Virus Specific RNA in Cells transformed by RNA Tumour Viruses

Virus specific RNA comprises 5% of the nuclear RNA and 0.5–1.0% of the cytoplasmic RNA of cells transformed by murine sarcoma viruses. Even cryptically transformed cells which possess no detectable virus or viral proteins synthesize detectable amounts of viral RNA.     

Tumour Induction by Simian Adenovirus SA7 DNA Fragments

DNA extracted from simian adenovirus SA7 can induce tumours in hamsters and is infectious in tissue culture^1,2. We now have evidence that fragments of the SA7 genome can initiate tumours in newborn hamsters.     

Induction of an altered DNA conformation by an inversion rearrangement in the 5'-flanking DNA of a U1 RNA gene

The anomalous electrophoretic behavior of a 686 base pair restriction fragment containing an in vitro-generated inversion mutation within the enhancer region of a chicken U1 RNA gene was investigated. This DNA fragment migrated with an abnormally slow mobility in polyacrylamide gels but migrated normally in agarose gels relative to the wild type fragment of identical size and base composition. In polyacrylamide gels, the degree of retardation was enhanced at low temperature, a phenomenon associated with bent DNA. A putative site of bending was localized at or near one end of the inverted region. These data suggest that the altered DNA conformation results from the juxtaposition of two normally remote DNA sequences.     

Hormone-activated Expression of the C-type RNA Tumour Virus Genome

THE concept of vertical transmission of specific viral information, particularly that possibly associated with the induction of malignancy in mice has been postulated. Moreover, it has been hypothesized that this genetic information may be expressed either in the form of whole virus in certain selected laboratory animal strains or as the operon involved in regulating cellular replication (oncogene)¹. To detect this proposed genetically transmitted message, one uses a group specific antisera against the C-type RNA tumour viruses having as one of its components, gs-3, first described by Gerring et al.². A similar group specific antigen has subsequently been reported by Schafer³, Gilden⁴ and Sarma et al.⁵ and designated “interspec”, meaning that the antigen is common to the internal components of the C-type RNA virion of several mammalian species.     

