Sendai virus gene sequences identified by oligonucleotide mapping

We have used Tl-oligonucleotide mapping to explore the nucleotide sequence organization of the Sendai virus genome. Duplexes of radioactive virus genes and unlabeled virus messenger RNA species, obtained by ribonuclease digestion of RNA hybrids, were separated by agarose gel electrophoresis. Identities of duplexes were deduced from their apparent molecular weights, relative to the molecular weights of the virus gene products. Five duplexes were assigned to the five genes that map at the 3′ side of the virus genome. However, oligonucleotide mapping revealed that there was not a simple relationship between genes and duplexes: (a) the 3′-terminal gene, NP, was present in two duplexes, the larger of which contained an additional oligonucleotide that may represent the template for the viral leader RNA; (b) the largest band in the group yielded more than twice as many oligonucleotides as any of the other duplexes and appears to represent a pair of duplexes containing the P and F₀ genes. The characteristic oligonucleotides of the L gene were identified separately; their abundance relative to the molecular weight of this gene suggested that the 5′ side of the viral genome is guanine-poor, compared to the 3′ side. Oligonucleotides of the full-length complement of the viral genome (50 S plus-strand) and of positive-sense virus messenger RNAs were also identified. These data provide a foundation for further structural analysis of the genome of this model paramyxovirus and its defective-interfering (DI) deletion mutants.     

Size and genetic content of virus-specific RNA in myeloblasts transformed by Avian Myeloblastosis Virus (AMV)

Radiolabeled complementary DNA probes representing sequences specific for avian myeloblastosis virus (AMV) and sequences of different regions of avian leukosis virus (ALV) genome were used to analyze the gene content and size distribution of viral-specific RNA in AMV-transformed myeloblasts. The producer myeloblasts contained 8–10 times more copies per cell of virus-specific RNA than those of nonproducer (NP) myeloblasts. In the producer myeloblast, 35 S, 34 S, and 21 S virus-specific RNAs were detected. The 35 S RNA contained gag, pol, env, and c sequences; representing both the genomic and the messenger RNA of the helper virus. The 34 S RNA contained gag, pol, AMV, and c sequences, representing the genomic and the messenger RNA of AMV. The 21 S RNA of producer cells contained two different species: one contained env and c sequences and represented the glycoprotein messenger RNA of the helper virus; another contained AMV and c sequences and represented the subgenomic messenger RNA of AMV. In NP cells, two virus-specific RNAs were found; a 34 S RNA containing gag, pol, AMV, and c sequences and a 21 S RNA containing AMV and c sequences. Both RNAs hybridized to a probe specific for 5′ sequences of ALV. About 15% of pol sequences of virus-specific RNA in NP cells were found to be deleted.     

Viral genome synthesis in BHK 21 cells persistently infected with Sendai virus

We have carried out daily analysis of the viral RNA species replicated by nucleocapsids present in the cytoplasm of two different BHK 21 cell lines persistently infected with Sendai virus. At each day of analysis (both before and after crises of cytopathology, and during long periods of stable growth) DI particle size RNA was the major species of viral RNA replicated within these nucleocapsids. The molar ratio of DI RNA to standard virus (50 S) RNA species within intracellular nucleocapsids was always extremely high (from 10/1 to 340/1). Thus, there is no evidence for cyclic variations in relative predominance of standard virus and DI nucleocapsids in populations of these persistently infected cells, although “cycling” could occur at the individual cell level at infrequent intervals. Furthermore, challenge of persistently infected cultures with standard Sendai virus or incubation at 33° also failed to significantly decrease the ratio of DI nucleocapsids to standard virus nucleocapsids. It was also found that multiple DI RNA species are present (most are not resolvable by sucrose gradient analysis), and that these evolve with time—some arising and increasing with time, while others are outcompeted. No selective advantage for DI RNA of any particular size was observed. Finally, by measuring the total amount of nucleocapsids present in these persistently infected cells (as estimated by level of NP protein in purified nucleocapsids), and the degree of synthesis of viral nucleocapsid (as determined by [³H]uridine incorporation in the presence of actinomycin D), we found that the rate of viral genome replication was 30- to 40-fold lower in the persistently infected cells than it was in St acutely infected cells. These findings may indicate that the accumulated nucleocapsids in persistently infected cells are built up over a period of many weeks of slow synthesis, although turnover times for NP protein and RNA of nucleocapsids have not yet been determined in persistently infected cells.     

Defective interfering particles of human parainfluenza virus 3

A cyclic pattern of virus production was observed when human parainfluenza virus 3 (HPIV3) was serially passaged nine times in LLC-MK2 cells. Viruses produced from serial passages 8 and 9 interfered with the replication of standard HPIV3. Three subgenomic RNA species (DI-1, DI-2, and DI-3) and virus genomic RNA were detected in the progeny virions produced from cells mixedly infected with standard virus and virus from either serial passages 5 or 8. Northern blot analysis with probes representing all six HPIV3 structural protein genes revealed that DI-1 and DI-2 RNAs contain sequences from the 5' end of the standard virus genome. DI-1 RNA contains L, HN, and F specific sequences, while DI-2 RNA contains only L and HN sequences. DI-3 RNA did not hybridize with any of the probes used. The possibility that DI-3 RNA contains sequences from the 5' end of the standard virus genome is discussed. These results demonstrate that 5' defective interfering particles are generated during serial passage of HPIV3.     

