Inhibition of vaccinia virus replication by N-(phosphonoacetyl)-L-aspartate: differential effects on viral gene expression result from a reduced pyrimidine nucleotide pool.

The replication of vaccinia virus was reduced by 3 logs in cells that had been treated before and during infection with a concentration of N-(phosphonoacetyl)-L-aspartate (PALA) which lowered the UTP and CTP to 5 and 20% of controls, respectively, without affecting cell viability. The antiviral activity of PALA was reversed with uridine, indicating that it was entirely due to the diminution in pyrimidine nucleotides. Analysis of viral proteins revealed prolonged synthesis of some early stage species but a drastic reduction in late stage species, even though the nucleotide concentrations remained relatively constant throughout the infection. Although the gene expression pattern resembled that caused by a potent inhibitor of DNA synthesis, viral DNA accumulation was reduced by only 60%. Very little of the DNA made in the presence of PALA was converted to genome length molecules. The effect of PALA on transcription of early genes was complex: there was a twofold increase in the amount of a relatively short mRNA of 500 nucleotides but a two- to threefold decrease in the amount of a 4300-nucleotide mRNA encoding the largest subunit of RNA polymerase. In contrast, PALA severely inhibited the accumulation of viral intermediate and late stage mRNAs. The extreme sensitivity of vaccinia virus to PALA and the differential effects of the drug on viral gene expression result from the cascade mechanism of viral gene regulation.

Evaluation of the antiviral activity of N-(phosphonoacetyl)-L-aspartate against paramyxoviruses in tissue culture and against respiratory syncytial virus in cotton rats.

N-(phosphonoacetyl)-L-aspartate (PALA), a potent inhibitor of L-aspartic acid transcarbamoylase, was evaluated for cytotoxicity and antiviral activity against three different paramyxoviruses in tissue culture, and for antiviral efficacy and toxicity in vivo using a cotton rat-respiratory syncytial virus (RSV) model. Significant in vitro cytotoxicity was observed in proliferating cultures of HEp-2 (IC50 = 250 micrograms/ml) and Vero cells (IC50 = 32 micrograms/ml), but was less evident in cultures containing confluent monolayers (i.e., stationary cells) of these cells, or in cultures of Madin Darby canine kidney (MDCK) cells (these IC50 values were all > or = 750 micrograms/ml, with 1000 micrograms/ml being the maximum concentration tested). Mean selective indices (ratio of the median cytotoxic dose: median efficacious dose) of 1, 72 and 146 were obtained against parainfluenza virus type 3, RSV and measles virus, respectively, when PALA was tested against these viruses using confluent HEp-2 and Vero cell monolayers. In cotton rats, significant reductions in pulmonary titers (0.8-1.4 log10/g lung) compared to pulmonary viral titers in placebo-treated control animals, were consistently seen in cotton rats given > or = 10 mg of PALA/kg/day (b.i.d.) intraperitoneally on days 1-3 postinfection with either subtype A or B RSV. No toxic effects were noted even in animals given 100 mg of PALA/kg/day for 7 consecutive days.

Inhibition of herpesvirus-induced thymidine kinase and DNA polymerase by beta-hydroxynorvaline.

Treatment of HSV-infected cells with 5-10 mM beta-hydroxynorvaline (Hnv), a threonine analog, specifically affects herpesvirus DNA replication: both the rate of and total DNA synthesis are reduced, the former approximately 15-fold by Hnv (6 h post-infection) and the latter by 12-fold (between 3 and 12 h post-infection). The effect on DNA replication was due to inhibition of HSV-1 thymidine kinase (TK) and DNA polymerase (DP) activities; the former is reduced by 75% and whereas DP returns to baseline levels (when compared to untreated and/or uninfected cells). Host cell TK and DP activities are unaffected. It is suggested that beta-hydroxynorvaline is incorporated into these enzyme(s), either close to or at the active site thus perturbing viral DNA synthesis. beta-Hydroxynorvaline should have unique utility as a targeted antiviral compound, acting on both membrane-mediated phenomena (fusion, penetration and attachment) and DNA replication.

Glycosylation inhibitors block the expression of LAV/HTLV-III (HIV) glycoproteins.

