Adenovirus fiber disrupts CAR-mediated intercellular adhesion allowing virus escape.

Adenovirus binds its receptor (CAR), enters cells, and replicates. It must then escape to the environment to infect a new host. We found that following infection, human airway epithelia first released adenovirus to the basolateral surface. Virus then traveled between epithelial cells to emerge on the apical surface. Adenovirus fiber protein, which is produced during viral replication, facilitated apical escape. Fiber binds CAR, which sits on the basolateral membrane where it maintains tight junction integrity. When fiber bound CAR, it disrupted junctional integrity, allowing virus to filter between the cells and emerge apically. Thus, adenovirus exploits its receptor for two important but distinct steps in its life cycle: entry into host cells and escape across epithelial barriers to the environment.

Regulated nuclear targeting of cauliflower mosaic virus.

The mature cauliflower mosaic virus (CaMV) capsid protein (CP), if expressed in the absence of other viral proteins, is transported into the plant cell nucleus by the action of a nuclear localization signal (NLS) close to the N terminus. In contrast, virus particles do not enter the nucleus, but dock at the nuclear membrane, a process inhibited by anti-NLS antibodies or by GTP gamma S, and apparently mediated by interaction of CP with host importin alpha. The very acidic N-terminal extension of the viral CP precursor inhibits nuclear targeting of the protein and hence the precursor is localized in the cytoplasm. We hypothesize that this provides a control mechanism which ensures that the CP precursor is used for virus assembly in the cytoplasm and that only mature virus particles reach the nuclear pore.

Reconstitution of cleavage of human immunodeficiency virus type-1 (HIV-1) RNAs.

A human immunodeficiency virus type 1 (HIV-1) particle contains approximately 1200 molecules of gag proteins and two copies of a 9.2-kb genomic RNA which has been reported to be dimerized and rapidly cleaved and to form a complex with a nucleocapsid protein, p7 (NCp7), during viral budding. These suggest that the cleavage can be reconstituted with gag proteins in vitro. Here we show that the p15(gag) coding region of viral RNA is fragmented in viral particles and that in vitro-synthesized RNA transcripts of HIV-1 undergo cleavage which is activated by NCp7 and other factors. Single-stranded oligoribonucleotides were cleaved between C and A or U and A, leaving 2',3'-cyclic phosphate and 5'-hydroxyl termini. These findings might explain the rapid degradation of genomic RNAs in HIV-1 particles.

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

Adenoviruses of six subgenera, namely, adenovirus 31 (Ad31) (subgenus A), Ad3, Ad7, Ad11p, Ad11a, and Ad35 (subgenus B), Ad5v and Ad5p (subgenus C), Ad37 (subgenus D), Ad4 (subgenus E), and Ad41 (subgenus F), were studied. The relative binding properties of different adenoviruses to 293 (human kidney embryonic cells) and A549 (human lung carcinoma cells) cells were compared by flow cytometry. All analyzed adenoviruses bound to cells in a dose-dependent manner. The binding capacity showed that Ad11p, Ad35 (subgenus B:2) with kidney tropism, and Ad4 (subgenus E), which can cause adenopharyngoconjunctivitis, bound strongly to both A549 and 293 cells. The other members of subgenus B and Ad37 of subgenus D manifested an intermediate binding capacity. The analyzed adenoviruses of subgenera A, C, and F manifested a low affinity. Adenoviruses of subgenera B:2 and E manifested high binding affinity to preparations of cell membranes from the epithelial cell lines. Reciprocal competition experiments using Ad11p and Ad4 demonstrated that the two viruses did not block each other. Antibodies against alphavbeta3 and alphavbeta5 reduced the binding of Ad5v virions and slightly impaired the binding of Ad4 but did not affect Ad11p binding to the A549 cell surface. Recombinant fiber proteins of Ad11p and Ad35 reciprocally blocked the binding of both viruses to the epithelial cells but they could not block Ad4. The hexon protein expression of Ad11p and Ad4 was 100 times more efficient than that of the Ad5 vector (pFG140), whereas the infectivity of Ad11p and Ad4 was 40- to 200-fold that of the commonly used Ad5v vector. Taken together, our findings demonstrate that Ad11p and Ad4 bind different receptor molecules and that the fibers of these two viruses provide the predominant high degree of binding, which obviously is a requirement for subsequent internalization and efficacious expression.

