Transfer Ribonucleic Acid in KB Cells Infected with Adenovirus Type 2

Populations of transfer ribonucleic acid (tRNA) extracted from control and type 2 adenovirus (Ad2)-infected KB cells were compared. No consistent differences in acceptor activity for 11 amino acids were observed. Comparison of methylated albumin-kieselguhr (MAK) elution profiles of arginyl-tRNA from control and infected cells revealed a minor modification in that the proportion of arginyl-tRNA eluting at high salt concentration was somewhat greater in infected cells. No similar differences were observed in MAK elution profiles of aspartyl-, isoleucyl-, leucyl-, phenylalanyl-, seryl-, tyrosyl-, and valyl-tRNA. Hybridization of 4S RNA from infected cells labeled by incorporation of ³H-uridine with Ad2 deoxyribonucleic acid revealed the presence of a complementary species of RNA in this preparation. Hybridization of ³H-arginyl-tRNA and of ³H-aminoacyl-tRNA labeled by charging with ³H-arginine or a ³H-mixture of amino acids, respectively, failed to detect the presence of virus-specific tRNA in Ad2-infected cells.     

Virus-specific Ribonucleic Acid Synthesis in KB Cells Infected with Herpes Simplex Virus

The production of virus-specific ribonucleic acid (RNA) was investigated in KB cells infected with herpes simplex virus. A fraction of RNA annealable to virus deoxyribonucleic acid (DNA) was found in these cells as early as 3 hr after virus inoculation. Production of this species of RNA increased up to 6 or 7 hr after infection, at which time elaboration of virus messenger RNA (mRNA) declined. At 24 hr after infection, the rate of incorporation of uridine into this RNA was approximately one-half of the rate present at 6 hr after inoculation. Nucleotide analysis of the RNA annealable to virus DNA was compatible with that expected for virus mRNA. Centrifugation showed considerable spread in the size of the virus-induced nucleic acid, the bulk of this RNA sedimenting between 12 and 32S. Incorporation of uridine into cell mRNA, ribosomal precursor RNA, and soluble RNA was suppressed rapidly after infection. As is the case with most other cytocidal viruses investigated to date, virus-induced suppression of cell RNA synthesis appears to be a primary mechanism of cell injury.     

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.     

Virus-Specific Ribonucleic Acid in the Nucleus and Cytoplasm of Rat Embryo Cells Transformed by Adenovirus Type 2

Nuclei were isolated from rat embryo cells transformed by adenovirus type 2. Nuclear and cytoplasmic virus-specific ribonucleic acids (RNA) were characterized and quantitated by deoxyribonucleic acid (DNA)-RNA hybrid formation with adenovirus DNA. The results indicate that most, if not all, virus-specific RNA molecules are synthesized in the cell nucleus and subsequently transported into cytoplasm where they degrade with a half-life of 1 to 2 hr. No difference in base sequences between nuclear and cytoplasmic virus-specific RNA species can be detected by hybridization competition experiment with viral DNA.     

Ribonucleic Acid Synthesis in Cells Infected with Herpes Simplex Virus: I. Patterns of Ribonucleic Acid Synthesis in Productively Infected Cells

HEp-2 cells were pulse-labeled at different times after infection with herpes simplex virus, and nuclear ribonucleic acid (RNA) and cytoplasmic RNA were examined. The data showed the following: (i) Analysis by acrylamide gel electrophoresis of cytoplasmic RNA of cells infected at high multiplicities [80 to 200 plaque-forming units (PFU)/cell] revealed that ribosomal RNA (rRNA) synthesis falls to less than 10% of control (uninfected cell) values by 5 hr after infection. The synthesis of 4S RNA also declined but not as rapidly, and at its lowest level it was still 20% of control values. At lower multiplicities (20 PFU), the rate of inhibition was slower than at high multiplicities. However, at all multiplicities the rates of inhibition of 18S and 28S rRNA remained identical and higher than that of 4S RNA. (ii) Analysis of nuclear RNA of cells infected at high multiplicities by sucrose density gradient centrifugation showed that the synthesis and methylation of 45S rRNA precursor continued at a reduced but significant rate (ca. 30% of control values) at times after infection when no radioactive uridine was incorporated or could be chased into 28S and 18S rRNA. This indicates that the inhibition of rRNA synthesis after herpesvirus infection is a result of two processes: a decrease in the rate of synthesis of 45S RNA and a decrease in the rate of processing of that 45S RNA that is synthesized. (iii) Hybridization of nuclear and cytoplasmic RNA of infected cells with herpesvirus DNA revealed that a significant proportion of the total viral RNA in the nucleus has a sedimentation coefficient of 50S or greater. The sedimentation coefficient of virus-specific RNA associated with cytoplasmic polyribosomes is smaller with a maximum at 16S to 20S, but there is some rapidly sedimenting RNA (> 28S) here too. (iv) Finally, there was leakage of low-molecular weight (4S) RNA from infected cells, the leakage being approximately three-fold that of uninfected cells by approximately 5 hr after infection.     