The Rate and Spectrum of Spontaneous Mutations in a Plant RNA Virus

Knowing mutation rates and the molecular spectrum of spontaneous mutations is important to understanding how the genetic composition of viral populations evolves. Previous studies have shown that the rate of spontaneous mutations for RNA viruses widely varies between 0.01 and 2 mutations per genome and generation, with plant RNA viruses always occupying the lower side of this range. However, this peculiarity of plant RNA viruses is based on a very limited number of studies. Here we analyze the spontaneous mutational spectrum and the mutation rate of Tobacco etch potyvirus, a model system of positive sense RNA viruses. Our experimental setup minimizes the action of purifying selection on the mutational spectrum, thus giving a picture of what types of mutations are produced by the viral replicase. As expected for a neutral target, we found that transitions and nonsynonymous (including a few stop codons and small deletions) mutations were the most abundant type. This spectrum was notably different from the one previously described for another plant virus. We have estimated that the spontaneous mutation rate for this virus was in the range 10⁻⁶−10⁻⁵ mutations per site and generation. Our estimates are in the same biological ballpark that previous values reported for plant RNA viruses. This finding gives further support to the idea that plant RNA viruses may have lower mutation rates than their animal counterparts.THE rate of spontaneous mutation is a key parameter to understanding the genetic structure of populations over time. Mutation represents the primary source of genetic variation on which natural selection and genetic drift operate. Although the exact value of the mutation rate is important for several evolutionary theories, accurate estimates are available only for a handful of organisms. RNA viruses show mutation rates that are orders of magnitude higher than those of their DNA-based hosts and in the range of 0.03–2 per genome and replication round (Drake et al. 1998; Drake and Holland 1999; Chao et al. 2002). This difference results from the lack of proofreading activity of the virus-encoded RNA-dependent RNA polymerases (Steinhauer et al. 1992). The evolutionary causes of such elevated mutation rates remain unknown and it is commonly accepted that they may be beneficial as a mechanism to escape from the strong selective pressures imposed by the host''s defense mechanisms, although not necessarily evolved in response to natural selection (Elena and Sanjuán 2005; Clune et al. 2008). Indeed, in the short term, a too high mutation rate has pernicious effects on viral fitness since most of the mutations produced are deleterious (Bonhoeffer et al. 2004; Sanjuán et al. 2004).In the case of plant RNA viruses, it has been repeatedly reported that their populations are highly genetically stable (Rodríguez-Cerezo et al. 1991; Fraile et al. 1997; Marco and Aranda 2005; Herránz et al. 2008) in comparison with their animal counterparts, although reports of higher substitution rates also exist (Fargette et al. 2008; Gibbs et al. 2008). This peculiar behavior might be due in part to stronger stabilizing selection, weaker immune-mediated positive selection (García-Arenal et al. 2001), the existence of strong bottlenecks during cell-to-cell movement and systemic colonization of distal tissues (Hall et al. 2001; Sacristán et al. 2003; Li and Roossinck 2004), severe bottlenecks during vector-mediated transmission (Ali et al. 2006; Moury et al. 2007; Betancourt et al. 2008), or differences in the replication mode compared to lytic animal viruses (French and Stenger 2003; Sardanyés et al. 2009). Another more obvious possibility is that, indeed, plant viruses have lower mutation rates than other RNA viruses. Indeed the only two available direct estimates of mutation rates for plant viruses are both in the lower side of the range usually accepted for animal riboviruses: 0.10–0.13 per genome and generation for Tobacco mosaic virus (TMV) (Malpica et al. 2002) and 0.28 for Tobacco etch virus (TEV) (Sanjuán et al. 2009). However, none of these estimates is perfect. Although in the TMV experiments particular care was taken to measure mutation rate in a long target protected from the action of purifying selection (hence deleterious mutations remain in the population), uncertainties related to the number of infection cycles elapsed during the mutation–accumulation phase and the fraction of mutations that produced a selectable phenotype exist. In the case of TEV, the estimate should be taken as an upper limit because selection was operating during the mutation–accumulation phase. Furthermore, the estimate is of the same order of magnitude as the methodological error.To further evaluate whether plant RNA viruses show unusually low mutation rates, we have developed a new empirical method that allows estimating the mutation rate and the spectrum of spontaneous mutations produced during an in vivo infectious process. The viral model system chosen for this experiment has been TEV (family Potyviridae, genus Potyvirus), a prototypical example of positive sense RNA virus that has also become a model for virus experimental evolution. The method is based on the analysis of the temporal accumulation of mutations in a 1536-nt-long neutral viral target. TEV genome size is 9539 nt long (GeneBank {"type":"entrez-nucleotide","attrs":{"text":"DQ986288","term_id":"115606139","term_text":"DQ986288"}}DQ986288) and encodes a large polyprotein of 346 kDa that self-processes into at least nine mature proteins. One of these proteins, the nuclear inclusion protein b (NIb) has RNA-dependent RNA-polymerase activity (Urcuqui-Inchima et al. 2001). This protein forms inclusions in the nucleus of infected plants and is required in the cytoplasm for replication complexes during viral RNA synthesis. NIb is the only protein that can be provided functionally in trans (Li and Carrington 1995). Taking advantage of this property, we infected Nicotiana tabacum transgenic plants expressing TEV NIb and followed the accumulation of mutations in the viral copy of NIb. This experimental system minimizes the effect of purifying selection on the virus-encoded NIb due to complementation by the transgene.     

Little Evidence for Synergism Among Deleterious Mutations in a Nonsegmented RNA Virus

Several models have been proposed to account for the segmentation of RNA viruses. One of the best known models suggests that segmentation, and mixing of segments during coinfections, is a way to eliminate deleterious mutations from the genome. However, for validity, this model requires that deleterious mutations interact in a synergistic way. That is, two mutations together should have a more deleterious effect than the result of adding their individual effects. Here I present evidence that deleterious mutations in foot-and-mouth disease virus produce a decline in fitness but that the relationship between the number of mutations fixed and the magnitude of fitness decline is compatible mainly with a nonsynergistic model. However, the statistical uncertainties associated with the data still give some room for the existence of very weak synergistic epistasis. Received: 2 November 1998 / Accepted: 19 April 1999     

Surface Alterations of Cells Carrying RNA Tumour Virus Genetic Information

CELLS transformed by the DNA tumour viruses, polyoma virus and SV40, are agglutinated by lectins such as wheat germ agglutinin¹, concanavalin A (Con A)² and soybean agglutinin³. Agglutination in these cases presumably reflects changes in the cell surface related to the transformed properties of the cell; studies with a temperature-dependent mutant of polyoma virus has shown that cell surface changes are controlled by viral genes⁴. Here we describe experiments in which we investigated the agglutinability of cells transformed by RNA tumour viruses. One recent report had suggested that cells transformed by RNA tumour viruses were not specifically agglutinated⁵, whereas a second more recent report claimed the specific agglutination of cells transformed by RSV⁶. We find that transformed rat, mouse and cat cells that replicate the sarcoma-leukaemia virus complex of murine (MSV) and feline (FeSV) origin are strongly agglutinated by Con A, but mouse and human cells that replicate the murine and feline leukaemia virus components alone are not agglutinated. The ability to agglutinate is rapidly acquired by normal mouse cells on infection with the murine sarcoma virus at a rate that parallels virus replication. In contrast to the results obtained with cells producing virus, non-virus-producing transformed hamster and mouse cells that synthesize virus-specific RNA are either not agglutinated or are agglutinated to a lesser degree. These results suggest that the cell surface alterations responsible for agglutination are not necessarily associated with the transformed state of the cell, but rather with the possession of sarcoma virus-specific information.     

