Recombination between temperature-sensitive and deletion mutants of reovirus.

In standard pairwise crosses there was no detectable recombination between defective reovirus lacking the largest genomic segment and prototypes of the seven known classes of ts mutants. However, in such crosses between R2A (201) and the various prototypes frequencies of ts+ recombinants between 2.6 and 6.1% were observed, as others have found (Fields, 1971; Fields and Joklik, 1969). An infectious center assay was devised to measure recombination in this system, and it was found that all mixedly infected cells gave rise to ts+ recombinants in crosses between prototype ts mutants, but no recombination was detectable when the defective virus was crossed with three different ts mutants. The ts mutation of mutant R2A (201) was efficiently rescued when crossed with UV-inactivated wild-type virus but not when crossed with UV-inactivated defective virus. It is concluded from these various experiments that if there is any recombination between these defective reovirions and any known class of ts mutants it is too low to be measured by methods presently available. The kinetics of recombination were measured in cells mixedly infected with R2A (201) and R2B (352) mutants. At the earliest time progeny virus could be found in the cells the frequency of ts+ recombinants was 4.5%, and this frequency remained unchanged despite a subsequent 1,000-fold increase in progeny virus.     

Complementation between temperature-sensitive and deletion mutants of reovirus.

A very low level of complementation has been found in conventional crosses between various classes of temperature-sensitive (ts) mutants of reovirus. A more definitive test for complementation was devised through a plaque assay on cell monolayers mixedly infected with defective reovirions lacking the L1 segment and prototype ts mutants from one or other of the known classes of reovirus mutants. An increase in the number of plaques on the mixedly infected plates over that on control plates infected with defective virions or ts mutants alone indicated that the ts mutant had been complemented by the defective virus. Class A, B, D, F, and G mutants were complemented at 39 C by the defective viruses, whereas class C and E mutants were not. In tests to determine whether complementation was reciprocal it was found that the defective virions were complemented by a class G mutant but not by the class C mutant. This and previous work (D.A. Spandidos and A. F. Graham, 1975) has therefore shown that of the seven known classes of ts mutants the class C mutant is the only one that neither complements nor is complemented by the defective virions. For this reason the class C ts mutation has been assigned to the L1 segment of the viral genome.     

Properties of T antigens induced by wild-type SV40 and tsA mutants in lytic infection

T antigen in extracts of cells infected with tsA mutants is 2 to 6 times more labile at 32°C or 41°C than the antigen in extracts of cells infected with wild-type SV40, as assayed by complement fixation. The stabilities of wild-type and mutant antigens are not altered by mixing the extracts, and thus the stability is an intrinsic property of each antigen and is not determined by another component of the extract. This observation indicates that T antigen is probably the virus-coded product of the A gene. In cells infected at the permissive temperature of 32°C with a high multiplicity of either wild-type or tsA mutant virus, the amounts of T antigen are approximately equivalent and increase logarithmically during the entire period of infection, up to 96 hr. Cells infected at 32°C for 96 hr with mixtures of wild-type and tsA virus produce T antigen with the stability of wild-type, even when the infection is carried out with up to a 5 fold excess of the mutant. The more stable wild-type antigen may repress, directly or indirectly, the synthesis of the more labile mutant antigen.     

Reaction in vitro of some mutants of RNase P with wild-type and temperature-sensitive substrates

The reaction of wild-type and two mutant derivatives of RNase P have been examined with wild-type and mutant substrates. We show that a mutant derivative of tRNA(Tyr)Su3, tRNA(Tyr)Su3A15, in which the G15.C48(57) base-pair essential for folding of the tRNA moiety is altered, is a temperature-sensitive suppressor in vivo. The precursor to tRNA(Tyr)Su3A15 is cleaved in a temperature-sensitive manner in vitro by RNase P and with a higher Km compared to the precursor to tRNA(Tyr)Su3. The precursor to tRNA(Tyr)Su3A2, another temperature-sensitive suppressor in vivo in which the G2.C71(80) base-pair in the acceptor stem is changed to A2.C71(80), behaves like the precursor to tRNA(Tyr)Su3 in vitro; that is, it is not cleaved in a temperature-sensitive manner. Therefore, there are at least two ways in which a suppressor tRNA can acquire a temperature-sensitive phenotype in vivo. One of the mutant derivatives of RNase P we have tested, rnpA49, which affects the protein cofactor of the enzyme, has a decreased kcat compared to wild-type, which can explain its phenotype in vivo.     

Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants

During endocytosis in Chinese hamster ovary (CHO) cells, Semliki Forest virus (SFV) passes through two distinct subpopulations of endosomes before reaching lysosomes. One subpopulation, defined by cell fractionation using free flow electrophoresis as "early endosomes," constitutes the major site of membrane and receptor recycling; while "late endosomes," an electrophoretically distinct endosome subpopulation, are involved in the delivery of endosomal content to lysosomes. In this paper, the pH-sensitive conformational changes of the SFV E1 spike glycoprotein were used to study the acidification of these defined endosome subpopulations in intact wild-type and acidification-defective CHO cells. Different virus strains were used to measure the kinetics at which internalized SFV was delivered to endosomes of pH less than or equal to 6.2 (the pH at which wild-type E1 becomes resistant to trypsin digestion) vs. endosomes of pH less than or equal to 5.3 (the threshold pH for E1 of the SFV mutant fus-1). By correlating the kinetics of acquisition of E1 trypsin resistance with the transfer of SFV among distinct endosome subpopulations defined by cell fractionation, we found that after a brief residence in vesicles of relatively neutral pH, internalized virus encountered pH less than or equal to 6.2 in early endosomes with a t1/2 of 5 min. Although a fraction of the virus reached a pH of less than or equal to 5.3 in early endosomes, most fus-1 SFV did not exhibit the acid-induced conformational change until arrival in late endosomes (t1/2 = 8-10 min). Thus, acidification of both endosome subpopulations was heterogeneous. However, passage of SFV through a less acidic early endosome subpopulation always preceded arrival in the more acidic late endosome subpopulation. In mutant CHO cells with temperature-sensitive defects in endosome acidification in vitro, acidification of both early and late endosomes was found to be impaired at the restrictive temperature (41 degrees C). The acidification defect was also found to be partially penetrant at the permissive temperature, resulting in the inability of any early endosomes in these cells to attain pH less than or equal to 5.3. In vitro studies of endosomes isolated from mutant cells suggested that the acidification defect is most likely in the proton pump itself. In one mutant, this defect resulted in increased sensitivity of the electrogenic H+ pump to fluctuations in the endosomal membrane potential.     

Genetic analysis of adenovirus type 2. I. Isolation and genetic characterization of temperature-sensitive mutants.

Temperature-sensitive mutants which replicate normally at 33 C but poorly at 39 C were isolated from nitrosoguanidine- or nitrous acid-mutagenized adenovirus 2 by (i) testing the cytopathic effect or inclusion body-forming capacity of random plaque isolates, or (ii) reduced plaque enlargement upon shifting from 33 to 39 C. Thirty-six mutants were isolated with 33 C/39 C plaque ratios varying from 20 to 10-5. Some of these mutants could be arranged into 13 groups by the complementation test. By means of recombination analysis a provisional linear genetic map was constructed.     

Complementation of defective reovirus by ts mutants.

Defective reovirions lacking the largest (L-1) of the normal 10 genomic segments grow only in association with helper reovirus. Because of the similarity in properties of defective and infectious virions, separation of the two populations by physical methods has been unseccessful. Controlled digestion of purified virus removes the outer capsomeres of the virions. The resulting core particles containing the viral genome have a buoyant density of 1.43/ml if derived from infectious virions and of 1.415g/ml if they originate in defectives, and this difference permits ready separation of the two types of cores. With the purpose of obtaining a pure population of defective virions, L cells were co-infected with defective cores and a class E temperature-sensitive mutant which has a mutation in an early function. After three serial passages at the permissive temperature (31 C) to build up the defective population, a fourth passage was made at 39 C, the nonpermissive temperature. The virus purified from this passage was predominantly defective; it contained practically no E mutant and had a low background of wild-type virus. Complementation was thus asymmetric; the L-1 function required for growth of defective virus was supplied by the E mutant and is thus a trans-function, while defective virus did not complement the E mutation which is thus in a cis-acting function. Defective virions were indistinguishable from infectious virions except for the absence of the L-1 genomic segment in the defectives. Such defective virions could be complemented at 39 C by class A and B temperature-sensitive mutants, both of which have lesions in late functions.     

Physiological characterization of temperature-sensitive mutants of mengovirus.

