The effects of cycloheximide, actinomycin D, and ethionine on the biliary excretion of labelled alpha-naphthylisothiocyanate in rats.

It has recently been shown that rats given a mixture of [³H-4-naphthyl] α-naphthylisothiocyanate (ANIT) and [¹⁴C-isothiocyanate] ANIT having a ${}^3\text{H}{}^{14}\text{C}$ ratio equal to 6.3, excreted more ³H than ¹⁴C into bile during the first 8 hours after ANIT administration, the ratio increasing to 7.5. However, in rats pretreated with cycloheximide, the ratio remained similar to that of the ANIT mixture administered (6.3) and data indicated that this inhibitor blocked this increase in ³H. Here we present further data showing that this effect by cycloheximide is dose-dependent, can be observed as late as 16 hours after ANIT administration, and is sensitive to early posttreatment times not to exceed 1 hour after ANIT. Actinomycin D and dl-ethionine also significantly blocked the increase in ${}^3\text{H}{}^{14}\text{C}$ ratio but it was found necessary to give both inhibitors 4 hours before ANIT administration in order to effect the decrease within 8 hours after ANIT administration. If given 30 minutes before ANIT, these inhibitors showed no effect until 9 hours after ANIT administration. These data further support the conclusion that cycloheximide, as well as actinomycin D and dl-ethionine, may act by inhibiting the formation of a toxic moiety of ANIT. The effects of these inhibitors on decreasing the excretion of a ³H moiety of ANIT in bile seem to coincide, on the basis of potency and protection, with their effects against hyperbilirubinemia and cholestasis.     

Ultrastructure of hepatic angiosarcoma in rats induced by vinyl chloride

The hepatic angiosarcoma studied was from a male Wistar rat having been exposed to vinyl chloride monomer (VCM) by the oral route for 120 weeks. Widened sinusoids lined by large electron-lucent, spindle-shaped tumor cells and membrane-bound nuclear-free structures were seen in the transitional zone between the tumor mass and the adjacent liver tissue. In areas merely consisting of tumor tissue the tumor cells had a more angular or irregular shape. The origin of the tumor cells is discussed in light of the characteristic differences between endothelial cells and Kupffer cells described in the literature. Although an endothelial origin of the tumor cells could not be established unequivocally, it was concluded that at present there is little reason to doubt such an origin.     

Studies on the generation and amplification of sendai virus defective-interfering genomes.

Sendai virus which had been cloned by successive plaque purification was found to generate a wide assortment of defective-interfering (DI) genomes on continued high-m.o.i. passage in eggs. No predisposition to generate a particular DI genome was noted. One stock of Sendai virus, which contained 13 different DI genomes varying in length from 670 to 7100 nucleotides was passaged undiluted in eggs for several more generations to see whether the smaller DI genomes could be selectively amplified. Our results indicate that the size of the DI genome per se does not confer a selective advantage and the implications of this finding are discussed.     

Studies on the DNA of an oncogenic papovavirus of the Syrian hamster.

The hamster papovavirus (HaPV) isolated from multiple skin tumors of the Syrian hamsters contains supercoiled, double stranded DNA with a G/C content of 39.2% and a molecular weight which is within the range of SV 40 and polyoma DNA. Spectrophotometric melting and annealing curves indicate a high thermal stability and a high sequence homogeneity of the HaPV-DNA. Restriction endonucleases BamH1, HindII, and EcoRI cleave HaPV-DNA into one, three, and four fragments, respectively, and the electrophoretic pattern of the digest products is distinctly different from that of SV 40 and all other known papovaviruses. The relative positions of the BamH1, HindII, and EcoRI restriction endonuclease sites have been established, taking the unique BamH1 site as a zero point.     

Adenovirus type 12 T antigen-related surface antigen detected by immunofluorescence on the membrane of transformed and infected cells

THA cells, derived from a hamster tumor induced by adenovirus type 12 (Ad12), were analyzed by immunofluorescence for the presence of virus-specific surface antigens. S antigen is a surface antigen detected on suspended living cells by a serum collected from hamsters immunized with Ad12. Besides S antigen, a new surface antigen (surface-T) was observed in formaldehyde-fixed cells treated with anti-T serum. Anti-T serum is not reactive against S antigen in living cells and anti-S serum did not detect surface-T antigen. The membrane location of surface-T antigen was confirmed with a Staphylococcus aureus binding assay. Surface-T antigen seems specific for Ad12, as it is not observed in hamster embryo cells, and is not recognized by antisera against SV40- or polyoma-T antigens nor by antiserum against hamster embryonic antigens. Surface-T antigen is also seen in infected KB cells but not in abortively infected RK13 cells. Anti-P serum, raised against an extract of infected RK13 cells, failed to detect surface-T antigen. Experiments with adsorbed anti-T and anti-S sera confirmed the specificity of surface-T antigen and its distinctiveness from S antigen.     

