Replication of Sindbis virus. I. Relative size and genetic content of 26 s and 49 s RNA

The genome of Sindbis virus, 49 s RNA, is a single, intact polynucleotide chain having a molecular weight of 4.3±0.3 × 10⁶ daltons. This has been determined using a variety of methods including polyacrylamide gel electrophoresis, sedimentation after reaction with formaldehyde, determination of the molecular weights of the double-stranded forms of Sindbis-specific RNA and hybridization competition. The second major species of single-stranded RNA made after infection with Sindbis, 26 s RNA, has been found to have a molecular weight of 1.6 × 10⁶ daltons as determined by sedimentation in dimethylsulfoxide. Hybridizationcompetition experiments carried out between these two species of RNA, using double-stranded forms of Sindbis RNA isolated from infected cells, showed that 26 s RNA contains only one-third of the base sequences in 49 s RNA and thus represents a unique fraction of the viral genome.     

Measurement of the Sequence Complexity of Cloned Moloney Murine Leukemia Virus 60 to 70S RNA: Evidence for a Haploid Genome

The sequence complexity of the 60-70S RNA complex from Moloney murine leukemia virus (M-MuLV) was determined by measuring the annealing rate of radioactively labeled virus-specific DNA with M-MuLV 60-70S RNA in conditions of vast RNA excess. The M-MuLV RNA annealing rate, characterized by the quantity C(r)t((1/2)), was compared with the C(r)t((1/2)) values for annealing of poliovirus 35S RNA (2.6 x 10(6) molecular weight) with poliovirus-specific DNA and Sindbis virus 42S RNA (4.3 x 10(6) molecular weight) with Sindbis-specific DNA. M-MuLV-specific DNA was prepared in vitro by the endogenous DNA polymerase reaction of M-MuLV virions, and poliovirus and Sindbis virus DNAs were prepared by incubation of viral RNA and DNA polymerase purified from avian myeloblastosis virus and an oligo deoxynucleotide primer. The poliovirus and Sindbis virus DNAs were sedimented through alkaline sucrose gradients, and those portions of the DNA with sizes similar to the M-MuLV DNA were selected out for the annealing measurements. M-MuLV was cloned on NIH-3T3 cells because it appeared possible that the standard source of M-MuLV for these experiments was a mixture of viruses. The annealing measurements indicated a sequence complexity of approximately 9 x 10(6) daltons for the cloned M-MuLV 60-70S RNA when standardized to poliovirus and Sindbis virus RNAs. This value supports the hypothesis that each of the 35S RNA subunits of M-MuLV 60-70S RNA has a different base sequence.     

Replication of Sindbis Virus V. Polyribosomes and mRNA in Infected Cells

Cells infected with wild-type Sindbis virus contain at least two forms of mRNA, 26S and 49S RNA. Sindbis 26S RNA (molecular weight 1.6 x 10(6)) constitutes 90% by weight of the mRNA in infected cells, and is thought to specify the structural proteins of the virus. Sindbis 49S RNA, the viral genome (molecular weight 4.3 x 10(6)), constitutes approximately 10% of the mRNA in infected cells and is thought to supply the remaining viral functions. In cells infected with ts2, a temperature-sensitive mutant of Sindbis virus, the messenger forms also include a third species of RNA with a sedimentation coefficient of 33S and an apparent molecular weight of 2.3 x 10(6). Hybridization-competition experiments showed that 90% of the base sequences in 33S RNA from these cells are also present in 26S RNA. Sindbis 33S RNA was also isolated from cells infected with wild-type virus. After reaction with formaldehyde, this species of 33S RNA appeared to be completely converted to 26S RNA. These results indicate that 33S RNA isolated from cells infected with either wild-type Sindbis or ts2 is not a unique and separate form of Sindbis RNA.     

Defective mutant of Sindbis virus with a smaller-molecular-weight form of the E1 glycoprotein.

