Characterization of varicella-zoster virus enhancement by the pesticide carbaryl.

Further characterization of the viral enhancement system of varicella-zoster virus by the pesticide carbaryl (1-naphthyl-N-methylcarbamate) is presented. It was necessary to expose cells to the enhancing chemical during the period of virus replication to detect enhancement. The optimum time for the pretreatment is 20 to 24 h. Maximum enhancement of virus expression occurs 48 to 72 h post-inoculation. Treated cells cannot pass on to daughter cells the ability to produce increased amounts of virus. alpha-Naphthol, a metabolite of carbaryl, is also capable of enhancing virus replication; Guthion, another pesticide tested, did not enhance varicella-zoster virus. The stable cell line HEP-2 can be used in place of human embryonic lung cells to detect enhancement. Differences in enhancement levels were not due to cell lot, cell passage, or a change in the stability of the cell membrane to sonic disruption.     

Enhancement of varicella-zoster virus replication in cultured human embryonic lung cells treated with the pesticide carbaryl

In studies designed to determine the factors responsible for control of herpesvirus replicaton in an infected cell, we examined the interaction of varicella-zoster (VZ) virus-infected human embryonic lung cells with the pesticide carbaryl. The replication of the cell-associated VZ virus was enhanced 2- to 13-fold as compared to control cultures in Sevin 4 Oil-treated cultures and in cultures treated with the pesticide's active ingredient, carbaryl. The replication of VZ virus in cultures teated with the base oil plus inert ingredients found in the pesticide formulation was not enhanced. Possible differences in cytotoxicity induced by Seven 4 Oil, pure carbaryl, or the base oil preparation were ruled out since treated and control cultures were shown to have similar numbers of viable cells when measured by trypan blue exclusion tests or by the ability of treated cells to form foci. A dose response study showed a decrease in viral enhancement in cells treated with decreasing carbaryl concentrations.     

Enhancement of varicella-zoster virus replication in cultured human embryonic lung cells treated with the pesticide carbaryl.

In studies designed to determine the factors responsible for control of herpesvirus replicaton in an infected cell, we examined the interaction of varicella-zoster (VZ) virus-infected human embryonic lung cells with the pesticide carbaryl. The replication of the cell-associated VZ virus was enhanced 2- to 13-fold as compared to control cultures in Sevin 4 Oil-treated cultures and in cultures treated with the pesticide's active ingredient, carbaryl. The replication of VZ virus in cultures teated with the base oil plus inert ingredients found in the pesticide formulation was not enhanced. Possible differences in cytotoxicity induced by Seven 4 Oil, pure carbaryl, or the base oil preparation were ruled out since treated and control cultures were shown to have similar numbers of viable cells when measured by trypan blue exclusion tests or by the ability of treated cells to form foci. A dose response study showed a decrease in viral enhancement in cells treated with decreasing carbaryl concentrations.     

Variation in human herpesvirus susceptibility to enhancement by the pesticide carbaryl

The ability of various human herpesviruses to be enhanced by the pretreatment of human embryonic lung cells with the pesticide carbaryl (1-naphthyl-N-methyl-carbamate) differs according to the virus tested. Different strains of varicella-zoster virus produced different patterns of susceptibility to enhancement. Laboratory-adapted strains were less sensitive to enhancement than were wild-type strains recently isolated from clinical specimens. The related human herpes simplex viruses types 1 and 2 and cytomegalovirus were negative for susceptibility to enhancement when either laboratory-adapted or wild-type strains were tested. No difference in the pattern of susceptibility was detected whether virus yields were determined by cell-associated or cell-free virus assay or when the input multiplicity was varied 10-fold.     

Variation in human herpesvirus susceptibility to enhancement by the pesticide carbaryl.

The ability of various human herpesviruses to be enhanced by the pretreatment of human embryonic lung cells with the pesticide carbaryl (1-naphthyl-N-methyl-carbamate) differs according to the virus tested. Different strains of varicella-zoster virus produced different patterns of susceptibility to enhancement. Laboratory-adapted strains were less sensitive to enhancement than were wild-type strains recently isolated from clinical specimens. The related human herpes simplex viruses types 1 and 2 and cytomegalovirus were negative for susceptibility to enhancement when either laboratory-adapted or wild-type strains were tested. No difference in the pattern of susceptibility was detected whether virus yields were determined by cell-associated or cell-free virus assay or when the input multiplicity was varied 10-fold.     

Enhancement of goldfish virus-2 in vitro replication by the pesticides carbaryl and toxaphene

Goldfish virus-2 replication was enhanced in vitro by pretreatment of CAR cells with subcytotoxic concentrations of carbaryl and toxaphene. This phenomenon was time and temperature dependent. Shortening of pretreatment with carbaryl eliminated enhancement, which was observed for toxaphene only with substantially increased concentrations. Decreasing the temperature of pretreatment (4 degrees C) abrogated any enhancement by carbaryl and resulted in enhancement by toxaphene only at increased concentrations. Increased absorption of input virus was ruled out as a mechanism for enhancement, as was stimulation of cell division in the presence of pesticides over that of control cultures. Pretreatment of virus rather than cells did not result in enhancement.     