Further studies on the association of herpes simplex virus type 1 DNA with host DNA during productive infection

Our previous finding of an association between herpes simplex virus type 1 (HSV-1) and African green monkey kidney (BSC-1) DNAs in productively infected cells has been extended by investigating the influence of the time course, temperature, and multiplicity of infection on the extent of this association. The interaction of host and viral DNAs was not only dependent on the concentration of intracellular viral DNA, but also upon the particular conditions of infection. An examination of the time course of infection revealed that the amount of free viral DNA per cell reached a maximum well before the amount of viral-host-associated DNA did so, while an examination of the effect of temperature revealed a threefold variation in the amount of viral-host-associated DNA over a temperature range (33.5-39.5 degrees ) in which the amount of free viral DNA was essentially constant. The quantity of HSV-1 DNA associated with host-cell DNA ranges from about 100 to 1500 genome equivalents per cell, depending on the conditions of infection, and it appears that between 5 and 40% of the association is alkali stable.     

The effect of mouse interferon on the transformation of NIH/3T3 mouse cells by murine sarcoma virus.

The effect of interferon (IF) on transformation of NIH/3T3 cells by murine sarcoma virus (MSV) was studied. Two assays of MSV transformation independent of virus replication were used, focus formation under conditions where foci developed from cells infected with MSV alone and colony formation in agar suspension medium. Pretreatment of cells with purified IF (10⁴ units/ml) substantially inhibited transformation upon infection with MSV by about 90%. Reconstruction experiments showed that such IF treatment did not block focus or colony formation by cells already stably transformed by MSV. These results indicate that there is an IF sensitive early step in the transformation process.     

Immunological evidence for the synthesis of all canine distemper virus polypeptides in chronic neurological diseases in dogs. Chronic distemper and old dog encephalitis differ from SSPE in man.

Immunoprecipitation of the polypeptides of canine distemper virus (CDV) from lysates of infected cells has revealed that the serum and cerebrospinal fluid (CSF) of dogs with chronic distemper and old dog encephalitis contain high levels of antibody to all the viral structural polypeptides, indicating that all of these polypeptides are being synthesized in the dogs. These findings, and the fact that infectious virus has been isolated from explants of brain tissue without cocultivation techniques, differ from those with measles virus in subacute sclerosing panencephalitis (SSPE) in which there is a lack of antibody to the virus membrane (M) protein and cocultivation of brain explants with permissive cells is required for virus isolation. These results indicate that CDV undergoes complete replication in the brain in the persistent infection resulting in chronic neurological disease, in contrast to the situation with measles virus in SSPE in which replication appears to be abortive.     

Translation of PVX RNA in vitro by wheat germ. I. Characterization of the reaction and product size

Potato virus X (PVX) RNA was an efficient mRNA for in vitro translation by the wheat germ system. Optimal incorporation of labeled amino acids occurred in reaction mixtures (50 μl) containing 3 mM Mg²⁺, 70 mM K⁺, and 3–4 μg of RNA. The presence of PVX RNA stimulated amino acid incorporation up to 40 times over background levels. Addition of spermine or spermidine further stimulated amino acid incorporation nearly twofold. PVX RNA translation was not inhibited by adding double-stranded RNA from Penicillium stoloniferum. The largest of several polypeptides obtained from PVX RNA translation reaction mixtures had an apparent molecular mass of 110,000 daltons. No polypeptide that coelectrophoresed with native PVX coat protein subunits was observed even when heat-treated PVX RNA was used, or when the products from spermine-stimulated reactions were analyzed.     

Herpes simplex virus thymidine kinase expression in infection of the trigeminal ganglion.

