Flat revertants of temperature-insensitive transformants induced by simian virus 40 tsA mutants lose their ability to express T-antigen.

Temperature-insensitive transformants that contained simian virus 40 sequences at only one or a few sites in the rat chromosome and that were induced by a temperature-sensitive A gene mutant of simian virus 40 were used to select flat revertants (revertants that had lost the transformed phenotype). The isolation was performed at the nonpermissive temperature so as not to select against temperature-sensitive transformants. Nonetheless, all of the revertants examined had lost their ability to express the T-antigen at both temperatures, and all contained rearrangements of the integrated simian virus 40 sequences. These results are most compatible with the hypothesis that the T-antigen of simian virus 40 is required for the maintenance of the transformed state even in temperature-insensitive cell lines.     

Selection of Temperature-Sensitive Mutants During Persistent Infection: Role in Maintenance of Persistent Newcastle Disease Virus Infections of L Cells

Virus mutants (NDV(pi)) recovered from L cells persistently infected with Newcastle disease virus (NDV, Herts strain) are temperature-sensitive (ts) at 43 C, although the wild-type virus (NDV(o)) which initiated the persistent infection replicates normally at that temperature. To study the relationship between the ts marker of NDV(pi) and the other properties which distinguish this virus from NDV(o), NDV(pi) ts(+) revertants were selected at the nonpermissive temperature and NDV(o) ts mutants were generated by treating NDV(o) with nitrous acid. Spontaneously-occurring ts mutants in the Herts NDV population were also isolated. The different virus populations were characterized with regard to plaque size, virulence for eggs, and thermal stability of infectivity, hemagglutinin, and neuraminidase. The NDV(pi) ts(+) revertants, although no longer temperature-sensitive, retained NDV(pi) properties, whereas both spontaneously-occurring and mutagen-induced ts mutants remained wild-type in their other properties. These findings showed that the properties which characterized NDV(pi) were independent of the ts marker. However, the ts marker and the other markers of NDV(pi) were coselected during the persistent infection, and the combination of those markers appeared to be important in the outcome of NDV infection of L cells. NDV(pi) replicated productively in L cells, whereas NDV(o), the NDV(pi) ts(+) revertants, and the spontaneously-occurring ts mutants all yielded covert infections in L cells. The role of the selection of ts mutants in persistent infection was confirmed as follows: L cells were persistently infected with NDV(pi) ts(+) revertants and NDV(o) ts mutants. Virus recovered from the persistently infected cultures after eight cell passages was always temperature-sensitive and of smaller plaque size than the parental virus in chicken embryo cell cultures. Similar results were obtained with virus recovered from L-cell cultures persistently infected with two other velogenic strains of NDV, the Texas-GB and Kansas-Man strains. These results strongly suggest that selection of ts mutants during the persistent infection was not random and played a role in establishment or maintenance of the persistent infection, or both.     

Analysis of rpsD mutations in Escherichia coli. I. Comparison of mutants with various alterations in ribosomal protein S4.

Summary Streptomycin-independent revertants were selected from streptomycin-dependent mutants. Twenty-five out of 150 such revertants were temperature sensitive. Ribosomal proteins from 18 temperature-sensitive and 10 temperature-insensitive revertants were analysed by SDS-polyacrylamide gel electrophoresis. Seventeen of the former but none of the latter category showed an alteration of protein S4. The mutated rpsD allele of 6 temperature-sensitive revertants was transduced into a rpsL ⁺ strain. In all cases an increased suppressibility of T4 amber phages was observed. Such suppressibility was not observed in the original rpsD, rpsL strains. All 18 temperature-sensitive mutants were disturbed in the processing of 17s to 16s RNA at non-permissive temperature and the accumulated 17s RNA was degraded. Temperature-insensitive rpsD revertants could be isolated, which had gained a second alteration in S4. Such revertants, which had lost the temperature-sensitive property, were also unable to suppress growth of T4 amber phages.It is concluded that temperature-sensitive growth, inability to process 17s RNA and to assemble 30S ribosomes at non-permissive temperature as well as increased translational ambiguity are highly correlated properties in rpsD mutants.     