The glycosylation inhibitors 2-deoxy-D-glucose (2-dGlc) and, to a lesser extent, beta-hydroxynorvaline blocked the formation of syncytia in HIV (LAV/HTLV-III)-infected cells. Using monospecific polyclonal antibodies against recombinant envelope proteins gp110 and gp41 or monoclonal antibodies against env gp110, we could demonstrate a marked reduction in the immunoreactivity of these antigens in HIV-infected cells exposed to the glycosylation inhibitors. There was concomitant accumulation of core proteins p15 and p24, as shown by a solid phase radio-immunoassay, and a decreased oligosaccharide synthesis of env proteins, as monitored by the incorporation of [6-3H]GlcNAc. The reverse transcriptase was not affected by the compounds. Glycosylation inhibitors may be considered for the chemotherapy of AIDS or AIDS-related complex, or chemoprophylaxis of HIV-positive individuals.

Efficacy of glycoprotein inhibitors alone and in combination with trifluridine in the treatment of murine herpetic keratitis.

The present study examined the anti-herpetic effect of the glycoprotein inhibitors, hydroxynorvaline and 2-deoxyglucose, alone and in combination with trifluridine on murine ocular herpes. Following ocular inoculation with a large dose of HSV-1 RE strain (10(6) pfu), ICR mice were treated during the acute infection with different therapeutic regimens, and their efficacy was evaluated by ocular virus titers, clinical grading of blepharo-conjunctivitis and histological evaluation of stromal keratitis and iridocyclitis. The results following a large dose HSV-1 inoculum demonstrated that trifluridine was the best single therapeutic agent. Hydroxynorvaline and 2-deoxyglucose had no effect at all. Combination therapy of the glycoprotein inhibitors with trifluridine was no better than trifluridine alone. The mouse HSV-1 keratitis model proved to be an effective, economical alternative to the rabbit model for the evaluation of new antiviral agents.

Galactose-rich glycoproteins are on the cell surface of herpes virus-infected cells. 1. Surface labeling and serial lectin binding studies of Asn-linked oligosaccharides of glycoprotein gC.

Cell-surface glycoproteins of mock-infected and herpes simplex virus type 1 (HSV-1)-infected BHK-21 and HEp-2 cells were radiolabeled by incubation with galactose oxidase followed by reduction with NaB3H4. The incorporation of radiolabel into glycoconjugates in both BHK-21 and HEp-2 cells was increased several fold following infection with HSV, showing an increase in surface-exposed Gal residues in the infected cells. This was further confirmed by an increase in binding of cell-surface-labeled glycoproteins gC and gB from HSV-infected BHK-21 cells to Ricinus communis agglutinin I, which is specific for beta-D-Gal residues. Prior treatment of cells with Clostridium perfringens neuraminidase enhanced the surface radiolabeling by the galactose oxidase/NaB3H4 method: HEp-2 cells exhibited over sixfold enhancement in labeling, while BHK-21 cells showed only a slight increase. HSV glycoprotein gC was the predominant cell-surface glycoprotein radiolabeled by the galactose oxidase/NaB3H4 method in virus-infected BHK-21 cells. The glycoprotein gC was purified by immunoaffinity column chromatography on monoclonal anti-gC-antibody-Sepharose. The radiolabel in the glycopeptides of gC was resistant to beta elimination, showing that it was associated only with Asn-linked oligosaccharides. A serial lectin affinity chromatography of glycopeptides on columns of concanavalin A-Sepharose, lentil (Lens culinaris) lectin-Sepharose, and Ricin I-agarose allowed the assignment of minimal oligosaccharide structures bearing terminal Gal residues in gC.

Inhibition of synthesis of herpesvirus (HSV-1) glycoproteins and endogenous fusion by beta-hydroxynorvaline in BHK-21 cells.

Treatment of HSV-infected BHK-21 cells with 5-10 mM of beta-hydroxynorvaline (Hnv), an analog of threonine which blocked attachment of oligosaccharides at the Asn-X-Thr sites, markedly inhibited the synthesis of all viral glycoproteins as well as the major capsid protein. However, the synthesis of host-specific dolichol-linked oligosaccharides was not significantly affected by Hnv. Treatment of cells with 10 mM reduced the yield of virus greater than 95% and completely blocked endogenous fusion. Inhibition of Hnv could be reversed by simultaneous addition of threonine to the culture medium. It is likely that the incorporation of Hnv into HSV polypeptides at Asn-X-Thr (in place of Thr) sites blocked transfer of N-linked oligosaccharides.

Enzymatic glucosylation of dolichol monophosphate and transfer of glucose from isolated dolichyl-D-glucosyl phosphate to ceramides by BHK-21 cell microsomes.