Rescue of a Sendai virus DI genome by other parainfluenza viruses: implications for genome replication.

Using a defective interfering Sendai virus stock (DIH4) freed of nondefective helper virus, we found that the closely related parainfluenza viruses 1 and 3 could substitute for the Sendai virus helper in replicating DIH4, creating chimeric nucleocapsids. The morbillivirus measles and the rhabdovirus VSV could not substitute. When DIH4 is incubated intracellularly for 5 days in the absence of help, the ability of PIV3 to rescue DIH4 at this time depended on fresh Sendai virus polymerase. The PIV3 polymerase apparently can only copy the chimeric template, but not that wrapped in the homologous Sendai NP protein. These results suggest that the cis-acting RNA sequences important for genome replication, e.g., the promoter and the encapsidation site, have been conserved among these viruses, but that the interactions between the polymerase and the template protein NP are unique for each virus.     

Sequence of the 5′ end of the sendai virus genome and its variable representation in complementary form at the 3′ ends of copy-back defective interfering RNA species: Identification of the L gene terminus

Direct sequencing showed that the 5' termini of several defective interfering RNA species, both fusion and copy-back types, were homologous to the 5' terminus of the Sendai virus genome. Size analyses of spontaneously formed terminal duplexes (stems) of three copy-back defective interfering RNA species revealed variable extents of terminal complementarity, ranging from about 155 to 210 nucleotides. Direct sequencing of the 3' terminus of one of these copy-back RNA species demonstrated its complementarity to the 5'-terminal sequence of the virus genome. This copy-back sequence contained, in complementary form, the 5' terminus of the L gene, comprising the same sequence, 3'-AUUCUUUUU-5', that was previously identified at the 5' ends of the five other major Sendai virus genes.     

De novo generation of defective interfering RNAs of tomato bushy stunt virus by high multiplicity passage

Defective interfering (DI) RNAs were generated de novo in each of 12 independent isolates of tomato bushy stunt virus (TBSV) upon serial passage at high multiplicities of infection (m.o.i.) in plants, but not in any of 4 additional isolates after 11 serial passages at low m.o.i. The DI RNAs were detected in RNA isolated from virus particles and in 2.3 M LiCl-soluble RNA fractions isolated from inoculated leaves. Symptom attenuation leading to persistent infections was closely correlated with the passage in which DIs first developed. Comparisons of nucleotide sequences of 10 cDNA clones from 2 DI RNA populations and with a previously characterized TBSV DI RNA revealed the same four regions of sequence from the TBSV genome were strictly conserved in each of the DI RNAs: the virus 5' leader sequence of 168 bases; a region of approximately 200-250 bases from the viral polymerase gene; approximately 70 bases from the 3' terminus of the viral p19 and p22 genes; and approximately 130 bases from the 3' terminal noncoding region. Conservation of the sequence motif present in all of the DIs suggests that there might be a common mechanism of DI formation as well as selection pressure to maintain sequences essential for replication and encapsidation.     

Defective interfering influenza RNAs of polymerase 3 gene contain single as well as multiple internal deletions

Defective interfering (DI) RNAs of influenza virus arise from polymerase genes by internal deletions. Utilizing the recombinant DNA cloning and sequencing techniques we have determined the nucleotide sequence of two DI RNAs of L clone of A/WSN/33 (L2a-7 and L2a-17) which are of polymerase 3 origin. L2a-7 DI RNA is 659 nucleotides long and contains a single internal deletion of 1682 nucleotides (nucleotide position 273 to 1954) of P3 gene. L2a-17 DI RNA (611 nucleotides long), on the other hand, contains two internal deletions: one of 1682 nucleotides at the identical position as that in L2a-7, the other 48 nucleotides at the nucleotide position 2032 to 2079 of P3 gene. Except for a few base mismatches the sequence of DI RNAs are identical to the corresponding portion of the P3 gene including the 5' and the 3' termini. Since these two DI RNAs contain one identical deletion but differ in the other deletion as well as in base mismatches, these two DI RNAs appear to originate from a progenitor DI RNA rather than independently from the progenitor P3 gene. The sequences around the deletion point do not reflect a consensus sequence for the origin of these deletions and suggest the role of multiple mechanisms in the generation and evolution of influenza DI RNAs.     