Mutagenesis in UV-irradiated simian virus 40 occurs predominantly at pyrimidine doublets

Phenotypic wild-type revertants from a UV-irradiated temperature-sensitive late mutant (ts BC245) of simian virus 40 (SV40) were isolated after replication in monkey cells at the nonpermissive temperature. The mutations occurring in 7 revertants were identified by DNA sequence analysis of the entire gene involved. All 10 mutations identified constituted single base substitutions, 7 of which occurred opposite pyrimidine doublets. Transitions were 3 times more abundant than transversions. Three base changes did not occur opposite pyrimidine-pyrimidine sequences. Exchange of a DNA fragment harbouring the altered base from a revertant with the corresponding fragment from the parental virus, showed that the base substitution was indeed responsible for the reversions to the wild-type phenotype (growth at the restrictive temperature). The data suggest that most base substitutions in highly UV-irradiated simian virus 40 are targeted at sites comprising two adjacent pyrimidines.     

Altered restriction endonuclease cleavage pattern of simian virus 40 DNA.

Three different groups of temperature-sensitive mutants of simian virus 40, isolated and characterized by Chou and Martin (J. Virol. 13:1101--1109, 1974), have been analyzed by using restriction endonucleases. Differences between the restriction endonuclease cleavage pattern of these mutants and that of the standard simian virus 40 strain have been mapped. These include the following observations: (i) tsD202 carries a defective HaeIII cleavage site at position 0.9 map units; (ii) tsB204 exhibits a defective HaIII site at position 0.21 and a defective HinIII site at 0.655 map units, and (iii) tsC219 carries a new HinIII site at position 0.15. We have isolated a few wild-type revertants from each of the temperature-sensitive mutant strains; each displays the endonuclease cleavage pattern of its parental temperature-sensitive strain.     

Genetics of resistance to phosphonoacetic acid in strain KOS of herpes simplex virus type 1.

A DNA- temperature-sensitive mutant of herpes simplex virus type 1 exhibiting thermolabile DNA polymerase activity, tsD9, was shown to be resistant to phosphonoacetic acid (PAA) when plated at the permissive temperature. ts+ revertants of tsD9 were PAA sensitive and exhibited DNA polymerase activity intermediate between that of the wild-type virus and tsD9, indicating that both temperature sensitivity and sensitivity to PAA are controlled by the same gene. Since the position of tsD9 on the existing herpes simplex virus type 1 linkage map is known, the locus for PAA resistance--and therefore for the structural gene for viral DNA polymerase--has been identified.     

Revertant analysis of a temperature-sensitive mutant of Newcastle disease virus with defective glycoproteins: implication of the fusion glycoprotein in cell killing and isolation of a neuraminidase-deficient hemagglutinating virus

Biological and molecular properties of a temperature-sensitive mutant (C1) of Newcastle disease virus and its revertants were analyzed. C1 exhibited three temperature-sensitive alterations (plaque formation, virion assembly, and cytopathogenicity) and several defects which were also present at the permissive temperature. C1 virions contained low amounts of hemagglutinin-neuraminidase glycopeptides and consequently were deficient in hemagglutinating and neuraminidase activities. These virions also contained defective fusion glycoproteins which rendered them poorly hemolytic and slow to penetrate cultured chicken embryo cells. The biological activities of the membrane glycoproteins were recovered sequentially in a series of plaque-forming revertants. The coreversion of hemolysis, membrane-penetrating activities, and cytopathogenicity in the first-step revertant (S1) suggested that fusion glycoproteins were major contributors to cellular destruction. This revertant also provided evidence of a role for fusion glycoproteins in virion assembly. From S1 we isolated a large-plaque-forming revertant (L1) that assembled wild-type amounts of biologically active hemagglutinin-neuraminidase glycoproteins into virions. Although it was normal for hemagglutination, L1 had less than 3% of the neuraminidase activity of the wild type, demonstrating that these two activities can be uncoupled genetically. The neuraminidase deficiency of L1 did not impair its virulence in ovo or its reproduction in cultured cells.     

Intracellular processing of the paramyxovirus F protein: critical role of the predicted amphipathic alpha helix adjacent to the fusion domain.

