Mapping oligonucleotides of Rous sarcoma virus RNA that segregate with polymerase and group-specific antigen markers in recombinants.

The RNase-T1-resistant oligonucleotides of two Prague Rous sarcoma viruses with temperature-sensitive (ts) DNA polymerases (DNA nucleotidyltransferases), termed ts LA 337 and 335 of one leukosis virus, RAV-6, and 20 of their recombinant progeny have been mapped relative to the 3' poly (A) terminus of the viral RNA. The resulting oligonucleotide maps have been ocrrelated with markers of the four known viral genetic elements encoded in the RNA of 10,000 nucleotides. In accord with previous results recombinant RNAs contained (i) oligonucleotides characteristic of the src gene, coding for sarcoma formation, between the poly(A) end and 2000 nucleotides and (ii) olignucleotides characteristic of the env gene, coding for the envelope glycoprotein, between 2500 and 5000 nucleo tides from the poly(A) end. (iii) A cluster of four oligonucleotides that mapped between 6000 and 8000 nucleotides from the 3' poly(A) end of each RNA was shared by both parental viruses and all recombinants. Since all other map segments of our recombinants failed to segregate with the ts- or wild-type markers of the parental DNA polymerase gene (pol), it was concluded that the ts pol lesion maps in this RNA segment. (iv) The 5' segment of each recombinant RNA contained a cluster of four to five oligonucleotides whose parental origin correlated with an electrophoretic marker of one of the parental virion proteins, p27, a major product of the viral gag gene. The gene order 5'-gag-pol-env-src-poly(A) is consistent with our data.     

Large T1 oligonucleotides of Moloney leukemia virus missing in an env gene recombinant, HIX, are present on an intracellular 21S Moloney viral RNA species.

HIX, a recombinant derived from Moloney leukemia virus, has an envelope glycoprotein different from that of the Moloney virus. HIX and Moloney viruses share the majority of the large T1 oligonucleotides derived from their genomes but each possesses a set of distinctive oligonucleotides that lie clustered in corresponding regions in the 3' halves of their oligonucleotide maps. These regions presumably contain envelope glycoprotein coding sequences. The type C viral envelope glycoprotein is believed to be translated from a 21S RNA. Thus, at least part of the region of the Moloney virus genome that is altered relative to HIX was expected to be present on such a species. To test this prediction, we purified an intracellular 21S Moloney viral RNA species and analyzed its large T1 oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. This RNA contains one T1 oligonucleotide that is probably derived from the 5' end of the Moloney virus genome, the Moloney virus T1 oligonucleotides that are missing in HIX, and those that lie to their 3' side on the Moloney virus T1 oligonucleotide map.     

Recombination between viral and cellular sequences generates transforming sarcoma virus

A series of sarcoma viruses has been obtained from tumors induced by transformation-defective (td) mutants of the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup A (SR-A). The RNA sequences of these "recovered avian sarcoma viruses" (rASVs) were compared with those of td mutants and of SR-A by oligonucleotide fingerprinting. Of six sarcoma-specific oligonucleotides present in SR-A RNA, three to six were missing in the RNAs of the four td mutants examined. All six isolates of rASV examined have regained these six oligonucleotides. In addition, most rASV RNAs have three new oligonucleotides not present in the RNA either of td mutants or of SR-A. The newly obtained oligonucleotides are located between 800 and 2600 nucleotides from the 3' end of rASV RNA, which corresponds to the src region of SR-A RNA mapped previously. Furthermore, viral RNAs of two td mutants isolated from a clone of rASV lack most src-specific oligonucleotides, including the three new ones. No differences were found among RNAs of td, SR-A, and rASV in the regions outside of src. Our results indicate that RNA sequences that rASVs have acquired from cells in the process of conversion from td virus to transforming virus are mapped within the src region and segregate with the transforming function. Some of the sequences are new and some are identical with those in SR-A RNA.     

