Induction of Antiviral Activity In Vivo and In Vitro by Human Placenta Ribonucleic Acid Treated with Nitrous Acid

Induction of antiviral activity and interferon by human placenta ribonucleic acid deaminated with sodium nitrite (NO₂-RNA) was studied in vitro and in vivo. (1) Viral multiplication in diploid cells from human kidney (HK cells) was depressed by pretreatment with NO₂-RNA, but not by pretreatment with the original placenta RNA. (2) NO₂-RNA showed an interferon-inducing activity in rabbits and mice. (3) NO₂-RNA sedimenting in 18 S and 28 S regions showed a higher antiviral activity than that sedimenting in 4 S region.     

The identification of globin messenger ribonucleic acid in newt erythropoietic cells.

Polyadenylated [poly(A)+]-RNA isolated from newt (Triturus cristatus) erythropoietic cells contained two main species sedimenting at 9S and 25S, and minor amounts of a 15-20S component. The 9S poly(A)+-RNA fraction induced synthesis of newt haemoglobin and globins in frog oocytes and in an mRNA-dependent rabbit reticulocyte lysate, confirming its identity as newt globin mRNA. Translation of 9S globin mRNA in reticulocyte lysate was concentration-dependent, the patterns of globin synthesis suggesting both preferential utilization and unequal amounts of the different globin mRNA subspecies. Globin mRNA activity was also evident in the 25S poly(A)+-RNA fraction whose localization in polyribosomes excluded its function as a nuclear globin mRNA precursor. Denaturation in formamide and estimation of its relative methyl content indicated that the 25S poly(A)+-RNA fraction contained equimolar amounts of 9S globin mRNA and 26S rRNA. Translation of the 25S fraction in reticulocyte lysate was less efficient than that of comparable amounts of 9S globin mRNA and induced a pattern of globin synthesis similar to that obtained with subsaturating amounts of 9S mRNA. The 25S mRNA-rRNA complex was considered to be a non-physiological aggregate generated by extraction of RNA in the presence of buffers of moderate to high ionic strength.     

Sedimentation Properties of Simian Virus 40-Specific Ribonucleic Acid Present in Green Monkey Cells During Productive Infection and in Mouse Cells Undergoing Abortive Infection

The size distribution of polyribosome-associated simian virus 40 (SV40) ribonucleic acid (RNA) was examined at various times after productive infection. Eight hours after infection, virus-specific RNA was detected in the 14 to 17S region of a sucrose gradient by deoxyribonucleic acid (DNA)-RNA hybridization; RNA present in fractions sedimenting more rapidly did not react with SV40 DNA. At successively later times, SV40 RNA was detected in more rapidly sedimenting regions. By 24 hr, a portion of the SV40 RNA was detected in the 28S region, sedimenting slightly more rapidly than a MS2 RNA marker. Nuclear SV40 RNA, prepared from cells 48 hr after infection, was distributed in more rapidly sedimenting regions of the gradient, peaking at about 32 to 34S. Some nuclear virus-specific RNA could be detected in the 45 to 50S region. During the abortive infection of mouse cells, the sedimentation profile of SV40 RNA was very similar to that observed during the early phases of the lytic cycle.     

Inhibition of Japanese encephalitis virus infection by nitric oxide: antiviral effect of nitric oxide on RNA virus replication.

The antiviral effects of nitric oxide (NO) on Japanese encephalitis virus (JEV), a member of the family Flaviviridae, were investigated in this study. In vitro, inhibition of replication of JEV in gamma interferon-activated RAW 264.7 murine macrophages was correlated to cellular NO production. When cocultured with infected murine neuroblastoma N18 cells, gamma interferon-activated RAW 264.7 cells also efficiently hindered JEV replication in contiguous bystanders, and this anti-JEV effect could be reversed by an NO synthase (NOS) inhibitor, N-monomethyl-L-arginine acetate. In vivo, the mortality rate increased as the NOS activity of JEV-infected mice was inhibited by its competitive inhibitor, N-nitro-L-arginine methyl ester. Moreover, when an organic donor, S-nitro-N-acetylpenicillamine (SNAP), was used, the NO-mediated antiviral effect was also observed in primarily JEV-infected N18, human neuronal NT-2, and BHK-21 cells, as well as in persistently JEV-infected C2-2 cells. These data reaffirm that NO has an effective and broad-spectrum antimicrobial activity against diversified intracellular pathogens. Interestingly, the antiviral effect of NO was not enhanced by treatment of N18 cells with SNAP prior to JEV infection, a measure which has been shown to greatly increase the antiviral effect of NO in infection by vesicular stomatitis virus. From biochemical analysis of the impact of NO on JEV replication in cell culture, NO was found to profoundly inhibit viral RNA synthesis, viral protein accumulation, and virus release from infected cells. The results herein thus suggest that NO may play a crucial role in the innate immunity of the host to restrict the initial stage of JEV infection in the central nervous system.     