We have isolated from a single plaque a mutant of Sindbis virus characterized by an E1 glycoprotein with higher electrophoretic mobility. This higher mobility is not attributable to a different extent of glycosylation of the protein nor to an altered proteolytic maturation pathway of the polypeptide precursor, but is the result of a deletion occurring during the replication of the viral RNA. The 26S RNA (the messenger for the Sindbis structural proteins) extracted from cells infected with the mutant is about 0.75 x 10(5) daltons smaller than the 26S RNA from the parental strain. As a consequence, in cells infected with the mutant, an E1 glycoprotein is synthesized with a polypeptide chain about 70 amino acids shorter. The biological relevance of this naturally occurring deletion of the viral genome is discussed.     

Gel Electrophoresis of Avian Leukosis and Sarcoma Viral RNA in Formamide: Comparison with Other Viral and Cellular RNA Species

Class a and class b 30 to 40S RNA subunits obtained by heat dissociation from the 60 to 70S RNA complex of avian tumor viruses were compared with several RNA standards by electrophoresis in formamide-polyacrylamide gels. Class a RNA was found to have a lower electrophoretic mobility and hence probably a higher molecular weight than class b RNA. The absolute molecular weight of class a and b RNA could not be determined with accuracy, because the relationship between logarithm of molecular weight and mobility of the RNA standards was not linear. The size of class a RNA fell into the range of 2.4 x 10(6) to 3.4 x 10(6) daltons and that of class b into the range of 2.2 x 10(6) to 2.9 x 10(6) daltons, depending on the standards used. The possible biological significance of this difference is discussed.     

Separation of Ten Reovirus Genome Segments by Polyacrylamide Gel Electrophoresis

Double-stranded ribonucleic acid (RNA) extracted from purified reoviruses of all three serotypes and from type 3 virus-infected cells was analyzed by polyacrylamide gel electrophoresis. It was calculated that each RNA includes 10 segments: 3 large, 3 intermediate, and 4 small fragments corresponding to molecular weights of about 2.5, 1.4, and 0.8 x 10(6) daltons, respectively, or a total of 15 x 10(6) daltons.     

Transcription In Vitro by Reovirus-Associated Ribonucleic Acid-Dependent Polymerase

Digestion of purified reovirus type 3 with chymotrypsin degrades 70% of the viral protein and converts the virions to subviral particles (SVP). The SVP contain 3 of the 6 viral structural proteins and all 10 double-stranded ribonucleic acid (RNA) genome segments but not adenine-rich, single-stranded RNA. An RNA polymerase which is structurally associated with SVP transcribes one strand of each genome segment by a conservative mechanism in vitro. The single-stranded products include large (1.2 x 10(6) daltons), medium (0.7 x 10(6) daltons), and small (0.4 x 10(6) daltons) molecules which hybridize exclusively with the corresponding genome segments. The enzyme obtained by heating virions at 60 C synthesizes similar products. Kinetic and pulse-chase studies indicate that the different-sized products are synthesized simultaneously but at rates which are in the order: small > medium > large.     

Synthesis and characterization of soluble concanavalin A oligomer

Concanavalin A, (Con A, MW 26,500/monomer unit) was crosslinked with glutaraldehyde to form soluble, high-molecular-weight (larger than MW 300,000) Con A Oligomers. After filtration to remove insoluble and low-molecular-weight portions (below 300,000 daltons), the size and molecular-weight distribution were characterized by laser light scattering and gel-filtration chromatography. The molecular-size determined by laser light scattering ranged from 870 to 4070 A, while the molecular weight determined by gel chromatography ranged from 6 x 10(5) to higher than 2 x 10(6) daltons. The affinity and kinetics of Con A oligomer binding to polysaccharide (glycogen) were evaluated by precipitation test and turbidity development, respectively. The binding with glycogen was strongest at neutral pH and showed similar activity to unmodified Con A molecules. The binding constants of alpha-D-glucose and succinyl-aminophenyl alpha-D glucopyranoside-insulin to Con A oligomer were 1.0 x 10(3)M(-1) and 4.5 x 10(4)M(-1), respectively and the binding capacity of the oligomer was nearly 85% to 95% of monomeric Con A. The complexes of saccharides and soluble Con A oligomer were stable for at least 7 days. (c) 1993 Wiley & Sons, Inc.     