Evidence for crossing-over between avian tumor viruses based on analysis of viral RNAs

The RNAs of several avian tumor virus recombinants that had inherited their focus-forming ability from a sarcoma virus and their host range marker from a leukosis virus were investigated. Electrophoretic analyses showed that the cloned sarcoma virus recombinants contained only size class a RNA, although they had acquired a marker that resided on class b RNA in the leukosis virus parent. Class a RNA of different recombinant clones, derived from the same pair of parental viruses and selected for the same biological markers, differed slightly in electrophoretic mobility from each other and from the parental sarcoma virus. They were also found to have different fingerprints of RNase T1-resistant oligonucleotides.The average complexity of the 60-70S RNA prepared from Prague Rous sarcoma virus of subgroup B was estimated to be 3.5 x 10(6) daltons from the size of 20 RNase T1-resistant oligonucleotides, which represented 3.9% of the RNA and that of a recombinant to be 3.3 x 10(6) daltons from 23 oligonucleotides, which represented 4.7% of the RNA. This result suggests that the genome of wild-type and of recombinant RNA tumor viruses is polyploid.The sum of these observations led us to propose that recombination among avian tumor viruses occurred by crossing-over between homologous pieces of nucleic acid.     

Distribution of envelope-specific and sarcoma-specific nucleotide sequences from different parents in the RNAs of avian tumor virus recombinants.

The distribution of leukosis-virus- and sarcoma-virus-specific oligonucleotide sequences was investigated in the RNAs of viral recombinants selected for an envelope gene (env) from a leukosis parent and a sarcoma gene (src) from a sarcoma parent. For this purpose 20 to 30 RNase-T1-resistant oligonucleotides were chemically analyzed and mapped within the 10,000 nucleotides of each viral RNA relative to the 3'-poly(A) end. The resulting oligonucleotide maps were compared. Proceeding from the 3' to the 5' end, the maps of four recombinants contained: (i) in a segment of 2000 nucleotides, three to four src-specific oligonucleotides, so identified because they were shared only with the sarcoma parent; and (ii) in a segment of 8000 nucleotides, 20 oligonucleotides shared with the leukosis parent, of which six to seven were also shared with the sarcoma parent. Two other recombinants contained: (1) in a segment of 2000 (one) or 3000 (the other) nucleotides, three src-specific oligonucleotides; (ii) in a segment of 3000 (one) or 2000 (the other) nucleotides, five (one) or four (the other) oligonucleotides, all or some of which are env-specific, because they were shared with the leukosis parent; (iii) in a segment of 5000 nucleotides (both), 11 functionally unidentified sarcoma-virus-derived oligonucleotides, of which seven were also shared with the leukosis parent. The map locations of parental oligonucleotides were not changed in recombinants and all viral strains tested shared six to eight highly conserved oligonucleotides at equivalent map locations. The partial map -env-src-poly(A) emerged from the analyses of these recombinants.     

Most sequence differences between the genomes of the Akv virus and a leukemogenic Gross A virus passaged in vitro are located near the 3'' terminus.

The 70S genomic RNA of nonleukemogenic AKR(Akv) virus was compared to that of an in vitro passaged, cloned, leukemogenic Gross A virus by fingerprint and sequence analysis. Fifty-seven of the large ribonuclease T1-resistant oligonucleotides of each virus have the same electrophoretic mobility and sequence. Thirteen large ribonuclease T1 oligo nucleotides are unique to the Gross A virus, whereas five are unique to Akv. Four of the oligonucleotides unique to each virus are related by one or two simple base changes. Five of the differences in oligonucleotides are located in the region of the genome that codes for the gag and pol genes. Eight of the differences are located near the 3' poly(A) terminus of the virus. The origins and biological consequences of these differences are discussed.     

Hybrid hepatitis B virus-host transcripts in a human hepatoma cell.

