Structural factors modulate the activity of antigenic poliovirus sequences expressed on hybrid hepatitis B surface antigen particles.

We have studied the functional expression of antigenic poliovirus fragments carried by various hybrid hepatitis B surface antigen (HBsAg) particles. Several constructions were made by using two different insertion sites in the HBsAg molecule (amino acid positions 50 and 113) and two different sequences, one derived from poliovirus type 1 (PV-1) and the other from PV-2. The inserted fragments each encompassed residues 93 to 103 of the capsid protein VP1, a segment which includes the linear part of the neutralization antigenic site 1 of the poliovirus. The antigenicity and immunogenicity of the hybrid particles were evaluated and compared in terms of poliovirus neutralization. A high level of antigenic and immunogenic activity of the PV-1 fragment was obtained by insertion at position 113 but not at position 50 of HBsAg. However, a cooperative effect was observed when two PV-1 fragments were inserted at both positions of the same HBsAg molecule. Antibodies elicited by the PV-2 fragment inserted at amino acid position 113 did not bind or neutralize the corresponding poliovirus strain. They did, however, bind a chimeric poliovirus in which the homologous antigenic fragment of PV-1 had been replaced by that of PV-2. The only virions that were neutralized by these antibodies were certain mutants carrying amino acid substitutions within the PV-2 fragment. These results show that position, constraints from the carrier protein, and nature of the inserted sequences are critically important in favoring or limiting the expression of antigenic fragments as viral neutralization immunogens.     

Construction of a poliovirus type 1/type 2 antigenic hybrid by manipulation of neutralization antigenic site II.

There are three serotypes of poliovirus, poliovirus type 1 (PV-1), PV-2, and PV-3. These viruses each display four distinct neutralization antigenic sites, designated N-AgI, N-AgII, N-AgIIIA, and N-AgIIIB. It has been demonstrated previously that part of N-AgI can be replaced with heterogeneous amino acid sequences, resulting in hybrid viruses expressing heterogeneous antigenic determinants. To study whether hybrid viruses could be constructed by modifying another antigenic site, a part of N-AgII (amino acids 158 to 173 of VP2) of PV-1(Mahoney) was replaced with the equivalent sequence from PV-2(Lansing). The resulting hybrid was viable and expressed both PV-1 and PV-2 antigenic determinants. When inoculated into rabbits, the hybrid induced neutralizing antibodies against both PV-1 and PV-2, showing that amino acids 158 to 173 of VP2 are able to function as an antigenic site independent of the rest of N-AgII. Manipulation of N-AgII represents a useful alternative method for the production of hybrid polioviruses.     

Engineering a poliovirus type 2 antigenic site on a type 1 capsid results in a chimaeric virus which is neurovirulent for mice.

Poliovirus type 2 (PV-2) Lansing strain produces a fatal paralytic disease in mice after intracerebral injection, whereas poliovirus type 1 (PV-1) Mahoney strain causes disease only in primates. Atomic models derived from the three-dimensional crystal structure of the PV-1 Mahoney strain have been used to locate three antigenic sites on the surface of the virion. We report here the construction of type 1-type 2 chimaeric polioviruses in which antigenic site 1 from the PV-1 Mahoney strain was substituted by that of the PV-2 Lansing strain by nucleotide cassette exchange in a cloned PV-1 cDNA molecule. These chimaeras proved to have mosaic capsids with composite type 1 and type 2 antigenicity, and induced a neutralizing response against both PV-1 and PV-2 when injected into rabbits. Moreover, a six-amino-acid change in PV-1 antigenic site 1 was shown to be responsible for a remarkable host-range mutation in so far as one of the two type 1-type 2 chimaera was highly neurovirulent for mice.     

