetically inserted at the C-terminus of the fiber knob were found to transduce MM-39 cells with a 10- to 20-fold higher efficiency than the original vectors, but the transduction of CF-KM4 was not significantly improved. Retargeting Ad5 to integrin receptors via RGD peptide ligands, inserted at the extremity of the fiber shaft, resulted in a transducing efficiency of 20- and 50-fold higher in MM-39 and KM4 cells, respectively, compared with Ad5 vectors carrying fibers terminated by their natural knob domain.

Abstract: BACKGROUND: We studied the ability of adenovirus type 5 (Ad5) to encapsidate new cellular ligands carried by their fibers to yield functional retargeted vectors for gene therapy. Recombinant Ad5 fibers containing shaft repeats 1 to 7 and an extrinsic trimerization motif, and terminated by its native knob or amino acid motifs containing RGD, have been rescued into infectious virions. METHODS: Polypeptide ligands of cell surface molecules, including single-chain antibodies or epidermal growth factor, were cloned into recombinant fibers. Phenotypic analysis of fiber constructs and rescuing into the Ad5 genome were performed. Recombinant viruses were characterized with reference to fiber content, growth rate and infectivity. RESULTS: A major limiting factor for recovering viable recombinant Ad5 carrying fiber-fused polypeptide ligands was apparently the ability of the ligand to fold correctly within the cellular cytoplasm. This constraint has previously not been systematically evaluated in the literature. Phenotypic analysis of the fiber-ligand fusions showed that their degree of cytoplasmic solubility correlated with their ability to yield viable Ad5 vectors. Our results suggested that the fiber manipulations diminish virus growth rate, probably through different, opposing effects: (i) the reduced shaft length increases fiber solubility in the absence of the knob but (ii) diminishes virus entry, and (iii) the absence of the knob alters the overall protein composition of the virion and decreases its fiber copy number. CONCLUSIONS: Based on our findings, cytoplasmic solubility and cytoplasmic ligand reactivity of fiber-ligand fusion proteins are the best prediction criterion for viability and recovery of genetically retargeted Ad vectors.

Abstract: For efficient and versatile use of adenovirus (Ad) as an in vivo gene therapy vector, modulation of the viral tropism is highly desirable. In this study, a novel method to genetically alter the Ad fiber tropism is described. The knob and the last 15 shaft repeats of the fiber gene were deleted and replaced with an external trimerization motif and a new cell-binding ligand, in this case the integrin-binding motif RGD. The corresponding recombinant fiber retained the basic biological functions of the natural fiber, i.e., trimerization, nuclear import, penton formation, and ligand binding. The recombinant fiber bound to integrins but failed to react with antiknob antibody. For virus production, the recombinant fiber gene was rescued into the Ad genome at the exact position of the wild-type (WT) fiber to make use of the native regulation of fiber expression. The recombinant virus Ad5/FibR7-RGD yielded plaques on 293 cells, but the spread through the monolayer was two to three times delayed compared to WT, and the ratio of infectious to physical particles was 20 times lower. Studies on virus tropism showed that Ad5/FibR7-RGD was able to infect cells which did not express the coxsackie-adenovirus receptor (CAR), but did express integrins. Ad5/FibR7-RGD virus infectivity was unchanged in the presence of antiknob antibody, which neutralized the WT virus. Ad5/FibR7-RGD virus showed an expanded tropism, which is useful when gene transfer to cells not expressing CAR is needed. The described method should also make possible the construction of Ad genetically retargeted via ligands other than RGD.

Abstract: A panel of nine independent mouse monoclonal antibodies (MAbs) against penton base capsomers of subgenus C adenovirus serotypes 2 (Ad2) and 5 (Ad5) were isolated and characterized. Two of them (1D2 and 5A5), raised against Ad5 virion as the immunogen, bound to sodium dodecyl sulfate (SDS)-resistant and subgenus C-specific epitopes that were not present in subgenus B Ad3 penton base. The 1D2 and 5A5 epitopes were mapped to two distinct regions that did not belong to the main variable region carrying the integrin-binding RGD motif at position 340. For the other seven MAbs, raised against recombinant Ad2 penton base protein (9S-pentamers), the epitopes were sensitive to SDS-denaturation, but reacted with native Ad2, Ad5, and Ad3 penton base. The epitopes recognized by the nine MAbs and by polyclonal antipenton base antibodies defined three major immunoreactive regions. One (I) mapped to the N-terminal domain (residues 116-165); the other two regions were almost symmetrically disposed on both sides of the integrin-binding RGD motif at position 340, within residues 248-270 (II), and within residues 368-427 (III) in the C-terminal domain. Region II overlapped the fiber-binding site in penton base (residues 254-260). None of the MAbs showed any detectable virus neutralization effect, but they all slightly augmented the efficiency of Ad-mediated gene transfer. Although none of their epitopes included the RGD-340 tripeptide, substitutions of the arginine residue in the RGD motif abolished the reactivity of six individual and distant epitopes, suggesting a major conformational role for the RGD-containing domain.