Novel strategy for inhibiting viral entry by use of a cellular receptor-plant virus chimera.

The plant virus cowpea mosaic virus (CPMV) has recently been developed as a biomolecular platform to display heterologous peptide sequences. Such CPMV-peptide chimeras can be easily and inexpensively produced in large quantities from experimentally infected plants. This study utilized the CPMV chimera platform to create an antiviral against measles virus (MV) by displaying a peptide known to inhibit MV infection. This peptide sequence corresponds to a portion of the MV binding site on the human MV receptor CD46. The CPMV-CD46 chimera efficiently inhibited MV infection of HeLa cells in vitro, while wild-type CPMV did not. Furthermore, CPMV-CD46 protected mice from mortality induced by an intracranial challenge with MV. Our results indicate that the inhibitory CD46 peptide expressed on the surface of CPMV retains virus-binding activity and is capable of inhibiting viral entry both in vitro and in vivo. The CD46 peptide presented in the context of CPMV is also up to 100-fold more effective than the soluble CD46 peptide at inhibiting MV infection in vitro. To our knowledge, this study represents the first utilization of a plant virus chimera as an antiviral agent.

The release of inflammatory cytokines from human peripheral blood mononuclear cells in vitro following exposure to adenovirus variants and capsid.

Preclinical and clinical studies with adenoviral vectors have clearly illustrated the potential advantages of this gene transfer system. However, many studies have also demonstrated potent immune responses directed at both vector and transduced cells. We examined in vitro responses of human peripheral blood mononuclear cells (PBMC) to virus exposure as a model for this host response. PBMC were isolated from normal donors and incubated with wild-type adenovirus (Ad5), Ad5 variants deleted for segments of E1 and/or E3, and empty viral capsids. Proinflammatory cytokine release was monitored for 96 hr. Induction of TNF-alpha by intact virions was low although stimulation by empty capsid gave a significant and sustained response. Induction of IL-6, GM-CSF, and a panel alpha- and beta-chemokines by intact virions was prominent, often approaching results obtained with 2.5 microg/ml of lipopolysaccharide (LPS). Responses were generally independent of virion genetic composition and were only partially blunted when UV-inactivated virus was used. Dose-response data showed 100-fold increases in virion concentration produced a maximum 3-fold increase in cytokine release, suggesting saturation. Surprisingly, prominent stimulation occurred after addition of empty capsid, which typically provoked responses equivalent to those seen with LPS stimulation. We present arguments that cellular signal transduction mechanisms activated by binding of virions/capsids stimulate transcription of proinflammatory cytokine genes.

A protein antibiotic in the phage Qbeta virion: diversity in lysis targets.

A(2), a capsid protein of RNA phage Qbeta, is also responsible for host lysis. A(2) blocked synthesis of murein precursors in vivo by inhibiting MurA, the catalyst of the committed step of murein biosynthesis. An A(2)-resistance mutation mapped to an exposed surface near the substrate-binding cleft of MurA. Moreover, purified Qbeta virions inhibited wild-type MurA, but not the mutant MurA, in vitro. Thus, the two small phages characterized for their lysis strategy, Qbeta and the small DNA phage phiX174, effect host lysis by targeting different enzymes in the multistep, universally conserved pathway of cell wall biosynthesis.

Potentiation of a recombinant oncolytic parvovirus by expression of Apoptin.

