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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.

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.

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., ((c) 2002 Elsevier Science (USA).)

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.

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.

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.

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.

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.

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., (Copyright 2001 S. Karger AG, Basel)

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., (Copyright 2000 Academic Press.)

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.

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., (Copyright 2000 Academic Press.)

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.

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.

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.

The membrane destabilizing and fusogenic properties of the synthetic peptide VP3(110-121), corresponding to an immunogenic sequence of the hepatitis A virus (HAV) VP3 capsid protein, were studied. By tryptophan fluorescence and acryalmide quenching it was demonstrated that the peptide binds liposomes of POPC-SM-DPPE (47 + 39 + 14) and POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) and penetrates the membrane, at both neutral and acidic pH (POPC = 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM = sphingomyelin; DPPE = 1,2-dipalmitoylphosphatidylethanolamine; DOTAP = 1,2-dioleoyl-3-trimethylammoniumpropane). VP3(110-121) did not have membrane-destabilizing properties at neutral pH. Acid-induced destabilization of the vesicles was demonstrated by fluorescence techniques and dynamic light scattering. VP3(110-121) induced aggregation of POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) vesicles, lipid mixing and leakage of vesicle contents, all consistent with fusion of vesicles. In POPC-SM-DPPE (47 + 39 + 14) vesicles, at acidic pH, VP3(110-121) induced membrane destabilization with leakage of contents but without aggregation of vesicles or lipid mixing. The peptide only showed fusogenic properties when bound to the vesicles at neutral pH before acidification to pH below 6.0, and no effect was seen if the peptide was added to vesicles already set at acidic pH. These results may have physiological significance in the mechanism of infection of host hepatic cells by HAV.

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.

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.

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., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

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.

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.

Nucleocapsid protein 7 (NCp7), the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, was shown to strongly potentiate the dimerization of the retroviral genomic RNA. This process involves the interaction of two retroviral RNA monomer subunits near their 5'-ends. A region located upstream from the splice donor site was recently identified as being responsible for the formation of dimeric HIV-1 RNA. This region appeared to be confined within a stem-loop structure, with an autocomplementary sequence in the loop. In an in vitro study of spontaneous dimer formation, we reported that the 77-402 RNA transcript forms two distinct dimers differing in their thermostability: D37 and D55. We identified D37 as a "kissing" complex structure, formed via a loop-loop interaction between the two monomers, and D55 as a double stranded structure involving all nucleotides of the stem-loop via canonical base pairing. In this report, we have characterized the role of NCp7 in the HIV-1Lai RNA dimerization process by using in vitro dimerization assays with RNA transcripts of different lengths and dimer thermal dissociation. Our results show that the nucleocapsid protein NCp7 activates RNA dimerization very likely through interaction with the kissing complex and converts it into a stable dimer. Furthermore, this NCp7-promoted conversion only occurs if the 240-280 stem-loop structure is present in HIV-1Lai RNA molecules and contains the autocomplementary G257CGCGC262 sequence. This study suggests that, under physiological conditions, an NCp7-mediated RNA conformational change is involved in the maturation of the HIV-1 RNA dimer.

Short RNA species that encompass the psi domain of the retroviral genome spontaneously form dimers in vitro, and the retroviral nucleocapsid protein activates this dimerization in vitro. Addition of gag RNA sequences downstream of the 3' end of the psi domain decreases the level of spontaneous dimerization. Here, we report the effects of RNA length on dimerization in vitro, studied with RNA fragments from Moloney murine leukaemia virus that contain the psi domain and all or part of the gag sequence. Extension of the RNA leads to progressive inhibition of the in vitro dimerization process. Sequences located downstream of the 3' end of the psi domain seem to stabilize the monomeric structures. This stabilization participates in dimerization of the RNA sequences involved in the recognition of two RNA molecules. We studied the ability of nucleocapsid protein 10 to promote dimerization of such long RNA fragments, and found that the protein greatly enhances their dimerization in vitro. We propose that nucleocapsid protein 10 stimulates the overall dimerization process by reduction of the energy barrier that must be overcome to allow dimer formation. Our results show that dimerization of RNA form Moloney murine leukaemia virus in vitro is enhanced by nucleocapsid protein 10. This finding is in agreement with the involvement of the nucleocapsid protein in RNA dimerization in vivo.

