Comparative electrophoresis of the 18-22S RNAs of Newcastle disease virus.

Between 80 and 90% of the 18-22S Newcastle disease virus intracellular RNA molecules contain poly(A) sequences. Electrophoresis of the 18S RNA in formamide-polyacrylamide gels resolves five species resolved by electrophoresis in aqueous gels. Thus, these five RNA species are probably unique size classes of RNA and not different conformations of the same RNAs. They are of sufficient size to code for the five smaller Newcastle disease virus proteins, and their combined molecular weights represent 60% of the viral genome-a value identical to that obtained by annealing 18-22S RNA with genome RNA. Formamide or heat treatment of the 22S RNA converts most of it into species with migration rates similar to those of the 18S species. Thus, the 22S RNA may not contain unique RNA species.     

Virus-specific RNA synthesis in the midgut of silkworm, Bombyx mori, infected with cytoplasmic-polyhedrosis virus

Virus-specific RNA synthesis in the midgut of silkworm infected with cytoplasmic-polyhedrosis virus was investigated under the condition inhibiting host RNA synthesis by actinomycin D injection. Two species of virus-induced RNA were formed; one was sensitive to ribonuclease (RNase) but the other was resistant. The resistant RNA had a sedimentation coefficient of 15 S and was considered as viral progeny with doublestranded RNA. The sensitive RNA, presumably single-stranded RNA, consisted of two classes with 15 S and 22 S sedimentation coefficients. Annealing the single-stranded RNA with heat-denatured CPV-RNA indicated that the single-stranded RNA was transcribed from viral genome RNA. The function of 22 S and 15 S single-stranded RNAs was discussed from the viewpoint of virus multiplication.     

Coronavirus multiplication strategy. II. Mapping the avian infectious bronchitis virus intracellular RNA species to the genome.

Avian infectious bronchitis virus, a coronavirus, directed the synthesis of six major single-stranded polyadenylated RNA species in infected chicken embryo kidney cells. These RNAs include the intracellular form of the genome (RNA F) and five smaller RNA species (RNAs A, B, C, D, and E). Species A, B, C, and D are subgenomic RNAs and together with the genome form a nested sequence set, with the sequences of each RNA contained within every larger RNA species (D. F. Stern and S. I. T. Kennedy, J. Virol 34:665-674, 1980). In the present paper we show by RNase T1 oligonucleotide fingerprinting that RNA E is also a member of the nested set. Partial alkaline fragmentation of the genome followed by sucrose fractionation, oligodeoxythymidylate-cellulose chromatography, and RNase T1 fingerprinting gave a partial 3'-to-5' oligonucleotide spot order. A comparison of the oligonucleotides of each of the five subgenomic RNAs with this spot order established that all of the RNAs are comprised of nucleotide sequences inward from the 3' end of the genome. This result is discussed in relation to the multiplication strategy both of coronaviruses and of other RNA-containing viruses.     

Avian erythroblastosis virus produces two mRNA''s.

We analyzed the viral mRNA's present in fibroblast nonproducer clones transformed by avian erythroblastosis virus. Two size classes of mRNA (28 to 30S and 22 to 24S) were identified by solution hybridization with both complementary DNA strong stop and complementary DNA made against the unique sequences of avian erythroblastosis virus. Based upon the kinetics of hybridization with complementary DNA made against the unique sequences of avian erythroblastosis virus, we estimated that there were 400 to 500 copies of the 28 to 30S RNA per cell and 200 to 250 copies of the 22 to 24S RNA per cell. Both RNA species were packaged in the virion. In vitro translation of the 28 to 30S virion RNA yielded a 75,000-dalton protein which was the 75,000-dalton gag-related polyprotein found in avian erythroblastosis virus-transformed cells. In vitro translation of the 22 to 24S virion RNA yielded two proteins (46,000 and 48,000 daltons). This indicates that there may be two genes in avian erythroblastosis virus, one coding for the 75,000-dalton gag-related polyprotein and the second coding for the 46,000- or 48,000-dalton protein or both.     

Characterization of Nucleic Acid of Pichinde Virus

The nucleic acid of Pichinde virus was found to be single-stranded ribonucleic acid (RNA) as determined by sensitivity to ribonuclease, by alkaline degradation, by buoyant density in cesium sulfate, and by analysis of the base composition. The RNA of the virion could be separated into five components which had sedimentation coefficients corresponding to 31S, 28S, 22S, 18S and 4 to 6S. The 28S, 18S, and possibly the 4 to 6S RNAs appear to be derived from host cell components incorporated into the virion, whereas the 31S and 22S components appear to represent the genome of the virus.     