Twenty-four temperature-sensitive mutants of mengovirus were characterized physiologically with respect to phenotype. The mutants were separated into four classes on the basis of viral RNA synthesis. L-67-S cells infected with five of the mutants synthesized little viral RNA at 39.5 C. These mutants are designated RNA-. One mutant is designated RNA* since its RNA synthesis is altered at both 39.5 and 31.5 C. The other mutants were divided into two groups, RNA plus or minus (25 TO 49% of wild-type RNA synthesis) and RNA plus (50 to 100% of wild-type RNA synthesis). The time of expression of the mutation in the RNA- mutants was estimated from the results of reciprocal temperature-shift experiments. The mutatation in ts12 appears to be expressed at the time RNA synthesis normally begins. The defect in three of the mutants was expressed 1 to 2 h before RNA synthesis is normally detectable. Protein synthesis is required before RNA synthesis begins when the cells are shifted from 39.5 to 31.5 C. The RNA polymerase synthesized by cells infected with these RNA- mutants at 31.5 C was stable and fully active when assayed at 39.5 C in vitro. The sedimentation profiles of the viral RNA synthesized by cells infected with RNA plus and RNA plus or minus mutants are similar to wild-type profiles with the exception of ts148. Cells infected with this RNA plus or minus mutant synthesize RNA that sediments in a sucrose gradient like replicative-intermediate RNA, but little mature viral RNA is evident. The results of step-up experiments indicate that the temperature-sensitive period for the majority of the RNA plus and RNA plus and minus mutants extends through most of the replicative cycle. The temperature-sensitive defect of four of the mutants, however, was expressed in the first hour, suggesting that some undefined early function is required for the eventual maturation of mengovirus. The virions of three of the RNA- mutants were more thermolabile than wild-type virions. Five of the RNA plus and RNA plus or minus mutants were also thermolabile. Genetic complementation at a significant level was not detectable in mixed infections of the mutants described.     

Properties of Bacillus megaterium temperature-sensitive germination mutants.

Bacillus megaterium mutants JV-9 and JV-10 are temperature sensitive for initiation of spore germination. At 46 C, they did not lose heat resistance, dipicolinic acid, or absorbance, indicating that the temperature-sensitive blocks are very early in the sequence of initiation reactions. Strain JV-9 was temperature sensitive for initiation by glucose alone, and strain JV-10 was temperature sensitive for initiation by glucose, L-leucine, L-proline, KBr, or calcium dipicolinate. The kinetics of initiation were followed after two kinds of temperature change (shift-up and shift-down) experiments. Mutant spores incubated for different times at 46 C and then shifted down to 30 C showed no significant differences in the rates of absorbance decrease, i.e., no stimulation or inhibition. Conversely, when mutant spores were incubated for different times at 30 C, a fraction of the population initiated germination, and after shift-up to 46 C an additional fraction continued initiation while a third fraction stopped. This latter fraction did initiate germination when the temperature was lowered to 30 C. The kinetics of initiation after shift-up and shift-down in temperature suggest that the early events in initiation reagents, whereas the other four initiated sensitivity for all of the above initiation reagents, whereas the other four initiated very poorly. It was suggested that the lesion in strain JV-10 may result in the formation of one temperature-sensitive protein. Revertants of strain JV-9 could not be isolated.     

Complementation analysis of measles virus temperature-sensitive mutants.

Two sets of independently isolated measles virus temperature-sensitive mutants were quantitatively tested for complementation. Analysis of the nine possible combinations of representative mutants indicated that only one pair of mutants is noncomplementing. Thus, the measles virus mutants studied to date define five complementation groups.     

Characterization of temperature-sensitive mutants of bacteriophage PM2: Membrane mutants

Summary In an effort to understand the genetic regulation of membrane morphogenesis, twenty-nine temperature-sensitive mutants of the membrane-containing bacteriophage PM2 were isolated. Characterization at restrictive temperature revealed groups showing no lysis (Groups I–IV), partial lysis (Groups V–VIII), and full lysis (Groups IX–XII) of the host Pseudomonas BAL-31. When the cell lysis data are considered in conjunction with data on stimulation of viral DNA synthesis, at least six mutant groups are defined. Analysis by gel electrophoresis of the pattern of viral proteins synthesized under restrictive conditions further divides the mutants into twelve groups. Temperature shift experiments delineate early, intermediate and late mutants. Complementation data support some of these groupings. The observed low levels of complementation and recombination are discussed in terms of gene product/genome restriction, bound to the membrane at the site of infection.It is of particular interest to membrane morphogenesis that under restrictive conditions late mutants in Groups II, III and IV make empty-appearing vesicles inside the cell that are the size of virus membranes as seen in thin sections of cells in the electron microscope. Mutants ts 1 (Group II) and ts 12 (Group III) show defects in their ability to incorporate into membranes viral structural proteins sp 13 and sp 6.6. The possibility is discussed that either of these proteins control the size and shape of the viral membrane.     

Epstein-Barr virus induces fragmentation of chromosomal DNA during lytic infection.

