Properties of a Soluble DNA Polymerase Isolated from Rous Sarcoma Virus

The DNA polymerase of the Prague strain of Rous sarcoma virus of subgroup C and of the Schmidt-Ruppin strain of subgroup A has been solubilized. DNA polymerase purified by sucrose gradient sedimentation and chromatography on DEAE-cellulose represented less than 2% of the soluble [(14)C]protein of the virus. The enzyme was separated from 90% of the viral glycoprotein; it is probably different from the viral group-specific antigen. The sedimentation coefficient (s(20, w)) of the soluble DNA polymerase was 8 S before, and 6 S after, incubation with pancreatic RNase. The molecular weight of the 8S DNA polymerase was estimated to be about 170,000, and that of the 6S DNA polymerase to be about 110,000.Purified DNA polymerase had a high activity with 60-70S viral RNA or salmon DNA as template, but it had a low activity with heat-dissociated 60-70S RNA, influenza virus RNA, or the RNA of tobacco mosaic virus as template. Neither the 8S nor the 6S DNA polymerase had endogenous template activity. The DNA-dependent and the RNA-dependent DNA polymerase activities of the Prague strain coincided in sucrose gradients, both in the 8S and the 6S form. It is concluded that the RNA-dependent and the DNA-dependent DNA polymerase activities of the avian tumor viruses are probably due to the same enzyme.     

Systematics of RNA Tumor Viruses and Virus-Like Particles of Human Origin

[(3)H]DNA copies of avian, feline, murine, and primate RNA tumor virus genomes were synthesized in vitro by an RNA-dependent DNA polymerase reaction. These DNAs were hybridized to 60-70S RNA that had been purified from the viruses. The amount of the [(3)H]DNA hybridized yielded a measure of the genetic relatedness among the DNA preparations synthesized by the viruses. When many combinations of DNA and RNA were analyzed, the pattern of hybridization showed in some cases that the DNA copies of the viral RNA were related to each other in the same way that the natural hosts of the viruses are phylogenetically related. This pattern was observed only among the RNA leukemia viruses. The sarcoma component in sarcoma-leukemia viruses from rats and primates appeared to be unusually closely related. The mouse mammary carcinoma virus and two unclassified viruses (MPMV and Visna) appeared to be genetically distinct.A similar analysis of DNA synthesized by an RNA-dependent DNA polymerase associated with a viral-like particle obtained from the cytoplasm of human leukemic white blood cells demonstrated that this DNA occupied a space in the affinity pattern of leukemia viruses which is expected of a nucleic acid from a primate-type-C RNA tumor virus. This observation strengthens earlier evidence that components of RNA tumor viruses are associated with human leukemia.     

The Genome of RNA Tumor Viruses Contains Polyadenylic Acid Sequences

The 70S genome of two RNA tumor viruses, murine sarcoma virus and avian myeloblastosis virus, binds to Millipore filters in buffer with high salt concentration and to glass fiber filters containing poly(U). These observations suggest that 70S RNA contains adenylic acid-rich sequences. When digested by pancreatic RNase, 70S RNA of murine sarcoma virus yielded poly(A) sequences that contain 91% adenylic acid. These poly(A) sequences sedimented as a relatively homogenous peak in sucrose gradients with a sedimentation coefficient of 4-5 S, but had a mobility during polyacrylamide gel electrophoresis that corresponds to molecules that sediment at 6-7 S. If we estimate a molecular weight for each sequence of 30,000-60,000 (100-200 nucleotides) and a molecular weight for viral 70S RNA of 3-12 million, each viral genome could contain 1-8 poly(A) sequences. Possible functions of poly(A) in the infecting viral RNA may include a role in the initiation of viral DNA or RNA synthesis, in protein maturation, or in the assembly of the viral genome.     

Virus-Specific Messenger RNA and Nascent Polypeptides in Polyribosomes of Cells Replicating Murine Sarcoma-Leukemia Viruses

We present evidence that virus-specific RNA is present in polyribosomes of transformed cells replicating the murine sarcoma-leukemia virus complex and that it serves as messenger RNA for the synthesis of viral-coded proteins. Both virus-specific RNA (detected by hybridization with the [(3)H]DNA product of the viral RNA-directed DNA polymerase) and nascent viral polypeptides (measured by precipitation with antiserum to purified virus) were found in membrane-bound and free polyribosomes. Membrane-bound polyribosomes contained a higher content of both virus-specific RNA and nascent viral polypeptides. From 60 to 70% of viral RNA sequences were released from polyribosomes with EDTA, consistent with a function as messenger RNA. Maximum amounts of both virus-specific RNA and nascent viral polypeptides were found in the polyribosome region sedimenting at about 350 S.     

