Poliovirus snapback double-stranded RNA isolated from infected HeLa cells is deficient in poly(A).

A portion of poliovirus double-stranded RNA (25 to 50%) isolated from infected HeLa cells contains hairpin loops at one end of the duplex structure. These structures rapidly reformed double-stranded molecules after denaturation and appeared as molecules of up to two times genome length upon electrophoresis in denaturing agarose gels. A second form of poliovirus double-stranded RNA was readily denaturable into genome length strands. When the hairpin RNA was treated with S1 nuclease, subsequent denaturation resulted in formation of strands of up to genome length. Hairpin molecules contained very little, if any, poly(A) sequences, suggesting that the hairpin forms after nucleolytic removal of the 3' end of plus-strand templates. We conclude that the hairpin double-stranded RNA found in infected cells is likely generated by intracellular nicking and self-priming and that it does not represent an intermediate in the process of RNA replication.     

Quantitation of bovine papilloma viral DNA in viral-induced tumors.

Bovine papilloma virus (BPV) DNA was labeled in vitro under conditions of repair synthesis and subsequently used as a "probe" in DNA-DNA reassociation studies to detect BPV-specific DNA sequences in a viral-induced calf meningioma and hamster fibroma. In vitro labeled BPV DNA had denaturation characteristics expected for duplex DNA and denatured DNA reassociated with apparent second-order kinetics. Analysis of in vitro labeled BPV DNA reassociation rates in the presence of excess tumor DNA revealed that the calf meningioma contained approximately 700 to 800 BPV genome equivalents per diploid cell whereas the hamster fibroma contained about 150 incomplete BPV genome equivalents per diploid cell. Thermal denaturation of in vitro labeled BPV DNA which reassociated in the presence of the two tumor DNA preparations indicated less than 1.5% base pair mismatching.     

RNA of simian sarcoma-associated virus type 1 produced in human tumor cells.

Simian sarcoma-associated virus type 1 propagated in human rhabdomyosarcoma cells exhibited characteristics typical of oncornaviruses but seemed to have several aberrant properties. It had a buoyant density of 1.14 g/cm3, had RNA-dependent DNA polymerase activity, seemed to be labile to high salt concentrations, and contained little 50 to 60S RNA but relatively large amounts of human ribosomal RNA. In addition to 50 to 60S RNA, purified virions contained smaller RNA molecules with sedimentation coefficients of 28 to 30S, 18 TO 20S, and 4 to 10S. Unlike the 50 to 60S RNA species, the smaller virion-associated RNAs lacked polyadenylic acid, and the 28 to 30S RNA had an average base composition similar to that of human ribosomal RNA. Upon heat denaturation, the native 50 to 60S RNA genome yielded polyadenylic acid-containing 28 to 30S subunits that degraded in to 18 to 20S molecules upon further heat treatment. The 50 to 60S viral RNA had a guanine plus cytosine content of 56%.     

Hybridization Selection and In Vitro Translation of Autographa californica Nuclear Polyhedrosis Virus mRNA

We isolated polyadenylated RNA from the cytoplasm of cells infected with Autographa californica nuclear polyhedrosis virus late after infection (21 h postinfection). At that time intracellular protein synthesis was directed almost exclusively toward infected cell-specific proteins. The polyadenylic acid-containing RNA sequences in the cytoplasm at 21 h postinfection were radiolabeled in vitro and hybridized to A. californica nuclear polyhedrosis virus DNA restriction fragments. The polyadenylic acid-containing RNA was derived from regions representing the entire viral genome. Translation in a reticulocyte cell-free protein-synthesizing system of cytoplasmic RNA selected by hybridization to viral DNA and polyadenylic acid-containing RNA produced almost identical polypeptide patterns, suggesting that late after infection almost all of the cytoplasmic polyadenylic acid-containing RNA present in infected cells was of viral origin. Polyhedrin protein (molecular weight, 33,000) and a number of virion structural proteins were among the translation products which were identified by immunoprecipitation and by comparing molecular weights. In addition, some tentative nonstructural infected cell-specific proteins were also detected. Using the hybridization selection technique, we determined that sequences complementary to the message coding for polyhedrin were located on EcoRI fragment I of A. californica nuclear polyhedrosis virus DNA, whereas sequences coding for a putative nonstructural protein (molecular weight, 39,000) were on EcoRI fragment J.     

