Isolation and characterization of Sendai virus DI-RNAs.

When passaged at high multiplicity, four strains of Sendai virus all showed evidence that they contained defective interfering (DI) particles. RNA isolated from nucleocapsids of cells infected with the high multiplicity passage stocks was found to consist of only minor amounts of nondefective genome length RNA and major amounts of smaller RNAs, the DI-RNAs. These DI-RNAs were found to have unusual and variable sedimentation properties in sucrose gradients, but were found to represent unique segments of the viral genome by length measurements in the electron microscope and by hybridization. A striking feature of the DI-RNAs is their ability to form circular structures, indicating that the ends of the DI-RNA are complementary. The implications of this finding in terms of the mechanism of genome replication is discussed.     

At least 104 nucleotides are transposed from the 5' terminus of the avian sarcoma virus genome to the 5' termini of smaller viral mRNAs.

Cells producing avian sarcoma virus (ASV) contain at least three virus-specific mRNAs, two of which are encoded within the 3' half of the viral genome. Each of these viral RNAs can hybridize with single-stranded DNA(cDNA5') that is complementary to a sequence of 101 nucleotides found at the 5' terminus of the ASV genome, but not within the 3' half of the genome. We proposed previously (Weiss, Varmus and Bishop, 1977) that this nucleotide sequence may be transposed to the 5' termini of viral mRNAs during the genesis of these RNAs. We now substantiate this proposal by reporting the isolation and chemical characterization of the nucleotide sequences complementary to cDNA5' in the genome and mRNAs of the Prague B strain of ASV. We isolated the three identified classes of ASVmRNA (38, 28 and 21S) by molecular hybridization; each class of RNA contained a "capped" oligonucleotide identical to that found at the 5' terminus of the ASV genome. When hybridized with cDNA5', each class of RNA gave rise to RNAase-resistant duplex hybrids that probably encompassed the full extent of cDNA5'. The molar yields of duplex conformed approximately to the number of virus-specific RNA molecules in the initial samples; hence most if not all of the molecules of virus-specific RNA could give rise to the duplexes. The duplexes prepared from the various RNAs all contained the capped oligonucleotide found at the 5' terminus of the viral genome and had identical "fingerprints" when analyzed by two-dimensional fractionation following hydrolysis with RNAase T1. In contrast, RNA representing the 3' half of the ASV genome did not form hybrids with cDNA5'. We conclude that a sequence of more than 100 nucleotides is transposed from the 5' end of the ASV genome to the 5' termini of smaller viral RNAs during the genesis of these RNAs. Transposition of nucleotide sequences during the production of mRNA has now been described for three families of animal viruses and may be a common feature of mRNA biogenesis in eucaryotic cells. The mechanism of transposition, however, and the function of the transposed sequences are not known.     

Evidence for the Identity of Shared 5′-Terminal Sequences Between Genome RNA and Subgenomic mRNA''s of B77 Avian Sarcoma Virus

The polyribosomal fraction from chicken embryo fibroblasts infected with B77 avian sarcoma virus contained 38S, 28S, and 21S virus-specific RNAs in which sequences identical to the 5'-terminal 101 bases of the 38S genome RNA were present. The only polyadenylic acid-containing RNA species with 5' sequences which was detectable in purified virions had a sedimentation coefficient of 38S. This evidence is consistent with the hypothesis that a leader sequence derived from the 5' terminus of the RNA is spliced to the bodies of the 28S and 21S mRNA's, both of which have been shown previously to be derived from the 3' terminal half of the 38S RNA. The entire 101-base 5' terminal sequence of the genome RNA appeared to be present in the majority of the subgenomic intracellular virus-specific mRNA's, as established by several different methods. First, the extent of hybridization of DNA complementary to the 5'-terminal 101 bases of the genome to polyadenylic acid-containing subgenomic RNA was similar to the extent of its hybridization to 38S RNA from infected cells and from purified virions. Second, the fraction of the total cellular polyadenylic acid-containing RNA with 5' sequences was similar to the fraction of RNA containing sequences identical to the extreme 3' terminus of the genome RNA when calculated by the rate of hybridization of the appropriate complementary DNA probes. This suggests that most intracellular virus-specific RNA molecules contain sequences identical to those present in the 5'-terminal 101 bases of the genome. Third, the size of most of the radioactively labeled DNA complementary to the 5'-terminal 101 bases of the genome remained unchanged after the probe was annealed to either intracellular 38S RNA or to various size classes of subgenomic RNA and the hybrids were digested with S1 nuclease and denatured with alkali. However, after this procedure some DNA fragments of lower molecular weight were present. This was not the case when the DNA complementary to the 5'-terminal 101 bases of the genome was annealed to 38S genome RNA. These results suggest that, although the majority of the intracellular RNA contains the entire 101-base 5'-terminal leader sequence, a small population of virus-specific RNAs exist that contain either a shortened 5' leader sequence or additional splicing in the terminal 101 bases.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses VII. Characterization of the Simian Virus 40 RNA Species Induced by Five Nondefective Hybrid Viruses