Antiradical and antioxidant properties of nonsteroidal anti-inflammatory agents--derivatives of pyridinecarboxylic acid

A number of pyridincarboxylic acid derivatives PV-1-4, 7 and emoxypine preparation antioxidative activity in yolk lipoprotein suspension was studied by a method of Fe(2+)-initiated biochemiluminescence. Lipid peroxidation in suspension was effectively inhibited by the studied compounds in various concentration ranges. PV 1, 3, 4, 7 inhibited lipid peroxidation at the concentrations 100-fold, then those of PV 2 and emoxypine. Antiradical activity of the studied compounds was demonstrated by their forming a complex with the stable diphenylpickrylhydrazyl radical. The effect of these compounds as antioxidants is discussed.     

Developmental regulation of PV-1 in rat lung: association with the nuclear envelope and limited colocalization with Cav-1

Plasmalemma vesicle protein-1 (PV-1) is a caveolae-associated protein that is enriched in lung endothelial cells. The PV-1 protein is first detected in the lung at embryonic day 12, before that of caveolin-1 (Cav-1). There is a postnatal rise in PV-1 and Cav-1 mRNA levels, reaching a peak at the time of weaning and declining to their lowest levels in the adult lung. In contrast, the PV-1 protein progressively increases during postnatal development with its highest levels in the adult lung; the Cav-1 protein remains relatively constant throughout this period. Alveolar endothelial cells express both PV-1 and Cav-1 proteins, but PV-1, unlike Cav-1, is also detectable in some bronchial epithelial cells. Endothelial cells transfected with a rat PV-1 construct show a punctate membrane distribution of PV-1, perinuclear accumulation, and an association with the nuclear envelope. In these cells, PV-1 exhibits only partial perinuclear colocalization with Cav-1 and F-actin. In summary, PV-1 is developmentally regulated in the rat lung and shows a divergent intracellular localization, with a limited caveolae/Cav-1 colocalization in cultured endothelial cells.     

Analysis of RNA synthesis of type 1 poliovirus by using an in vitro molecular genetic approach.

Membranous crude replication complexes (CRC) were isolated from poliovirus-infected HeLa cells as recently described (N. Takeda, R.J. Kuhn, C.-F. Yang, T. Takegami, and E. Wimmer, J. Virol. 60:43-53, 1986). Viruses used to produce the CRC were poliovirus type 1 (Mahoney), [PV-1(M)], poliovirus type 1 (Sabin) [PV-1(S)], and four in vitro recombinants that were constructed from infectious cDNA clones. RNA synthesis in CRC was studied. No end-linked, full-length double-stranded poliovirus RNA was detected in CRC regardless of whether nonionic detergent (Nonidet P-40) was added prior to incubation. Synthesis of VPg-pU and VPg-pUpU, two nucleotidyl proteins presumed to be involved in the initiation of RNA synthesis, was slower at 30 degrees C in CRC induced by PV-1(S) than by PV-1(M). This observation was used to design a pulse-chase experiment whose result suggested that synthesis of VPg-pUpU occurred by uridylylation of VPg-pU. Synthesis of VPg-pU(pU) was thermosensitive in CRC induced by PV-1(S). With CRC of recombinant viruses, the thermosensitive block covaried to nucleotide substitutions in PV-1(S) that mapped to the virus-induced RNA polymerase 3Dpol. We conclude that plus-stranded RNA synthesis in CRC does not proceed via hairpin structures. The results of VPg-pU----VPg-pUpU synthesis are consistent with a model in which VPg-pU is the primer of RNA synthesis mediated by 3Dpol. The data suggest that uridylylation of VPg or a precursor thereof may be catalyzed by 3Dpol itself, a mechanism resembling events occurring in adenovirus DNA replication.     