The genomic RNA of avian reticuloendotheliosis virus REV

Purified virus obtained from a subline of chicken bone marrow cells transformed by avian reticuloendotheliosis virus (REV) was found to contain the RNA of REV in excess over the RNA of its associated helper virus REV-A. Electrophoretic and sedimentation analyses resolved these RNAs into a 28 S and a 34 S component, respectively. Comparison of these RNA species with the RNA obtained from plaque-purified preparations of REV-A confirmed that the 28 S RNA represents the genome of transforming REV. The small size of 28 S REV RNA suggests that the defectiveness of REV is due to a deletion of replicative sequences. Hybridization experiments indicated that about 25–30% of REV RNA sequences are unrelated to REV-A. These may include the putative transforming sequences of REV. REV shared 12–15 of 42 identifiable large RNase T₁-resistant oligonucleotides with REV-A. The 28 S REV RNA did not contain the transformation-specific oligonucleotides which are largely conserved among avian acute leukemia viruses MC29, MH2, and CMII or the src-specific oligonucleotides of avian sarcoma viruses. It is concluded that the sequences which are unique for REV contain a new class of avian tumor virus transforming genes.     

Paradoxical effects of Sendai virus DI RNA size on survival: inefficient envelopment of small nucleocapsids

In the assembly of nonsegmented negative-stranded RNA viruses, such as Sendai virus, the envelopment process allows extensively deleted genomes to survive by transmission from cell to cell in virus particles. To assess the impact of the sizes of such defective-interfering (DI) genomes on their survival, we performed competition tests among various species. Among copy-back DI RNAs, a 450-base species was gradually eliminated from DI virions by a 1200-base species, and the latter was independently eliminated by a 2800-base species. In each case, the smaller RNA species was synthesized and encapsidated at least as efficiently as the larger species, revealing that the level of competition was at the envelopment step in virus assembly. In contrast to the results obtained with the copy-back DI RNAs, repeated high multiplicity passage of a family of four internally deleted RNAs eliminated all but the smallest species, comprising about 1600 bases. Both sets of findings can be reconciled by the hypothesis that the efficiency of DI nucleocapsid envelopment decreases progressively when the RNA is smaller than about 1600 bases.     

Avian acute leukemia virus MC29: conserved and variable RNA sequences and recombination with helper virus.

The RNAs and proteins of five laboratory strains of defective avian acute leukemia virus MC29, differing in passage history and in some cases in their helper viruses and oncogenic spectra, were investigated for genetic variation. Earlier work based on homology with nondefective viruses of the avian tumor virus group has distinguished in 5.7-kb (kilobase) MC29 RNA a 5′ group-specific section, an internal MC29-specific section, and a 3′ group-specific section, and has determined that 5′ and internal sections together are translated into a nonstructural, gag gene-related 110K (kilodaltons) protein. The RNAs of the five MC29 strains analyzed here shared 20 conserved T₁-oligonucleotides and differed in specific sets selected from 10 variable oligonucleotides. Since some, but not all, variable and some conserved MC29-oligonucleotides were shared with helper viruses, and since the 5′ cap-oligonucleotides of MC29 RNA rescued from the same nonproducer cell varied with that of the helper virus RNA, it is concluded that genetic variation of MC29 includes recombination with helper virus. Oligonucleotide maps of the five MC29 RNAs located 8 variable oligonucleotides in their 3′ group-specific sections, only conserved oligonucleotides in their internal-specific section, and mostly conserved oligonucleotides in their 5′ group-specific section (except for variable 5′ cap-oligonucleotides and the lack of an internal oligonucleotide in one strain). The 110K proteins of the MC29 strains were electrophoretically indistinguishable, which is in accord with the conserved nature of their templates. The ratio of MC29 RNA to helper virus RNA in virions was found to reflect, by a constant, the ratio of 110K MC29 to helper viral proteins in transformed cells. MC29 RNA is proposed to consist of two genetic units: one made of the highly conserved 5′ and internal-specific RNA sections, which codes for the 110K protein and appears to serve a primary function in oncogenicity; the other made of the variable 3′ group-specific RNA sections which may affect oncogenicity indirectly.     

Intracellular equine arteritis virus (EAV)-specific RNAs contain common sequences

Equine arteritis virus (EAV) is a nonarthropod-borne togavirus. Six virus-specific RNA species have been found in EAV-infected cells having the following molecular weights: 4.3 X 10(6) (RNA1), 1.3 X 10(6) (RNA2), 0.9 X 10(6) (RNA3), 0.7 X 10(6) (RNA4), 0.3 X 10(6) (RNA5), and 0.2 X 10(6) (RNA6). RNA1 comigrates with the viral genome (M. F. Van Berlo, M. C. Horzinek, and B. A. M. Van der Zeijst, 1982, Virology 118, 345-352). All RNAs hybridized with a radio-labeled cDNA probe representing RNA6, indicating that they contain common sequences. To study this homology in more detail, RNase T1 oligonucleotide fingerprinting of the RNAs was undertaken. This confirmed the presence of common sequences and showed more specifically that the intracellular viral RNAs form a nested set. The number of oligonucleotides in RNA1, however, is only one-third of the expected value. In all aspects studied the replication mechanism of EAV differs from that of other known positive-stranded RNA viruses.