Polyadenylate Sequences on Newcastle Disease Virus mRNA Synthesized In Vivo and In Vitro

Polyadenylate [poly(A)] sequences are associated with the 35 and 50S Newcastle disease virus (NDV)-specific RNAs as well as all six to seven of the 18-22S NDV-specific messenger RNAs extracted from infected chicken embryo cells. The poly(A) associated with the 18-22S RNA has an average size of 120 to 130 nucleotides. The 18-22S RNA synthesized in vitro by NDV's virion-bound polymerase contains six to seven species of the same size and relative proportions as its intracellular counterpart. This in vitro synthesized 18-22S RNA also contains covalently linked poly(A) sequences which, although variable in size, are usually larger and more heterogeneous than those from the infected cell. In vitro RNA synthesis is supported not only by magnesium (at an optimal concentration of mM) but by manganese (at an optimal concentration of 0.5 to 1.0 mM) as well. However, the major product made in the presence of manganese, although sedimenting at 18 to 22S, differs somewhat from the product made in the presence of magnesium.     

Nucleic acids of respiratory syncytial virus.

Analysis of purified respiratory syncytial virus revealed that the virion RNA was composed of 50S, 28S, 18S, and 4S species. The 18S and 28S species were presumed to represent host rRNA since virus grown in actinomycin D-treated cells contained only 50S and 4S RNAs. Actinomycin D treatment stimulated production of infectious respiratory syncytial virus 5- to 10-fold. The 50S virion RNA was shown to hybridize with polyadenylated mRNA's isolated from infected cells, indicating that respiratory syncytial virus RNA is of negative-strand sense. Six mRNA's were identified by polyacrylamide gel electrophoresis.     

Common and distinct regions of defective-interfering RNAs of Sindbis virus

Defective-interfering (DI) particles are helper-dependent deletion mutants which interfere specifically with the replication of the homologous standard virus. Serial passaging of alphaviruses in cultured cells leads to the accumulation of DI particles whose genomic RNAs are heterogeneous in size and sequence composition. In an effort to examine the sequence organization of an individual DI RNA species generated from Sindbis virus, we isolated and sequenced a representative cDNA clone derived from a Sindbis DI RNA population. Our data showed that: (i) the 3' end of the DI RNA template was identical to the 50 nucleotides at the 3' end of the standard RNA; (ii) the majority (75%) of the DI RNA template was derived from the 1,200 5'-terminal nucleotides of the standard RNA and included repeats of these sequences; and (iii) the 5' end of the DI RNA template was not derived from the standard RNA, but is nearly identical to a cellular tRNAAsp (S. S. Monroe and S. Schlesinger, Proc. Natl. Acad. Sci. U.S.A. 80:3279-3283, 1983). We have also utilized restriction fragments from cloned DNAs to probe by blot hybridization for the presence of conserved sequences in several independently derived DI RNA populations. These studies indicated that: (i) a 51-nucleotide conserved sequence located close to the 5' end of several alphavirus RNAs was most likely retained in the DI RNAs; (ii) the junction region containing the 5' end of the subgenomic 26S mRNA was deleted from the DI RNAs; and (iii) the presence of tRNAAsp sequences was a common occurrence in Sindbis virus DI RNAs derived by passaging in chicken embryo fibroblasts.     

Structural analysis of the intracellular RNAs of murine mammary tumor virus.

We have characterized murine mammary tumor virus (MuMTV)-specific RNA in several types of cells in which viral DNA is transcribed into RNA: cultured GR mouse mammary tumor cells, S49 lymphoma cells from BALB/c mice, lactating mammary glands from C57BL/6 mice, and mink lung cells infected in vitro with MuMTV. In all cell types studied, there are three distinct species of intracellular viral RNA, with sedimentation coefficients of 35S, 24S, and 13S (or molecular weights of 3.1 X 10(6), 1.5 X 10(6), and 0.37 X 10(6), as determined by rate-zonal sedimentation in sucrose gradients and by electrophoresis in agarose gels under denaturing conditions. These three viral RNA species appear to be present regardless of viral RNA concentration, responsiveness to glucocorticoid hormones, production of extracellular virus, and use of either endogenous or acquired MuMTV proviral DNA as template. The three viral RNAs display characteristics of mRNAs in that they are polyadenylated, associated with polyribosomes, and released from polyribosomes by treatment with EDTA; hence all three species presumably direct the synthesis of virus-coded proteins. The two larger species of viral RNA are probably responsible for synthesis of the structural proteins of the virion, but the function of the 13S RNA is not known. Both of the subgenomic RNAs contain sequences found at the 3' terminus of 35S (or genomic) RNA. However, only the 24S RNA (not the 13S RNA) contains sequences which are located at the 5' terminus of 35S RNA and are apparently transposed during RNA synthesis of maturation, as described for subgenomic mRNA's of other retroviruses.     