Selective ion binding and membrane activity of synthetic cyclopeptides

Four cyclic peptides related to the membrane-active complexones PV, cyclo-(L-Pro-Lval-D-Pro-D-Val)3, and valinomycin were synthesized: (1) cyclo-(L-Pro-L-Ala-D-Val)3 or PVPA, (2) cyclo-(L-Ala-L-Val-D-Pro-D-Val)3 or PVAV, (3) cyclo-(L-Pro-L-Val-D-Pro-D-Val)2-L-Pro-D-Val or PV-10, (4) cyclo-(L-Pro-L-Val-D-Pro-D-Val)2 or PV-8. In a two-phase extraction assay the affinity of PV and PVPA for alkali picrates was about three orders or magnitude greater than that of valinomycin. It was about equal to valinomycin for PVAV and much lower for PV-10 and PV-8. PV, PVPA and PVAV showed a selectivity sequence similar to that of valinomycin, namely K+ approximately Rb+ greater than Cs+ greater than Na+ greater than Li+. In the series PV, PV-10, PV-, the preference for K+ over Na+ was 700, 5 and less than 1, respectively. Thus, it was possible to reverse the selectivity of PV for K+ over Na+ by reducing the ring size from 12 to 8 amino acid residues. In sheep red cell lipid bilayer membranes PVPA increased the membrane conductance significantly in the presence of either KCl or NaCl but it was less potent than PV. PV-10, PV-8 and PVAV on the other hand were ineffective in this assay. The inactivity of PVAV as a potassium carrier in membrane was in contrast to its high affinity for potassium picrate in two-phase assays. Such a behaviour may be observed of a compound that has too low an aqueous cation binding constant to use the solution-complexation mechanism of PV (Davis et al. (1976) Biochemistry 15, 768--774 and Pinkerton et al. (1969) Biochem. Biophys. Res. Commun. 35, 512--518) and too slow binding and release kinetics to use the interfacial-complexation mechanism of valinomycin.     

Isolation, cloning, and localization of rat PV-1, a novel endothelial caveolar protein.

By using an immunoisolation procedure (Stan, R.-V., W.G. Roberts, K. Ihida, D. Predescu, L. Saucan, L. Ghitescu, and G.E. Palade. 1997. Mol. Biol. Cell. 8:595-605) developed in our laboratory, we have isolated a caveolar subfraction from rat lung endothelium and we have partially characterized the proteins of this subfraction which include an apparently caveolae-specific glycoprotein we propose to call PV-1 (formerly known as gp68). The isolation and partial sequencing of PV-1, combined with the cloning of the full length PV-1 cDNA led to the following conclusions: (a) PV-1 is a novel single span type II integral membrane protein (438 amino acids long) which forms homodimers in situ; (b) the transmembrane domain of PV-1 is near the NH2 terminus defining a short cytoplasmic endodomain and a large COOH-terminal ectodomain exposed to the blood plasma; (c) PV-1 is N-glycosylated and its glycan antennae bear terminal nonreducing galactosyl residues in alpha1-3 linkage. PV-1 is expressed mostly in the lung but both the messenger RNA and the protein can be detected at lower levels also in kidney, spleen, liver, heart, muscle, and brain. No signal could be detected in testis and two lower molecular weight forms were detected in brain. Immunocytochemical studies carried out by immunodiffusion on rat lung with an anti-PV-1 polyclonal antibody directed against a COOH-terminal epitope reveal a specific localization of PV-1 to the stomatal diaphragms of rat lung endothelial caveolae and confirm the extracellular orientation of the PV-1 COOH terminus.     

PV-1 Labels Trans-Cellular Openings in Mouse Endothelial Cells and is Negatively Regulated by VEGF

The PV-1 protein is endogenously expressed from a single mRNA in the mouse pancreatic MS-1 endothelial cell line as a 60-kDa N-glycosylated and 50-kDa non-glycosylated protein that form DTT sensitive oligomers. In the absence of cell permeabilization, PV-1 labels transcellular openings of variable size, many that penetrate through the cytosol with circular openings on the free and attached surface of the plasma membrane. Intracellular PV-1 is localized in perinuclear aggregates that can extend as a fibrous network through the cytosol and often surround the nuclear compartment. In some cells, PV-1 is organized as a large unipolar spindle-like structure that is often associated with severe deformation of the nucleus. The VEGF-R2 inhibitor SU5614 increased the PV-1 protein in a dose-dependent manner and inhibited MS-1 cell growth, without inducing apoptosis. This report provides compelling evidence for a functional role of PV-1 in the formation of large transendothelial channels and modulation of nuclear shape. Moreover, these data suggest the PV-1 protein is negatively regulated by VEGF.     