Ribonucleic Acid Synthesis in Cells Infected with Influenza Virus

Virus-specific ribonucleic acid (RNA), synthesized in influenza virus-infected cells from 3.5 to 7.5 hr after infection, was studied. After velocity centrifugation in sucrose, three peaks of virus-specific RNA could be identified: 34S, 18S, and 11S. These RNA species are predominantly single-stranded and consist of 90% viral (plus) and 10% complementary (minus) RNA strands. Most (75%) of the complementary RNA is single-stranded, i.e., not part of RNA duplexes or replicative intermediates. The 34S RNA species is an aggregate of 18S and 14S RNA species. Both 18S and 11S RNA species are relatively heterogenous compared to 18S ribosomal RNA, and these species probably contain different RNA molecules having closely related sedimentation coefficients.     

Reovirus-directed Ribonucleic Acid Synthesis in Infected L Cells

Reovirus replication in L-929 mouse fibroblasts was unaffected by 0.5 mug of actinomycin per ml, a concentration which inhibited cell ribonucleic acid (RNA) synthesis by more than 90%. Under these conditions of selective inhibition, the formation of both single-stranded and double-stranded virus-specific RNA was detected beginning at 6 hr after infection. The purified double-stranded RNA was similar in size and base composition to virus RNA and presumably was incorporated into mature virus. The single-stranded RNA formed ribonuclease-resistant duplexes when annealed with denatured virus RNA but did not self-anneal, thus indicating that it includes copies of only one strand of the duplex. The single-stranded RNA was polyribosome-associated and may function as the virus messenger RNA. Production of both types of virus-induced RNA required protein synthesis 6 to 9 hr after infection. At later times in the infectious cycle, only double-stranded RNA synthesis was dependent on continued protein formation.     

Quantitative Determination and Location of Newly Synthesized Virus-Specific Ribonucleic Acid in Chicken Cells Infected with Rous Sarcoma Virus

A sensitive and quantitative nucleic acid hybridization assay for the detection of radioactively labeled avian tumor virus-specific RNA in infected chicken cells has been developed. In our experiments we made use of the fact that DNA synthesized by virions of avian myeloblastosis virus in the presence of actinomycin D (AMV DNA) is complementary to at least 35% of the sequences of 70S RNA from the Schmidt-Ruppin strain (SRV) of Rous sarcoma virus. Annealing of radioactive RNA (either SRV RNA or RNA extensively purified from SRV-infected chicken cells) with AMV DNA followed by ribonuclease digestion and Sephadex chromatography yielded products which were characterized as avian tumor virus-specific RNA-DNA hybrids by hybridization competition with unlabeled 70S AMV RNA, equilibrium density-gradient centrifugation in Cs(2)SO(4) gradients, and by analysis of their ribonucleotide composition. The amount of viral RNA synthesized during pulse labeling with (3)H-uridine could be quantitated by the addition of an internal standard consisting of (32)P-labeled SRV RNA prior to purification and hybridization. This quantitative assay was used to determine that, in SRV-infected chicken cells labeled for increasing lengths of time with (3)H-uridine, labeled viral RNA appeared first in a nuclear fraction, then in a cytoplasmic fraction, and still later in mature virions. This observation is consistent with the hypothesis that RNA tumor virus RNA is synthesized in the nucleus of infected cells.     

Electron Microscopy of Cells Infected with Adenovirus Type 2

An electron microscope study of two strains of human adenovirus type 2 revealed the production of intranuclear paracrystalline formations by one of the strains. The crystals were composed of cylindrical tubules 25.0 nm in diameter arranged in a crystalline lattice with a periodicity of 75.0 nm. They appeared at 36 hr postinfection in nuclei which contained viral particles. Prolonged treatment with proteolytic enzyme only partially digested the crystals. The relationship of these crystals to similar nonviral crystals found in association with other virus infected-cells was discussed.     

Ribonucleic Acid Synthesis in Cells Infected with Herpes Simplex Virus VI. Polyadenylic Acid Sequences in Viral Messenger Ribonucleic Acid

Evidence is presented by use of radiolabeling and pancreatic and T1 ribonuclease digestion that some of the ribonucleic acid specified by herpes simplex virus contains polyadenylic acid sequences. The polyadenylic sequences are not transcribed from viral DNA.     