Conversion of human immunodeficiency virus type 1 (HIV-1) genomic RNA to viral DNA is a requisite step in the virus life cycle. This conversion is catalyzed by reverse transcriptase (RT) associated with a large nucleoprotein complex composed of several viral proteins including nucleocapsid (NC). To better characterize the biochemical mechanisms of viral DNA synthesis, we overexpressed and purified recombinant HIV-1 NC and studied its effect on the activity and processivity of HIV-1 RT during polymerization of HIV-1 template sequences in vitro. The effect of NC on steady-state RT activity was dependent on the order of addition of reaction components. Addition of NC prior to formation of RT-primer.template-dNTP ternary complexes inhibited primer extension and reduced total product yields by slowing steady-state RT turnover. In contrast, addition of NC to preformed ternary complexes resulted in efficient primer extension and increased RT processivity at specific DNA template sites. NC stimulated polymerization (2-4 times) through eight of 13 sites examined in the cRRE region of HIV-1 env and increased the rate of polymerization through the D3/CTS region of HIV-1 pol 10 times. The data suggest that NC affects RT processivity by facilitating polymerization through regions of template secondary structure. Thus, NC functions as a single-strand binding (SSB)-like accessory replication factor for RT in vitro and may be part of a multicomponent retroviral replication complex.

Apoptin, a small protein derived from chicken anemia virus (CAV), induces apoptosis in human tumor cell lines regardless of whether these express p53 or not. We examined whether the small adenovirus 5 E1B protein of 21 kDa (E1B-21kD), also called E1B-19kD) and Bcl-2 could inhibit apoptin-induced apoptosis in human tumor cell lines and compared this with p53-induced apoptosis. E1B-21kD, but not Bcl-2 was found to inhibit apoptin-induced apoptosis in the osteosarcoma cell lines U2OS and Saos-2. However, neither expression of E1B-21kD nor of Bcl-2 resulted in inhibition of apoptin-induced apoptosis in Hep3B hepatoma cells and kidney rhabdoid tumor G401 cells. Both Bcl-2 and Ad5 E1B-21kD were able to inhibit p53-induced apoptosis in the human tumor cell lines Saos-2 and Hep3B. In Saos-2 and U2OS, but not in Hep3B and G401, expression of E1B-21kD leads to retention of apoptin in the cytoplasm, in that way preventing its nuclear function. These results indicate that proteins inhibiting the p53-induced apoptotic pathway do not block apoptin-induced apoptosis or do so only in a cell type-specific manner. The apoptin-induced apoptotic pathway is distinct from that induced by p53 and, therefore, apoptin is a potential antitumor agent.

The capacity of the rabies virus superantigen (SAg) and the nucleocapsid (NC) to activate human tonsil lymphocytes was analyzed by studying the capacity of NC to cause lymphocytes to proliferate and secrete Ig and cytokines. NC activation was compared to that obtained with the Staphylococcus-derived SAg, SEE, and TSST-1. Despite a weak T lymphocyte mitogenic activity restricted to CD4+ T cells, NC triggers tonsil B lymphocytes to produce IgG in quantities and frequencies similar to those of SEE and TSST-1. In the same way as these two SAg, NC induces IgG production only in the presence of T cells and optimally with a T/B ratio of 1/5. However, unlike SEE and TSST-1, NC does not trigger IgM production. The pattern of cytokines produced upon NC activation, IL-4 and IL-10, weak IL-2 production, and no IFN-gamma, suggests that rabies SAg stimulates Th2 rather than Th1 lymphocytes. In contrast, the pattern of cytokines produced upon TSST-1 activation, IL-2, IFN-gamma, and no IL-4, suggests that TSST-1 induces rather a Th1 response. The specific Th2 triggering by NC could explain the unique capacity of the rabies SAg to increase the in vivo antibody response to a simultaneously injected antigen.

A virus-based vector was used for the transient expression of the alfalfa mosaic virus coat protein (CP) gene in protoplasts and plants. The accumulation of wild-type CP conferred strong protection against subsequent alfalfa mosaic virus infection, enabling the efficacy of CP mutants to be determined without developing transgenic plants. Expression of the CP mRNA alone without CP accumulation conferred weaker protection against infection. The activity of the N-terminal mutant CPs in protection did not correlate with their activities in genome activation. The activity of a C-terminal mutant suggested that encapsidation did not have a role in protection. Our results indicate that interaction of the CP with alfalfa mosaic virus RNA is not important in protection, thereby leaving open the possibility that interactions with host factors lead to protection.

Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it is in tight association with genomic RNA and the tRNA primer. NC protein is necessary for the dimerization and encapsidation of genomic RNA, the annealing of the tRNA primer to the primer binding site (PBS) and the initial strand transfer event. Due to the general nature of NC protein-promoted annealing, its use to improve nucleic acid interactions in various reactions can be envisioned. Parameters affecting NC-promoted nucleic acid annealing of NCp7 from HIV-1 have been analyzed. The promotion of RNA:RNA and RNA:DNA annealing by NCp7 is more sensitive to the concentration of MgCl2 than the promotion of DNA:DNA hybridization. Stimulation of complex formation for all three complexes was efficient at 0-90 mM NaCl, between 23 and 55 degrees C and at pH values between 6.5 and 9.5, inclusive. Parameters affecting NCp7-promoted hybridization of tRNA(Lys,3) to the PBS, which appears to be specific for NC protein, will be discussed. Results implicate the basic regions of NCp7, but not the zinc fingers, in promoting the annealing of complementary nucleic acid sequences. Finally, NCp7 strand transfer activity aids the formation of the most stable nucleic acid complex.

Binding and signaling proteins based on Escherichia coli alkaline phosphatase (AP; EC 3.1.3.1) were designed for the detection of antibodies. Hybrid proteins were constructed by using wild-type AP and point mutants of AP [Asp-101 --> Ser (D101S) and Asp-153 --> Gly (D153G)]. The binding function of the hybrid proteins is provided by a peptide epitope inserted between amino acids 407 and 408 in AP. Binding of anti-epitope antibodies to the hybrid proteins modulates the enzyme activity of the hybrids; upon antibody binding, enzyme activity can increase to as much as 300% of the level of activity in the absence of antibody or can decrease as much as 40%, depending on the presence or absence of the point mutations in AP. The fact that modulation is altered from inhibition to activation by single amino acid changes in the active site of AP suggests that the mechanism for modulation is due to structural alterations upon antibody binding. Modulation is a general phenomenon. The properties of the system are demonstrated by using two epitopes, one from the V3 loop of human immunodeficiency virus type 1 gp120 protein and one from hepatitis C virus core protein, and corresponding monoclonal antibodies. The trend of modulation is consistent for all hybrids; those in wild-type AP are inhibited by antibody, while those in the AP mutants are activated by antibody. This demonstrates that modulation of enzyme activity of the AP-epitope hybrid proteins is not specific to either a particular epitope sequence or a particular antibody-epitope combination.

In a group of immunized cattle with a variety of MHC class II types, T-cell responses were detected to a synthetic peptide (FMDV15) proposed as a basis for a vaccine against foot-and-mouth disease. This peptide combines the loop region of VP1 with the C-terminal sequence connected by a spacer (PPS). Two major immunodominant regions of FMDV15 for bovine T cells were detected, one within the loop region and the other around the spacer. A substantial proportion of the T-cell response to FMDV15 was directed to the spacer region in 5/12 animals, and in vitro generation of FMDV15-specific T cells preferentially selected for spacer-specific T cells. This pattern of response was associated with a particular MHC class II type which is very common in cattle. Focusing of the T-cell response to non-native virus epitopes may explain why FMDV15 is inefficient at inducing protection.

Infection of BHK cells by Sindbis virus leads to rapid inhibition of host cell protein synthesis and cytopathic effects (CPE). We have been studying these events to determine whether the expression of a specific viral gene is required and, in the present study, have focused our attention on the role of the structural proteins--the capsid protein and the two membrane glycoproteins. We tested a variety of Sindbis viruses and Sindbis virus replicons (virus particles containing an RNA that is self-replicating but with some or all of the viral structural protein genes deleted) for their abilities to inhibit host cell protein synthesis and cause CPE in infected BHK cells. Our results show that shutoff of host cell protein synthesis occurred in infected BHK cells when no viral structural proteins were synthesized and also under conditions in which the level of the viral subgenomic RNA was too low to be detected. These results support the conclusion that the early steps in viral gene expression are the ones required for the inhibition of host cell protein synthesis in BHK cells. In contrast, the Sindbis viruses and Sindbis virus replicons were clearly distinguished by the time at which CPE became evident. Viruses that synthesized high levels of the two membrane glycoproteins on the surface of the infected cells caused a rapid (12 to 16 h postinfection) appearance of CPE, and those that did not synthesize the glycoprotein spikes showed delayed (30 to 40 h) CPE.