Inhibitors of the RNA and DNA Dependent Polymerase Activities of RNA Tumour Viruses

RNA viruses of several animal leukaemias and sarcomas possess what seems so far to be a unique enzyme—an RNA dependent DNA polymerase^1–6. Specific inhibitors of the viral enzyme will not only be useful in the analysis of its possible role in neoplasia, but might provide drugs for leukaemia and cancer therapy.     

Mutations That Increase DNA Binding by the Processivity Factor of Herpes Simplex Virus Affect Virus Production and DNA Replication Fidelity

The interactions of the herpes simplex virus processivity factor UL42 with the catalytic subunit of the viral polymerase (Pol) and DNA are critical for viral DNA replication. Previous studies, including one showing that substitution of glutamine residue 282 with arginine (Q282R) results in an increase of DNA binding in vitro, have indicated that the positively charged back surface of UL42 interacts with DNA. To investigate the biological consequences of increased DNA binding by UL42 mutations, we constructed two additional UL42 mutants, including one with a double substitution of alanine for aspartic acid residues (D270A/D271A) and a triple mutant with the D270A/D271A and Q282R substitutions. These UL42 mutants exhibited increased and prolonged DNA binding without an effect on binding to a peptide corresponding to the C terminus of Pol. Plasmids expressing any of the three UL42 mutants with an increased positive charge on the back surface of UL42 were qualitatively competent for complementation of growth and DNA replication of a UL42 null mutant on Vero cells. We then engineered viruses expressing these mutant proteins. The UL42 mutants were more resistant to detergent extraction than wild-type UL42, suggesting that they are more tightly associated with DNA in infected cells. All three UL42 mutants formed smaller plaques on Vero cells and replicated to reduced yields compared with results for a control virus expressing wild-type UL42. Moreover, mutants with double and triple mutations, which contain D270A/D271A mutations, exhibited increased mutation frequencies, and mutants containing the Q282R mutation exhibited elevated ratios of virion DNA copies per PFU. These results suggest that herpes simplex virus has evolved so that UL42 neither binds DNA too tightly nor too weakly to optimize virus production and replication fidelity.Processivity factors of DNA polymerases promote long-chain DNA synthesis by preventing dissociation of the DNA polymerase from the primer/template. Processivity factors also can influence DNA replication fidelity, as indicated by numerous in vivo and in vitro studies (1-3, 5, 6, 11, 12, 18, 28, 36). A major class of processivity factors known as “sliding clamps” includes proliferating cell nuclear antigen (PCNA) of eukaryotic cells (23) and gp45 of T4 bacteriophage (27). Sliding clamps are homodimers or homotrimers that encircle DNA and interact with the catalytic subunits (Pols) of their cognate DNA polymerases to promote processive DNA synthesis.A second class of processivity factors includes those encoded by herpesviruses and is exemplified by herpes simplex virus (HSV) UL42. UL42 forms a heterodimer with the HSV Pol. Both subunits are essential for production of infectious virus and for viral DNA replication (20, 26). UL42 can stimulate long-chain DNA synthesis by Pol, and template challenge experiments established that this stimulation is due to increased processivity (15). In addition to its interaction with Pol, which is mediated by the C terminus of Pol, UL42 also binds DNA directly with high affinity (14, 15, 30, 37). This mode of DNA binding differs from that of sliding clamps, which do not form high-affinity direct interactions with DNA (13) but must be loaded onto DNA with the aid of ATP-dependent clamp loaders for their normal functioning (16). Nevertheless, the structure of UL42 is very similar to a monomer of the sliding clamp PCNA (39). Like other processivity factors, UL42 also plays a role in maintaining DNA replication fidelity both in vivo and in vitro (5, 18).The “back face” (opposite face to the side that binds Pol) of a UL42 molecule contains several positively charged residues. By titrating the effects of cations on UL42 DNA binding, it was determined that charge-charge interactions are involved in the interaction (22). Substitutions of alanine for any of four arginine residues on the back face of UL42 resulted in substantial reductions in DNA binding without affecting the binding to peptide corresponding to the C terminus of Pol in vitro (31), while substitutions of lysine for arginine had little or no effect on DNA binding affinity (22). A UL42 mutant (Q282R) containing a substitution of arginine for a negatively charged glutamine residue on the back face of UL42 exhibited a fourfold increase in DNA binding without altering the interaction with the Pol C-terminal peptide in vitro (22). Therefore, the positively charged surface of UL42 is important for the interaction between UL42 and DNA. A question raised by these studies is whether UL42 could bind DNA so tightly as to affect HSV replication.Mutant viruses engineered to encode individual arginine-to-alanine substitution mutations in UL42 exhibit several phenotypes, including a delayed onset of viral DNA replication, reduced virus yields, and reduced fidelity of DNA replication (18). Recombinant viruses expressing UL42 with multiple substitutions of alanine for arginine residues exhibit even greater effects on viral DNA replication and virus yields (19). Thus, reducing DNA binding by UL42 deleteriously affects viral growth and DNA replication fidelity. However, these studies did not address whether increasing DNA binding by UL42 would have any effects on viral DNA replication, replication fidelity, or virus production.In this study we engineered two new UL42 mutant proteins (with the D270A/D271A or Q282R/D270A/D271A mutations) that contain less negative charge on the back face and examined the effects of these substitutions on DNA and Pol peptide binding. In addition, recombinant viruses were constructed to examine the effect of these multiple substitutions and the single Q282R substitution on virus production, DNA replication, and the fidelity of DNA replication.     