Abstract: The best-characterized receptors for adenoviruses (Ads) are the coxsackievirus-Ad receptor (CAR) and integrins alpha(v)beta(5) and alpha(v)beta(3), which facilitate entry. The alpha(v) integrins recognize an Arg-Gly-Asp (RGD) motif found in some extracellular matrix proteins and in the penton base in most human Ads. Using a canine adenovirus type 2 (CAV-2) vector, we found that CHO cells that express CAR but not wild-type CHO cells are susceptible to CAV-2 transduction. Cells expressing alpha(M)beta(2) integrins or major histocompatibility complex class I (MHC-I) molecules but which do not express CAR were not transduced. Binding assays showed that CAV-2 attaches to a recombinant soluble form of CAR and that Ad type 5 (Ad5) fiber, penton base, and an anti-CAR antibody partially blocked attachment. Using fluorescently labeled CAV-2 particles, we found that in some cells nonpermissive for transduction, inhibition was at the point of internalization and not attachment. The transduction efficiency of CAV-2, which lacks an RGD motif, surprisingly mimicked that of Ad5 when tested in cells selectively expressing alpha(v)beta(5) and alpha(v)beta(3) integrins. Our results demonstrate that CAV-2 transduction is augmented by CAR and possibly by alpha(v)beta(5), though transduction can be CAR and alpha(v)beta(3/5) independent but is alpha(M)beta(2), MHC-I, and RGD independent, demonstrating a transduction mechanism which is distinct from that of Ad2/5.

Abstract: Adenovirus (Ad) tropism is mediated in part through the fibre protein. The common coxsackie B virus and Ad receptor (CAR) was recently identified as the major receptor for subgroup C Ad serotype 5 (Ad5) and serotype 2 (Ad2) fibres. Effects of mutations in the Ad5 fibre gene were studied to assess domains of the fibre capsomer that could alter virus tropism without altering virus assembly and replication. All mutants that accumulated as fibre monomers failed to assemble with a penton base and proved lethal for Ad5 which suggests that the absence of infectious virions resulted in part from a defect in fibre penton base assembly. Cell binding capacity of all fibre mutants was investigated in cell binding competition experiments with adenovirions using CHO-CAR cells (CHO cells that have been transfected with CAR cDNA and express functional CAR). The results suggest that the R-sheet of the Ad5 fibre knob monomer contains binding motifs for CAR and that beta-strands E and F, or a region close to them, may also be involved in receptor recognition.

Abstract: The efficiency of human adenovirus serotype 5 (Ad5) transgene delivery was tested on several human and animal cell lines in vitro, by using a bimodular 35-mer oligopeptide carrying two peptide domains with different ligand specificities. One domain mimicked the fiber knob-binding region of the alpha2 domain of human MHC-1 molecules (MH20), and the other corresponded to the gastrin-releasing peptide (GRP). Two synthetic peptides with different configurations were analyzed in Ad-mediated gene transfer assays using Ad5Luc3 vector carrying the luciferase reporter gene. One peptide (GRP-MH20) had the GRP domain on the N-terminal side of MH20, while the other (MH20-GRP), the C-terminally amidified GRP, was on the C-terminal side of MH20. The GRP-MH20 peptide, but not MH20-GRP, was capable of enhancing luciferase gene delivery to Ad-susceptible cells in a GRP receptor-dependent manner. More importantly, GRP-MH20 could also confer susceptibility to Ad infection to normal or cancer cells that lack fiber receptors for the virus. Our data suggested that GRP receptors could function efficiently as alternative attachment receptors for Ad5, but that Ad5 bound to GRP receptors still depended, at least partially, on the penton base-mediated endocytotic pathway for subsequent cell entry. Gene delivery by a human adenovirus serotype 5 (Ad5) vector was assayed with a bimodular oligopeptide carrying two peptide domains of different binding specificities. One domain was a high-affinity peptide ligand of the Ad5 fiber knob (MH20), and the other corresponded to the gastrin-releasing peptide (GRP). The synthetic peptide GRP-MH20 was found to be capable of enhancing Ad-mediated gene transfer to Ad-susceptible cells in a GRP receptor-dependent manner. More importantly, GRP-MH20 could also confer susceptibility to Ad infection to normal or cancer cells that lack fiber receptors. Our data suggested that GRP receptors could function efficiently as alternative attachment receptors for Ad5, but virus bound to GRP receptors still depended partially on the penton base-mediated pathway for cell entry.