Synthesis and evaluation of in vitro antiviral activity of novel phenoxy acetic acid derivatives

Several substituted phenoxy acetic acid derived pyrazolines were synthesized by the reaction between 2-{4-[3-(2,4-dihydroxyphenyl)-3-oxo-1-propenyl]-2-methoxyphenoxy} acetic acid and substituted acid hydrazides and were tested for their in vitro cytotoxicity and antiviral activity. None of the compounds showed any specific antiviral activity [50% antivirally effective concentration (EC(50)) > or = 5-fold lower than minimum cytotoxic concentration]. The most cytotoxic of the series was 2-{4-[3-(2,4-dihydroxyphenyl)-1-(2-hydroxybenzoyl-4,5-dihydro-1H-5-pyrazolyl]-2-methoxyphenoxy}acetic acid (3(j)), with a minimum cytotoxic concentration of 0.16 microg/mL in human embryonic lung (HEL) cells.     

mRNA for the plasminogen activator of the urokinase type: translation in oocytes of Xenopus laevis

Poly(A)+-RNA from human kidney and human embryonal lung fibroblasts fractionated by sucrose gradient centrifugation was translated in Xenopus oocytes. Assay for plasminogen-dependent fibrinolytic activity detected synthesis of secreted plasminogen activator and revealed the active fraction of poly(A)+-RNA with a sedimentation coefficient of approximately 23S. Translation products of the active fraction were immunoadsorbed by antiurokinase monoclonal antibodies immobilized on sepharose. Gel electrophoretic analysis of the protein products showed that the 23S fraction of poly(A)+-RNA from human kidney contains mRNA for single-chain urokinase-type plasminogen activator with apparent molecular weight of approximately 50 kDa.     

Single amino acid substitutions at conserved residues of human interferon-alpha can effect antiviral specific activity

Site-specific in vitro mutagenesis was used to direct various amino acid substitutions at conserved positions within the sequence of human interferon-alpha 1 (IFN-alpha 1). The antiviral specific activity of IFN-alpha 1, expressed in M13 as a fusion protein [IFN-alpha 1 (phi WT)], could be altered by single amino acid substitutions. The substitution of glycine for tyrosine at position 123 results in a loss of more than 99% of the antiviral specific activity on human cells, but causes no significant change in the antiviral specific activity on primary bovine cells. The tyrosine at position 123 is thus implicated in determining human cell specificity. Based on analysis of IFN-alpha 2, IFN-alpha 1 contains two dulsulphide bridges between cysteine residues 29 and 139 and cysteine residues 1 and 99. IFN-alpha 1 also contains a fifth cysteine residue at position 86. IFN-alpha 1 (phi WT) carrying three serine for cysteine substitutions at positions 1, 86 and 99 retains 23% of the antiviral specific activity of IFN-alpha 1 (phi WT) on human cells. However, the antiviral activity on bovine cells is not significantly affected by this modification. The presence of the disulphide bridge between residues 1 and 99 thus appears to be required for full antiviral activity on human but not bovine cells. A single serine for cysteine substitution at position 29 reduces the antiviral specific activity on both human and bovine cells by some 95%. This data shows that the disulphide bridge between residues 29 and 139 is critical for the antiviral activity of IFN-alpha's.     

In Vitro Product of a Ribonucleic Acid Polymerase Induced by Influenza Virus

The ribonucleic acid (RNA)-dependent RNA polymerase induced in the microsomal fraction of cells infected with influenza virus synthesized a mixture of single-and double-stranded RNA in vitro. The single-stranded RNA sedimented mainly in the 8S region on sucrose density gradients, with a smaller proportion of the RNA sedimenting at 18S. This sedimentation pattern corresponds closely to that of incomplete influenza virus RNA. The double-stranded RNA formed in vitro sedimented at 11S, but molecules which may be replicative intermediate, sedimenting at 14 to 20S, were also detected in the in vitro reaction product. Similar species of RNA were detected in vivo by pulse-labeling infected cells at the time of polymerase harvest, but the proportion of each RNA species was different, most of the RNA being single-stranded and sedimenting in the 18S region. An 11S double-stranded RNA was also synthesized in vivo. Pulse chase analysis of the double-stranded RNA synthesized in vitro showed that most is stable, and only a small proportion turns over during the reaction. A proportion of the RNA formed in vitro could be annealed to RNA formed in infected cells and to RNA extracted from purified virus.     