At a nonpermissive temperature, the group D temperature-sensitive mutants of Newcastle disease virus strain Australia-Victoria (AV) are defective in plaque formation, in inducing infected cells to fuse, and in incorporating the cleaved fusion glycoprotein, F1 + F2, into virus particles. In this study, the F protein of AV, expressed in chicken embryo cells, was able to complement these mutants in a plaque assay, identifying the F gene as the gene containing the group D temperature-sensitive lesions. The F genes of mutants D1, D2, and D3 were found to contain single mutations relative to the AV sequence, clustered within a predicted amphipathic alpha helix (AAH) adjacent to the hydrophobic amino terminus of F1. These mutant F proteins were inefficiently processed at the permissive temperature, a problem that was exacerbated at the nonpermissive temperature. Surprisingly, the AV F protein was also found to be partially temperature sensitive in processing. Its AAH is predicted to contain a break in the helix close to the D mutation sites, which are themselves predicted to further weaken the helix at this point. Interestingly, six revertants of the group D mutants were found to have an additional lesion in the AAH, repairing both the AV and mutant helices, resulting in a predicted perfect helix. The F protein of these revertants had overcome both the processing defects of the mutants and the temperature sensitivity of AV, indicating that the AAH region is critical for F protein processing. The lesions of a second group of revertants were localized within F2, suggesting an interaction with the F1 AAH region containing the original lesion.     

Gene amplification as a mechanism of reversion at the HPRT locus in V79 Chinese hamster cells

Spontaneous phenotypic revertants of hypoxanthine phosphoribosyl-transferase (HPRT) temperature-sensitive V79 Chinese hamster cells were selected by plating a temperature-sensitive mutant in HAT medium at 39 degrees C. The incidence of such revertants was approximately 2 X 10(-4) per cell. The majority of the revertants examined had increases of between three- and tenfold in their specific activity of the enzyme, and they were able to grow continuously in the presence of HAT medium at 39 degrees C. When the revertants were cultivated in the absence of HAT, they recovered their HAT-sensitive phenotype and their lowered level of HPRT. Three of the revertants were examined for their temperature inactivation profiles, and all were found to have profiles identical to the ts parent, and quite different from the V79 wild type. The kinetic properties of the cell lines were studied: the Km for both PRPP and hypoxanthine was significantly different in the temperature-sensitive cells but was not significantly altered in the revertants with respect to the ts mutants. A specific antibody to Chinese hamster brain HPRT was employed in immunoprecipitation experiments. By measuring the point at which the immunoprecipitation of the antibody to HPRT was overcome by increasing concentrations of cell supernatant, it was possible to estimate the relative amount of enzyme molecules in the cell lines. From these data, it could be concluded that the revertants overproduced an enzyme with the same immunological properties as the ts line. Southern blots of the Hind III restricted DNA from the ts mutant and two revertant cell lines were examined with an HPRT cDNA probe. This established that the HPRT gene was amplified twofold in one of the revertants, and threefold in the other. However, if the revertants were reintroduced into nonselective medium, the gene copy number declined to one. Finally, northern blots of RNA extracted from the various cell lines demonstrated that the HPRT mRNA was augmented 1.5-fold in one revertant and 1.4-fold in the other. Reintroduction into non-selective medium resulted in a decline in mRNA level for the second mutant, whereas the first mutant appeared to be stabilized. We conclude that gene amplification and concomitant amplification of messenger RNA and enzyme levels are mechanisms of phenotypic reversion at the HPRT locus in Chinese hamster cells.     

The effect of cell irradiation on mutation in ultraviolet-irradiated and intact simian virus 40

The induction of phenotypic wild-type revertants in the progeny of an unirradiated or UV-irradiated temperature-sensitive late mutant of simian virus 40 was studied after low multiplicity passages in normal or UV-irradiated confluent monkey kidney cells. The production of wild-type revertants in the progeny of undamaged tsBC245 was followed by infecting the cells at distinct times after irradiation of the cells. Mutation frequencies reached a maximum when infection was delayed for 3--4 days after irradiation of the host cells, and declined gradually thereafter. Virus grown in unirradiated cells did not show such an alteration in mutation frequency. The temporarily higher mutation frequency of virus in UV-pretreated cells is due to a transient mutator activity operating in these cells rather than to an increased number of replications performed in UV-irradiated cells. A similar time course was found for the reactivation of UV-damaged SV40. This might suggest that reactivation and mutagenesis are manifestations of the same process. The yield of mutants due to irradiation of the virus alone was enhanced when infection was delayed for some days after the cells reached confluency; UV pretreatment of the host cells did not enhance the level of mutation obtained by UV irradiation of the virus.     