The oncotropic and oncolytic behaviors of certain autonomous rodent parvoviruses make them promising vectors for anticancer gene therapies. However, these parvoviruses are often not potent enough to kill all tumor cells equally well. With the aim of enhancing the intrinsic antitumor effect and the range of natural parvoviruses, a recombinant H1 parvovirus vector was constructed that produces the Apoptin protein, a tumor cell-specific, p53-independent, Bcl-2-insensitive apoptotic effector. We compared the apoptotic activity exerted by a recombinant hH1/Apoptin virus with that of a Green Fluorescent Protein (GFP)-transducing recombinant virus, hH1/GFP, in three human tumor cell lines differing in their susceptibility to wild-type parvovirus H1-induced killing. We found that in cells that were rather resistant to the basal cytotoxic effect of wild-type H1 or the GFP recombinant virus, a parvovirus that expressed Apoptin caused a pronounced, additional cytotoxic effect. In contrast to its enhanced cytotoxicity toward tumor cells, hH1/Apoptin virus was not more toxic to normal human fibroblasts than was the wild-type H1 virus. Taken together, these data indicate that enhancing the oncotropic behavior of wild-type H1 parvoviruses with the tumor-specific apoptotic potency of Apoptin should lead to an effective replicative parvoviral vector.

Recombinant parvovirus B19 empty capsids inhibit fetal hematopoietic colony formation in vitro.

Erythroid lineage cells are target cells for human parvovirus B19, and a natural infection often results in transient anemia. To determine whether recombinant B19 capsid proteins (VP1/VP2) also inhibit human hematopoietic progenitor growth, a model system was set up. The B19 capsids were inoculated into primary cultures of hematopoietic stem cells derived from human fetal liver, resulting in a 70-95% reduction of BFU-E (burst-forming unit erythroid cells) as compared with the medium control. A similar effect was seen in human hematopoietic stem cell cultures derived from cord blood and adult bone marrow. Preincubation of the B19 capsids with either a monoclonal antibody to the virus or with B19 IgG positive human sera reduced the inhibitory effect. Furthermore, the inhibitory effect could be reduced by preincubating the target cells with a monoclonal antibody to the cellular receptor for the virus, the P antigen. These findings thus show that the inhibition of colony formation of human hematopoietic stem cells can occur in the absence of parvovirus B19 nonstructural proteins. We speculate that B19 capsid could provide a possible strategy to downregulate indigenous hematopoiesis in fetal stem cell transplantations.

Plasmids encoding foot-and-mouth disease virus VP1 epitopes elicited immune responses in mice and swine and protected swine against viral infection.

VP1 is a capsid protein of foot-and-mouth disease virus (FMDV) and contains epitopes of the virus. Plasmids encoding two VP1 epitopes (amino acid residues 141-160 and 200-213) and a host-self immunoglobulin molecule were constructed to produce a new type of FMD DNA vaccine. Two plasmids, namely, pCEIM and pCEIS, containing mouse immunoglobulin (IgG) or swine IgG were subjected to immunogenicity testing in mice and swine, respectively. In mice administrated pCEIM in the abdomen using a genegun, both FMDV-specific T-cell proliferation and neutralizing antibodies were detected. In swine immunized with pCEIS at the back of the ear, immune responses were achieved after the second administration. Swine showed a T-cell proliferative response with a stimulation index (SI) of up to 8.1 and a neutralizing antibody response that was able to protect suckling mice from 10(2) LD(50) (lethal dose 50) FMDV challenge. To compare the immunogenicity of the DNA-based vaccine candidate, versus the protein-based vaccine candidates, a second group of swine was immunized with the protein F1-scIgG, which was encoded by the plasmid pCEIS. Injection with F1-scIgG elicited a T-cell proliferative response of SI < 1.7 and a neutralizing antibody response that protected suckling mice from up to 10(5) LD(50) FMDV challenge. In the challenge test, three of three swine immunized with pCEIS were fully protected from FMDV challenge.

Modified adenovirus penton base protein (UTARVE) as a non-replicating vector for delivery of antisense oligonucleotides with antiviral and/or antineoplastic activity.

