RESUMEN

DNA from HSV-1-infected cells was separated by pulsed-field gel electrophoresis into two virus-specific bands: one that migrated as the linear monomer genome (152 kb) and another that remained at the origin of the gel. The latter band contained the replicating HSV-1 DNA, as determined by pulse-labeling with [3H]thymidine. To investigate the structure of this "gel origin" DNA, we constructed a HSV-1 KOS mutant bearing a unique PacI restriction site (HSV-1 PAC1DTK). Partial digestion of gel origin PAC1DTK DNA at late times postinfection (24-48 hr) demonstrated the presence of linear concatemers on pulsed-field gel electrophoresis. Within each concatemer, the long (L) regions of adjacent monomer genomes were found in the two possible orientations. In addition, shorter-than-unit-size fragments that corresponded in size to the left end fragments of the viral genome were detected with the UL region in the two possible orientations. At early times postinfection (8-12 hr), digestion with PacI released only a trace of linear fragments, and most of the gel origin DNA did not migrate on pulsed-field gel electrophoresis. Multiple cuts with EcoRI (a restriction enzyme that cuts the HSV-1 KOS genome 12 times) were necessary to release linear fragments that migrated from the origin of the gel. These results indicate that replicative intermediates of HSV-1 DNA are linked in a large network that needs to be unraveled before packaging takes place. This network may be composed of linear molecules linked together by frequent recombination events or of products of a mode of replication other than simple rolling circle (e.g., theta replication).

Asunto(s)

RESUMEN

Paramyxovirus membrane (M) protein specifically binds to cellular actin but not to bovine serum albumin or myoglobin, as determined by affinity chromatography and enzyme-linked immunosorbent assay. The binding site for M protein on actin is different from the binding sites for antiactin antibodies. The interaction of M protein with actin resulted in production of antibodies to several new antigenic sites on the actin molecule. Five rabbits immunized with actin alone produced antibodies against the N-terminal sequence (residues 1 to 39). Another five rabbits immunized with a mixture of M protein and actin produced antibodies against a C-terminal fragment and a central region as well as the N-terminal fragment. By immunoblotting with proteolytic fragments of actin, the new antigenic sites were located between amino acid residues 40 to 113, 114 to 226, and 227 to 375. Antisera taken from some patients with recent measles virus infections demonstrated antiactin antibodies to sites other than the N-terminal fragment of actin (amino acids 1 to 39). The interaction of paramyxovirus M protein with actin and the subsequent production of antibodies to new antigenic sites may serve as a model for one of the mechanisms of virus-induced autoimmunity.

Asunto(s)

RESUMEN

A series of new 5-(1-hydroxy-2-iodoethyl)-2'-deoxyuridine and uridine compounds (11, 16) was synthesized by the regiospecific addition of HOI to the vinyl substituent of 5-vinyl-2'-deoxyuridine (10a), 5-vinyl-2'-fluoro-2'-deoxyuridine (10b), 5-vinyluridine (10c), and (E)-5-(2-iodovinyl)-2'-deoxyuridine (4b). Treatment of the iodohydrins 11a-c with methanolic sulfuric acid afforded the corresponding 5-(1-methoxy-2-iodoethyl) derivatives (12a-c). In contrast, reaction of 5-(1-hydroxy-2-iodoethyl)-2'-deoxyuridine (11a) with sodium carbonate in methanol afforded a mixture of 5-(1-hydroxy-2-methoxyethyl)-2'-deoxyuridine (13) and 2,3-dihydro-3-hydroxy-5-(2'-deoxy-beta-D-ribofuranosyl)- furano[2,3-d]pyrimidin-6(5H)-one (14). The most active compound, 5-(1-methoxy-2-iodoethyl)-2'-deoxyuridine (12a, ID50 = 0.1 micrograms/mL), which exhibited antiviral activity (HSV-1) 100-fold higher than that of the 5-(1-hydroxy-2-iodoethyl) analogue (11a), was less active than IVDU or acyclovir (ID50 = 0.01-0.1 micrograms/mL range). The C-5 substituent in the 2'-deoxyuridine series was a determinant of cytotoxic activity, as determined in the in vitro L1210 screen, where the relative activity order was CH(OH)CHI2 (16) greater than CH(OMe)CH2I (12a) greater than CH(OH)CH2I (11a) congruent to CH(OH)CH2OMe (13). The 2'-substituent was also a determinant of cytotoxic activity in the 5-(1-hydroxy-2-iodoethyl) (11a-c) and 5-(1-methoxy-2-iodoethyl) series of compounds, where the relative activity profile was 2'-deoxyuridine greater than 2'-fluoro-2'-deoxyuridine greater than uridine (11a greater than 11b greater than or equal to 11c; 12a greater than 12b greater than 12c). The most active cytotoxic agent (16), possessing a 5-(1-hydroxy-2,2-diiodoethyl) substituent (ED50 = 0.77 micrograms/mL), exhibited an activity approaching that of melphalan (ED50 = 0.15 micrograms/mL). All compounds tested, except for 13 and 14, exhibited high affinity (Ki = 0.035-0.22 mM range relative to deoxyuridine, Ki = 0.125) for the murine NBMPR-sensitive erythrocyte nucleoside transport system, suggesting that these iodohydrins are good permeants of cell membranes.