Infection of the trigeminal ganglion was investigated using standard thymidine kinase-positive (TK⁺) herpes simplex virus (HSV) and two TK^? mutants. After corneal inoculation of mice with TK^? HSV, the incidence of acute and latent trigeminal ganglion infection was markedly decreased compared to TK⁺ virus. When cell-free virus was titered from mice 1 hr to 5 days post-corneal inoculation, ocular replication of TK^? HSV was found to be similar to TK⁺ HSV, but whereas TK⁺ HSV replicated well and was found in substantial amounts in trigeminal ganglia (2 × 10³ PFU/mg ganglion tissue), TK^? HSV did not replicate in the ganglia. Mean TK^? trigeminal ganglion virus titers were 10,000-fold less than TK⁺ titers. When a TK⁺ revertant of TK^? mutant virus was tested, however, trigeminal ganglion virus replication was similar to that with the parental TK⁺ virus. The results obtained were interpreted as being consistent with impaired axonal transport of TK^? HSV from cornea to trigeminal ganglion neurons or more likely, with impaired replication of TK^? HSV in ganglionic neurons.     

Production and characterization of antisera specific for the erb-portion of p75, the presumptive transforming protein of avian erythroblastosis virus

The polypeptide pattern of intracytoplasmic A particles, isolated from mammary tumors of ICRC mice, displayed a prominant doublet of about 75,000 molecular weight (Ap75) and a lesser band of 42,000 molecular weight (Ap42). A particle proteins were analyzed by tryptic fingerprint mapping and compared to similarly treated core proteins (p10, p14, p23, and p28) of murine mammary tumor virus (MuMTV) purified from the milk of ICRC mice. Comparison of the peptide maps showed that almost all the major spots of the MuMTV core proteins could be located within the map of Ap75, and that Ap42 contained peptides of only p28 and p14. Since tryptic peptide maps of MuMTV core proteins have been shown to be strain-specific markers, we conclude that the A particles present in mammary tumors of ICRC mice are coded for by the same genes that code for the virions present in the milk of these mice.     

Molecular basis of the am8H1 lesion in bacteriophage M13.

The nucleotide sequence of the mutant region of am8H1, the only conditional lethal mutant in gene VIII of the filamentous phages, has been determined and compared to the wild-type sequence. The amber mutation lies adjacent to the codons specifying the cleavage site for the procaryotic signal peptidase (1). The precoat protein of this mutant appears to be processed normally in the amber-suppressing strain K37, which inserts a serine residue in place of a glutamic acid residue present in the wild-type precoat protein. The amino acid compositions of wild-type and mutant phage are in agreement with the sequence data.     

Comparative studies on tomato aspermy and cucumber mosaic viruses. VI. Partial compatibility of genome segments from the two viruses

Isolated RNA genomic segments (RNAs 1, 2, and 3) of tomato aspermy virus and of three strains of cucumber mosaic virus (CMV) were inoculated to leaves of Chenopodium amaranticolor Costs and Reyn in all combinations. The data presented demonstrate that whereas RNA 3 is readily interchangeable between the two viruses to produce infections resulting in lesion development, RNAs 1 and 2 are not, although they are interchangeable between the three CMV strains. The possible significance of these observations to virus taxonomy is discussed.     

Alterations in the protein synthetic apparatus of cells infected with herpes simplex virus.

The effect of infection with herpes simplex virus (HSV) on the rate of protein synthesis, the distribution of ribosomes, and the time required to complete nascent peptides (transit time) in either actively growing or stationary phase Vero cells was examined. It was shown that the rate of protein synthesis per polysomal ribosome decreased continuously throughout the early portion of the infection cycle. This occurred despite an increase in the accumulation of ribosomes in polysome-like structures that accompanies the onset of synthesis of HSV-specified proteins. Transit-time measurements demonstrated that the rate of polypeptide elongation was not altered after infection. These data are discussed in light of a model that suggests that polysome-like structures are present but not functioning (or functioning very poorly) in HSV infected-cells.     

Biochemical transformation of LM(TK-) cells by hybrid plasmids containing the coding region of the herpes simplex virus type 1 thymidine kinase gene