Abstract: An Ad2 capsid component, the penton base, expressed as recombinant protein, was found to be capable of affecting the entire entry pathway of adenovirion in HeLa cells, i.e., cell attachment, endocytosis, vesicular escape, intracytoplasmic movement, and translocation through the nuclear pore complex. Data with pentamerization-defective mutants suggested that none of these successive steps depended upon penton base pentamer status, indicating that the peptide domains responsible for these functions were carried by the monomer. Observations performed with wild-type (WT) and an integrin-binding-site double-mutant (K288E340) suggested that the penton base could enter the cell via an alternative, RGD- and LDV-independent, pathway. Of three mutants that were found to be defective in nuclear addressing in insect cells, only one, W165H, was also altered in nuclear transport in HeLa cells. The other two, W119H and RRR547EQQ, showed a WT pattern of nuclear localization in HeLa cells, suggesting that the region including tryptophan-119 and the basic signal at position 547 did not act as a nuclear localization signal in the human cell context. The integrity of cellular structures and the cytoskeleton seemed to be required for the vectorial movement and nuclear import of WT penton base, as suggested by experiments using permeabilized HeLa cells, isolated nuclear membranes, and cytoskeleton-targeted drugs.

Abstract: heed, the human homolog of mouse eed and Drosophila esc, two members of the trithorax (trx) and Polycomb group (Pc-G) of genes, was isolated by screening an activated lymphocyte cDNA library versus the immunodeficiency virus type 1 (HIV-1) MA protein used as a bait in a two-hybrid system in yeast. The human EED protein (HEED) had 99. 5% identity with the mouse EED protein and contained seven WD repeats. Two heed gene transcripts were identified, with a putative 407-nucleotide-long intron, giving rise to two HEED protein isoforms of 535 and 494 residues in length, respectively. The shorter HEED isoform, originated from the unspliced message, lacked the seventh WD repeat. HEED was found to bind to MA protein in vitro, as efficiently as in vivo in yeast cells. Site-directed mutagenesis and phage biopanning suggested that the interaction between HEED and MA involved the N-terminal region of the MA protein, including the first polybasic signal, in a MA conformation-dependent manner. In the HEED protein, however, two discrete linear MA-binding motifs were identified within residues 388-403, overlapping the origin of the fifth WD repeat. Deletion of the C-terminal 41 residues of HEED, spanning the seventh WD repeat, as in the 494-residue HEED protein, was detrimental to HEED-MA interaction in vivo, suggesting the existence of another C-terminal binding site and/or a conformational role of the HEED C-terminal domain in the MA-HEED interaction. MA and HEED proteins co-localized within the nucleus of co-transfected human cells and of recombinant baculovirus co-infected insect cells. This and the failure of HEED to bind to uncleaved GAG precursor suggested a role of HEED at the early stages of virus infection, rather than late in the virus life cycle.

Abstract: Human immunodeficiency virus type 1 (HIV-1) wild-type (WT) virion infectivity factor (Vif) protein (Vifwt) and full-length Gag precursor (Pr55Gag) were found to be co-encapsidated into extracellular, membrane-enveloped virus-like particles released by budding from Sf9 cells co-expressing the two recombinant proteins in trans, with an average copy number of 3.5+/-0.6 Vifwt per 100 Pr55Gag molecules. No preferential localization at the plasma membrane was observed for recombinant Vif in the absence of Gag expression, and a significant proportion of Vif accumulated within the nucleus. Two conserved motifs, W89RKRRY94 and P156KKIKP161, seemed to act as nuclear addressing signals. The Pr55Gag and Vifwt interacting domains were analysed by biopanning of a phage-displayed hexapeptide library. The Vif-binding domain, which spanned residues H421-T470 in Pr55Gag, corresponded to the C-terminal region of nucleocapsid (NC), including the second zinc finger, the intermediate spacer peptide sp2 and the N-terminal half of the p6 domain. Deletions in these Gag domains significantly decreased the Vif encapsidation efficiency, and complete deletion of NC abolished Vif encapsidation. In Vif, four discrete Gag-binding sites were identified, within residues T68-L81 (site I) and W89-P100 (site II) in the central domain, and within residues P162-R173 (III) and P177-M189 (IV) at the C terminus. Substitutions in site I and deletion of site IV were detrimental to Vif encapsidation, whereas substitution of basic residues for alanine in sites III and IV had a positive effect. The data suggest a direct intracellular Gag-Vif interaction and the occurrence of a Pr55Gag-mediated membrane-targeting pathway for Vif in Sf9 cells.