Rotavirus infection of monkey kidney cells has been reported to result in a significant increase in the concentration of intracellular calcium. This increase in intracellular calcium was associated with viral protein synthesis and cytopathic effects in infected cells. We tested the effect of individual rotavirus proteins on intracellular calcium concentrations in insect Spodoptera frugiperda (Sf9) cells. Insect cells were infected with wild-type baculovirus or baculovirus recombinants that contained an individual rotavirus gene. The cells were harvested at different times postinfection, and the intracellular calcium concentration was measured by using fura-2 as a fluorescent calcium indicator. We found that the concentration of intracellular calcium was increased nearly fivefold in infected Sf9 cells that expressed the nonstructural glycoprotein (NSP4) of group A rotavirus, and this increase in intracellular calcium concentration coincided with NSP4 expression. A similar result was observed in insect cells expressing NSP4 from a group B rotavirus, suggesting the conservation of this function among rotavirus groups. Expression of the other 10 rotavirus proteins or of wild-type baculovirus proteins in Sf9 cells did not significantly increase intracellular calcium levels. These results suggest that the nonstructural glycoprotein NSP4 is responsible for the increase in cytosolic calcium observed in rotavirus-infected cells.

Chicken anemia virus (CAV) causes cytopathogenic effects in chicken thymocytes and cultured transformed mononuclear cells via apoptosis. Early after infection of chicken mononuclear cells, the CAV-encoded protein VP3 exhibits a finely granular distribution within the nucleus. At a later stage after infection, VP3 forms aggregates. At this point, the cell becomes apoptotic and the cellular DNA is fragmented and condensed. By immunogold electron microscopy VP3 was shown to be associated with apoptotic structures. In vitro, expression of VP3 induced apoptosis in chicken lymphoblastoid T cells and myeloid cells, which are susceptible to CAV infection, but not in chicken embryo fibroblasts, which are not susceptible to CAV. Expression of a C-terminally truncated VP3 induced much less pronounced apoptosis in the chicken lymphoblastoid T cells.

In a previous study using reassortant viruses, bovine rotavirus B223 VP7 protein enhanced the neutralization titers of some VP4 specific cross-reactive monoclonal antibodies (MAbs) (Xu and Woode, Virology, 1993). In this report, the influence of the B223 VP7 gene on the growth of reassortants was studied. The growth curves of three B223/69M reassortants with or without the B223 VP7 gene were compared. Reassortant M8, which has B223 VP7 on the genetic background of 69M, replicated as fast and to a similar titer as B223 and better than 69M. In contrast, the growth of reassortants B9, which has 69M VP7 on the B223 background, and of M46, which has B223 VP4 and VP6 on the 69M background, were poorer than B223. As B223 VP7 appeared to provide replication advantages, B223 was cultured as co-infections with each of the following viruses: Wa, 69M, H-2, SA11-4F, and NCDV followed by 20 subpassages. For co-infections with Wa, 69M, H-2, and SA11-4F, B223 VP7 gene was selected, in contrast to the B223/NCDV co-infection, when NCDV VP7 gene was selected. Other genes were also selected non-randomly, but varied among the five co-infection pairs. These data suggest in particular that B223 or NCDV VP7 protein can provide replication advantages to reassortant viruses, possibly by modifying VP4 to make it adsorb more efficiently to the cell receptors.

Infant mice (NMRI strain) showed the inhibition of hepatic extramedullary haemopoiesis by oral inoculation of a 100 ID50 dose of EB rotavirus and nucleoprotein of SA-11 rotavirus (serotype 3). The extramedullary haemopoiesis was observed by oral inoculation of surface protein VP7 of SA-11 rotavirus and in control (placebo administered) mice.

Antinociceptive activity of seven pentapeptide fragments of human adenovirus type 2 (Ad2) virion proteins: Thr-Val-Pro-Pro-Arg (1), Thr-Arg-Pro-Pro-Arg (2), Thr-Gly-Pro-Pro-Thr (3), Pro-Arg-Pro-Pro-Thr (4), Phe-Val-Pro-Pro-Arg (5), Ala-Arg-Pro-Pro-Ala (6), Tyr-Gly-Pro-Pro-Lys (7)--analogs of known tuftsin inhibitor Thr-Lys-Pro-Pro-Arg, was measured by hot-plate procedure. Also two tuftsin-like fragments of epitopes of HIV-1 and HIV-2: Thr-Lys-Ala-Lys (8), Thr-Lys-Glu-Lys (9), and tuftsin analog Thr-Lys-Asp-Lys (10) were tested. In the control experiments the effects of tuftsin and pentapeptide tuftsin inhibitor were also studied. The peptides 2, 4 and 5 were found to produce very strong analgesia after the icv injection. It was observed that pretreatment with peptide 8 remarkably diminished the antinociceptive effect induced by tuftsin.