The human PLC/PRF/5 hepatoma cell line (the Alexander cell) contains at least seven copies of hepatitis B virus (HBV) DNA integrated in its genome; but it selectively expresses the HBV surface antigen (HBsAg) gene and perhaps low levels of the core gene. We have prepared a cDNA library from PLC/PRF/5 cell poly(A)+ RNA and isolated clones containing HBV sequences. Hybridization experiments show that the great majority of HBV-specific RNAs in this cell line contain HBsAg coding sequences and are presumably derived from the HBsAg gene. Primer extension experiments show that these HBsAg mRNAs are, however, derived from multiple initiation sites in the HBsAg gene and involve two promoters: one at the 5' end of the gene that can produce a protein of 45 kDa, and one located in the pre-S region that can produce two proteins of 31 kDa and the mature HBsAg, 25 kDa, respectively. The HBV RNAs are hybrid RNA species that contain HBV sequences at their 5' ends and host DNA sequences at the 3' ends. The great majority of these hybrid RNAs are transcribed from two closely related yet distinct HBV integrants. The viral-host sequences of these two related hybrid RNAs suggest that the related HBV sequences were generated from a parental fragment via duplication, translocation, and mutagenesis. These processes may play a role in HBV-related oncogenesis.     

5''-Terminal nucleotide sequence of Semliki forest virus 18S defective interfering RNA is heterogeneous and different from the genomic 42S RNA

An 18S defective interfering (DI) RNA population was isolated from the cytoplasm of baby hamster kidney (BHK-21) cells infected with Semliki Forest virus from the 10th undiluted passage. The RNA was approximately 2000 nucleotides long and contained a 5'-terminal cap with the structure 7mGpppAp and a poly(A) tract. The DI RNA contained large TI oligonucleotides derived from both the 42S RNA-specific region and the 3' one-third of the genome common to 42S and 26S RNA. Several of the large oligonucleotides were present in nonequimolar ratios, suggesting that the RNA population is heterogeneous. As this population is approximately uniform in size, this suggests that the DI RNAs may be generated by internal deletions involving different regions of the genome. The 5'-terminal cap-containing RNase T1 oligonucleotide was isolated by two-dimensional gel electrophoresis from uniformly 32P-labeled RNA and shown to be heterogeneous. Five T1 caps with the structure 7mGpppA-U(A-U)nC-A-U-G(n = 4-8) were identified. The two major T1 caps (n = 4 and 6) comprised about 75% of the total yield of T1 caps. The T1 caps were different from the genomic 42S RNA T1 cap (7mGpppA-U-G), suggesting that the extreme 5' end of the genome is not conserved in this defective interfering RNA.     

A host dicer is required for defective viral RNA production and recombinant virus vector RNA instability for a positive sense RNA virus

Defective interfering (DI) RNAs, helper virus-dependent deletion mutant RNAs derived from the parental viral genomic RNA during replication, have been described for most RNA virus taxonomic groups. We now report that DI RNA production in the chestnut blight fungus, Cryphonectria parasitica, persistently infected by virulence-attenuating positive sense RNA hypoviruses, depends on one of two host dicer genes, dcl-2. We further report that nonviral sequences that are rapidly deleted from recombinant hypovirus RNA virus vectors in wild-type and dicer gene dcl-1 deletion mutant strains are stably maintained and expressed in the Deltadcl-2 mutant strain. These results establish a requirement for dcl-2, the C. parasitica dicer gene responsible for antiviral defense and generation of virus-derived small interfering RNAs, in DI RNA production and recombinant virus vector RNA instability.     

Characterization of the transforming gene of Fujinami sarcoma virus.