Antisense oligonucleotides that selectively inhibit gene expression are a genetic approach for disease treatment and prevention. However, their use as therapeutic agents is complicated by their low rate of transport across cellular membranes and their sequestration within endocytic-like vesicles. We report that the adenovirus type-2 penton base protein modified to include the fusogenic peptide of the influenza virus hemagglutinin protein is a non-replicating vector (designated UTARVE) that improves delivery of antisense oligonucleotides. Approximately 10-18% of the input vector was internalized by A549 and HeLa cells as determined by immunoblotting. It was cleared by proteolysis within 48 h. The vector had endosome disruptive potential as evidenced by erythrocyte lysis activity at low pH and a primarily diffuse cytoplasmic distribution in treated cells. Despite concentration and time-dependent cell detachment, UTARVE was not cytotoxic in the dye release assay. We used R1T1, an antisense oligonucleotide that inhibits expression of the multifunctional herpes simplex virus type-2 (HSV-2) R1 protein, HSV-2 growth and the proliferation of R1 PK transformed cells to examine vector-mediated delivery. Conjugated FITC-labeled R1T1 was rapidly (15-30 min) internalized by all cells treated at low (80 nM) concentration and the oligomer was intracellularly dissociated from the vector. This compares to 65-83% of cells internalizing the unconjugated R1T1 when treated for 24 h. In antiviral assays, the IC50 and time required to inhibit HSV-2 growth were significantly lower for the conjugated (2 nM; 30 min) as compared to unconjugated (100 nM; 24 h) R1T1. The data indicate that the bioavailability and biological activity of R1T1 were significantly increased by its delivery with UTARVE.

Selection of a specific peptide from a nona-peptide library for in vitro inhibition of grass carp hemorrhage virus replication.

Grass carp hemorrhage virus (GCHV), a member of Reoviridae, causes severe hemorrhagic disease of grass carp (Ctenopharyngodon idellus) in China. Icosahedral virions of GCHV were used as to assay the effect of specific peptides for the inhibition of GCHV infectivity. A random nona-peptide library displayed on phage fUSE5 was constructed, and the expressed peptides were fused onto the amino terminus of the minor coat protein III. By biopanning, the fused peptides were bound to the biotinylated GCHV. Phages containing specific peptides bound to GCHV were eluted and amplified in Escherichia coli K91. Three rounds of affinity selection enriched the pool of inhibiting peptides. Sixteen clones which inhibited the replication of GCHV in a grass carp kidney cell line were selected. The TCID(50) of GCHV was decreased over 10000x. Six clones having the strongest inhibitory effect shared the same DNA sequence, with a deduced amino acid sequence of NH(2)-Leu-Trp-Val-Gly-Gly-Gly-Arg-Asn-Ala-COOH. A synthesized nona-peptide of identical sequence exhibited similar inhibitory activity towards GCHV replication in vitro.

Targeting a recombinant adenovirus vector to HCC cells using a bifunctional Fab-antibody conjugate.

We developed a specific adenoviral gene delivery system with monoclonal antibody (mAb) AF-20 that binds to a 180 kDa antigen highly expressed on human hepatocellular carcinoma (HCC) cells. A bifunctional Fab-antibody conjugate (2Hx-2-AF-20) was generated through AF-20 mAb crosslinkage to an anti-hexon antibody Fab fragment. Uptake of adenoviral particles and gene expression was examined in FOCUS HCC and NIH 3T3 cells by immunofluorescence; beta-galactosidase expression levels were determined following competitive inhibition of adenoviral CAR receptor by excess fibre knob protein. The chimeric complex was rapidly internalized at 37 degrees C, and enhanced levels of reporter gene expression was observed in AF-20 antigen positive HCC cells, but not in AF-20 antigen negative NIH 3T3 control cells. Targeting of recombinant adenoviral vectors to a tumor associated antigen by a bifunctional Fab-antibody conjugate is a promising approach to enhance specificity and efficiency of gene delivery to HCC.

Cytomegalovirus MHC class I homologues and natural killer cells: an overview.