Five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses have been isolated and found to contain segments of SV40 DNA covalently linked to Ad2 DNA. The quantity of SV40 DNA present is a stable characteristic of each hybrid virus, and varies from less than 5% (in Ad2(+)ND(3)) to more than 30% (in Ad2(+)ND(4)) of the SV40 genome. We have characterized the SV40 portions of these hybrids by relating the SV40-specific RNA sequences transcribed in cells infected with each hybrid virus to those transcribed in cells infected with each of the other hybrid viruses and with SV40 itself. RNA-DNA hybridization-competition experiments indicate that the number of unique SV40 RNA sequences transcribed in infected cells is proportional to the size of the SV40 DNA segment contained within each hybrid and, in the case of the three hybrids which induce detectable SV40-specific antigens, to the number of SV40 antigens induced. Furthermore, the SV40-specific RNA sequences transcribed from any one of the hybrids are completely represented in the RNA transcribed from all other hybrids with longer SV40 segments. Thus, the SV40 DNA regions in the five hybrid viruses appear to contain some nucleotide sequences in common. The SV40-specific RNA transcribed from Ad2(+)ND(4), the hybrid containing the largest SV40 segment, is qualitatively similar to the SV40-specific RNA transcribed early (i.e., prior to viral DNA replication) in SV40 lytic infection. Thus, it appears that no significant amount of late SV40 DNA is transcribed during infection by any of the five nondefective Ad2-SV40 hybrid viruses.     

Sequence of 3,687 nucleotides from the 3' end of Sendai virus genome RNA and the predicted amino acid sequences of viral NP, P and C proteins.

The sequence of 3,687 nucleotides from the 3' end of the Sendai virus genome (Z strain) was determined by a molecular cloning technique followed by rapid sequence analysis. Two large open reading frames, one consisting of 1,572 nucleotides and the other of 1,704 nucleotides, were observed in the region, that is OP-1 and OP-2 from the 3' end of the genome. The amino acid sequences of the gene products were predicted from the observed sequence. Determination of amino acid compositions of viral proteins, P, HN, Fo, NP and M, led us to conclude that NP and P are the gene products of OP-1 and OP-2, respectively. An additional open reading frame consisting of 612 nucleotides (OP-3) was discovered in the 3' most proximal region of OP-2. The predicted product of OP-3 was considered to be viral non-structural protein C. The leader sequence of 51 nucleotides at the 3' terminal of the genome and consensus sequences at 3' and 5' ends of each gene for proteins NP and P were identified.     

Genomic and copy-back 3'' termini in Sendai virus defective interfering RNA species

Direct sequencing of nine Sendai virus defective interfering RNA species revealed two kinds of 3'-terminal sequences. Six RNA species had 3' termini identical to the virus genome (negative strand), confirming that internal deletions are a frequent cause of Sendai virus defectiveness. The other three RNA species had 3'-terminal sequences identical to that described as the complement of the 5' terminus of the virus genome (R. A. Lazzarini, J. D. Keene, and M. Schubert, Cell 26:145-154, 1981), indicating that they are of the copy-back type. Extensive homology between these two types of 3' sequences evidently accounts for the ability of the copy-back sequence to function as an initiation signal for viral RNA replication. There may not be a selective advantage of one type of terminus over the other, since one defective interfering strain possessed two RNA species, one of which had the genomic 3' terminus and the other copy-back type.     

Isolation of vesicular stomatitis virus defective interfering genomes with different amounts of 5''-terminal complementarity.

I isolated at least 30 different vesicular stomatitis virus defective interfering (DI) genomes, distinguished by chain length, by five independent undiluted passages of a repeatedly cloned virus plaque. Labeling of the 3' hydroxyl ends of these DI genomes and RNase digestion studies demonstrated that the ends of these DI genomes were terminally complementary to different extents (approximately 46 to 200 nucleotides). Mapping studies showed that the complementary ends of all of the DI genomes were derived from the 5' ends of the nondefective minus-strand genome. Regardless of the extent of terminal complementarity, all of the DI genomes synthesized the same 46-nucleotide minus-strand leader RNA.     