Abstract: Adenovirus serotype 5 (Ad5) fiber receptor was investigated using reverse antibody biopanning of a phage-displayed hexapeptide library, and virus-neutralizing monoclonal antibodies (mAbs 1D6.3 and 7A2.7) raised against recombinant Ad5 fiber knob. Both mAbs inhibited attachment of Ad5 to HeLa cells. Mimotopes of 1D6.3 showed homology with the C-terminal segment of the alpha2 domain of the heavy chain of human MHC class I molecules (MHC-I alpha2), and mimotopes of 7A2.7 were consensus to human fibronectin type III (FNIII) modules. In vitro, GST-fused MHC-I alpha2- and FNIII-derived oligopeptides interacted with recombinant fibers in a subgroup-specific manner. In vivo, the MHC-I alpha2 synthetic icosapeptide RAIVGFRVQWLRRYFVNGSR showed a net neutralization effect on Ad5 in HeLa cells, whereas the FNIII icosapeptide RHILWTPANTPAMGYLARVS significantly increased Ad5 binding to HeLa cells. Daudi cells, which lack surface expression of HLA class I molecules, showed a weak capacity for Ad5 binding. In beta2-microglobulin-transfected Daudi cells, Ad5 attachment and permissivity were restored to HeLa cell levels, with 4000 receptors per cell and a binding constant of 1.4x10(10)/M. The results suggested that the conserved region of MHC-I alpha2-domain including Trp167 represents a high affinity receptor for Ad5 fiber knob, whereas ubiquitous FNIII modules would serve as auxiliary receptors.

Abstract: Discrete domains involved in structural and functional properties of adenovirus type 2 (Ad2) penton base were investigated with site-directed mutagenesis of the recombinant protein expressed in baculovirus-infected cells. Seventeen substitution mutants were generated and phenotyped for various functions in insect and human cells as follows. (i) Pentamerization of the penton base protein was found to be dependent on three amino acid side chains, the indole ring of Trp119, the hydroxylic group of Tyr553, and the basic group of Lys556. (ii) Arg254, Cys432, and Trp439, the stretch of basic residues at positions 547 to 556, and Arg340 of the RGD motif played a critical role in stable fiber-penton base interactions in vivo. (iii) Nuclear localization of penton base in Sf9 cells was negatively affected in mutants W119H or W165H, and, to a lesser extent, by substitutions in the consensus polybasic signal at positions 547 to 549. (iv) Penton base mutants were also assayed for HeLa cell binding, cell detachment, plasmid DNA internalization, and Ad-mediated gene delivery. The results obtained suggested that the previously identified integrin-binding motifs RGD340 and LDV287 were functionally and/or topologically related to other discrete regions which include Trp119, Trp165, Cys246, Cys432, and Trp439, all of which were involved in penton base-cell surface recognition, endocytosis, and postendocytotic steps of the virus life cycle.

Abstract: A filamentous phage-displayed random hexapeptide library was screened on the adenovirus type 2 (Ad2) penton capsomer and its separate domains, penton base, full-length fiber, fiber shaft and fiber knob. Affinity supports were designed to immobilize the penton ligate with a preferred orientation, via immuno-adsorption to pre-coated antibody. Three classes of phagotopes were distinguished in the eluates from the penton and fiber domains. (i) The first class represented peptide sequences identified in certain Ad2 capsid proteins, protein IIIa, protein pVIII, penton base and penton fiber. Data from specific ligand elution of phages bound to fiber and penton base wild-types and mutants suggested that the region overlapping the RLSNLLG motif at residues 254-260 in the penton base and the FNPVYP motif at residues 11-16 in the fiber tail formed mutual interacting sites in the penton capsomer. (ii) The second class consisted of phagotopes homologous to peptide sequences found in host cell membrane proteins involved in receptor or adhesion functions. One of the most abundant species corresponded to a conserved motif present in the beta-strand B of type III modules of human fibronectin. In addition, phages which were screened for their failure to bind to penton base RGD mutants were found to carry consensus motifs to peptide sequences present in the RGD recognition site of human integrin beta subunits. (iii) The third class comprised peptide motifs common to both viral and cellular proteins, suggesting that a mechanism of ligand exchange could occur during virus entry and uncoating, and virus assembly and release.