Genomic complexities of murine leukemia and sarcoma, reticuloendotheliosis, and visna viruses.

The genetic complexities of several ribodeoxyviruses were measured by quantitative analysis of unique RNase T1-resistant oligonucleotides from 60-70S viral RNAs. Moloney murine leukemia virus was found to have an RNA complexity of 3.5 x 10(6) daltons, whereas Moloney murine sarcoma virus had a significantly smaller genome size of 2.3 x 10(6). Reticuleondotheliosis and visna virus RNAs had complexities of 3.9 x 10(6), respectively. Analysis of RNase A-resistant oligonucleotides of Rous sarcoma virus RNA gave a complexity of 3.6 x 10(6), similar to that previously obtained with RNase T1-resistant oligonucleotides. Since each of these viruses was found to have a unique sequence genomic complexity near the molecular weight of a single 30-40S viral RNA subunit, it was concluded that ribodeoxyvirus genomes are at least largely polyploid.     

The 5'' ends of yeast killer factor RNAs are pppGp.

The 5' nucleotides of the double-stranded RNAs of yeast killer factor have been isolated by digestion with pancreatic, T1 and T2 RNase followed by two-dimensional electrophoresis. They were identified by bacterial alkaline phosphatase and snake venom phosphodiesterase digestions. Both the larger double-stranded RNA (L, of 2.5 x 10(6) daltons) and the smaller double-stranded RNA (M, of 1.4 x 10(6) daltons) have the 5' end groups pppGp. These 5' ends are dissimilar to those of the double-stranded RNAs of animal viruses but may be characteristic of the 5' ends of the double-stranded RNAs of fungal viruses.     

DNA of herpesvirus pan, a third member of the Epstein-Barr virus-Herpesvirus papio group

The DNA of herpesvirus pan, a primate B-lymphotropic herpesvirus, shares about 40% well-conserved sequence relatedness with Epstein-Barr virus (EBV) and herpesvirus papio DNAs. Labeled cloned fragments from the EBV recombinant DNA library were cross hybridized to blots of EcoRI, XbaI, and BamHI restriction endonuclease fragments of herpesvirus pan DNA to identify and map homologous sequences in the herpesvirus pan genome. Regions of colinear homology were demonstrated between 6 x 10(6) daltons and 108 x 10(6) daltons in the DNAs. The structural organization of herpesvirus pan DNA was similar to the format of Epstein-Barr virus and herpesvirus papio DNAs. The DNA consists of two domains of largely unique sequence complexity, a segment US of 9 x 10(6) daltons and a segment UL of 88 x 10(6) daltons. US and UL are separated by a variable number of tandem repetitions of a sequence IR (2 x 10(6) daltons). There was homology between DNA which mapped at 26 to 28 x 10(6) daltons and 93 to 95 x 10(6) daltons in UL. The terminal reiteration component, TR, of herpesvirus pan DNA and sequences which mapped to the left of 6 x 10(6) daltons and to the right of 108 x 10(6) daltons had no detectable homology with the corresponding regions of Epstein-Barr virus DNA.     

Transcription of the Influenza Ribonucleic Acid Genome by a Virion Polymerase II. Nature of the In Vitro Polymerase Product

The properties of the ribonucleic acid (RNA) synthesized by the influenza (WSN) virion polymerase have been investigated. Most of the product RNA is synthesized in association with virion RNA species from which it can be released by heat treatment as single-stranded, ribonuclease-sensitive polynucleotides (average molecular weight, 2-hr sample, about 10(5) daltons). At least 95% of the product is complementary to the viral RNA species. On the basis of the molar ratio of the RNA species isolated from a (3)H-uridine-labeled virus preparation, it was calculated that the WSN genome consists of seven pieces of RNA with a sum molecular weight of about 5 x 10(6) daltons.     