RNA-DNA Covalent Bonds Between the RNA Primers and the DNA Products Formed by RNA Tumor Virus DNA Polymerase

Initiation of DNA synthesis by endogenous RNA primer molecules was studied with three different RNA tumor viruses. The influence of the method of virus disruption on the observed RNA-DNA bonds was ascertained. Ether disrupted virions of both murine leukemia virus (MuLV) and the B77 strain of avian sarcoma virus (B77 virus) have rC-dC and rA-dA covalent linkages between RNA primers and newly synthesized DNA. None of the 14 other possible bonds were formed. Ether-disrupted virions of avian myeloblastosis virus (AMV) have rU-dC and rA-dA linkages. In contrast, work reported herein and from other laboratories shows that Nonidet P-40 (NP-40)-disrupted virions of all three viruses have only the rA-dA junction. Studies with virus particles which were first disrupted with ether and then treated with NP-40 indicated that the detergent treatment disallowed the formation of the ribopyrimidine-dC internucleotide bond. The same transfers are found with AMV in the presence or absence of actinomycin D, where only single-stranded DNA is formed. This finding is consistent with the notion that virtually all of the significant primers have been recognized. In contrast to mature virions, transfer experiments with ether-disrupted early harvest (5 min) MuLV showed only the rC-dC bond; the rA-dA bond was absent. The short-time harvest contains a significantly higher proportion of infectious virions than 24-h harvests. Also, since the RNA from early harvest virus is appreciably more homogenous than the RNA of mature MuLV, it is concluded that the ribopyrimidine-dC linkage is the more significant initiation event from a biochemical standpoint.     

Nucleotide Composition of RNA hybridized to Homologous DNA from Cells transformed by Avian Tumour Viruses

PART of the evidence which indicates that RNA tumour viruses replicate through a DNA intermediate¹ was the detection of DNA which is complementary to the viral RNA in leukaemic cells transformed by avian myeloblastosis virus (AMV)² and in cells transformed in vitro by avian sarcoma viruses, Schmidt-Ruppin (SR-RSV) and B-77 (ref. 3). If this DNA serves as a template for the viral RNA, it must be a copy of the entire viral genome. One of the necessary requirements for this function is that the homologous DNA has the same nucleotide composition as the viral RNA. In this study, the average base composition of the RNA which had been hybridized to homologous DNA from transformed cells was compared with the base composition of the input viral RNA. Two experimental conditions had to be met: (1) the recovery of all the ribonucleotides which had been hybridized and (2) the absence of partially hybridized ribonucleotide sequences. The first requirement called for the deletion of the treatment of DNA-RNA hybrids with pancreatic ribonuclease fraction A and ribonuclease T₁ which had been used in our previous experiments because such a treatment can cause the non-random loss of hybridized nucleotides⁴. The second requirement called for a hybridization and washing procedure in which only specifically hybridized ribonucleotide sequences would remain bound to the filters. Both of these conditions were met by using fragmented viral RNA and a modified washing procedure which excluded the use of ribonuclease. The results show that the average nucleotide composition of the hybridized RNA is identical to that of the input viral RNA.     

The effect of the Primary Structure of DNA on Induction of Mutations by Alkylating Agents

Analysis and comparison of mutation spectra is one of the major tasks of molecular biology, since mutation spectra often reveal important properties of various mutagens and proteins involved in the repair/replication systems. Mutability is known to vary significantly along the nucleotide sequence. Mutations are abundant at certain positions (mutation hotspots). In this work, we applied regression analysis based on the basic logic patterns to understand the role of the nucleotide sequence context in mutation induction. The spectra of mutations induced by various alkylating agents were studied. The nucleotide bases at positions –2, –1, +1 and +2 were shown to have the most significant effect in G : C A : T replacements.