The nucleocapsid protein (NC) is the major genomic RNA binding protein that plays integral roles in the structure and replication of all animal retroviruses. In this report, select biochemical properties of recombinant Mason-Pfizer monkey virus (MPMV) and HIV-1 NCs are compared. Evidence is presented that two types of saturated Zn2 NC-polynucleotide complexes can be formed under conditions of low [NaCl] that differ in apparent site-size (n = 8 vs. n = 14). The formation of one or the other complex appears dependent on the molar ratio of NC to RNA nucleotide with the putative low site-size mode apparently predominating under conditions of protein excess. Both MPMV and HIV-1 NCs kinetically facilitate the renaturation of two complementary DNA strands, suggesting that this is a general property of retroviral NCs. NC proteins increase the second-order rate constant for renaturation of a 149-bp DNA fragment by more than four orders of magnitude over that obtained in the absence of protein at 37 degrees C. The protein-assisted rate is 100-200-fold faster than that obtained at 68 degrees C, 1 M NaCl, solution conditions considered to be optimal for strand renaturation. Provided that sufficient NC is present to coat all strands, the presence of 400-1,000-fold excess nonhomologous DNA does not greatly affect the reaction rate. The HIV-1 NC-mediated renaturation reaction functions stoichiometrically, requiring a saturated strand of DNA nucleotide:NC ratio of about 7-8, rather than 14. Under conditions of less protein, the rate acceleration is not realized. The finding of significant nucleic acid strand renaturation activity may have important implications for various events of reverse transcription particularly in initiation and cDNA strand transfer.

Cotton rats (Sigmodon hispidus) were inoculated intranasally with 10(2.0) to 10(10.0) PFU of human adenovirus type 5. The virus replicated to a high titer in pulmonary tissues, with the peak titer being proportional to the input dose. The 50% lethal dose was 10(9.4) PFU. Histopathologic changes were proportional to the infecting inoculum and included the infiltration of interstitial and intra-alveolar areas, moderate damage to bronchiolar epithelium, and cellular infiltration of peribronchiolar and perivascular regions. These changes could be divided into two phases: an early phase (affecting alveoli, bronchiolar epithelium, and peribronchiolar regions) with an infiltrate consisting primarily of monocytes-macrophages and neutrophils, with occasional lymphocytes, and a later phase (affecting peribronchiolar and perivascular regions) with an infiltrate consisting almost exclusively of lymphocytes. In both phases, the predominant process was the response of the host to infection, rather than direct viral damage to infected cells. An infecting inoculum of 10(8.0) PFU or larger caused severe damage to type II alveolar cells, which were swollen, showed a loss of lamellar bodies, and were surrounded by polymorphonuclear leukocytes and macrophages. No evidence of complete viral replication was found in type II alveolar cells.

RGD-containing peptides from the immunodominant region of VP1 between amino acids 135-160 from foot-and-mouth disease virus (FMDV) type O1 Kaufbeuren (O1K) prevented virus adsorption to piglet kidney (PK) cells. The highly conserved amino acid RGD sequence (Arg.-Gly.-Asp.) was a prerequisite of this effect. To prevent infection with 100-200 TCID50 in 10(6) PK cells, 20-250 micrograms of each peptide should have been added.

To study the biological function of the NS protein of vesicular stomatitis virus (VSV), we prepared 21 species of synthetic oligopeptides with 11-21 amino acid residues, corresponding to every portion of the amino acid sequence of NS protein (Indiana serotype), and tested their effects on the VS virion (VSV) transcriptase activity in vitro. Only one peptide affected the virion-associated transcriptase activity of VSV Indiana, by reducing the incorporation of [3H]GMP into acid-insoluble fraction (IC50 = 26 microM). This peptide, the amino acid sequence of which corresponded to the carboxy (C)-terminal region of NS protein, also inhibited the New Jersey serotype virus transcriptase activity, as expected from a high degree of homology found between the amino acid sequences of the C-terminal regions of NS protein of both serotype viruses. Electrophoretic analysis on acrylamide gels of RNA transcripts revealed that the inhibitory synthetic peptide decreased the frequency of the initiation of transcription with no apparent effect on the chain-elongation process of viral transcription. As expected from its highly conserved amino acid sequence, these results suggest that the C-terminal domain of VSV NS protein is involved in initiating viral RNA synthesis.

