Sedimentation characteristics of virion RNA of Machupo virus reproducing in the presence of actinomycin D

Actinomycin D treatment (0.005-05 g/ml) of Vero and BHK-21 cells infected with Machupo virus suppressed the synthesis of ribosomal RNAs but did not affect the production of infectious Machupo virus. Virion RNAs contained 3 high molecular weight RNA species: 28-31 S, 22-24 S and 18 S. In the presence of actinomycin D [3H]-uridine incorporated only in 30-31 S and 22-24 S RNA species. The data are supported by previous results which show that Machupo virus genome contains two RNA species: "large" (30-31 S) and "small" (22-24 S).     

Heterogeneity of the 3'' end of minus-strand RNA in the poliovirus replicative form.

The 3' terminus of the strand (minus strand) complementary to poliovirion RNA (plus strand) has been examined to see whether this sequence extends to the 5'-nucleotide terminus of the plus strand, or whether minus-strand synthesis terminates prematurely, perhaps due to the presence of a nonreplicated nucleotide primer for initiation of plus-strand synthesis. The 3' terminus was labeled with 32P using [5'-32P]pCp and RNA ligase, and complete RNase digests were performed with RNases A, T1, and U2. 32P-oligonucleotides were analyzed for size by polyacrylamide-urea gel electrophoresis. The major oligonucleotide products formed were consistent with the minus strand containing 3' ends complementary and flush with the 5' end of the plus strand. However, a variable proportion of the isolated minus strands from different preparations were heterogeneous in length and appeared to differ from each other by the presence of one, two, or three 3'-terminal A residues.     

Sequence-specific inhibition of RNA elongation by actinomycin D

We have developed a method to localize specific sites where RNA elongation is arrested due to DNA-bound ligands. The method was used to determine apparent binding sites for actinomycin D. We have found 14 strong RNA hindrance sites along nucleotide sequence of T7 and D111 T7 DNA of 380 nucleotides full length under low actinomycin D concentration conditions. Nucleotide sequence of all the sites is described by general formula XGCY where X ‡ G and Y ‡ C.     

Replication of mouse hepatitis virus: negative-stranded RNA and replicative form RNA are of genome length

There are seven virus-specific mRNA species in mouse hepatitis virus-infected cells (Lai et al., J. Virol. 39:823-834, 1981). In this study, we examined virus-specific negative-stranded RNA to determine whether there are corresponding multiple negative-stranded RNAs. Intracellular RNA from mouse hepatitis virus-infected cells was separated by agarose gel electrophoresis, transferred to nitrocellulose membranes, and hybridized to positive-stranded genomic 60S [32P]RNA. Only a single RNA species of genomic size was detected under these conditions. This RNA was negative stranded. No negative-stranded subgenomic RNA was detected. We also studied double-stranded replicative-form RNA in the infected cells. Only one replicative-form of genomic size was detected. When the double-stranded RNA isolated without RNase treatment was analyzed, again only one RNA species of genomic size was detectable. Furthermore, most of the virus-specific mRNAs could be released from this RNA species upon heating. These results suggest that all of the mouse hepatitis virus-specific RNAs are transcribed from a single species of negative-stranded RNA template of genomic size.     

Stimulated growth of respiratory syncytial virus in the presence of actinomycin D

The growth of respiratory syncytial virus in HeLa cells, during the first day, was stimulated tenfold in the presence of 0.05 to 0.5 μg/ml of actinomycin D despite visible cytotoxicity of the drug over this range of concentrations. However, the ultimate virus yield, after three days, was suppressed. It is concluded that, as with other pseudo- and paramyxoviruses, the growth of respiratory syncytial virus is not dependent on the production of a new cell-coded protein.     

Inhibition of measles virus replication and RNA synthesis by actinomycin D

Measles virus replication and RNA synthesis in Vero cells are inhibited by actinomycin at concentrations which inhibit cellular RNA synthesis. Drug present from the 2nd to the 24th hr post infection inhibited infectivity but not hemagglutinating activity or cell fusion. Infectivity was much less sensitive to drug added during the second 24-hr period, and 52S RNA was labeled and incorporated into virions during this later time interval.     

Temperature-dependent actinomycin D effect on RNA synthesis during synchronous development in Neurospora crassa

Actinomycin D at a concentration of 5 g/ml of medium inhibited DNA-dependent RNA synthesis by 92% at 35 C, 42% at 30 C, and 28% at 25 C in Neurospora crassa. This concentration also inhibited the development of conidiophores and conidia at 35 C, but not at 30 or 25 C. Mycelia which were induced to synchronous development formed conidiophores in 2.5 hr and conidia in 4.5 hr at 35 C in the absence of drug additives. Addition of actinomycin D to synchronously developing mycelia at zero time and at 0.5-hr intervals thereafter at 35 C indicated that RNA synthesis required for conidiophores occurred before 0.5 hr and for conidia before 2 hr. Addition of cycloheximide at the same times to another synchronous mycelial series at 35 C indicated that protein synthesis required for conidiophores occurred before 2 hr and for conidia before 3.5–4 hr.This work was supported in part by U.S. Public Health Service Training Grant 1 TO1 GM 01968 01.     

Synthesis of ribonucleic acid in baby-hamster kidney cells in the presence of actinomycin D

1. RNA molecules with sedimentation values in sucrose gradients of 12–20s are synthesized in baby-hamster kidney cells even after prolonged incubation in medium containing 1μg. of actinomycin D/ml. 2. The rate of formation of this RNA is dependent on the age of the cultures and is greatest during the exponential phase of growth. 3. Growth of cells on nutritionally poor medium causes degradation and inhibits the synthesis of these RNA fractions. 4. Replacement of the nutritionally poor medium with a rich medium stimulates the synthesis of actinomycin-resistant RNA. This stimulation is blocked by cycloheximide. 5. The base composition of this RNA is characterized by low cytidine and high guanosine values.