Simian virus 40 large T antigen oligomers: analysis of electrophoresis in the absence of detergent.

Large T antigen of simian virus 40 is found as monomeric and oligomeric species in transformed cells. These can be demonstrated in cell extracts by velocity centrifugation in sucrose gradients. We analyzed them further in a transformed human line cell (SV80) and a transformed mouse line cell (SVT2). Individual fractions from sucrose gradients were subjected to polyacrylamide gel electrophoresis in the absence of detergent. T-antigen species were then detected by protein blotting and antibody overlay with polyclonal anti-D2 T antibody or monoclonal Pab419, Pab101, or Pb1700 antibody. The rapidly sedimenting species (14S and larger) of large T antigen from both cell lines reproducibly showed two major bands with estimated molecular weights of 670,000 and 850,000. A third band of 1,200,000 was more prominent in SVT2 cells than in SV80 cells. In SV80 cells the slowly sedimenting species of large T antigen (5S to 11S) contained two reproducible bands. A band with a molecular weight of 95,000 was the predominant one in all fractions between 5S and 11S. A relatively minor band with a molecular weight of 230,000 was found in fractions between 9S and 11S. The low-molecular-weight forms were seen in SVT2 cells only when a prominent peak at 5S to 7S was present, that is, when extracts were stored before analysis. In fresh extracts, the low-molecular-weight bands and slowly sedimenting forms were absent.     

In Vitro Formation of Nitrate Reductase Using Extracts of the Nitrate Reductase Mutant of Neurospora crassa, nit-1, and Rhodospirillum rubrum

In vitro formation of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-nitrate reductase (NADPH: nitrate oxido-reductase, EC 1.6.6.2) has been attained by using extracts of the nitrate reductase mutant of Neurospora crassa, nit-1, and extracts of either photosynthetically or heterotrophically grown Rhodospirillum rubrum, which contribute the constitutive component. The in vitro formation of NADPH-nitrate reductase is characterized by the conversion of the flavin adenine dinucleotide (FAD) stimulated NADPH-cytochrome c reductase, contributed by the N. crassa nit-1 extract from a slower sedimenting form (4.5S) to a faster sedimenting form (7.8S). The 7.8S NADPH-cytochrome c reductase peak coincides in sucrose density gradient profiles with the NADPH-nitrate reductase, FADH(2)-nitrate reductase and reduced methyl viologen (MVH)-nitrate reductase activities which are also formed in vitro. The constitutive component from R. rubrum is soluble (both in heterotrophically and photosynthetically grown cells), is stimulated by the addition of 10(-4) M Na(2)MoO(4) and 10(-2) M NaNO(3) to cell-free preparations, and has variable activity over the pH range from 3.0 to 9.5. The activity of the constitutive component in some extracts showed a threefold stimulation when the pH was lowered from 6.5 to 4.0. The constitutive activity appears to be associated with a large molecular weight component which sediments as a single peak in sucrose density gradients. However, the constitutive component from R. rubrum is dialyzable and is insensitive to trypsin and protease. These results demonstrate that R. rubrum contains the constitutive component and suggests that it is a low molecular weight, trypsin- and protease-insensitive factor which participates in the in vitro formation of NADPH nitrate reductase.     

Studies on the immunomodulatory effect of extract of Cyanodon dactylon in shrimp, Penaeus monodon, and its efficacy to protect the shrimp from white spot syndrome virus (WSSV)

The present study investigates the protection of shrimp Penaeus monodon against white spot syndrome virus (WSSV) using antiviral plant extract derived from Cyanodon dactylon and the modulation of the shrimp non-specific immunity. To determine the antiviral activity, the shrimp were treated by both in vitro (intramuscular injection) and in vivo (orally with feed) methods at the concentration of 2mg per animal and 2% of the plant extract incorporated with commercially available artificial pellet feed, respectively. The antiviral activity of C. dactylon plant extract was confirmed by PCR, bioassay and Western blot analysis. In the present study, anti-WSSV activity of C. dactylon plant extract by in vivo and in vitro methods showed strong antiviral activity and the immunological parameters such as proPO, O(2)(-), NO, THC and clotting time were all significantly (P<0.05) higher in the WSSV-infected shrimp treated with plant extract when compared to control groups. These results strongly indicate that in vivo and in vitro administration of C. dactylon plant extract enhances immunity of the shrimp. Based on the present data and the advantages of plant extract available at low price, we believe that oral administration of C. dactylon plant extract along with the pellet feed is a potential prophylactic agent against WSSV infection of shrimp.     