Comparative Properties of RNA and DNA Templates for the DNA Polymerase of Rous Sarcoma Virus

Several natural RNAs were compared with respect to their template activities for the DNA polymerase of Rous Sarcoma Virus during a 2-hr incubation period. 60-70S viral RNA was found to be a 5- to 10-fold better template than heat-dissociated Rous viral RNA, influenza virus RNA, tobacco mosaic virus RNA, or ribosomal RNA. Denatured salmon DNA is a little better, and poly(dAT) is 2-4 times better as a template for the enzyme than is 60-70S Rous viral RNA. The 60-70S RNAs of different strains of avian tumor viruses have very similar template activities for a given avian tumor virus DNA polymerase. Oligo(dT) or oligo(dC) were found to enhance the template activity of heat-dissociated Rous viral RNA 20- to 30-fold, and that of other natural RNAs tested one- to several-fold. DNA syntheses of 1-24% were obtained during a 2-hour incubation of the enzyme with the above RNA templates. The results suggest that the enzyme prefers partially doublestranded or hybrid regions of RNAs for optimal DNA synthesis, but certain regions of single-stranded RNA can also serve as templates.Poly(dAT) competes with viral RNA for purified DNA polymerase during DNA synthesis, as would be expected if RNA- and DNA-dependent DNA synthesis was performed by at least one common active site of the same enzyme.     

Specificity of the DNA product of RNA-dependent DNA polymerase in type C viruses. II. Quantitative analysis

A number of mammalian Type C viruses were analyzed for relatedness by the technique of DNA.RNA hybridization. Viral DNAs were prepared in single-stranded form from complexes with 70S viral RNA formed during endogenous polymerase reactions. Extent of hybridization was assayed with the single-strand nuclease (S-1) from Aspergillus oryzae. Results obtained indicated a high degree of viral specificity, with significant cross-reactions being observed only with viruses obtained from within a species, as in the case of mouse and cat viruses, or in the special case of woolly monkey-gibbon comparisons. Comparisons of RD-114 virus, recently determined to be of feline origin, and conventional feline Type C viruses (FeLV), revealed minimal relatedness, especially when feline virus was grown on human cells, thus indicating the possibility of coexistence of greatly disparate Type C viruses within one species. A rat-specific virus, recovered from tumors induced by murine sarcoma virus, was found to contain genetic material common to both the original mouse virus and viruses indigenous to the rat, even though only rat-specific proteins have been detected during infection by this virus.     

DNA in Uninfected and Virus-Infected Cells Complementary to Avian Tumor Virus RNA

The 70S RNA component of several avian tumor viruses was hybridized with DNA extracted from avian tumor virus-infected and uninfected chicken and Japanese quail cells. Tritium-labeled 70S RNAs from Rous sarcoma virus (RSV), Rous associated virus-1 (RAV-1), RAV-60, and Schmidt-Ruppin-RSV (SR-RSV) hybridize from 3 to 10 times more with DNA from uninfected chicken cells than with DNA from Escherichia coli, calfthymus, or baby hamster kidney cells. After infection of chicken cells with RSV(RAV-1), SR-RSV, or RAV-2, the amount of 70S avian tumor virus [(3)H]RNA hybridized increases by 1.6 times. The specificity of the hybridization reaction was shown by the specific competition of 70S SR-RSV [(3)H]RNA with 70S RNA from RSV(RAV-1), and not with RNA from Sendai virus or chicken cells. There was no difference in the hybridization of 70S RNA from RSV (RAV-1), RAV-1, or RAV-60 with DNA either from chicken cells that contain RAV-60 in a nonreplicating form or from chicken cells that do not appear to contain RAV-60. These results indicate that both types of uninfected chicken cells contain DNA that is complementary to RNA from several avian tumor viruses and that the amount of complementary DNA increases in such cells after infection with an avian tumor virus. The RNAs of genetically different avian tumor viruses appear to have indistinguishable base sequences by this technique.     

Detection and characterization of RNA tumor virus-specific DNA in cells.