The src gene present in all avian sarcoma viruses is not present in the genome of Fujinami sarcoma virus, a potent sarcoma-inducing virus in chickens. Fujinami virus is defective and requires helper virus for replication. RNA from a mixture of helper and transforming viruses consists of two components, 35S and 28S. Oligonucleotide fingerprinting of each RNA component revealed that the 35S component was identical to the RNA of the helper virus. Thus, the genome of Fujinami virus must be the 28S RNA, which corresponds approximately to a molecular weight of 1.7 x 10(6) or 5300 nucleotides. Fujinami viral RNA shares several oligonucleotides with helper viral RNA at both 3' and 5' ends but contains a unique sequence of at least 3000 nucleotides in the middle of the genome. Fujinami viral RNA contains no src-specific oligonucleotides of the Rous sarcoma virus genome and did not hybridize with DNA complementary to the src sequences. The 60,000-dalton src protein of Rous sarcoma virus was undetectable in Fujinami virus-transformed cells. Instead, these transformed cells contain a protein of 140,000 daltons precipitable by antisera against virion proteins, which is likely to be the transforming protein of this virus.     

Segment-specific inverted repeats found adjacent to conserved terminal sequences in wound tumor virus genome and defective interfering RNAs.

Defective interfering (DI) RNAs are often associated with transmission-defective isolates of wound tumor virus (WTV), a plant virus member of the Reoviridae. We report here the cloning and characterization of WTV genome segment S5 [2613 base pairs (bp)] and three related DI RNAs (587-776 bp). Each DI RNA was generated by a simple internal deletion event that resulted in no sequence rearrangement at the deletion boundaries. Remarkably, although several DI RNAs have been in continuous passage for more than 20 years, their nucleotide sequences are identical to that of corresponding portions of segment S5 present in infrequently passaged, standard, transmission-competent virus. The positions of the deletion breakpoints indicate that the minimal sequence information required for replication and packaging of segment S5 resides within 319 bp from the 5' end of the (+)-strand and 205 bp from the 3' end of the (+)-strand. The terminal portions of segment S5 were found to contain a 9-bp inverted repeat immediately adjacent to the conserved terminal 5'-hexanucleotide and 3'-tetranucleotide sequences shared by all 12 WTV genome segments. The presence of a 6- to 9-nucleotide segment-specific inverted repeat immediately adjacent to the conserved terminal sequences was found to be a feature common to all WTV genome segments. These results reveal several basic principles that govern the replication and packaging of a segmented double-stranded RNA genome.     

Slow virus infection: replication and mechanisms of persistence of visna virus in sheep.

The influence of the age and immune status of the host on the slow replication and persistence of visna virus in sheep was studied. Twenty-five randomly bred fetal American lambs were inoculated intracerebrally with visna virus. Eight of these fetuses were immunosuppressed by thymectomy and antiserum to lymphocytes before inoculation. Fetuses were sacrificed sequentially, and tissues were processed for viral quantitation. No exponential increase of virus occurred in either the normal or immunosuppressed fetuses, and virus was recovered mainly by explanation of tissues. This finding indicated that the viral genome was present in tissue cells but that the extent of replication in the early phase of infection was restricted by factors unassociated with maturation or immune status of the host. In addition, virus isolated from the peripheral blood leukocytes of a sheep one year after inoculation was antigenically distinct from the plaque-purified virus used for inoculation. This distinction suggested that a major antigenic shift of the agent had occurred and provided another mechanism for the maintenance of the persistent infection.     

A chimeric plasmid from cDNA clones of poliovirus and coxsackievirus produces a recombinant virus that is temperature-sensitive.

We have inserted a 405-nucleotide fragment from the 5' noncoding region of the coxsackievirus B3 genome into an infectious cDNA copy of the poliovirus RNA genome. Transfection of plasmid DNA containing this hybrid genome construct into cultured monkey cells produced infectious virus. Recombinant virus stocks displayed a temperature-sensitive phenotype for growth at 37 degrees C. We found that there is a dramatic reduction in the level of viral proteins and viral RNAs in HeLa cells infected with the recombinant at 37 degrees C compared to that obtained at 33.5 degrees C. Thus, insertion of a portion of the coxsackievirus genome into the poliovirus genome produces a temperature-sensitive recombinant virus. That this substitution occurs in a region of the poliovirus genome that, to date, has not been shown to have any coding function suggests that RNA sequences involved in replicase recognition or ribosome binding may contribute to the temperature-sensitive phenotype of the recombinant virus.