Intralesional Injection of Rose Bengal Induces a Systemic Tumor-Specific Immune Response in Murine Models of Melanoma and Breast Cancer

Intralesional (IL) injection of PV-10 has shown to induce regression of both injected and non-injected lesions in patients with melanoma. To determine an underlying immune mechanism, the murine B16 melanoma model and the MT-901 breast cancer model were utilized. In BALB/c mice bearing MT-901 breast cancer, injection of PV-10 led to regression of injected and untreated contralateral subcutaneous lesions. In a murine model of melanoma, B16 cells were injected into C57BL/6 mice to establish one subcutaneous tumor and multiple lung lesions. Treatment of the subcutaneous lesion with a single injection of IL PV-10 led to regression of the injected lesion as well as the distant B16 melanoma lung metastases. Anti-tumor immune responses were measured in splenocytes collected from mice treated with IL PBS or PV-10. Splenocytes isolated from tumor bearing mice treated with IL PV-10 demonstrated enhanced tumor-specific IFN-gamma production compared to splenocytes from PBS-treated mice in both models. In addition, a significant increase in lysis of B16 cells by T cells isolated after PV-10 treatment was observed. Transfer of T cells isolated from tumor-bearing mice treated with IL PV-10 led to tumor regression in mice bearing B16 melanoma. These studies establish that IL PV-10 therapy induces tumor-specific T cell-mediated immunity in multiple histologic subtypes and support the concept of combining IL PV10 with immunotherapy for advanced malignancies.     

Molecular characterization of mouse-virulent poliovirus type 1 Mahoney mutants: involvement of residues of polypeptides VP1 and VP2 located on the inner surface of the capsid protein shell.

Most poliovirus (PV) strains, including PV PV-1/Mahoney, are unable to cause paralysis in mice. Determinants for restriction of PV-1/Mahoney in mice have been identified by manipulating PV-1 cDNA and located on the viral capsid protein VP1. These determinants consist of a highly exposed amino acid sequence on the capsid surface corresponding to the B-C loop (M. Murray, J. Bradley, X. Yang, E. Wimmer, E. Moss, and V. Racaniello, Science 241:213-215, 1988; A. Martin, C. Wychowski, T. Couderc, R. Crainic, J. Hogle, and M. Girard, EMBO J. 7:2839-2847, 1988) and of residues belonging to the N-terminal sequence located on the inner surface of the protein shell (E. Moss and V. Racaniello, EMBO J. 10:1067-1074, 1991). Using an in vivo approach, we isolated two mouse-neurovirulent PV-1 mutants in the mouse central nervous system after a single passage of PV-1/Mahoney inoculated by the intracerebral route. Both mutants were subjected to two additional passages in mice, plaque purified, and subsequently characterized. The two cloned mutants, Mah-NK13 and Mah-NL32, retained phenotypic characteristics of the parental PV-1/Mahoney, including epitope map, heat lability, and temperature sensitivity. Mah-NK13 exhibited slightly smaller plaques than did the parental virus. The nucleotide sequences of the mutant genomes were determined, and mutations were identified. Mutations were independently introduced into the parental PV-1/Mahoney genome by single-site mutagenesis. Mutated PV-1/Mahoney viruses were then tested for their neurovirulence in mice. A single amino acid substitution in the capsid proteins VP1 (Thr-22-->Ile) and VP2 (Ser-31-->Thr) identified in the Mah-NK13 and Mah-NL32 genomes, respectively, conferred the mouse-virulent phenotype to the mouse-avirulent PV-1/Mahoney. Ile-22 in VP1 was responsible for the small-plaque phenotype of Mah-NK13. Both mutations arose during the first passage in the mouse central nervous system. We thus identified a new mouse adaptation determinant on capsid protein VP1, and we showed that at least one other capsid protein, VP2, could also express a mouse adaptation determinant. Both determinants are located in the inside of the three-dimensional structure of the viral capsid. They may be involved in the early steps of mouse nerve cell infection subsequent to receptor attachment.     