Viruses that establish a persistent infection with their host have evolved numerous strategies to evade the immune system. Consequently, they are useful tools to dissect the complex cellular processes that comprise the immune response. Rapid progress has been made in recent years in defining the role of cellular MHC class I molecules in regulating the response of natural killer (NK) cells. Concomitantly, the roles of the MHC class I homologues encoded by human and mouse cytomegaloviruses in evading or subverting NK cell responses has received considerable interest. This review discusses the results from a number of studies that have pursued the biological function of the viral MHC class I homologues. Based on the evidence from these studies, hypotheses for the possible role of these intriguing molecules are presented.

The viral protein Apoptin induces apoptosis in UV-C-irradiated cells from individuals with various hereditary cancer-prone syndromes.

Apoptin, a protein derived from chicken anemia virus, has previously been shown to induce apoptosis in a p53-independent and Bcl-2-stimulated manner in transformed and tumorigenic human cells but not in normal diploid human cells, suggesting that it is a potential agent for tumor therapy. Here we report that Apoptin can induce apoptosis in UV-C-irradiated diploid skin fibroblasts from individuals with various hereditary cancer-prone syndromes that are characterized by a germ-line mutation in a tumor suppressor gene. The same effect is found when these cells are irradiated with X-rays. In contrast, diploid skin fibroblasts from healthy donors or from individuals with DNA repair disorders are not responsive to Apoptin-induced apoptosis upon UV-C or X-ray irradiation. After transfection of untreated cells, Apoptin is found predominantly in the cytoplasm, whereas in UV-C-exposed Apoptin-responsive cancer-prone cells, it migrates to the nucleus, where it causes rapid apoptosis. Apoptin remains localized in the cytoplasm after UV-C treatment of diploid cells from healthy individuals. The induction of apoptosis by Apoptin in cancer-prone cells with a germ-line mutation in a tumor suppressor gene is UV dose-dependent and transient, just like many other UV-induced processes. These results suggest that Apoptin may be used as a diagnostic tool for detection of individuals with an increased risk for hereditary cancer and premalignant lesions.

Silent mutations in the Escherichia coli ompA leader peptide region strongly affect transcription and translation in vivo.

In order to test the effect of silent mutations on the regulation of gene expression, we monitored several steps of transcription and translation of the ompA gene in vivo , in which some or all codons between codons 6 and 14, frequently used in Escherichia coli , had been exchanged for infrequent synonymous codons. Northern blot analysis revealed an up to 4-fold reduction in the half-life of the mutated messengers and a >10-fold reduction in their steady-state amounts. Western blot analysis showed a 10-fold reduction in the amount of OmpA protein. Use of a system expressing a Rho-specific anti-terminator allowed us to detect a strong transcription polarity effect in the silent mutants. These results demonstrate that silent mutations can severely inhibit several steps of gene expression in E. coli and that code degeneracy is efficiently exploited in this species for setting signals for gene control and regulation.

Apoptin specifically causes apoptosis in tumor cells and after UV-treatment in untransformed cells from cancer-prone individuals: a review.

Tumor formation is caused by an imbalance between cell replication and apoptosis, which is a physiological form of cell death. For instance, UV damage can result in tumor formation due to mutations of the tumor-suppressor gene p53, a major apoptosis-inducing protein. Over-expression of the proto-oncogene Bcl-2, due to chromosomal translocation, can also inhibit apoptosis resulting in, e.g., lymphomas and leukemias. Anti-tumor therapies are often based on induction of apoptosis mediated via p53 and/or inhibited by Bcl-2, which explains the frequently poor results of anti-tumor treatment. The avian-virus-derived protein 'Apoptin', induces apoptosis in a p53-independent way, is stimulated by Bcl-2 and is insensitive to BCR-ABL, another inhibitor of chemotherapeutic agents. Apoptin induces apoptosis in human transformed/tumorigenic cells but not in normal diploid cells. Co-synthesis of SV40 large T antigen and Apoptin results in induction of apoptosis, illustrating that the establishment of a stable transformed state is not required. UV-irradiation causes an aberrant SOS-response in primary diploid cells from cancer-prone individuals and renders such cells susceptible to Apoptin-induced apoptosis. All these features make Apoptin a potential candidate as a therapeutic and diagnostic tool in cancer treatment.