Mutations in the Ion gene of E. coli K12 phenotypically suppress a mutation in the sigma subunit of RNA polymerase

We have isolated spontaneous temperature-resistant revertants of a temperature-sensitive mutation (rpoD800) in the sigma subunit of E. coli K12 RNA polymerase. These revertants still contained the rpoD800 allele. They were mucoid, and sensitive to ultraviolet light and the radiomimetic agent nitrofurantoin, which are characteristics of lon mutants. One revertant, Tr29, was mapped to the lon region of the chromosome. Lon- rpoD800 double mutants were constructed, and were phenotypically indistinguishable from the spontaneous temperature-resistant revertant. It is the degradation-deficient property of lon mutants that is responsible for the suppression of the temperature-sensitive phenotype. We show that the rpoD800 sigma polypeptide is a substrate for the ion proteolytic system, and that mutations in lon decrease the rate of mutant sigma degradation. The rate of synthesis of mutant sigma was also affected in lon- strains. The net effect of lon-mutations was to increase the concentration of mutant sigma. We conclude that the temperature-sensitive phenotype results from insufficient concentration, rather than altered function, of the mutant protein.     

Mutant of herpes simplex virus type 2 with temperature-sensitive lesions affecting virion thermostability and DNase activity: identification of the lethal mutation and physical mapping of the nuc-lesion.

We had previously shown that a temperature-sensitive (ts) mutant of herpes simplex virus type 2 strain HG52, ts13, induced a heat-labile DNase activity in infected cells (B. Francke, H. Moss, M. C. Timbury, and J. Hay, J. Virol. 26:209-213, 1978). Earlier work indicated that the mutant also possessed temperature-sensitive infectivity (I. W. Halliburton and M. C. Timbury, J. Gen. Virol. 30:207-221, 1976). In this study temperature-stable revertants of ts13 have been isolated; examination of them revealed that ts13 is a double mutant, with genetically distinct temperature-sensitive lesions affecting nuclease activity and particle stability. The lethal mutation, in the cell system studied, is the latter. Revertants, which all maintain the nuclease lesion, grew well at a high temperature. Physical mapping of the nuclease lesion placed it between 0.12 and 0.21 (fractional length) on the virus genome, quite distant from the lethal mutation at 0.64 to 0.70.     

Temperature-Sensitive Pleiotropic Revertants from a Mutant in the AROM Gene Cluster of NEUROSPORA CRASSA

Genetical and biochemical studies have been performed with revertants induced in a polyaromatic mutant (No. 58) in the arom gene cluster of Neurospora crassa. In addition to complete and partial revertants able to grow on minimal at both 25 degrees and 35 degrees , temperature-sensitive revertants capable of growth on minimal at 25 degrees but not at 35 degrees have been recovered. One of these revertants has been shown to lack biosynthetic dehydroquinase activity at both temperatures (utilizing the inducible catabolic isozyme for growth at 25 degrees ), to have dehydroshikimate reductase activity only at 25 degrees , and to form an arom aggregate having a molecular weight approximately one-half that of wild type. These results are interpreted as indicating that pleiotropic mutants in the arom gene cluster can result from missense mutations, as well as from nonsense mutations as indicated in previous studies.     

Identification of the Sites for Suppressor Mutations on the Hemagglutinin Molecule to Temperature-Sensitive Phenotype of the Influenza Virus

A temperature-sensitive (ts) mutant of the influenza virus A/WSN/ 33 strain, ts-134, possessed a defect in intracellular transport at the nonpermissive temperature and marked thermolability of hemagglutinin (HA) activity at 51 C. These were caused by a change at amino acid residue 157 from tyrosine to histidine in the HA protein. We isolated 37 spontaneous revertant clones from ts-134 at the nonpermissive temperature and determined their HA sequences. The deduced amino acid sequences demonstrated that one was a true revertant and the others were revertants with suppressor mutations, each of which had an additional amino acid change besides those of ts-134. The changed amino acids were located at 14 positions on the HA molecule, and eight of them were found in multiple revertants. These were located in five to six distinct regions on the three-dimensional structure of the HA molecule. However, the heat stability of HAs in the revertants was recovered differently depending on the sites of the changed amino acids. The kinetics of transport of the HA protein in the revertants were slightly delayed compared to the wild-type both at permissive and nonpermissive temperatures.     