Structure and origin of a novel class of defective interfering particle of vesicular stomatitis virus

The genome structure and terminal sequences of a 'copyback' defective interfering (DI) particle ST1, and a novel complexly rearranged 'snapback' DI particle ST2 of vesicular stomatitis virus have been determined. The ST1 DI genome RNA possesses 54 base long inverted complementary termini, the 5' end of which is homologous to the standard virus genome 5' end. Following this region of inverted complementarity the DI RNA 5' end continues to be homologous to standard virus RNA 5' sequences, whereas the 3' end diverges into sequences within the virus L gene internal sequences. ST2 DI genome RNA does not contain colinear covalently linked plus and minus sense RNA copies of the standard infectious virus RNA 5' terminus as predicted from the prototype snapback DI structure, but instead appears to be a hairpin copy of the ST1 DI RNA genome. This is the first evidence suggesting that DI particles may be generated from RNA templates other than the standard virus RNA. Generation models and the implications of these findings for RNA virus evolution are discussed.     

Comparison of ribonucleotide sequences from the genome of vesicular stomatitis virus and two of its defective interfering particles.

RNA genomes from standard vesicular stomatitis virus and two defective interfering (DI) particles dI 0.33 (DI-T) and DI 0.52, were purified and digested with RNase T1. The resulting oligonucleotides were labeled at the 5' end with [32P]ATP and separated by two-dimensional electrophoresis in polyacrylamide gels. All of the major oligonucleotides containing 20 or more nucleotides were sequenced. Those oligonucleotides that were thought to be in common by their migration on polyacrylamide gels actually did have identical sequences. Those oligonucleotides thought to be unique to the DI RNAs either differed by only one nucleotide from oligonucleotides of the standard RNA or contained new sequences which were complementary to known sequences at the 5' end. These data indicate that RNAs from DI particles are not simple deletions but contain point mutations and additional complementary sequences.     

Analysis of efficiently packaged defective interfering RNAs of murine coronavirus: localization of a possible RNA-packaging signal.

We have previously shown that most of the defective interfering (DI) RNA of mouse hepatitis virus (MHV) are not packaged into virions. We have now identified, after 21 serial undiluted passages of MHV, a small DI RNA, DIssF, which is efficiently packaged into virions. The DIssF RNA replicated at a high efficiency on its transfection into the helper virus-infected cells. The virus released from the transfected cells interfered strongly with mRNA synthesis and growth of helper virus. cDNA cloning and sequence analysis of DIssF RNA revealed that it is 3.6 kb and consists of sequences derived from five discontinuous regions of the genome of the nondefective virus. The first four regions (domains I to IV) from the 5' end are derived from gene 1, which presumably encodes the RNA polymerase of the nondefective virus. The entire domain I (859 nucleotides) and the first 750 nucleotides of domain II are also present in a previously characterized DI RNA, DIssE, which is not efficiently packaged into virions. Furthermore, the junction between these two domains is identical between the two DI RNAs. The remaining 77 nucleotides at the 3' end of domain II and all of domains III (655 nucleotides) and IV (770 nucleotides) are not present in DIssE RNA. These four domains are derived from gene 1. In contrast, the 3'-most domain (domain V, 447 nucleotides) is derived from the 3' end of the genomic RNA and is also present in DIssE. The comparison of primary sequences and packaging properties between DIsse and DIssF RNAs suggested that domains III and IV and part of the 3' end of domain II contain the packaging signal for MHV RNA. This conclusion was confirmed by inserting these DIssF-unique sequences into a DIssE cDNA construct; the in vitro-transcribed RNA from this hybrid construct was efficiently packaged into virion particles. DIssF RNA also contains an open reading frame, which begins from domain I and ends at the 5'-end 20 bases of domain III. In vitro translation of DIssF RNA and metabolic labeling of the virus-infected cells showed that this open reading frame is indeed translated into a 75-kDa protein. The structures of both DIssE and DIssF RNAs suggest that a protein-encoding capability is a common characteristic of MHV DI RNA.     