RNA tumor virus-specific DNA in cells can be detected by its capacity to 1) alter the reassociation kinetics of labeled double-stranded product of viral RNA-directed DNA polymerase; 2) anneal single-stranded DNA (cDNA) synthesized by viral polymerase; or 3) hybridize labeled viral 70S (genomic) RNA. Duplexes formed with these procedures can be analyzed for fidelity of base pairing, and the integration of viral DNA into the host genome can be established with a simple but stringent technique. We illustrate this methodology as applied to detection of Rous sarcoma virus (RSV)-specific DNA in XC cells and of mouse mammary tumor virus (MMTV)-specific DNA in murine and human tissues.     

Further Characterization of the Cellular DNA Hybridizing with the RNA of Rous Sarcoma Virus

Both 70S and 4S RNAs of Rous sarcoma virus (RSV) contain nucleotide sequences homologous to those in cellular DNA. Preparations of these RNAs were found to include some adventitious cellular sRNA. Nevertheless, both 70S and 4S RSV-RNAs were found by competition techniques to contain sequences distinct from those in cellular ribosomal RNA and 4S RNA. Competition experiments also reveal substantial homology between 70S and 4SRSV-RNA. In hybridization experiments performed in formamide at low temperatures, the melting profiles of hybrids between 70S RSV-RNA and cellular DNA, and 4S RSV-RNA and cellular DNA, are essentially superimposable. It is suggested that there are at least two origins of 4S RSV-RNA in RSV-RNA preparations: degradation of 70S RSV-RNA and inclusion of adventitious cellular 4S RNA. The possibility of a specific viral 4S RNA is not excluded.     

用3'非编码区通用引物通过RT-PCR检测登革1～4型病毒RNA

目的 　根据登革病毒 (DEN) 3 端非编码区的一段高度保守序列 ,设计登革 1～ 4型通用引物 ,采用逆转录聚合酶链反应 (RT PCR)检测登革病毒 1～ 4型RNA。方法　用C6/ 3 6细胞培养登革病毒。用热酚法提取病毒RNA ,进行RT PCR扩增 ,并对扩增产物测序。结果　DEN 2 NGC株可扩增出至少 10TCID5 0 的病毒 ,测序结果与已知序列一致。DEN 1～ 4型标准株和DEN -2 -0 4与DEN 2 4 3株均能够扩增出唯一的一条特异条带。结论　应用通用引物RT PCR法可从病毒浓度少至 10TCID5 0 的感染细胞培养液中检出DEN 2 NGC株病毒 ,并且该对通用引物对于其他 3型登革病毒亦具有很好的通用性 ,其方法的特异性和敏感性亦较强。     

THE ISOLATION OF TWO ENZYME-RIBONUCLEIC ACID COMPLEXES INVOLVED IN THE SYNTHESIS OF FOOT-AND-MOUTH DISEASE VIRUS RIBONUCLEIC ACID

The foot-and-mouth disease virus-RNA polymerase complex was released from membrane particulates present in the cytoplasm of infected baby hamster kidney cells. The soluble polymerase complex was fractionated by zonal centrifugation in sucrose gradients. Two polymerase complexes (RNA and protein complex) active in the cell-free system were isolated and had S-rate ranges of 20-70S and 100-300S, respectively. The light polymerase complex contained 20S double-stranded RNA; and the heavy polymerase complex contained a polydisperse, partially RNase-resistant RNA. The cell-free product of these two polymerase complexes was analyzed by zonal centrifugation in sucrose gradients. The light polymerase complex synthesized only 20S double-stranded RNA. The product of the heavy polymerase complex contained no detectable 20S double-stranded RNA and only a peak of single-stranded RNA with S-rate corresponding to 37S viral RNA. A third polymerase complex was isolated with S-rate greater than 300S, and it contained a polydisperse, partially RNase-resistant RNA. This third polymerase complex synthesized both 37S viral RNA and 20S double-stranded RNA in the cell-free system, and it is probably the native polymerase complex still bound to cellular particulates.     