Asunto(s)

RESUMEN

5-(1-Hydroxy-2-haloethyl)- (4), 5-(1-methoxy-2-haloethyl)- (5) and 5-(1-hydroxy-2-methoxyethyl)uracils (6) (see Figure 2 for structures) were synthesized to investigate the effect of the C-5 substituents on cytotoxic and antiviral activity. The bromo compounds (4b and 5b) exhibited greater cytotoxic activity than the chloro or iodo analogues in the in vitro L1210 assay. Replacement of the hydroxyl substituent of 4b (bromo) and 4c (iodo) by a methoxyl substituent (5b-c), or substitution of their halogen substituents by methoxyl (providing 6) increased the potency. However, the cytotoxic activity of all the compounds was weak, the most active (6) producing a 45% decrease in cell survival at a concentration of 50 micrograms/ml, as compared with a 97% decrease when the reference standard (melphalan) was tested at 1 microgram/ml. They were inactive antiviral agents against herpes simplex virus type 1 (HSV-1) infected Vero cells at 10 micrograms/ml; in the same test, the reference standard (acyclovir) exhibited an ID50 of 0.01 micrograms/ml.

Asunto(s)

RESUMEN

A series of new 5-(1-hydroxy-2-haloethyl)-2'-deoxyuridines (3, 6, 8) were synthesized in 60-70% yields by addition of HOX (X = Br, Cl, I) to the vinyl substituent of the respective 5-vinyl-2'-deoxyuridines (2, 5, 7). Treatment of 3a,b with methanolic sulfuric acid afforded the corresponding 5-(1-methoxy-2-haloethyl)-2'-(deoxyuridines (4a,b). The 5-(1-hydroxy-2-chloroethyl) (3b), 5-(1-methoxy-2-bromoethyl) (4a), 5-(1-hydroxy-2-bromo-2-(ethoxycarbonyl)ethyl) (6a), and 5-(1-hydroxy-2-iodo-2-(ethoxycarbonyl)ethyl) (6b) derivatives exhibited in vitro antiviral activity (ID50 = 0.1-1 microgram/mL range) against herpes simplex virus type 1 (HSV-1). 5-(1-Hydroxy-2-bromo-2-(ethoxycarbonyl)-ethyl)-2'-deoxyuridine (6a) was the most active cytotoxic agent in the in vitro L1210 screen exhibiting an ED50 of 11 micrograms/mL relative to melphalan (ED50 = 0.15 micrograms/mL).

Asunto(s)

RESUMEN

Synthesis of (E)-5-(2-iodovinyl)-1-(2-deoxy-2-fluoro-beta-D-ribofuranosyl)uracil (IVFRU; 4a), and its [125I] and [131I] derivatives (4b and 4c) are described. Compared with IVDU, IVFRU had comparable antiviral activity (MIC50 = 0.01-0.1 microgram/ml), and a greater ethanol/water partition coefficient; its affinity for the murine erythrocyte nucleoside transporter system was greater than that of 2'-deoxyuridine. The [125I] derivative (4b) was selectively trapped within rabbit kidney cells (27.9 and 41.2% at 4 and 24 hr, respectively) infected in vitro by thymidine kinase-positive (TK+) herpes simplex virus (HSV-1), but not within either HSV (TK-) or mock-infected cells where uptake was less than 1%. It was resistant to glycosidic bond cleavage by pyrimidine nucleoside phosphorylases, but underwent catabolism to an unidentified metabolite and iodide during in vivo plasma and urinary excretion studies in mice. We conclude that the [123I] derivative of IVFRU warrants further evaluation as a non-invasive radiopharmaceutical agent for the diagnosis of herpes simplex encephalitis (HSE), and that IVFRU warrants further evaluation as an antiviral agent.