The virulent satellite RNA of turnip crinkle virus has a major domain homologous to the 3' end of the helper virus genome

RNA C (355 bases), RNA D (194 bases) and RNA F (230 bases) are small, linear satellite RNAs of turnip crinkle virus (TCV) which have been cloned as cDNAs and sequenced in this study. These RNAs produce dramatically different disease symptoms in infected plants. RNA C is a virulent satellite that intensifies virus symptoms when co-inoculated with its helper virus in turnip plants, while RNA D and RNA F are avirulent. RNA D and RNA F, the avirulent satellites, are closely related to each other except that RNA F has a 36-base insert near its 3' end, not found in RNA D. The 189 bases at the 5' end of RNA C, the virulent satellite, are homologous to the entire sequence of RNA D. However, the 3' half of RNA C, is composed of 166 bases which are nearly identical to two regions at the 3' end of the TCV helper virus genome. Hence, the virulent satellite is a composite molecule with one domain at its 5' end homologous to the other avirulent satellites and another domain at its 3' end homologous to the helper virus genome. All four TCV RNAs, RNAs C, D and F and the helper virus genome have identical 7 bases at their 3' ends. The secondary structure of RNA C deduced from the sequence can be folded into two separate domains — the domain of helper virus genome homology and the domain homologous to other TCV satellite RNAs. Comparative sequences of several different RNA C clones reveal that this satellite is a population of molecules with sequence and length heterogeneity.     

Interference among defective interfering particles of vesicular stomatitis virus

Three defective interfering (DI) particles of vesicular stomatitis virus (VSV), all derived from the same parental standard San Juan strain (Indiana serotype), were used in various combinations to infect cells together with the parental virus. The replication of their RNA genomes in the presence of other competing genomes was described by the hierarchical sequence: DI 0.52 particles greater than DI 0.45 particles less than or equal to DI-T particles greater than standard VSV. The advantage of one DI particle over another was not due simply to multiplicity effects nor to the irreversible occupation of limited cellular sites. Interference, however, did correlate with a change in the ratio of plus and minus RNA templates that accumulated intracellularly and with the presence of new sequences at the 3' end of the DI genomes. DI 0.52 particles contained significantly more nucleotides at the 3' end that were complementary to those at the 5' end of its RNA than did DI-T or DI 0.45 particles. The first 45 nucleotides at the 3' ends of all of the DI RNAs were identical. VSV and its DI particles can be separated into three classes, depending on their terminal RNA sequences. These sequences suggest two mechanisms, one based on the affinity of polymerase binding and the other on the affinity of N-protein binding, that may account for interference by DI particles against standard VSV and among DI particles themselves.     

Terminally redundant sequences in cellular intracisternal A-particle genes.

The sequences coding for intracisternal A-particle RNA form a family of related but not identical genetic elements which are present in 650 to 1,000 copies within the mouse genome. We showed that different intracisternal A-particle genes had a terminally redundant sequence of about 400 base pairs, one-half of which arose from the 3' end of the intracisternal A-particle RNA. A second portion of the redundant region did not contain 3-related sequences and was probably derived from the 5' end of intracisternal A-particle RNA. Thus, there were endogenous intracisternal A-particle genes in the cellular DNA-3'-5'--3'-5'-cellular DNA configuration identified for type B and C retroviruses. This indicated that the initial integration of intracisternal A-particle genes into the Mus musculus genome occurred by the same mechanism as the integration of other retroviruses. Two types of heterogeneity were identified among the 5' sequences of the two genes.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses. VI. Characterization of the DNA from Five Nondefective Hybrid Viruses

Certain biophysical characteristics of the DNA from each of the five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses (Ad2(+)ND(1), Ad2(+)ND(2), Ad2(+)ND(3), Ad2(+)ND(4), Ad2(+)ND(5)) have been determined. The guanine plus cytosine content varied from 55 to 57% and was not significantly different from that of nonhybrid Ad2 (56%), and the hybrid DNA molecules had mean molecular lengths which were similar to that of the standard, Ad2. The Ad2 and SV40 components of each hybrid were linked by alkali-resistant, presumably covalent bonds. The percentage of SV40 DNA in each hybrid virus was determined by hybridization with SV40 complementary RNA in a calibrated system. The results indicate that each hybrid virus DNA contains a different percentage of SV40 nucleotide sequences. The estimated size of the SV40 DNA component varies from 48,000 daltons for Ad2(+)ND(3) to 840,000 daltons for Ad2(+)ND(4), the latter being equivalent to between one-fourth and one-third of the SV40 genome.