Three Size-Classes of Intracellular Adenovirus Deoxyribonucleic Acid

When human adenovirus type 2 or 12 infects cells, either productively or non-productively, three classes of viral deoxyribonucleic acid (DNA) are found within the cells: (i) viral DNA which cosediments with DNA extracted from infectious adenovirions at 31.3S for adenovirus type 2 and at 29.0S for adenovirus type 12, (ii) viral DNA which sediments at about 18S, and (iii) viral DNA which sediments at >45S and is apparently integrated into the cellular DNA. A precursor-product relationship is suggested as a working hypothesis; the intact viral DNA is hydrolyzed to slowly sedimenting DNA and the slowly sedimenting DNA is integrated into the cellular DNA. Both the parental and the newly synthesized viral DNA are altered by this route. The intact viral DNA within the cells apparently is cleaved into the slowly sedimenting DNA by a preformed enzyme.     

Interferon-induced transfer of viral resistance by human B and T lymphocytes

Enriched human B lymphocytes cocultivated with mouse L cells produced human leukocyte interferon (IFN-alpha) and shortly thereafter transferred antiviral activity to the recipient cells (99% inhibition of expected virus yield). In contrast, cocultivation of enriched T-cell populations with mouse L cells resulted in no IFN production or transfer of antiviral activity. In addition, both T and B lymphocytes pretreated with exogenous IFN or stimulated in vitro by mitogens could transfer antiviral activity to human WISH cells. The transfer of antiviral activity was not blocked by antibodies to IFN. The data indicate that both T and B cells can be recruited by IFN to transfer antiviral activity. Thus, once cells are recruited by IFN they can transfer antiviral activity in the absence of IFN and protect cells locally or distally from the site of infection.     

Down-regulation of inducible nitric oxide synthase by lysophosphatidic acid in human respiratory epithelial cells

Viral infection generally results in the activation of inducible nitric oxide synthase (iNOS or NOS2) in respiratory epithelial cells by inflammatory cytokines. Activated NOS2 catalyzes synthesis of nitric oxide (NO), which in excess can cause cellular injury. On the other hand, lysophosphatidic acid (LPA), a lipid mediator released from epithelial cells, platelets, and fibroblasts in injured tissue, functions in repair of cell injury. However, details of the mechanism for repair by LPA remain unknown. We demonstrated one effect of LPA favoring repair, specifically inhibition by LPA of cytokine-induced NOS2 protein and mRNA expression by human respiratory epithelial cells in vitro. NO production by LPA-treated, cytokine-stimulated cells was also reduced. These decreases were prevented by Rho kinase inhibition with Y-27632. Thus, down-regulation by LPA of cytokine-induced increases in NOS2 activity is likely to involve a Rho-dependent signaling pathway. Harmful biologic effects of NO in viral respiratory infection might be modified by therapeutic manipulations involving LPA or Rho.     

Effects of mutation in hepatitis C virus nonstructural protein 5A on interferon resistance mediated by inhibition of PKR kinase activity in mammalian cells

The IFN-induced double-stranded RNA (dsRNA)-activated protein kinase PKR is one of the key molecules in the antiviral effects of IFN. To clarify the effects of hepatitis C virus nonstructural protein 5A (NS5A) on antiviral activity of IFN, in particular on PKR kinase activity, in mammalian cells, we established inducible NS5A-expressing cell lines derived from human osteosarcoma (Saos-2). The cells expressing NS5A derived from an IFN-resistant clone (NS5A-lb) that interacted with endogenous PKR in vitro, showed a suppressive effect on IFN function as determined by interference with vesicular stomatitis virus (VSV) infection, whereas NS5A (NS5A-2a) from an IFN-sensitive clone did not block the antiviral effect of IFN. A mutant with deletion of the IFN sensitivity determining region (ISDR) in NS5A-1b (NS5A-AISDR) also interacted with PKR and suppressed its activity in vitro. However, neither NS5A-2a nor the C-terminal truncated mutant of NS5A-1b (NS5A-deltaC) blocked PKR activity. These observations confirmed the previous report that the inhibitory effect of NS5A on IFN activity is mediated at least in part by the repression of PKR. In addition, we showed that IFN sensitivity was determined not only by the ISDR but that the involvement of the C-terminal region of NS5A-1b is important for the suppression of PKR activity.     