Asunto(s)

RESUMEN

1-(2-Deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) and the 5-iodo (FIRU), 5-bromo (FBrRU), 5-chloro (FClRU) and 5-fluoro (FFRU) analogues of 1-(2-deoxy-2-fluoro-beta-D-ribofuranosyl)uracil were examined in four in vitro tests designed to evaluate their potential as radiopharmaceuticals in a non-invasive diagnostic test for herpes simplex encephalitis (HSE). The tests involved, (a) cellular uptake studies--which showed that all five nucleosides are selectively trapped in rabbit kidney cells infected with herpes simplex virus, type I, which codes for its own thymidine kinase; (b) incubation studies--which showed that, in human platelets, FIAU, FIRU and FFRU are not degraded by thymidine phosphorylase (an enzyme which catalyzes the glycosidic bond cleavage of physiological nucleosides); (c) a transport study, using mouse erythrocytes--which indicated that all five nucleosides have good affinity for a NBMPR-sensitive nucleoside transporter, with inhibition constants (Ki values in the range of 0.177-0.41; and (d) determination of octanol/water partition coefficients--which (log P = -0.14 to -1.67) indicated that FIAU is the most lipophilic of the five compounds studied, and may therefore cross the blood-brain barrier most readily. We conclude from the results that FIAU is the most promising compound for further evaluation in HSE animal models. The combination of tests described in this study provide a useful screening protocol for evaluation of other nucleoside analogues of potential use in a diagnostic test for HSE.

Asunto(s)

RESUMEN

Selective uptake of nucleoside analogues by herpes simplex virus infected cells may serve as the basis for a specific non-invasive diagnostic test for herpes simplex encephalitis. We have examined the effect of acyclovir on the selective uptake of [131I] 1-(2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl)-5-iodouracil in herpes simplex virus infected primary rabbit kidney cells. Infected cells treated with acyclovir continued to concentrate [131I] 1-(2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl)-5-iodouracil for up to 24 h after the addition of the antiviral agent. These results indicated that therapy with acyclovir for as long as 24 h would not prevent the selective trapping of nucleoside analogues. This has important implications for the use of nucleoside analogues in diagnostic brain scans to detect herpes simplex encephalitis.

Asunto(s)

RESUMEN

E-5-(2-Iodovinyl)-2'-deoxyuridine (IVdU) is a potent inhibitor of herpes simplex virus type 1 replication in vitro. The selective antiviral activity of IVdU is due to preferential phosphorylation by the herpes simplex virus type 1-encoded thymidine kinase. This selective sequesteration provided the rationale for the development of radioiodinated IVdU as a potential radiopharmaceutical compound for use in noninvasive diagnosis of herpes simplex virus encephalitis. We studied the pharmacokinetics and the in vivo metabolism of [131I]IVdU in dogs. The radioactive components in plasma were characterized and quantitated by radio high-pressure liquid chromatography. During incubation with dog blood, [131I]IVdU was metabolized to the corresponding base (E)-5-(2-iodovinyl)uracil. 131I-labeled (E)-5-(2-iodovinyl)uracil accounted for 73% of the total radioactivity present in plasma after 2 h of incubation, suggesting that phosphorolysis of the nucleoside is the major degradation pathway of IVdU in blood. The in vivo studies showed that there was an initial rapid clearance of the tracer from blood, followed by a second very slow clearance phase. Evaluation of the renal excretion of the radiotracer showed that only 8% of the injected dose was excreted by kidneys over an 8-h period. IVdU was rapidly metabolized to three radioactive compounds. Two of these metabolites, the base (E)-5-(2-iodovinyl)uracil and iodide, were characterized. The radioactivity associated with these metabolites was responsible for the slow clearance phase. Our results suggest that the development of [131I]IVdU as a radiopharmaceutical compound will require measures to prevent its rapid degradation in vivo.

Asunto(s)

RESUMEN

Peripheral blood lymphocytes from MS patients and non-MS patients were induced to produce interferon by incubation with canine distemper, mumps, Sendai, herpes simplex I, and two strains of measles viruses. Lymphocytes from MS patients produced as much pH 2 stable interferon as did those from non-MS patients in response to induction with any of these viruses. In addition, there were no significant differences in amounts of interferon produced by lymphocytes from MS patients in three different clinical stages of the disease.