Enzymatic glucosylation of dolichol monophosphate (dolichol-P) from UDP-D-[3H]glucose was studied using the microsomal fraction of BHK-21 cells. The reaction product was separated by preparative thin-layer chromatography, further purified by DEAE-cellulose acetate column chromatography, and characterized as dolichyl-beta-D-glucosyl phosphate (Dol-P-Glc). The microsomal fraction of BHK cells catalyzed the incorporation of glucose from UDP-[3H]glucose into ceramides (endogenous and exogenous) and Dol-P; both reactions required Mn2+. Maximal glucosylation of Dol-P was achieved at pH 5.6-5.8 in the presence of a non-ionic detergent, Zonyl A. Glucosylation of exogenous Dol-P, from UDP-Glc, was non-competitively inhibited by exogenous ceramides. Incubation of Dol-P-[3H]Glc or Dol-P-[14C]Glc with liposomes (containing ceramides) and the microsomal fraction of BHK-21 cells resulted in the formation of a radioactive glucolipid which comigrated with the same RF value as glucosylceramide (Glc-Cer) on silica gel thin-layer chromatography. Transfer of [14C]glucose from Dol-P-[14C]Glc to exogenous ceramides was confirmed by double-labeling techniques. The pH dependence for transfer of radio-labeled glucose from Dol-P-[3H]Glc to ceramides was multi-phasic (optima at pH 4.0 and 7.0); glycosylation occurred within 5 min and Zonyl A was absolutely essential for the transfer reaction. These results indicate that Dol-P-Glc may also participate in the synthesis of ceramide hexosides.

Binding of influenza virus to a reconstituted receptor complex containing glycophorin.

Membrane particles possessing receptor activity for influenza virions have been reconstituted following solubilization of human erythrocyte membranes with octylglucoside (OG) and fractionation on a DEAE-cellulose column. Fractions that eluted with 1.5 M NaCl yielded, after removal of OG, reconstituted membrane particles (RMP) which could bind virus and inhibit hemagglutination. RMP contained essentially two membrane proteins (glycophorin and a nonglycosylated protein of molecular weight 29,000), two phospholipids (sphingomyelin and phosphatidylcholine), cholesterol, and gangliosides. Incubation of influenza virus with RMP at 4 degrees resulted in the formation of a virus-RMP complex (liposomes). The specificity of the receptor was demonstrated by inhibition of viral attachment when RMP were treated with neuraminidase or preincubated with rabbit anti-M or anti-N antiserum, suggesting that both the carbohydrates and peptide moiety may play a role in attachment. Calculations suggest that there are 6 X 10(3) N-acetyl neuraminic acid residues per attached virion. This system provides a simple and gentle means of reconstituting membrane components to study receptor activity.

Enzymatic deoxyglucosylation of ceramides by microsomes of BHK-21 cells. The effect of deoxyglucose treatment and herpesvirus infection.

The microsomal fractions of cultured hamster fibroblasts (BHK-21 cells) catalyze the incorporation of glucose from UDPglucose or of deoxyglucose from UDPdeoxyglucose into a reaction mixture with liposomes consisting of ceramide and phosphatidylcholine. The microsomal fractions also catalyze the transfer of glucose from UDPglucose to endogenous acceptors. The specific activity of ceramide deoxyglucoside or ceramide glucoside formation was significantly higher when microsomal preparations obtained from deoxyglucose-treated or herpesvirus-infected BHK-21 cells were used as the glucosyltransferase source. Deoxyglucose was incorporated from UDPdeoxyglucose into hydroxy- and nonhydroxy-fatty acid-containing ceramides at approximately the same rate. Competitive inhibition of deoxyglucosylation of ceramides by UDPglucose suggests that both reactions were catalyzed by the same enzyme, viz. UDPglucose:ceramide glucosyltransferase. This inhibition of glycosphingolipid synthesis may account, in part, for the inhibitory effect of deoxyglucose on lipid-containing viruses.

Historical findings in subjects from a high socioeconomic group who have genital infections with herpes simplex virus.