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.     

Hybridization selection and cell-free translation of mRNA''s encoded within the inverted terminal repetition of the vaccinia virus genome

Early polypeptides encoded within the 10,000-base pair terminally repeated region of the vaccinia virus genome were mapped by cell-free translation of mRNA that was selected by hybridization to restriction fragments and to separated strands of a recombinant lambda phage. The results, which were confirmed by hybrid arrest of translation, indicated that polypeptides of 7,500 (7.5K), 19,000 (19K), and 42,000 (42K) daltons mapped at approximately 3.2 to 4.3, 6.5 to 7.2, and 7.2 to 8.3 kilobase pairs from the end of the genome, respectively. mRNA's for the 42K and 7.5K polypeptides were transcribed towards the end of the genome, whereas mRNA for the 19K polypeptide was transcribed in the opposite direction. Including polyadenylic acid tails, the lengths of the mRNA's for the 7.5K, 19K, and 42K polypeptides, determined by gel electrophoresis of denatured RNA, hybridization selection, and cell-free translation, were approximately 1,200, 680, and 1,280 nucleotides, respectively. mRNA's for the 42K and 19K polypeptides were only about 100 nucleotides longer than the minimums required to code for their respective polypeptides, whereas mRNA for the 7.5K polypeptide contained 900 nucleotides of untranslated sequence. This long untranslated portion of the latter mRNA was probably located near the 3' end, because this gene was only inactivated by high doses of UV irradiation. This small target size also excluded certain models for RNA processing involving formation of the mRNA's for the 42K and 7.5K polypeptides from a common promoter. Rabbitpox virus, which has an inverted terminal repetition approximately half that of vaccinia virus, was also shown to encode mRNA's that hybridized to the cloned terminal segment of vaccinia virus DNA.     

Molecular cloning of the terminal hairpin of vaccinia virus DNA as an imperfect palindrome in an Escherichia coli plasmid

The genome of vaccinia virus is a linear duplex molecule of approximately 185 kb with hairpins at each end that link the complementary strands. The hairpins, which exist in two forms that are inverted and complementary in sequence, were isolated as XbaI restriction fragments and converted to a linear intermolecular duplex structure by denaturation and reannealing. The latter was then stably cloned as a 142-bp imperfect palindrome in an Escherichia coli plasmid. The insert was excised from the plasmid and the palindrome was extended on both sides by ligating it to the adjacent vaccinia virus DNA segment. The resulting fragment was cloned as a 278-bp imperfect palindrome. Restriction endonuclease analysis and DNA sequencing indicated the absence of any deletions or rearrangements. After excision from the plasmid, the palindrome was converted by heating and rapid cooling to the original two hairpin forms. In this manner, large quantities of vaccinia virus telomeres may be obtained for physical and biochemical studies.     

Mechanism of Synthesis of Vaccinia Virus Double-Stranded Ribonucleic Acid In Vivo and In Vitro

The synthesis of vaccinia virus double-stranded ribonucleic acid (RNA) in infected HeLa cells was sensitive to actinomycin D, suggesting that a deoxyribonucleic acid dependent reaction is involved. Some double-stranded RNA was made in the presence of cytosine arabinoside in infected cells. Double-stranded and complementary RNA were synthesized in vitro by using vaccinia cores. These two observations indicate that some of the double-stranded RNA is read from "early" genes. The double-stranded RNA synthesized in vitro had the same properties as that made in vivo. At least 70% of the double-stranded RNA made in vivo was in ribonuclease-resistant form prior to sodium dodecyl sulfate-phenol extraction. In addition, there was a complementary RNA in infected cells which could be converted to double-stranded RNA by annealing.     