Transformation of the ground cover after surface fires and estimation of pyrogenic carbon emissions in the dark-coniferous forests of Central Siberia

Ground fuel loads and structure in dark-coniferous forests with the dominance of Siberian pine have been studied in the Central Siberian State Nature Biosphere Reserve located in the central-taiga zone of Central Siberia. The impacts of surface fires of various forms and severity on the living ground cover are examined. It is found that fires of low to moderate severity reduce ground fuel loads from 35–49 t/ha to 26–28 t/ha, while fires of moderate to high severity reduce them to 17–18 t/ha. Consumption of down woody debris varies from 3 to 29 t/ha, depending on the prefire fuel characteristics and fire form and severity. Steady fires spreading with the fire danger index PV-1 of 3919 ± 482 result in carbon emissions of 14.0 tC/ha from fires of low to moderate severity and 24.6 tC/ha from fires of moderate to high severity. The lowest carbon emissions (10.1 tC/ha) are noted for fast-moving fires spreading with PV-1 of 1167 ± 386.     

Effects of chlorophyllin on replication of poliovirus and bovine herpesvirus in vitro

Aims:  Chlorophyllin (CHLN), a synthetic derivative of chlorophyll, was assayed in the replication of poliovirus (PV-1) and bovine herpesvirus (BoHV-1) in HEp-2 cell cultures.
Methods and Results:  Virucidal activity of CHLN was evaluated and the time-of-addition assay was performed as follows: before the infection (−1 and −2 h), at the time of the infection (0 h) and after the infection (1 and 2 h). Plaque reduction assay (PRA) showed that CHLN inhibited BoHV-1 and PV-1 infection and the 50% inhibitory concentrations (IC₅₀) against BoHV-1 and PV-1 infection were 8·6 and 19·8 μg ml⁻¹, respectively. The time-of-addition study demonstrated that the CHLN was effective inhibiting viral replication in 51% and 66·5% for PV-1 and BoHV-1, respectively, at the highest concentration of 20·0 μg ml⁻¹, when added during the infection. The directed effect of CHLN on viral strains demonstrated an inhibition of 62% and 66·4% for PV-1 and BoHV-1, respectively, by PRA.
Conclusions:  These results demonstrated that CHLN could be used as an antiviral suggesting directed activity on virus particles and on virus-receptor sites to BoHV. For poliovirus, CHLN also demonstrated virucide activity, moreover, showed to inhibit early steps of the replication cycle.
Significance and Impact of the Study:  CHLN demonstrated promising selectivity index for both virus strains; therefore, it can be used for the development of an antiviral agent.     

Infectivity of RNA from inactivated poliovirus

During inactivation of poliovirus type 1 (PV-1) by exposure to UV, hypochlorite, and heat (72 degrees C), the infectivity of the virus was compared with that of its RNA. DEAE-dextran (1-mg/ml concentration in Dulbecco's modified Eagle medium buffered with 0.05 M Tris, pH 7.4) was used to facilitate transfecting PV-1 RNA into FRhK-4 host cells. After interaction of PV-1 RNA with cell monolayer at room temperature (21 to 22 degrees C) for 20 min, the monolayers were washed with 5 ml of Hanks balanced salt solution. The remainder of the procedure was the same as that for the conventional plaque technique, which was also used for quantifying the PV-1 whole-particle infectivity. Plaque formation by extracted RNA was approximately 100,000-fold less efficient than that by whole virions. The slopes of best-fit regression lines of inactivation curves for virion infectivity and RNA infectivity were compared to determine the target of inactivation. For UV and hypochlorite inactivation the slopes of inactivation curves of virion infectivity and RNA infectivity were not statistically different. However, the difference of slopes of inactivation curves of virion infectivity and RNA infectivity was statistically significant for thermal inactivation. The results of these experiments indicate that viral RNA is a primary target of UV and hypochlorite inactivations but that the sole target of thermal inactivation is the viral capsid.     