The coat protein of bacteriophage MS2 is a translational repressor. It inhibits the synthesis of the viral replicase by binding a specific RNA structure that contains the replicase translation initiation region. In order to begin a genetic dissection of the repressor activity of coat protein, a two-plasmid system has been constructed that expresses coat protein and a replicase-beta-galactosidase fusion protein from different, compatible plasmids containing different antibiotic-resistant determinants. The coat protein expressed from the first plasmid (pCT1) represses synthesis of a replicase-beta-galactosidase fusion protein encoded on the other plasmid (pRZ5). Mutations in the translational operator or in coat protein result in constitutive synthesis of the enzyme. This permits the straightforward isolation of mutations in the coat sequence that affect repressor function. Because of the potential importance of cysteine residues for RNA binding, mutations were constructed that substitute serines for the cysteine residues normally present at positions 46 and 101. Both of these mutations result in translational repressor defects. Chromatographic and electron microscopic analyses indicate that the plasmid-encoded wild-type coat protein forms capsids in vivo. The ability of the mutants to adopt and/or maintain the appropriate conformation was assayed by comparing them to the wild-type protein for their ability to form capsids. Both mutants exhibited evidence of improper folding and/or instability as indicated by their aberrant elution behavior on a column of Sepharose CL-4B. Methods were developed for the rapid purification of plasmid-encoded coat protein, facilitating future biochemical analyses of mutant coat proteins.

The present study demonstrates that subcutaneous injection of purified adenovirus 12 fibre protein (FP) to 4-week-old Balb/c mice, 4, 2 and 1 day(s) before intramuscular injection of murine sarcoma virus (MSV-M) or 2 and 1 day before and 1 day after MSV-M injection causes significant inhibition of tumour growth (P less than 0.001). The evidence suggests that treatment with FP leading to tumour inhibition is due to neither an interferon mechanism nor to inhibition of virus multiplication of FP in the reticuloendothelial system. It is postulated that immune functions, probably against MSV-induced cell antigens, might be the critical mechanisms underlying inhibition of tumour growth.

It has been reported previously that ammonium chloride, chloroquine, monensin, and adenovirus-2 potentiate the cytotoxicity of several protein toxins conjugated with various targeting molecules. We have tested whether these agents, and protein components of adenovirus-2, would enhance the cytotoxicity of conjugates of gelonin with J5, an antibody directed against common acute lymphoblastic leukemia-associated antigen, with 5E9, an antibody directed against human transferrin receptor, or with ricin B-chain. We found that none of these agents affected the cytotoxicity of gelonin conjugates to any significant extent. For example, monensin moderately (3-fold) enhanced the cytotoxicity of 5E9-gelonin for Namalwa cells but showed no effect when 5E9-gelonin was tested on HeLa cells. The potentiating effects of these agents for the cytotoxicity of free gelonin varied from marked to nonexistent, depending on the type of cells. In particular, adenovirus-2 potentiated the cytotoxicity of gelonin for HeLa cells but not for Namalwa cells. The three major adenoviral capsid proteins, penton, hexon, and fiber, were isolated. It was shown that penton potentiated the cytotoxicity of gelonin for HeLa cells and that hexon and fiber had no measurable effect on the cytotoxicity of gelonin. However, like the whole virus, penton was not able to affect the cytotoxicity of gelonin conjugates.

The in vitro resistance of an Sdi- mutant of vesicular stomatitis virus to interference by wild-type defective interfering (DI) particles was expressed quantitatively in a cell-free replication system derived from mutant-infected cells. Added wild-type DI particle templates were replicated very poorly by extracts of Sdi- mutant-infected cells. However, the addition of purified viral polymerase (a complex of L and NS proteins) from wild-type vesicular stomatitis virus allowed efficient replication of wild-type DI particle genomes in these cell extracts. Added wild-type NS protein alone did not complement DI particle genome replication in these cell extracts, but it did complement a defect in the in vitro transcriptional activity of Sdi- mutant virus. These results clearly implicate the vesicular stomatitis virus polymerase complex in the inability of Sdi- mutants to replicate DI particles and in the quantitative escape from DI particle interference in evolving virus populations.

Simian adenovirus 7, either complete virus or its capsid subunits, agglutinates Rat (Sprague-Dowley) red blood cells in the presence of heterotypical antiserum. Haemagglutination takes place at 4 degrees C and room temperature. The antigen could not be eluted and its haemagglutinin properties are heat-stable. The reaction is specific. It is inhibited by homologous antiserum only. This property and its characteristics permit a camparison of this strongly oncogenic adenovirus to the human adenovirus of subgroup III of Rosen.