System beta and system A amino acid transporters in the feline endotheliochorial placenta

There is no knowledge of the transport mechanisms by which solutes cross the cat placenta or any other endotheliochorial placenta. Here, we investigated whether the amino acid transport systems beta and A are present in the cat placenta using a placental fragment uptake technique. Data were compared with studies in the human placenta, in which the presence of these two transport systems has been well established. A time course of [(3)H]taurine (substrate for system beta) and [(14)C]MeAIB (nonmetabolizable substrate for system A) uptake was determined in the term cat and human placental fragments in the presence and absence (choline substituted) of Na(+), and further studies were carried out over 15 min. Taurine uptake into both cat and human placenta fragments was found to be Na(+) and Cl(-) dependent, and Na(+)-dependent taurine uptake was blocked by excess beta-alanine. MeAIB uptake was found to be Na(+) dependent, and Na(+)-dependent MeAIB uptake was blocked by excess MeAIB or glycine. Western blotting and immunohistochemistry performed on cat and human placenta showed expression of TAUT and ATA2 (SNAT2), proteins associated with system beta and system A activity, respectively. This study therefore provides the first evidence of the presence of amino acid transport systems beta and A in the cat placenta.     

Antiviral Activity in Tissue Culture Systems of bis-Benzimidazoles, Potent Inhibitors of Rhinoviruses

(S,S)-1,2-bis(5-methoxy-2-benzimidazolyl)-1,2-ethanediol showed antiviral activity in monolayer tissue culture systems against 55 strains of rhinovirus, three types of poliovirus, and strains of type A and B coxsackieviruses. Neither the compound nor any of the analogues tested showed virucidal activity. Its antiviral activity was not associated with interference with viral attachment to or penetration into the cell. At a concentration of 0.1 mg/ml, this group of compounds was generally nontoxic to WI-38, primary bovine kidney, and African green monkey kidney cells and had antiviral activity with 100% inhibition of virus-induced cytopathic effects (CPE). At antiviral levels, these compounds prevented CPE of up to 10(6) median tissue culture infective dose units of virus and completely inhibited formation of new infective virions. The compounds showed antiviral activity both prophylactically and therapeutically against rhinoviruses. Infected cultures could be cleared of CPE up to 90 hr after infection.     

In vitro cleavage of 45S pre-ribosomal RNA and of giant heterogeneous RNA extracted from human leukemic cells

45S ribosomal precursor RNA and large heterogeneous RNA molecules (>45S) extracted from human leukemic cells were incubated in vitro with purified RNase III, which specifically attacks double-helical RNA regions.About 50% of the ribosomal precursor was cleaved into two major fragments sedimenting at 28S and 32S respectively. A limited number of cleavages was also introduced in about 40% of heterogeneous RNA molecules sedimenting faster than 45S, causing a partial shift to a polydisperse distribution in the 10S–45S range.     

Synthesis and biological evaluation of furoxan-based nitric oxide-releasing derivatives of glycyrrhetinic acid as anti-hepatocellular carcinoma agents

A series of novel furoxan-based nitric oxide (NO)-releasing derivatives of glycyrrhetinic acid (GA) were designed, synthesized, and evaluated for their in vitro cytotoxicity against human hepatocellular carcinoma (HCC) and non-tumor liver cells. Five furoxan/GA hybrids, 7b-d, 7f, and 7g, displayed potent cytotoxicity against HCC cells (IC(50): 0.25-1.10 μM against BEL-7402 cells and 1.32-6.78 μM against HepG2 cells), but had a little effect on the growth of LO2 cells, indicating that these compounds had selective cytotoxicity against HCC cells. Furthermore, these compounds produced high concentrations of NO in HCC cells, but low in LO2 cells and treatment with hemoglobin partially reduced the cytotoxicity of the hybrid in HCC cells. Apparently, the high concentrations of NO produced by NO donor moieties and the bioactivity of GA synergistically contribute to the cytotoxicity, but the NO is a major player against HCC cells in vitro. Potentially, our findings may aid in the design of new chemotherapeutic reagents for the intervention of human HCC at clinic.