Isolation of Chinese hamster ovary cells that overproduce asparaginyl-tRNA synthetase.

Temperature-resistant revertants, derived from the temperature-sensitive CHO asparaginyl-tRNA synthetase mutant, Asn-5, were isolated and characterized. Several lines of evidence indicate that the temperature-resistant phenotype of the revertants is due to their overproducing the same altered enzyme present in the Asn-5 parent.     

Spontaneous temperature-sensitive mutations in bacteriophage T7.

Attempts to recover temperature-sensitive mutations affecting genes 13 and 14 (virion proteins) in bacteriophage T7 by analysis of amber revertants were confounded by the frequent occurrence of spontaneous temperature-sensitive mutations in other genes. These incidental temperature-sensitive mutations are physically distinct from but may be functionally related to genes 13 and 14, as shown by complementation and recombination studies. The possibility that these incidental temperature-sensitive mutations represent secondary-site suppressors of the pseudonormal suppressed amber products is discussed.     

Transformation-defective virus mutants in a class of morphologic revertants of sarcoma virus transformed nonproducer cells

In studies of the viral and cellular functions involved in expression of transformation by murine sarcoma virus, selective methods have led to the isolation of morphologic revertants following mitomycin C mutagenization of nonproductively transformed mouse cells. The revertants exhibit normal growth properties, yet still contain the sarcoma virus. Further, they are as susceptible as normal cells to exogenous sarcoma virus infection. In the present studies, these revertants are shown to contain levels of sarcoma viral RNA quantitatively and qualitatively indistinguishable from that present in the parental transformed clone. Following rescue with helper leukemia virus, they release low levels of wild-type transforming virus and a large excess of transformation-defective sarcoma virus as measured by molecular hybridization. The defective viruses can be transmitted to new cells in the absence of morphologic alteration. These results provide strong evidence that the revertants contain mutant viruses defective in transforming functions. The release of wild-type sarcoma virus by cells in a revertant culture appears to occur concomitantly with the spontaneous appearance of retransformed cells. This suggests that the reversion of mutant virus to wild-type within the cell occurs as a result of reversion of a point mutation in the integrated sarcoma viral genome. The present sarcoma virus mutants appear to be the first obtained by spontaneous or chemically-induced genetic alteration of stably integrated virus in eucaryotic cells.     

Effects of temperature and antibody on the cyclid growth of vesicular stomatitis virus.

To see the effects of temperature on the interrelated cyclic production of standard and defective interfering (DI) particles of vesicular stomatitis virus, a temperature-sensitive (ts) G114 mutant was passaged successively at different temperatures and the production of the two types of viral particles as well as the ability of Chinese hamster ovary cells to survive each passage was continuously monitored. When the temperature was nonpermissive for standard virus, the synthesis of both standard and defective interfering particles was inhibited. When revertants appeared in the population, their ability to take over the infection depended on the permissiveness of the temperature for the temperature-sensitive mutant. At permissive temperatures periodic inhibition of both types of standard viruses was maintained by the production of defective interfering particles. Reverents did not become a majority of the population due to this periodic inhibition. When the conditions were nonpermissive for the mutant, revertants became the major standard virus in the population within a few passages. These findings can be understood if conditions of high and low multiplicities are dissected out together with a thorough understanding of the individual properties of each of the viral particles and of the result of interactions between them. In the presence of antiserum which neutralized only 90% of the viral particles, cyclic production of standard virus occurred, with a decline in the total amount of virus produced after each cycle. Therefore, in the presence of limiting concentrations of antiserum, the virus appeared to be able to establish a persistent cyclic growth pattern.     

Isolation and Characterization of Revertant Cell Lines VI. Susceptibility of Revertants to Retransformation by Simian Virus 40 and Murine Sarcoma Virus

The susceptibility of two classes of revertants of Simian virus 40 (SV40)-transformed 3T3 cells to retransformation by SV40 or murine sarcoma virus (MSV) was studied. Both serum-sensitive and density-sensitive revertants are not retransformable by SV40. MSV can transform both types of revertants. The MSV-transformed revertants grow to high cell densities and form colonies when suspended in semi-solid methylcellulose medium, but are unable to grow in 1% calf serum. The MSV-transformed revertants produce infectious MSV and murine leukemia virus and possess the same number of chromosomes as the untransformed revertants.