Recombinant plasmid pAGO codes for herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) and consists of a 2-kbp HSV-1 DNA fragment inserted at the unique PvuII cleavage site of plasmid pBR322. A hybrid plasmid, designated pMH110, has been derived from plasmid pAGO by deleting the 1689-bp pBR322 nucleotide sequence of pAGO, which extends from the BamHI to the PvuII cleavage site, and the 250-bp HSV-1 nucleotide sequence of pAGO, which extends from the PvuII to the BglII cleavage site. Plasmid pMH110 biochemically transformed LM(TK^?)cells to the TK⁺ phenotype. The biochemically transformed cell lines had the following properties: (i) they were resistant to the growth-inhibiting effects of 1 mM thymidine; and (ii) they expressed an HSV-1-specific TK activity. This HSV-1 TK activity was purified after labeling biochemically transformed cell lines [LM(TK^?)/TF pMH110 E2 and LM(TK^?)/TF pMH110 Hc2] with [³⁵S]methionine. Immunoprecipitation experiments revealed that the TK polypeptides made in the biochemically transformed cells had molecular weights of about 39,000 to 40,000, which are about the same as the molecular weights of the TK polypeptides previously purified from HSV-1-infected LM(TK^?) cells and other biochemically transformed cell lines. The experiments support the hypothesis that the functional coding region of the HSV-1 TK gene is 3′ to the BglII cleavage site, and they also suggest that the HSV-1 TK messenger RNA may have been initiated in cells transformed by HincII- and EcoRI-cleaved pMH110 DNA at a site in cellular (or plasmid) DNA upstream from the HSV-1 DNA BglII cleavage site.     

Proteins of murine cytomegalovirus: identification of structural and nonstructural antigens in infected cells.

The synthesis of viral proteins in tertiary mouse embryo cells infected with murine cytomegalovirus (MCMV) has been studied by polyacrylamide gel electrophoresis. Three immediate-early proteins were detected by 4 hr postinfection (p.i.), but between 4 hr and the onset of viral DNA synthesis at 8–10 hr, no further viral proteins could be distinguished. The major structural proteins appeared after viral DNA synthesis had commenced and continued to be made until 36 hr p.i. or later. Host translation occurred until 24 hr p.i. but was inhibited between then and 30 hr p.i. at which time viral protein synthesis predominated. At this time 52 proteins could be labeled in infected cells and, of these, 30 could be precipitated with viral-specific antisera. The proteins precipitated included 22 with electrophoretic mobilities similar to structural viral proteins and a further 8 which were not present in purified MCMV. These, together with the three immediate-early proteins, were the only nonstructural proteins which were detected in the infected cell.     

Receptors for encephalomyocarditis virus on murine and human cells

The attachment kinetics of radiolabeled encephalomyocarditis virus were studied using established murine and human cell lines and murine and human erythrocytes. Unlabeled virus completely blocked the binding of labeled virus and virus subjected to pH 6.0 treatment bound at only one-tenth the rate of native virus. The initial rate constant at 0° calculated for human erythrocytes was 3.2 × 10^?10 cm³ min^?1 cell^?1. The rate constants for Friend leukemia cells and HeLa cells were, respectively, 200 and 2000 times faster. In contrast, no binding was observed to murine erythrocytes. The attachment of the virus to HeLa cells was found to be temperature independent over the range 0 to 40°. The attachment to murine cells (L-929 and Friend leukemia), however, was progressively reduced with increasing temperatures (0 to 40°) to about 0.01 of the rate at 0°. The decrease in binding at temperatures greater than 0° appears to be due to an increased rate of dissociation of the virus. It is proposed that both receptor number and viral affinity can influence pathogenicity.     

Studies on interferon priming: cellular response to viral and nonviral inducers and requirement of protein synthesis

Preexposure of L cells to mouse interferon (priming) and subsequent induction by Newcastle disease virus (NDV) resulted in earlier emergence of interferon and its mRNA by 3–4 hr, and a reduction of the final yield of secreted interferon. The effect of priming was most prominent in L cells stimulated by the addition of poly(I)-poly(C). Only cells that had been primed produced interferon and its mRNA in detectable amounts. When protein synthesis was blocked by the addition of cycloheximide, interferon mRNA was synthesized at the usual time and tended to accumulate in the cytoplasm, providing an estimate of the level of the primed state. By using this system, we were able to show that interferon priming was dependent on cellular protein synthesis.     

Enhanced poliovirus replication in cytomegalovirus-infected human fibroblasts.

Replication of poliovirus in human cytomegalovirus (CMV)-infected cells is enhanced 5-to 10-fold over replication in uninfected cells. Enhanced poliovirus replication in dually infected cells was not due to a difference in adsorption on infected cells and was supported by evidence of increased synthesis of polio-specific RNA. A functional CMV genome appeared to be required for the enhancement of polio replication since enhanced replication was not seen in cells infected with uv-irradiated CMV or in cultures treated with the inhibitors of CMV replication. Enhanced polio replication in CMV-infected cells may be due to the enhanced cellular metabolism in these cells.