Sequence relationships between adenovirus 2 early RNA and viral RNA size classes synthesized at 18 hours after infection.

Synthesis of cytoplasmic viral RNA was studied during infection of cultured human (KB) cells with adenovirus 2. At 6 h, before viral DNA synthesis began 5% of the poly(A)-containing RNA hybridized to viral DNA; by 12 h and at later times more than 80% was virus specified. At 18 h after infection, four major size classes of cytoplasmic viral RNA were identified among the poly(A)-containing molecules. These size classes migrated as 27S, 24S, 19S, and 12 to 15S in polyacrylamide gels. The three larger size classes could also be identified in denaturing formamide gels. Hybridization of the 27S, 24S, and 19S viral RNAs was not inhibited by RNA harvested from cells at early times in infection. Therefore, these three major RNAs must code for late viral proteins. Hybridization of the 12 to 15S RNA was partially inhibited by RNA from cultures harvested at early times, suggesting that in this size class some of the RNA labeled at 18 h codes for early viral proteins.     

Epstein-barr virus-specific RNA. II. Analysis of polyadenylated viral RNA in restringent, abortive, and prooductive infections.

The complexity and abundance of Epstein-Barr (EBV)-specific RNA in cell cultures restringently, abortively, and productively infected with EBV has been analyed by hybridization of the infected cell RNA with purified viral DNA. The data indicate the following. (i) Cultures containing productively infected cells contain viral RNA encoded by at least 45% of EBV DNA, and almost all of the species of viral RNA are present in the polyadenylated and polyribosomal RNA fractions. (ii) Restringently infected Namalwa and Raji cultures, which contain only intranuclear antigen, EBNA, and enhanced capacity for growth in vitro, contain EBV RNA encoded by at least 16 and 30% of the EBV DNA, respectively. The polyadenylated and polyribosomal RNA fractions of Raji and Namalwa cells are enriched for a class of EBV RNA encoded by approximately 5% of EBV DNA. The same EBV DNA sequences encode the polyadenylated and polyribosomal RNA of both Raji and Namalwa cells. (iii) After superinfection of Raji cultures with EBV (HR-1), the abortively infected cells contain RNA encoded by at least 41% of EBV DNA. The polyadenylated RNA of superinfected Raji cells is enriched for a class of EBV RNA encoded by approximately 20% of EBV HR-1 DNA. Summation hybridization experiments suggest that the polyadenylated RNA in superinfected Raji cells is encoded by the same DNA sequences as encode RNA present in Raji cells before superinfection, most of which is not polyadenylated. That the same EBV RNA sequences are present in the polyadenylated and polyribosomal fractions of two independently derived, restringently infected cell lines suggests that these RNAs may specify functions related to maintenance of the transformed state. The complexity of this class of RNA is adequate to specify a sequence of a least 5,000 amino acids. That only some RNA species are polyadenylated in restringent and abortive infection suggests that polyadenylation or whatever determines polyadenylation may play a role in the restricted expression of the EVB genome.     

Synthesis of human adenovirus early RNA species is similar in productive and abortive infections of monkey and human cells.

Northern (RNA) blot analysis has been used to show that synthesis of early mRNA species is similar in monkey cells productively or abortively infected with human adenovirus. mRNA species from all five major early regions (1A, 1B, 2, 3, 4) are identical in size and comparable in abundance whether isolated from monkey cells infected with adenovirus type 2 or with the host range mutant Ad2hr400 or coinfected with adenovirus type 2 plus simian virus 40. The mRNA species isolated from monkey cells are identical in size to those isolated from human cells. Production of virus-associated RNA is also identical in productive and abortive infections of monkey cells. Synthesis of virus-associated RNA is, however, significantly greater in HeLa cells than in CV1 cells at late times after infection regardless of which virus is used in the infection.     

Two Classes of Cytoplasmic Viral RNA Synthesized Early in Productive Infection with Adenovirus 2