Enhancement of goldfish virus-2 in vitro replication by the pesticides carbaryl and toxaphene.

Goldfish virus-2 replication was enhanced in vitro by pretreatment of CAR cells with subcytotoxic concentrations of carbaryl and toxaphene. This phenomenon was time and temperature dependent. Shortening of pretreatment with carbaryl eliminated enhancement, which was observed for toxaphene only with substantially increased concentrations. Decreasing the temperature of pretreatment (4 degrees C) abrogated any enhancement by carbaryl and resulted in enhancement by toxaphene only at increased concentrations. Increased absorption of input virus was ruled out as a mechanism for enhancement, as was stimulation of cell division in the presence of pesticides over that of control cultures. Pretreatment of virus rather than cells did not result in enhancement.     

Enhancement of Goldfish Virus Type 2 Replication by 1- Naphthol, the Major Hydrolysis Product of the Pesticide Carbaryl

The major hydrolysis product of the pesticide carbaryl, 1-naphthol, enhanced the replication of goldfish virus type 2 in piscine cell culture at concentrations below any detectable cytotoxicity. Enhancement indices of 2.3, 3.7, and 7.1 were observed at 1, 5, and 10 ppm (μl/liter), respectively. Replication at 0.5 ppm was equivalent to that of controls.     

Cellular laminin-binding protein and Venezuelan equine encephalomyelitis virus replication in Vero, 293 and 9S2 cells

The expression of the laminin-binding protein (LBP) on cellular membranes in different cell lines has been studied. A high level of replication of Venezuelan equine encephalomyelitis (VEE) virus was registered in Vero cells with high levels of LBP on the cell surface. The treatment of Vero cells with monoclonal antibodies to human LBP reduced VEE virus replication by a factor of more than 200. A low level of LBP expression on the surface of 293 cells was increased via transfection by plasmid with gene for human LBP. The VEE virus replication in transfected cells (9S2) was increased by more that 2000 times compared to the 293 cells. The results demonstrated the principal role of cellular LBP in the entry of VEE virus into mammalian cells. It is proposed that LBP is a key cellular protein for the early stage of the VEE virus replication in cells. LBP may be a target protein for the development of a new generation of antiviral drugs capable of inhibiting (enhancing) the alphavirus replication in human cells.     

Influenza virus replication in human cells exposed to the pesticide emulsifier Atlox

The emulsifier Atlox was capable of enhancing the infectivity of influenza virus in Chang human conjunctiva cells. Prior to infection, pre-treatment of cells with 2.5 to 10 ppm of Atlox for 6 to 8 h was necessary to detect an increase in virus production. Although there was no difference in the amount of adsorbed virus, the number of successfully infected cells was at least 1 log higher in Atlox treated cells as compared to the controls. Monitoring the 51Cr release from Atlox treated cells indicated a temporary change in membrane structure of the cells as one of the mechanisms of virus enhancement.     

Construction of varicella-zoster virus recombinants from parent Oka cosmids and demonstration that ORF65 protein is dispensable for infection of human skin and T cells in the SCID-hu mouse model

We generated an ORF65 deletion mutant by using a cosmid system constructed from the genome of a low-passage clinical isolate (P-Oka). Using the SCID-hu mouse model, we demonstrated that the ORF65 protein is dispensable for viral replication in skin and T cells, which are critical host cell targets during primary varicella-zoster virus infection.     

Suppression of interferon synthesis by the pesticide carbaryl as a mechanism for enhancement of goldfish virus-2 replication

Interferon production was demonstrated by the goldfish-derived CAR cell line in response to infection by goldfish virus-2. Supernatants of infected cultures provided antiviral protection to CAR cells and another cell line derived from goldfish, ABIII. The protective factor retained activity after ultracentrifugation, dialysis, freezing and thawing, acid treatment (pH 2), or heating to 56 degrees C but was sensitive to trypsin. Supernatants of infected cultures did not affect adsorption of virus. Previous studies have shown that replication of goldfish virus type 2 is enhanced by pretreatment of cultures with subcytotoxic concentrations of carbaryl. In the present study, pesticide-treated cultures were found to synthesize reduced levels of interferon.     

Suppression of interferon synthesis by the pesticide carbaryl as a mechanism for enhancement of goldfish virus-2 replication.