Rubella virus 40S genome RNA specifies a 24S subgenomic mRNA that codes for a precursor to structural proteins.

We have analyzed the structure of the rubella virus genome RNA and the virus-specific RNA species synthesized in B-Vero cells infected with rubella virus. A single-stranded, capped, and polyadenylated RNA species sedimenting at 40S in a sucrose gradient was released from purified virions treated with sodium dodecyl sulfate. This RNA species migrated with an Mr of about 3.8 X 10(6) in an agarose gel after denaturation with glyoxal and dimethyl sulfoxide. Infected cells labeled with [3H]uridine in the presence of actinomycin D contained, in addition to the 40S RNA, a single-stranded polyadenylated 24S RNA species as shown by sucrose gradient analysis. In a Northern blot analysis, this RNA hybridized to a cDNA probe derived from the 3' portion of the genomic 40S RNA. In vitro translation of the 24S RNA species yielded a 110,000-dalton polypeptide, in addition to some smaller products which were immunoprecipitated with an antiserum prepared against the structural proteins E1, E2a, E2b, and C. Since the sum of the molecular weights of the nonglycosylated envelope proteins and the capsid protein has been estimated to be about 116,000 (C. Oker-Blom et al., J. Virol. 46:964-973, 1983), these results suggest that the 24S RNA species represents a subgenomic mRNA coding for a precursor (p110) to the structural proteins of rubella virus. Thus, the strategy of gene expression of rubella virus appears to be similar to that of the alphaviruses.     

Structure and metabolism of adenovirus RNAs containing sequences from the fiber gene

The body of adenovirus fiber messenger RNA is specified by viral r-strand co-ordinates 86.2 to 91.2. Since this mRNA is transcribed from the major late promoter at map position 16, nuclear precursors to the mRNA could be as large as 84% of the length of the 35,000 nucleotide genome. This study identified and characterized polyadenylated nuclear RNAs that contain fiber sequences and therefore are possible processing intermediates. These nuclear RNAs were characterized by hybridization of [³H]RNA preparations and by electron microscopy of RNA-DNA hybrids. Three size classes of RNAs containing fiber sequences were identified: (1) a 22 S species maps from 86.2 to 90.3. This RNA has essentially the same co-ordinates as fiber mRNA. (2) Two 28 S species have co-ordinates of 80.1 to 90.4 and 85.9 to 96.9, respectively. Thus one species has a 5′ terminus coincident with that of the mRNA body, and one has a 3′ terminus coincident with that of the 3′ end of the mRNA body. The polyadenylated terminus at 96.9 does not coincide with the 3′ end of any known mRNA. (3) There are at least two 35 S species. The 3′ end of one species is coincident with that of fiber mRNA. The 3′ terminus of the second RNA is at approximately 96.9.The labeling kinetics of each of these polyadenylated nuclear RNAs were investigated. In continuous label experiments, the two 35 S RNAs and the 85.9 to 96.9 28 S RNA became uniformly labeled in approximately 60 minutes. The 22 S RNA and the 80.1 to 90.4 28 S species continued to accumulate for at least several hours. These results are consistent with a precursor function for the 35 S RNAs and the 85.9 to 96.9 28 S species. The structures of the putative precursors imply that processing of the 3′ end is not a prerequisite for 5′ cleavage.     

Studies of defective interfering RNAs of Sindbis virus with and without tRNAAsp sequences at their 5'' termini.

Three of six independently derived defective interfering (DI) particles of Sindbis virus generated by high-multiplicity passaging in cultured cells have tRNAAsp sequences at the 5' terminus of their RNAs (Monroe and Schlesinger, J. Virol. 49:865-872, 1984). In the present work, we found that the 5'-terminal sequences of the three tRNAAsp-negative DI RNAs were all derived from viral genomic RNA. One DI RNA sample had the same 5'-terminal sequence as the standard genome. The DI RNAs from another DI particle preparation were heterogeneous at the 5' terminus, with the sequence being either that of the standard 5' end or rearrangements of regions near the 5' end. The sequence of the 5' terminus of the third DI RNA sample consisted of the 5' terminus of the subgenomic 26S mRNA with a deletion from nucleotides 24 to 67 of the 26S RNA sequence. These data showed that the 5'-terminal nucleotides can undergo extensive variations and that the RNA is still replicated by virus-specific enzymes. DI RNAs of Sindbis virus evolve from larger to smaller species. In the two cases in which we followed the evolution of DI RNAs, the appearance of tRNAAsp-positive molecules occurred at the same time as did the emergence of the smaller species of DI RNAs. In pairwise competition experiments, one of the tRNAAsp-positive DI RNAs proved to be the most effective DI RNA, but under identical conditions, a second tRNAAsp-positive DI RNA was unable to compete with the tRNAAsp-negative DIs. Therefore, the tRNAAsp sequence at the 5' terminus of a Sindbis DI RNA is not the primary factor in determining which DI RNA becomes the predominant species in a population of DI RNA molecules.     