The RNA sequences and RNA size classes transcribed early in productive infection with adenovirus 2 were analyzed by RNA-DNA hybridization. Two independent procedures demonstrated that early cytoplasmic viral RNA is composed of two sequence classes, class I which is absent or present in greatly reduced quantities at 18 h, and class II which persists throughout the infection. When the sequences in early viral RNA were analyzed by hybridization-inhibition studies, the hybridization of early [(3)H]RNA was inhibited only 50% by RNA from cultures harvested late (18 h) in infection. Liquid hybridizations with radioactive viral DNA confirmed that early RNA includes two classes. Duplex formation of RNA with (32)P-labeled viral DNA was assayed by hydroxylapatite chromatography and resistance to S(1) nuclease digestion. Both methods showed that the cytoplasmic RNA present early in infection annealed 12 to 15% of the viral DNA; late cytoplasmic RNA hybridized 21 to 25% of the DNA. Mixtures of early plus late cytoplasmic RNAs hybridized 30 to 34% of the viral DNA, demonstrating the reduced concentration of early class I RNA in the late RNA preparations. Experiments were performed to correlate class I and class II early RNA with size-fractionated cytoplasmic RNA synthesized early in infection. Fractionation of RNA by gel electrophoresis or sucrose gradient centrifugation confirmed three major size classes, 12 to 15S, 19 to 20S, and 26S. Total cytoplasmic RNA and RNA selected on the basis of poly(A) content contained the same size classes of viral RNA. In standard electrophoresis conditions, the 19 to 20S viral RNA could be resolved into two size classes, and the distribution of 12 to 15S RNA also indicated the presence of more than one size component. Hybridization-inhibition studies under nonsaturating conditions were performed with 26S, 19 to 20S, and 12 to 15S viral RNAs fractionated by gel electrophoresis. Late RNA inhibited the hybridization of 26S RNA only 20%, 19 to 20S RNA was inhibited 45%, and 12 to 15S RNA was inhibited 50%. When 18 to 19S and 12 to 15S viral RNAs purified by sucrose gradient centrifugation were similarly analyzed, late RNA inhibited hybridization of 18 to 19S RNA 50%, and the annealing of 12 to 15S RNA was inhibited 70%.     

Transcription and Transport of Virus-Specific Ribonucleic Acids in African Green Monkey Kidney Cells Abortively Infected with Type 2 Adenovirus

The techniques of deoxyribonucleic acid-ribonucleic acid (DNA-RNA) hybridization and immunological precipitation were used to compare the synthesis of adenovirus-specific macromolecules in African green monkey kidney (AGMK) cells infected with adenovirus, an abortive infection, and coinfected with both adenovirus and simian virus 40 (SV40), which renders the cells permissive for adenovirus replication. When viral protein synthesis was proceeding at its maximum rate, the incorporation of (14)C-amino acids into adenovirus structural proteins was about 90 times greater in the doubly infected cells than in cells infected only with adenovirus. However, the rates of synthesis of virus-specific ribonucleic acid appeared to be comparable in the two infections at all times measured. A time-dependent increase in the rate of RNA synthesis observed late in the abortive infection was dependent upon the prior replication of viral DNA. Moreover, all virus-specific RNA species that are normally made late in a productive adenovirus infection (i.e., the true late and class II early RNA species) were also detected in the abortive infection. Adenovirus-specific RNA was detected by molecular hybridization in both the cytoplasm and nuclei of abortively infected cells. Comparable amounts of viral RNA were found in the cytoplasmic fractions of AGMK cells infected either with adenovirus or with both adenovirus and SV40. The results of hybridization-inhibition experiments clearly showed that there was a class of virus-specific RNA molecules, representing about 30% of the total, in the nucleus that was not transported to the cytoplasm. This class of RNA was also identified in similar amounts in productively infected human KB cells. The difference in the abilities of cytoplasmic and nuclear RNA to inhibit the hybridization of virus-specific RNA from whole cells was shown not to be due to a difference in the molecular size of the RNA species from the two cell fractions or to the specific loss of a cytoplasmic species during RNA extraction procedures.     

Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis.

The temporal sequence of coronavirus plus-strand and minus-strand RNA synthesis was determined in 17CL1 cells infected with the A59 strain of mouse hepatitis virus (MHV). MHV-induced fusion was prevented by keeping the pH of the medium below pH 6.8. This had no effect on the MHV replication cycle, but gave 5- to 10-fold-greater titers of infectious virus and delayed the detachment of cells from the monolayer which permitted viral RNA synthesis to be studied conveniently until at least 10 h postinfection. Seven species of poly(A)-containing viral RNAs were synthesized at early and late times after infection, in nonequal but constant ratios. MHV minus-strand RNA synthesis was first detected at about 3 h after infection and was found exclusively in the viral replicative intermediates and was not detected in 60S single-stranded form in infected cells. Early in the replication cycle, from 45 to 65% of the [3H]uridine pulse-labeled RF core of purified MHV replicative intermediates was in minus-strand RNA. The rate of minus-strand synthesis peaked at 5 to 6 h postinfection and then declined to about 20% of the maximum rate. The addition of cycloheximide before 3 h postinfection prevented viral RNA synthesis, whereas the addition of cycloheximide after viral RNA synthesis had begun resulted in the inhibition of viral RNA synthesis. The synthesis of both genome and subgenomic mRNAs and of viral minus strands required continued protein synthesis, and minus-strand RNA synthesis was three- to fourfold more sensitive to inhibition by cycloheximide than was plus-strand synthesis.