Interferon production was demonstrated by the goldfish-derived CAR cell line in response to infection by goldfish virus-2. Supernatants of infected cultures provided antiviral protection to CAR cells and another cell line derived from goldfish, ABIII. The protective factor retained activity after ultracentrifugation, dialysis, freezing and thawing, acid treatment (pH 2), or heating to 56 degrees C but was sensitive to trypsin. Supernatants of infected cultures did not affect adsorption of virus. Previous studies have shown that replication of goldfish virus type 2 is enhanced by pretreatment of cultures with subcytotoxic concentrations of carbaryl. In the present study, pesticide-treated cultures were found to synthesize reduced levels of interferon.     

CK2beta gene silencing increases cell susceptibility to influenza A virus infection resulting in accelerated virus entry and higher viral protein content

Background

Influenza A virus (IVA) exploits diverse cellular gene products to support its replication in the host. The significance of the regulatory (β) subunit of casein kinase 2 (CK2β) in various cellular mechanisms is well established, but less is known about its potential role in IVA replication. We studied the role of CK2β in IVA-infected A549 human epithelial lung cells.

Results

Activation of CK2β was observed in A549 cells during virus binding and internalization but appeared to be constrained as replication began. We used small interfering RNAs (siRNAs) targeting CK2β mRNA to silence CK2β protein expression in A549 cells without affecting expression of the CK2α subunit. CK2β gene silencing led to increased virus titers, consistent with the inhibition of CK2β during IVA replication. Notably, virus titers increased significantly when CK2β siRNA-transfected cells were inoculated at a lower multiplicity of infection. Virus titers also increased in cells treated with a specific CK2 inhibitor but decreased in cells treated with a CK2β stimulator. CK2β absence did not impair nuclear export of viral ribonucleoprotein complexes (6 h and 8 h after inoculation) or viral polymerase activity (analyzed in a minigenome system). The enhancement of virus titers by CK2β siRNA reflects increased cell susceptibility to influenza virus infection resulting in accelerated virus entry and higher viral protein content.

Conclusion

This study demonstrates the role of cellular CK2β protein in the viral biology. Our results are the first to demonstrate a functional link between siRNA-mediated inhibition of the CK2β protein and regulation of influenza A virus replication in infected cells. Overall, the data suggest that expression and activation of CK2β inhibits influenza virus replication by regulating the virus entry process and viral protein synthesis.     

Bile acids specifically increase hepatitis C virus RNA-replication

Background

Hepatitis C virus (HCV) patients with high serum levels of bile acids (BAs) respond poorly to IFN therapy. BAs have been shown to increase RNA-replication of genotype 1 but not genotype 2a replicons. Since BAs modulate lipid metabolism including lipoprotein secretion and as HCV depends on lipids and lipoproteins during RNA-replication, virus production and cell entry, BAs may affect multiple steps of the HCV life cycle. Therefore, we analyzed the influence of BAs on individual steps of virus replication.

Methods

We measured replication of subgenomic genotype (GT) 1b and 2a RNAs as well as full-length GT2a genomes in the presence of BAs using quantitative RT-PCR and luciferase assays. Cell entry was determined using HCV pseudoparticles (HCVpp). Virus assembly and release were quantified using a core-specific ELISA. Replicon chimeras were employed to characterize genotype-specific modulation of HCV by BAs. Lunet CD81/GFP-NLS-MAVS cells were used to determine infection of Con1 particles.

Results

BAs increased RNA-replication of GT1b replicons up to 10-fold but had no effect on subgenomic GT2a replicons both in Huh-7 and HuH6 cells. They did not increase viral RNA translation, virus assembly and release or cell entry. Lowering replication efficiency of GT2a replicons rendered them susceptible to stimulation by BAs. Moreover, replication of full length GT1b with or without replication enhancing mutations and GT2a genomes were also stimulated by BAs.

Conclusions

Bile acids specifically enhance RNA-replication. This is not limited to GT1, but also holds true for GT2a full length genomes and subgenomic replicons with low replication capacity. The increase of HCV replication by BAs may influence the efficacy of antiviral treatment in vivo and may improve replication of primary HCV genomes in cell culture.     

Mutational analysis of the role of glycoprotein I in varicella-zoster virus replication and its effects on glycoprotein E conformation and trafficking.

The contributions of the glycoproteins gI (ORF67) and gE (ORF68) to varicella-zoster virus (VZV) replication were investigated in deletion mutants made by using cosmids with VZV DNA derived from the Oka strain. Deletion of both gI and gE prevented virus replication. Complete deletion of gI or deletions of 60% of the N terminus or 40% of the C terminus of gI resulted in a small plaque phenotype as well as reduced yields of infectious virus. Melanoma cells infected with gI deletion mutants formed abnormal polykaryocytes with a disrupted organization of nuclei. In the absence of intact gI, gE became localized in patches on the cell membrane, as demonstrated by confocal microscopy. A truncated N-terminal form of gI was transported to the cell surface, but its expression did not restore plaque morphology or infectivity. The fusogenic function of gH did not compensate for gI deletion or the associated disruption of the gE-gI complex. These experiments demonstrated that gI was dispensable for VZV replication in vitro, whereas gE appeared to be required. Although VZV gI was dispensable, its deletion or mutation resulted in a significant decrease in infectious virus yields, disrupted syncytium formation, and altered the conformation and distribution of gE in infected cells. Normal cell-to-cell spread and replication kinetics were restored when gI was expressed from a nonnative locus in the VZV genome. The expression of intact gI, the ORF67 gene product, is required for efficient membrane fusion during VZV replication.     