Virion nucleic acid of Ebola virus.

The virion nucleic acid of Ebola virus consists of a single-stranded RNA with a molecular weight of approximately 4.0 x 10(6). The virion RNA did not bind to oligodeoxythymidylic acid-cellulose under conditions known to bind RNAs rich in polyadenylic acid and was not infectious under conditions which yielded infectious RNA from Sindbis virus, suggesting that Ebola virus virion nucleic acid is a negative-stranded RNA.     

Characterization of Bluetongue Virus Ribonucleic Acid

An improved purification procedure yielded bluetongue virus free from any single-stranded ribonucleic acid (RNA) component. Double-stranded RNA obtained from purified virus or isolated from infected cells was fractionated into 5 components by means of sucrose gradient sedimentation analysis, and into 10 components by electrophoresis on polyacrylamide gels. The size of these components vary from 0.5 x 10(6) to 2.8 x 10(6) daltons, with a total molecular weight estimate of about 1.5 x 10(7) for the viral nucleic acid. The denaturation of the genome and separation of the resulting fragments are also discussed.     

Characterization and mapping of RNase III cleavage sites in VSV genome RNA.

Ribonuclease III cleaves the genome RNA of vesicular stomatitis virus (VSV) to yield an array of fragments which range in size from 3.5 to 0.1 x 10(6) daltons under partial digestion conditions. The locations of the RNase III cleavage sites which give rise to these fragments have been ordered relative to the 3' end of the virion RNA by digestion of 3' end-labeled RNA. Based on a map of the cleavage sites we predicted that fragments having the same size could be generated which contain information from each gene. Annealing of individual VSV mRNA probes to Northern blots of the separated RNase III-generated fragments confirmed that fragments having the same size are, in fact, generated which contain information from each coding region of the VSV genome. Analysis of maps of partial digestion products indicates that fragments having the same size arise repeatedly along the 3' half of the genome. The cleavage of VSV RNA by RNase III can be detected only if the nuclease treated molecules are denatured. This suggest that the structure features in VSV RNA which signal cleavage involve areas of higher order RNA structure.     

Functional defects of RNA-negative temperature-sensitive mutants of Sindbis and Semliki Forest viruses.

Defects in RNA and protein synthesis of seven Sindbis virus and seven Semliki Forest virus RNA-negative, temperature-sensitive mutants were studied after shift to the restrictive temperature (39 degrees C) in the middle of the growth cycle. Only one of the mutants, Ts-6 of Sindbis virus, a representative of complementation group F, was clearly unable to continue RNA synthesis at 39 degrees C, apparently due to temperature-sensitive polymerase. The defect was reversible and affected the synthesis of both 42S and 26S RNA equally, suggesting that the same polymerase component(s) is required for the synthesis of both RNA species. One of the three Sindbis virus mutants of complementation group A, Ts-4, and one RNA +/- mutant of Semliki Forest virus, ts-10, showed a polymerase defect even at the permissive temperature. Seven of the 14 RNA-negative mutants showed a preferential reduction in 26S RNA synthesis. The 26S RNA-defective mutants of Sindbis virus were from two different complementation groups, A and G, indicating that functions of two viral nonstructural proteins ("A" and "G") are required in the regulation of the synthesis of 26S RNA. Since the synthesis of 42S RNA continued, these functions of proteins A and G are not needed for the polymerization of RNA late in infection. The RNA-negative phenotype of 26S RNA-deficient mutants implies that proteins regulating the synthesis of this subgenomic RNA must have another function vital for RNA synthesis early in infection or in the assembly of functional polymerase. Several of the mutants having a specific defect in the synthesis of 26S RNA showed an accumulation of a large nonstructural precursor protein with a molecular weight of about 200,000. One even larger protein was demonstrated in both Semliki Forest virus- and Sindbis virus-infected cells which probably represents the entire nonstructural polyprotein.