Induction of anti foot and mouth disease virus T and B cell responses in cattle immunized with a peptide representing ten amino acids of VP1.

We previously demonstrated that the immunization of cattle with a synthetic peptide representing the amino acid sequence of foot and mouth disease virus (FMDV) type O1 Campos VP1 residues 135-160 (p135-160), containing immunodominant T and B epitopes, was able to induce a strong neutralizing antibody (NA) response. The epitope mapping of p135-160 identified T and B epitopes in the area restricted to amino acid residues 135-144 (Zamorano et al. 1994, Virology 201; 1995, Virology 212). We are now reporting that, although immunization with a synthetic peptide covering amino acids 135-144 (p135-144) failed to elicit an anti-FMDV response, a synthetic peptide representing a tandem duplication of the VP1 epitope 135-144 (p135-144 x 2) was very efficient in inducing a strong NA response in cattle. Both the antibody and T cell responses elicited by p135-144 x 2 were highly specific for the VP1 135-144 sequence since no reactivity was detected against synthetic peptides representing the 140-160 sequence of VP1. Additionally, both responses to B and T epitopes were long lasting in the immunized cattle. These results constitute a good example of the improvement of the immune response by rational handling of precisely identified B and T epitopes. To our knowledge, this is the shortest native amino acid sequence to induce a significant NA response to FMDV in cattle.

Cationic liposomes enhance adenovirus entry via a pathway independent of the fiber receptor and alpha(v)-integrins.

The ability of adenoviral vectors to mediate efficient gene delivery both in vitro and in vivo is limited by the availability of specific cell surface receptors and alpha(v)-containing integrins. We tested whether this limitation could be overcome by enhancing viral entry with cationic liposomes. In cultured vascular smooth muscle cells, delivery of adenoviral vectors in the presence of cationic liposomes increased vector-encoded transgene expression up to 20-fold. The increase in transgene expression was associated with the formation of adenovirus-lipid aggregates and an increase in the amount of vector DNA in the cells, suggesting that enhanced viral entry was responsible for the increase in gene expression. Treatment of the cells with an RGD-containing peptide or adenovirus type 5 fiber protein did not diminish liposome enhancement of transgene expression, indicating that liposomes increase viral entry via a pathway independent of the fiber receptor and of alpha(v) integrin-assisted endocytosis. Liposomes also significantly enhanced transgene expression from adenoviral vectors delivered to cells deficient in alpha(v)-containing integrins. The magnitude of liposome enhancement of transgene expression in cultured smooth muscle cells was greatest during brief periods of virus-cell contact and at low concentrations of virus. Despite these promising in vitro results, addition of liposomes did not improve in vivo adenoviral gene delivery into injured rat carotid arteries. Liposomes can improve adenoviral gene delivery in vitro; however, application of this observation to accomplish improved in vivo gene delivery remains a challenge.

An interaction between penton base and alpha v integrins plays a minimal role in adenovirus-mediated gene transfer to hepatocytes in vitro and in vivo.

Studies in cultured cell lines have shown that adenovirus infection involves binding of adenovirus fiber to its cell surface receptor and binding of penton base to alpha v integrins. However, much less is known about the role of these interactions in cells that are targets for adenovirus-mediated gene transfer. Earlier work showed that hepatocytes are readily infected by adenovirus, making them an attractive target for gene therapy in several diseases. We found that addition of fiber protein blocked adenovirus infection of primary cultures of hepatocytes. This suggests an important role for fiber and its receptor. However, mutation of the integrin-binding motif in penton base did not inhibit infection of hepatocytes, even though the mutation impaired infection of HeLa cells. Hepatocytes had undetectable amounts of alpha v integrins on their cell surface and showed no specific adherence to vitronectin, the natural substrate of alpha v integrins. Adenovirus with an intact penton base enhanced infection of liver following intravenous injection, but only by three-fold as compared with virus in which the integrin-binding motif was disrupted. These studies suggest that interactions between cell surface integrins and penton base are not required for adenovirus infection of hepatocytes in vitro, but the interaction enhances infection to a small degree in vivo.