Stimulation of adenovirus replication in simian cells in the absence of a helper virus by pretreatment of the cells with iododeoxyuridine.

Pretreatment of African green monkey kidney cells with 50 mu g of 5'-iododeoxyruidine (IUdR) per ml can modify their susceptibility to the replication of human adenovirus type 7 in the absence of simian virus 40 (SV40) although this enhancement of adenovirus replication is not as efficient as that of the helper SV40 virus. Since the number of infectious centers remains unchanged after IUdR pretreatment whereas the burst size of virus from each infected cell increases, the IUdR appears to allow each infected cell to produce more virus. Cell DNA synthesis appears to be stimulated in IUdR pretreated cells infected with adenovirus 7, but the host cell DNA synthesized is small enough to remain in the Hirt supernatant fluid. The modification of susceptibility to adenovirus replication and the changed pattern of cell DNA synthesis is stable for at least two additional cell passages of the pretreated cells.     

T-cell tropism and the role of ORF66 protein in pathogenesis of varicella-zoster virus infection

The pathogenesis of varicella-zoster virus (VZV) involves a cell-associated viremia during which infectious virus is carried from sites of respiratory mucosal inoculation to the skin. We now demonstrate that VZV infection of T cells is associated with robust virion production and modulation of the apoptosis and interferon pathways within these cells. The VZV serine/threonine protein kinase encoded by ORF66 is essential for the efficient replication of VZV in T cells. Preventing ORF66 protein expression by stop codon insertion (pOka66S) impaired the growth of the parent Oka (pOka) strain in T cells in SCID-hu T-cell xenografts in vivo and reduced formation of VZV virions. The lack of ORF66 protein also increased the susceptibility of infected T cells to apoptosis and reduced the capacity of the virus to interfere with induction of the interferon (IFN) signaling pathway following exposure to IFN-gamma. However, preventing ORF66 protein expression only slightly reduced growth in melanoma cells in culture and did not diminish virion formation in these cells. The pOka66S virus showed only a slight defect in growth in SCID-hu skin implants compared with intact pOka. These observations suggest that the ORF66 kinase plays a unique role during infection of T cells and supports VZV T-cell tropism by contributing to immune evasion and enhancing survival of infected T cells.     

Spindle disturbances in mammalian cells. II. Induction of viable aneuploid/polyploid cells and multiple chromatid exchanges after treatment of V79 Chinese hamster cells with carbaryl. Modifying effects of glutathione and S9

Treatment of V79 cells with carbaryl increased the frequency of cells with elevated chromosome numbers (greater than 22). This effect of carbaryl was inhibited by simultaneous addition of glutathione or S9. Although selective forces seemed to act against cells with increased chromosome numbers, such cells were still significantly more frequent in treated compared with control cultures 74 h after treatment. In addition, a high adaptive value for these remaining aneuploid/polyploid cells was indicated because they were able to go through 2 successive rounds of replication immediately before fixation to the same extent as cells with chromosome numbers considered normal (21/22) for this cell line. Multiple chromatid exchanges were also observed after carbaryl treatment. The lack of single exchange events indicates that the effect may be systemic. However, additional experiments are required before this hypothesis can be confirmed or discarded. Considering the results obtained and the possible importance of gene dosage for tumor promotion, it is suggested that carbaryl should be tested for tumor promotion in vivo.     

Glucocorticoids enhance viral transformation of mammalian cells

The effect of glucocorticoids on the replication of murine sarcoma virus (MSV) in mammalian cells were examined. Glucocorticoids, hydrocortisone (1 to 10 micrograms/ml) and dexamethasone (5 micrograms/ml), enhance transformation induced by the Kirsten strain of MSV (Ki-MSV) in normal rat kidney and human cells 10- to 30-fold. The enhancing effect was much more pronounced in normal human colonic mucosal epithelial-like cells. On the other hand, the hormones estradiol, testosterone, and progesterone had no effect (5 micrograms/ml). Individual foci appeared earlier and were larger in hydrocortisone-treated cells compared with untreated cells. This enhancing effect is further evidenced by the increased virus yield and murine leukemia virus complement-fixing antigen production in the test system. However, such enhancement of hydrocortisone on the Ki-MSV-induced transformation was not observed in mouse embryo cells as previously reported.