Specific RNA sequences and gene products of MC29 avian acute leukemia virus

The 28S RNA of the defective avian acute leukemia virus MC29 contains two sets of sequences: 60% are hybridized by DNA complementary to other avian tumor virus RNAs (group-specific cDNA) and 40% are hybridized only by MC29-specific cDNA. Specific and group-specific sequences of viral RNA, defined in terms of their large RNase T(1)-resistant oligonucleotides, were located on a map of all large T(1) oligonucleotides of viral RNA. Oligonucleotides representing MC29-specific sequences of viral RNA mapped between 0.4 and 0.7 unit from the 3'-poly(A) end. Oligonucleotides of group-specific sequences mapped between 0 and 0.4 and between 0.7 and 1 map unit. Cell-free translation of viral RNA yielded three proteins with approximate molecular weights of 120,000, 56,000, and 37,000, termed P120(mc), P56(mc), and P37(mc). P120(mc) contained both MC29-specific peptides and serological determinants and peptides of the conserved, internal group-specific antigens of avian tumor viruses. P120(mc) is translated only from full-length 28S RNA. Furthermore, MC29 RNA contains sequences related to the group-specific antigen gene (gag), near the 5' end, which are followed by MC29-specific sequences. We conclude that this protein is translated from the 5' 60% of the RNA, and that it includes a segment translated from the specific sequences. It is suggested that the transforming (onc) gene of MC29 may consists of the specific and some group-specific RNA sequences and that P120(mc), which is also found in transformed cells, may be the onc gene product.     

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.     

Endogenous Guinea Pig Virus: Equability of Virus-Specific DNA in Normal, Leukemic, and Virus-Producing Cells

The kinetics of hybrid formation between the RNA of BrdU-activated endogenous guinea pig virus and the DNA of leukemic, normal, or BrdU-activated guinea pig cells were measured by the technique of RNA.DNA hybridization in DNA excess. The results suggest that virus-specific sequences representing some 60-70% of the viral genome are unique (2-3 copies per haploid cell genome), while the remainder (30-40%) are reiterated (147 copies), and that the reiterated virus-specific DNA may be composed of more than one species, each having a different reiteration frequency. No difference was found in the quantity of viral DNA sequences contained in normal, leukemic, or bromodeoxyuridine-activated guinea pig cells. These data are considerably different from those reported for exogenous (infectious) oncornaviruses, where cells infected or transformed by exogenous RNA tumor viruses have been shown to contain increased amounts of virus-specific DNA. The data reported here are consistent with the contention that preexisting viral genes are activated by bromodeoxyuridine treatment. Results of hybridization experiments done at different DNA/RNA ratios suggest that although the virus-specific DNA is partly unique and partly reiterated, the viral RNA does not contain any detectable internal reiteration. Total mass of the viral RNA sequences is around 0.7 to 1 x 10(7) daltons.     

Mutation Rates,Mutation Frequencies,and Proofreading-Repair Activities in RNA Virus Genetics

The error rate displayed during template copying to produce viral RNA progeny is a biologically relevant parameter of the replication complexes of viruses. It has consequences for virus–host interactions, and it represents the first step in the diversification of viruses in nature. Measurements during infections and with purified viral polymerases indicate that mutation rates for RNA viruses are in the range of 10⁻³ to 10⁻⁶ copying errors per nucleotide incorporated into the nascent RNA product. Although viruses are thought to exploit high error rates for adaptation to changing environments, some of them possess misincorporation correcting activities. One of them is a proofreading-repair 3′ to 5′ exonuclease present in coronaviruses that may decrease the error rate during replication. Here we review experimental evidence and models of information maintenance that explain why elevated mutation rates have been preserved during the evolution of RNA (and some DNA) viruses. The models also offer an interpretation of why error correction mechanisms have evolved to maintain the stability of genetic information carried out by large viral RNA genomes such as the coronaviruses.     

Detection of La Crosse and snowshoe hare viral nucleic acids by in situ hybridization

A molecular hybridization technique was developed to detect bunyavirus RNA in cells. Complementary DNAs (cDNAs) to the small (S) RNA segment of La Crosse (LAC) virus and to a portion of the middle (M) RNA segment of snowshoe hare (SSH) virus were used as probes to detect LAC or SSH viral RNA by in situ hybridization. Protocols were developed and standardized using radiolabeled DNA probes, and adapted for use with biotin labeled probes. The in situ hybridization procedure detected an estimated 3,600 copies of viral S RNA/cell at 24 hr postinfection. In growth curve studies, LAC nucleocapsid antigen was detectable slightly before S RNA. LAC S RNA synthesis was first seen about the nucleus. By 12 hr postinfection, hybridization signal was detected throughout the cytoplasm of the cell. The LAC S RNA probe was group-specific and cross-hybridized to 5 other California group viruses. The SSH M RNA probe was type-specific.