Infectivity of RNA from Inactivated Poliovirus

During inactivation of poliovirus type 1 (PV-1) by exposure to UV, hypochlorite, and heat (72°C), the infectivity of the virus was compared with that of its RNA. DEAE-dextran (1-mg/ml concentration in Dulbecco's modified Eagle medium buffered with 0.05 M Tris, pH 7.4) was used to facilitate transfecting PV-1 RNA into FRhK-4 host cells. After interaction of PV-1 RNA with cell monolayer at room temperature (21 to 22°C) for 20 min, the monolayers were washed with 5 ml of Hanks balanced salt solution. The remainder of the procedure was the same as that for the conventional plaque technique, which was also used for quantifying the PV-1 whole-particle infectivity. Plaque formation by extracted RNA was approximately 100,000-fold less efficient than that by whole virions. The slopes of best-fit regression lines of inactivation curves for virion infectivity and RNA infectivity were compared to determine the target of inactivation. For UV and hypochlorite inactivation the slopes of inactivation curves of virion infectivity and RNA infectivity were not statistically different. However, the difference of slopes of inactivation curves of virion infectivity and RNA infectivity was statistically significant for thermal inactivation. The results of these experiments indicate that viral RNA is a primary target of UV and hypochlorite inactivations but that the sole target of thermal inactivation is the viral capsid.     

Effect of gamma irradiation on human cortical bone transplants contaminated with enveloped and non-enveloped viruses.

In the production of bone grafts intended for transplantation, basic safety measures to avoid the transmission of pathogens are selection and serological screening of donors for markers of virus infections. As an additional safety tool we investigated the effect of gamma irradiation on the sterility of human bone diaphysis transplants and evaluated its impact on the virus safety of transplants. Model viruses were included in the study to determine the dose necessary to achieve a reduction factor for the infectivity titres of at least 4 log(10) at a temperature of -30+/-5 degrees C. The following viruses were used: human immunodeficiency virus type 2 (HIV-2), hepatitis A virus (HAV), and poliovirus (PV-1), and the following model viruses: pseudorabies virus (PRV) as a model for human herpesviruses, bovine viral diarrhoea virus (BVDV) for HCV, and bovine parvovirus (BPV) for parvovirus B19. A first approach was to determine the D(10) values (kGy) for the different viruses (virus inactivation kinetics: BPV 7.3; PV-1 7.1; HIV-2 7.1; HAV 5.3; PRV 5.3; BVDV <3.0 kGy). Based on these results, inactivation of these viruses was studied in experimentally contaminated human bone transplants (femoral diaphyses). For BPV, the most resistant one of the viruses studied, a dose of approximately 34 kGy was necessary to achieve a reduction of infectivity titres of 4 log(10). We therefore recommend a dose of 34 kGy for the sterilisation of frozen bone transplants.     

Analysis of the role of calcium in analgesic action of non-narcotic analgesics and calcium channel blockers]

In the mice hot-plate test we have compared analgesic effect of calcium channel blockers and new non-narcotic analgesic antiinflammatory agent PV-107: verapamil > fenigidin > PV-107. Simultaneously we have shown strong correlation (r - 0.82) between analgesic effect and 45Ca2+ efflux of cardiac membrane in depolarizing media in vitro.