Synthesis of Ribonucleic Acid in Cells Infected with LSc Poliovirus at Elevated Temperatures

The synthesis of viral ribonucleic acid (RNA) was detected within 2 hr after infection with LSc poliovirus at 35 C. This RNA eluted as a single peak with 0.9 m NaCl on methylated albumin celite columns, was sensitive to ribonuclease, precipitated in the presence of 2 m LiCl, and had an S(20) value at 34 +/- 2 in linear sucrose gradients. When cells were infected at 39 to 40 C, there was also early synthesis of RNA. However, 2 hr after infection this synthesis was drastically inhibited. The absence of net RNA synthesis at 39 to 40 C during the late stages of infection was not caused by rapid degradation of newly formed RNA, since the RNA produced between 1 and 2 hr at 39 to 40 C was still present 3.5 hr after infection. There was a 3 log(10) inhibition in the production of infectious virus when p-fluorophenylalanine was present in the medium at a concentration of 25 mug/ml. This concentration of analogue had little effect upon the production of viral polymerase and viral RNA. Virus grown in the presence of analogue at a concentration of 10 mug/ml exhibited increased heat sensitivity compared to control virus. However, viral polymerase exhibited no change in sensitivity to heat or manganese when cells were exposed to 25 mug of p-fluorophenylalanine per ml during infection. p-Fluorophenylalanine had a relatively selective effect on viral capsid protein but did not reverse the inhibition of synthesis of viral RNA at 39 to 40 C.     

Intracellular Components Associated with Chikungunya Virus-Specific Ribonucleic Acids in Infected BHK21 Cells1

Cytoplasmic extracts of BHK21 cells infected with Chikungunya virus were analyzed by sucrose gradient sedimentation. Besides 140 S nucleocapsid, 65 S component associated with 26 S single-stranded ribonucleic acid was labeled with ³H-uridine in actinomycin-treated infected cells. The 65 S component, demonstrable in ethylenediaminetetraacetic acid-containing buffers, appeared to accumulate when protein synthesis was inhibited. After glutaraldehyde fixation, this component had a density of 1.48–1.50 g/ml in CsCl. Pulse-chase experiments did not indicate that the 65 S component was a precursor of the 140 S nucleocapsid.     

Deoxyribonucleic Acid Synthesis in Synchronized Mammalian KB Cells Infected with Herpes Simplex Virus

We examined the patterns of host cell and virus deoxyribonucleic acid (DNA) synthesis in synchronized cultures of KB cells infected at different stages of the cell cycle with herpes simplex virus (HSV). We found that the initiation of HSV DNA synthesis, we well as the production of new infectious virus, is independent of the S, G1, and G2 phases of the mitotic cycle of the host cell. This is in contrast to data previously found with equine abortion virus. Because HSV replicates independently of the cell cycle, we were able to establish conditions that would permit the study of rates of HSV DNA synthesized in logarithmically growing cells in the virtual absence of cellular DNA synthesis. This eliminates the need for separation of viral and cellular DNA by isopycnic centrifugation in CsCl. We found that HSV DNA synthesis was initiated between 2 to 3 hr after infection. The rate of DNA synthesis increased rapidly, reaching a maximum 4 hr after infection, and decreased to 50% of maximum by 8 hr. Evidence is also presented which suggests that HSV infection can inhibit both the ongoing synthesis of host DNA as well as the initiation of the S phase.     

Adenovirus Type 2 DNA Replicated in Arginine-Starved KB Cells Is Infectious

Adenovirus type 2 DNA from extracts of KB cells rendered nonpermissive by arginine starvation is infectious.     

Ribonucleic Acid Polymerase Induced in Cells Infected with Sendai Virus

A ribonucleic acid (RNA)-dependent RNA polymerase was induced in chick embryo fibroblast cells after infection with Sendai virus (parainfluenza 1 virus). The enzyme was associated with the microsomal fraction of infected cells and reached maximum detectable activity at 18 hr after virus infection. The activity of the enzyme in vitro was dependent on the presence of added magnesium ions and all four nucleoside triphosphates and was not inhibited by actinomycin D. The RNA synthesized by the enzyme in vitro was sensitive to ribonuclease and consisted of a complex mixture of RNA species including 34S, 24S, and 18S components. Similar RNA components were detected in the microsomal fraction of Sendai virus-infected cells by labeling with (3)H-uridine from 17 to 18 hr postinfection in the presence of actinomycin D. Of the RNA synthesized by Sendai virus-induced RNA polymerase in vitro, 98% became insensitive to ribonuclease after annealing with RNA extracted from purified Sendai virus particles.     

Transfection of KB Cells by Liposomes Containing Adenovirus Type 2 DNA

Adenovirus type 2 DNA was entrapped in liposomes which were then used to transfect KB cells with an efficiency of ~4,000 plaques per μg of encapsulated DNA.     

Ribonucleic Acid Transcriptases in Sendai Virions and Infected Cells

Sendai virions contain an enzyme which catalyzes the incorporation of ribonucleotides into ribonucleic acid (RNA). Enzyme activity was optimal at pH 8.0 and 28 C; otherwise conditions were similar to those reported for Newcastle disease virion (NDV) RNA polymerase. The initial rate of RNA synthesis by the Sendai virion enzyme was about 10 pmoles per mg of protein per hr, but after 3 hr of incubation the rate increased about fivefold. The virion enzyme was compared with an RNA polymerase in the microsomal fraction of infected cells. Both enzymes made predominantly single-stranded RNA which was complementary in base sequences to 50S virion RNA. Most of the RNA synthesized by the virion polymerase sedimented at 16S, but the product of the microsomal enzyme sedimented at about 8S.