Abstract: Two substitution mutants of the human immunodeficiency virus type 1 gag gene product were isolated after nitrous acid mutagenesis of a recombinant baculovirus expressing a non-N-myristylated, p6-deleted Gag precursor (Pr49). Both mutants failed to assemble intracellular Gag virus-like particles, as does the parental recombinant, and therefore expressed a self-assembly defective (Sad) phenotype in insect cells. The mutations consisted of nonconservative changes involving highly conserved hydrophobic residues in the p24 domain, Leu to Pro at position 268 (L268P) and Leu to Ser at amino acid 322 (L322S). Experimental data suggested that the two mutated residues belonged to functionally different regions of the Gag precursor. (i) A partial complementation effect between the two mutants for Gag precursor assembly was observed in coinfection experiments. (ii) The two mutations showed different phenotypes when placed in the N-myristylated context, of which only the L268P mutation abolished extracellular budding and release of Gag particles at the plasma membrane. Both L268P and L322S mutants had a trans-dominant negative effect on the intracellular assembly of a non-N-myristylated, full-length (Pr55) Gag precursor expressed by a coinfecting recombinant. None of the mutants, however, showed any detectable effect in trans on membrane targeting and budding of the coexpressed N-myristylated wild-type Gag precursor.

Abstract: The level of synthesis and extracellular release of human immunodeficiency virus type 1 Gag by insect cells was analyzed, using eight different recombinants of Autographa californica nuclear polyhedrosis virus harboring various constructs of the gag gene, cloned under the polyhedrin promoter. The results obtained suggested that gag expression was mainly regulated at the transcriptional level and was not significantly influenced by posttranslational events, e.g., Gag self-assembly, nuclear transport, or extracellular release. Two different forms of Gag were found in the culture medium of recombinant-infected cells. One form consisted of membrane-enveloped, corelike particles released by budding at the plasma membrane; the other of nonparticulate, soluble Gag polyprotein molecules. Both forms coexisted in recombinants expressing Gag with an intact N-terminal myristylation signal, whereas recombinants expressing nonmyristylated Gag released solely the soluble form. This suggested that myristylation of the N terminus was not a prerequisite for efficient extracellular release of Gag by insect cells, which could proceed via two independent but simultaneous mechanisms.

Abstract: Seven recombinants of AcNPV harboring various forms of complete or truncated gag gene from HIV-1 were constructed to determine which functional domains of the gag polyprotein are implicated in its self-assembly and cellular localization. The p6 carboxy-terminal portion of the p15 NCgag domain appeared to be dispensable for assembly, budding, and release of gag particles by insect cells. However, all the morphopoietic information was not entirely confined to the p9 NC domain, as N-myristylation could compensate for p15 NC deletion in gag assembly and the budding process. The two consensus karyophilic signals situated in the p17 MAgag domain were inefficient for targeting nonmyristylated forms of gag polyprotein to the nucleus when the p6 NC domain was deleted. In the presence of p6, or with a third, baculovirus-specific, karyophilic signal added at its N-terminus, gag particles relocated in the nucleus. These data suggested that p6 played a critical role in the conformation of gag polyprotein.

Abstract: Virus-induced vesicles evolved early in the Kunjin virus replication cycle around 9 to 10 h p.i. just before the end of the latent period in infected Vero cells. About 2 h following the appearance of the vesicles, microtubule paracrystals were also formed. These two virus-induced structures seemed interlinked and have essential roles in Kunjin virus replication. A viral protein NS3 was found to be associated with the microtubule component of the cells. When vinblastine sulphate was added to the cells immediately after infection, formation of the paracrystals was delayed by two hours, and the affiliation of NS3 protein was also observed to be rearranged.

Abstract: Induction of microtubulin paracrystals (Pc) by Kunjin (KUN) virus occurred after 15 hours post-infection and were often associated with convoluted membranes (cm) and virus particles. Vinblastine sulphate which disrupts microtubulin, had an inhibitory effect on the virus production when added during the viral latent period. When infected samples were extracted with Triton X-100 and analysed by SDS-PAGE, four viral proteins were observed.