A highly motivated, self-selected group of 1,535 men and 1,607 women of middle-to-high socioeconomic class who had recurring genital herpes were surveyed in an assessment of the historical characteristics of these subjects and their disease. All subjects lived in the continental United States and were members of the national herpes organization, HELP, sponsored by the American Social Health Association. Subjects were predominantly well-educated white persons (mean educational level, 15.2 years) earning > $20,000 per year. The mean ages of acquisition of genital herpes were 26.9 years for women and 30.8 years for men. The mean durations of infection were 3.9 years for women and 5.1 years for men. Women usually acquired genital herpes between the ages of 20 and 29 years, whereas substantial numbers of men experienced their initial episodes of infection when they were in their thirties. The population studied was predominantly heterosexual. Many of the subjects, especially the men, had experienced other sexually transmitted diseases such as gonococcal or nongonococcal urethritis. Over two-thirds of the subjects experienced more than five relapses every year, and the percentage of subjects with more than five recurrences yearly did not decrease with time.

Successful treatment of human genital herpes infections with 2-deoxy-D-glucose.

Thirty-six women with genital herpes infections (proved by virological or cytological means) were treated in a double-blind placebo-controlled study with the glucose analogue 2-deoxy-D-glucose for a three-week period. In initial mucocutaneous cases, 89% were cured, with two recurrences after 24 months; in the case of recurrent or secondary infections, 90% had a notable improvement manifested by no or less-frequent recurrences, fewer lesions, or shortened duration of symptoms. In initial infections, discomfort cleared within 12 to 72 hours of therapy; 90% of the patients were asymptomatic within four days. In both cases, virus shedding was notably reduced by 2-deoxy-D-glucose. Concomitant controls treated with placebos failed to respond within this time frame. The use of 2-deoxy-D-glucose provides a simple and unique approach to the treatment of genital herpesvirus infections.

Lipids of rabies virus and BHK-21 cell membranes.

The lipid composition of highly purified Flury strain of rabies virus (HEP) propagated in BHK-21 cells in a chemically defined medium was observed to be 6.7% neutral lipids, 15.8% phospholipids, and 1.5% glycolipids. In the virion, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were the most abundant phospholipids, accounting for 90% of the total, and the molar ratio of cholesterol to phospholipid was 0.48. Uninfected BHK-21 cell membranes were obtained by nitrogen cavitation techniques and separated by density gradient centrifugation, and the membranes were assayed for purity using 5'-nucleotidase, cytochrome oxidase, and reduced nicotinamide adenine dinucleotide phosphate diaphorase activities. Lipids of the plasma membrane were enriched in cholesterol, phosphatidylcholine, and phosphatidylethanolamine. In contrast, membranes of the endoplasmic reticulum were enriched in phosphatidylcholine, but contained smaller amounts of phosphatidylethanolamine and sphingomyelin. Comparison of the fatty acyl chains of virus and membranes from uninfected cells revealed the virion to have the lowest ratio of C18:1 to C18:0 (1.771), compared with values of about 3.0 for the plasma membrane and endoplasmic reticulum. Total polyenoic fatty acids were enriched in the plasma membrane, whereas the virus contained higher amounts of total saturates than either of the two membrane preparations. Analysis of the polar and neutral lipid fractions as well as the acyl chain analysis suggests the virion has a lipid composition that is intermiediate to that of the plasma membrane and endoplasmic reticulum and is consistent with the view that numerous viral particles are synthesized de novo by not utilizing a preexisting membrane template. From the ratio of cholesterol to phospholipid of 0.48, we calculated that 1.92 X 10(5) molecules of lipid would cover 4.14 X 10(4) nm2 in the form of a bilayer. Considerations of the molecular dimensions of the rabies envelope (total surface area, 5 X 10(4) nm2) as a bilayer suggest that some penetration of lipids by envelope proteins (M and G) is necessary.

Composition of the neutral lipids of bovine meilbomian secretions.

Lipids secreted form the bovine meibomian glands were assigned to following classes: cholesteryl esters (A) (fatty acyl chain lengths from 15 to 27 carbon atoms), 41%; wax esters, 29%; triacylglycerols, 10%; and cholesteryl ester (B) (fatty acyl chain lengths from 14 to 18 carbons), 15%. The remaining 5% consisted of cholesterol, fatty acids, and highly polar material. Analysis of the lipids showed that only the cholesteryl esters (A) contained fatty acyl chains greater than or equal to C20:0. One third of the fatty acyl chains of the wax esters and the cholesteryl esters (B) was iC15:0. The fatty alcohol moieties of the wax esters were found to be branched chain, C23:0-C27:0. A computer-based programmable gas-liquid chromatographic procedure using 10% apolar 10C on Gas Chrom Q could be used to distinguish both iso and anteiso fatty alcohols and esters of fatty acids.