Asunto(s)

RESUMEN

Evidence for an interaction of the membrane (M) protein of Newcastle disease and Sendai viruses with cellular actin was obtained by three different techniques. M protein linked to Sepharose 4B was found to bind actin, but not myoglobin or bovine serum albumin, and to selectively remove actin from a mixture of these three proteins. Sedimentation of a mixture of M protein and F-actin through a sucrose gradient resulted in sedimentation of M protein with actin. Control proteins, bovine serum albumin and cytochrome c, did not sediment with actin. In circular dichroism studies, M protein added to actin in a 1:1 complex resulted in a significant increase in negative ellipticity at 220 nm, which corresponds to an increase in alpha-helix and a decrease in beta-structure and random coil. This is indicative of an interaction between M protein and actin. It is possible that the frequent identification of cellular actin in a number of enveloped viruses may be attributed to the interaction of actin and M protein or its equivalent.

Asunto(s)

RESUMEN

Serial passage of Sindbis at high multiplicities of infection resulted in cyclical variations in virus titer. Decreases in virus titer were correlated with the appearance of smaller-sized virions, interference and truncated viral RNA. The smaller particles were 37 nm in diameter, exclusive of the hemagglutinin spikes as compared with a diameter of 50 nm for standard virions. Passages which contained 37-nm partilces also interfered with infectious center formation by standard, plaque-purified virus. Polyacrylamide gel analysis of RNA isolated from virions present in interfering passages demonstrated the sequential appearance of three RNA species smaller than standard RNA with approximate molecular weights of 3.3 X 106, 2.7 X 106, and 2.2 X 106. The 3.3 X 106 RNA was evident in passage 5, by passage 8 both the 3.3 X 106 and 2.7 X 106 RNAs were present, and by passage 13 all three were present with the 2.2 X 106 RNA predominating.

Asunto(s)

Asunto(s)

RESUMEN

Encephalomyocarditis (EMC) virus ribonucleic acid (RNA) stimulated the incorporation of (14)C-amino acids into polypeptides in cell-free systems using preincubated S10 extracts from L cells. Incorporation was linear for over 2 hr. Analysis of the tryptic peptides derived from the polypeptide products formed in response to EMC RNA showed them to be virus specific. The major product, a polypeptide of 140,000 in molecular weight, migrated on sodium dodecyl sulfate-polyacrylamide gels with one of the virus-specific polypeptides present in EMC-infected cells. A minor component of molecular weight about 230,000 may correspond to the product of complete translation of the EMC virus genome. Little or no effect of interferon or vaccinia virus infection was observed in the preincubated, cell-free system. The EMC RNA-stimulated incorporation of (14)C-amino acids into polypeptides was not inhibited in extracts derived from L cells early in virus infection, from interferon-treated cells, or from cells subjected to both treatments. Interferon treatment did appear to have a slight inhibitory effect on chain elongation in this system. However, treatment of cells with highly purified interferon before virus infection caused a decrease of about 80% in the capacity of non-preincubated cell extracts to translate added EMC RNA. This effect did not extend to the translation of polyuridylic acid and could be reversed by preincubation of the extracts at 37 C for 20 min. The inhibition of translation was manifest at interferon concentrations as low as 5IU/ml, and in this respect closely paralleled the inhibition of virus growth. Inactivation of the antiviral activity of the interferon by heating or digestion with trypsin also abolished the effect on cell-free protein synthesis. The EMC-specific polypeptides formed in reduced amounts in extracts of interferon-treated vaccinia-infected cells were smaller than those formed in extracts of untreated, vaccinia-infected cells. Thus, inhibition of initiation or elongation of polypeptides, or both, can be demonstrated in cell-free systems employing non-preincubated extracts from interferon-treated, virus-infected cells. These results indicate that antiviral activity of interferon is directed against the translation of viral messenger RNA.

Asunto(s)

RESUMEN

The polypeptide products synthesized at different times in a cell-free system from Krebs mouse ascites tumor cells in response to the addition of encephalomyocarditis (EMC) virus ribonucleic acid (RNA) were characterized by electrophoresis on polyacrylamide gels and fingerprint analysis of their tryptic peptides. Translation of the EMC RNA genome with time occurred in a nonrandom fashion in these systems, to yield products containing sequences characteristic of both virion capsid polypeptides and EMC-specific polypeptides present only in the infected cell. The molecular weights of the products fell in a series from 20,000 to 140,000 daltons, although occasionally traces of larger polypeptides were also observed. All of the major polypeptides appeared to arise from partial or complete translation of about 60% of the EMC RNA genome. They were not formed by cleavage of a large precursor molecule. It is suggested that they are artifacts generated by premature "termination" of nascent polypeptide chains at preferred sites.

Asunto(s)

RESUMEN

The presence of serum in suspensions of Sendai-induced human leukocytes is necessary for the synthesis of significant amounts of interferon. Very little interferon is obtained from serum-free suspensions. Cow's milk or milk casein can substitute for serum in the production of high yields of human leukocyte interferon.

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)