Characterization of RNA from equine infectious anemia virus.

The genome of equine infectious anemia virus, a nononcogenic retrovirus, has been characterized by velocity sedimentation, electrophoresis in polyacrylamide gels, buoyant density in CS2SO4, and susceptibility to nuclease digestion. The nucleic acid of purified virus was resolved by sedimentation analysis into a fast-sedimenting genome component, which comprises about two-thirds of the virion RNA, and a slow-sedimenting RNA, which is probably comprised of host-derived tRNA and a trace amount of 5S RNA. The fast-sedimenting RNA had a sedimentation coefficient of 62S and a molecular weight of 5.4 X 10(6) to 5.6 X 10(6), as determined by sedimentation velocity and electrophoretic mobility. Upon heat denaturation, [3H]uridine-labeled 62S RNA dissociated into material comprised of 90 to 95% single-stranded species, sedimenting predominantly at 34S, with a molecular weight of 2.7 X 10(6) to 2.9 X 10(6) and 5 to 10% 4S RNA. The 62S RNA was predominantly single-stranded but contained double-stranded regions, as indicated by partial resistance to RNase IA and SI nuclease and by a lower buoyant density in CS2SO4 than that of the single-stranded 34S RNA derived by heat denaturation. These data indicated that the viral genome consisted of two 34S subunits of single-stranded RNA held in a high-molecular-weight complex with 4S RNA by a mechanism involving a small degree of base pairing. Thus, the structure of equine infectious anemia virus RNA is similar to that of other retroviruses.     

Homologous terminal sequences of the genome double-stranded RNAs of bluetongue virus.

The 3'-terminal sequences of the 10 double-stranded RNA genome segments of bluetongue virus (serotypes 10 and 11) were determined. The double-stranded RNAs were 3' labeled with [5'-32P]pCp and resolved into 10 segments by electrophoresis. After denaturation, the two complementary strands of segments 4 through 10 were resolved into fast- and slow-migrating species by polyacrylamide gel electrophoresis, and their 3' end sequences were determined. Complete RNase T1 digestion of the individual 3'-labeled double-stranded RNA segments yielded two labeled oligonucleotides, one of which migrated faster than the other on 20% polyacrylamide-7 M urea gels. Sequence analyses of the two oligonucleotides of segments 4 through 10 confirmed the corresponding RNA sequence data. For RNA segments 1 through 3 the oligonucleotide analyses gave comparable results. The 3'-terminal sequences of the fast-migrating RNA species were HOCAAUUU. . . ; those of the slow-migrating RNA species were HOCAUUCACA. . . . Similar results were obtained for double-stranded RNA from bluetongue virus serotypes 10 and 11. Beyond the common termini, the sequences for each segment varied considerably.     

DNA-dependent RNA polymerase subunits encoded within the vaccinia virus genome.

Antiserum to a multisubunit DNA-dependent RNA polymerase from vaccinia virions was prepared to carry out genetic studies. This antiserum selectively inhibited the activity of the viral polymerase but had no effect on calf thymus RNA polymerase II. The specificity of the antiserum was further demonstrated by immunoprecipitation of RNA polymerase subunits from dissociated virus particles. The presence in vaccinia virus-infected cells of mRNA that encodes the polymerase subunits was determined by in vitro translation. Immunoprecipitable polypeptides with Mrs of about 135,000, 128,000, 36,000, 34,000, 31,000, 23,000, 21,000, 20,000, and 17,000 were made when early mRNA was added to reticulocyte extracts. The subunits were encoded within the vaccinia virus genome, as demonstrated by translation of early mRNA that hybridized to vaccinia virus DNA. The locations of the subunit genes were determined initially by hybridization of RNA to a series of overlapping 40-kilobase-pair DNA fragments that were cloned in a cosmid vector. Further mapping was achieved with cloned HindIII restriction fragments. Results of these studies indicated that RNA polymerase subunit genes are transcribed early in infection and are distributed within the highly conserved central portion of the poxvirus genome in HindIII fragments E, J, H, D, and A.