Pulsed-field agarose gel electrophoresis showed that fragmentation of chromosomal DNA in Raji cells was induced by infection with the P3HR-1 strain of Epstein-Barr virus (EBV). S1 nuclease treatment of the agarose plugs containing cells suggested that the majority of DNA fragments did not contain single-strand gaps. Chromosomal DNA fragmentation was inhibited by cycloheximide, indicating that protein synthesis was required for DNA fragmentation. Phosphonoacetic acid, an inhibitor of EBV DNA polymerase, did not inhibit fragmentation of chromosomal DNA. These findings suggest that EBV-specific early proteins participate in fragmentation of chromosomal DNA. Chromosomal DNA of P3HR-1 cells was also fragmented by treatment with n-butyrate plus 12-O-tetradecanoylphorbol-13-acetate (TPA), which induced activation of latent EBV genome following viral replication. In addition, fragmentation of DNA preceded cell death during lytic infection. These results suggest that fragmentation of chromosomal DNA is generally induced during EBV replication and probably contributes to the cytopathic effect of EBV. The role of DNA fragmentation in death of infected cells is discussed in relation to apoptosis.     

Toxoplasma gondii: isolation and preliminary characterization of temperature-sensitive mutants.

Toxoplasma gondii, strain RH, produced plaques in human fibroblast tissue cultures over the temperatures 30–41 C. Muta?enesis with N-methyl-N′-nitro-N-nitrosoguanidine yielded seven temperature-sensitive mutants that had lost the ability to form plaques at 40 C but still grew well at 33 C. No spontaneous mutants were detected. The temperature-sensitive mutants were not markedly thermolabile and adsorbed normally to tissue culture cells at 40 C. Three mutants differed from one another in their temperatures for optimal growth, and in their ability to remain infectious within cells incubated at 40 C. Both mutants that were tested were found to be markedly less virulent for mice than was the wild type RH strain.     

Isolation and genetic characterization of ethanol-resistant reovirus mutants.

To better understand the mechanism(s) by which viruses respond to chemical or physical treatments, we isolated a series of mutant strains of reovirus type 3 Dearing that exhibit increased ethanol resistance. Following exposure to 33% ethanol for 20 min, the parental strain exhibited a 5 log10 decrease in infectivity. The mutant strains, however, exhibited a 2 to 3 log10 decrease in titer following identical treatment. Through the use of reassortant viruses, we mapped this increased ethanol resistance mutation to the M2 gene segment, which encodes a major outer capsid protein, mu1C. Sequence analysis of mutant M2 genes revealed that six of seven unique mutants possessed single-point mutations in this gene. In addition, the change in six of seven mutants caused a predicted amino acid change in a 35-amino-acid region of the gene product between amino acids 425 and 459. The identification of ethanol resistance mutations within a discrete region of this outer capsid protein identifies that portion of the protein as important in reovirus stability. The presence of viral particles possessing altered stability also suggests that subpopulations of viruses may possess altered environmental stability, which, in turn, could affect viral transmission.     

Flagellar assembly in Salmonella typhimurium: analysis with temperature-sensitive mutants.

The process of flagellar assembly in Salmonella typhimurium was investigated by using temperature-sensitive mutants. The mutants were grown at the restrictive temperature and then at the permissive temperature, with radiolabel supplied in the first phase of the experiment and not the second, or vice versa. Flagellar hook-basal body complexes were then purified and analyzed by gel electrophoresis and autoradiography. The extent to which a given protein was labeled in the two phases of the experiment provided information as to whether it preceded or followed the block caused by the mutant protein. We conclude the following concerning flagellar assembly. The M-ring protein (FliF) is stably incorporated in the earliest stage detected, along with two previously unknown proteins, with apparent molecular masses of 23 and 26 kilodaltons, respectively, and possibly one of the switch components, FliG. Independent of that event and all other events, the P-ring and L-ring proteins (FlgI and FlgH) are synthesized and exported to the periplasm and outer membrane by the primary cellular export pathway. Rod assembly occurs by export (via the flagellum-specific pathway) of subunits of four proteins, FlgB, FlgC, FlgF, and FlgG, and their incorporation, probably in that order, into the rod structure; this stage requires the flhA and fliI genes, perhaps because they encode part of the export apparatus. Once rod assembly is complete, the FlgI and FlgH proteins assemble around the rod to form the P and L rings. The rod structure, which is only metastable while it is being constructed, becomes stable upon P-ring addition. Export (via the flagellum-specific pathway) and assembly of hook protein, hook-associated proteins, and filament protein then occur successively. A number of flagellar proteins, whose genetic origin and structural role are not yet known, were identified on the basis of their dependence on the flagellar master operon for expression.