Dexamethasone Induction of the Intracellular RNAs of Mouse Mammary Tumor Virus

We have studied the kinetics of dexamethasone induction of mouse mammary tumor virus (MMTV) RNAs and proteins in virus-infected rat XC cells and GR mouse mammary tumor cells. A detectable increase in viral RNA in infected XC cells was present within 10 min after hormone addition, and half-maximal induction was achieved in less than 2 h. The increase in viral RNA concentration was apparent first in nuclear RNA and later in the cytoplasm. Within the first 15 min of induction, only genome-sized RNA (35S, 7.8 kilobases) was present in augmented amounts, whereas the major subgenomic RNA (24S, 3.8 kilobases) did not appear until at least 30 to 60 min postinduction. The sequential appearance of these RNAs, the probable mRNA's for the gag and env proteins, paralleled the order of appearance of the gag and env proteins, respectively, after hormone treatment. An additional species of viral RNA (20S, 2.5 kilobases) was detected during these induction experiments, but the role of this RNA is not known. Both subgenomic RNAs contain sequences derived from both the 5′ and 3′ termini of genomic RNA and are presumably spliced. After dexamethasone induction of infected XC cells, we detected two smaller env-related proteins which were not found in full hormone induction. The functional role of these smaller proteins is not known. A previously reported smaller species of RNA (13S, 1.0 kilobase) did not appear to be induced and was shown to be cellular rather than viral in origin. In the fully induced infected XC and GR mammary tumor cells, the only viral RNAs present were the 35S and 24S RNAs. In addition, mammary tumors contained only these two viral RNAs. Thus, tumor cells appear to contain only the viral RNAs which direct the synthesis of the gag, pol, and env proteins of the virion.     

Accumulation of bacteriophage T7 head-related particles in an Escherichia coli mutant.

We have analyzed the structure of the late cytoplasmic RNAs made after infection with wild-type simian virus 40 and a set of viable mutants, four of which have deletions and one an insertion within the nucleotide sequence specifying the leader segment of the 16S and 19S mRNA's. The principal findings are: (i) simian virus 40 16S and 19S mRNA's made during infections with wild-type virnds and possibly in the nucleotide sequence comprising the "leader" segments. (II) "Spliced" 16S and 19S mRNA's are made during infections with each of the mutants although, in some cases, the ratio of 19S to 16S mRNA species is reduced. (iii) The deletion or insertion of nucleotides within the DNA segment defined by map position 0.70 to 0.75 causes striking alterations in the types of leader structures in the late mRNAs. (iv) Many of the late RNA leader segments produced after infection with the mutants appear to be multiply spliced, i.e., instead of the major 200- to 205-nucleotide-long leader segment present in wild-type 16S mRNA, the RNAs produced by several of the deletion mutants have leaders with whort discontiguous segments.     

Chapare virus, a newly discovered arenavirus isolated from a fatal hemorrhagic fever case in Bolivia

A small focus of hemorrhagic fever (HF) cases occurred near Cochabamba, Bolivia, in December 2003 and January 2004. Specimens were available from only one fatal case, which had a clinical course that included fever, headache, arthralgia, myalgia, and vomiting with subsequent deterioration and multiple hemorrhagic signs. A non-cytopathic virus was isolated from two of the patient serum samples, and identified as an arenavirus by IFA staining with a rabbit polyvalent antiserum raised against South American arenaviruses known to be associated with HF (Guanarito, Machupo, and Sabiá). RT-PCR analysis and subsequent analysis of the complete virus S and L RNA segment sequences identified the virus as a member of the New World Clade B arenaviruses, which includes all the pathogenic South American arenaviruses. The virus was shown to be most closely related to Sabiá virus, but with 26% and 30% nucleotide difference in the S and L segments, and 26%, 28%, 15% and 22% amino acid differences for the L, Z, N, and GP proteins, respectively, indicating the virus represents a newly discovered arenavirus, for which we propose the name Chapare virus. In conclusion, two different arenaviruses, Machupo and Chapare, can be associated with severe HF cases in Bolivia.