Isolation of heat- and cold-sensitive mutants of chinese hamster lung cells affected in their ability to express the transformed state.

A clone of spontaneously transformed Chinese hamster lung cells was exposed to N-methyl-N'-nitro-N-nitroso-guanidine (MNNG), and six heat-sensitive and three cold-sensitive mutants were isolated after selection for inability to form colonies in soft agar at 39.5 degrees C and 34.5 degrees C, respectively. The heat-sensitive mutants had growth characteristics of transformed cells at 34.5 degrees C, but exhibited a normal phenotype at 39.5 degrees C. By contrast, cold-sensitive mutants displayed the characteristics of the normal cells at 34.5 degrees C and converted to a transformed phenotype at 39.5 degrees C. Transformed parent cells exhibited no obvious temperature-dependent properties. Temperature shift experiments showed that the colony-forming ability of both types of mutants was fully reversible. All of the mutants were able to grow well at both permissive and nonpermissive temperatures when grown on the surface of plastic dishes. Such mutants will be useful in analysis of factors involved in the expression of the transformed state or the maintenance of the nontransformed state.     

Isolation and preliminary characterization of a phosphonoacetic acid-resistant and temperature-sensitive mutant of herpes simplex virus type 1.

A group of 43 phosphonoacetic acid (PAA)-resistant mutants of herpes simplex virus type 1 was isolated after the mutagenesis of infected cells with nitrosoguanidine. One of these mutants, designated PAA1rts1, was found to be temperature sensitive (ts), that is, unable to replicate at 39.5 degrees C, the nonpermissive temperature. Recombination analysis of PAA1rts1 indicated that the PAA1r mutation and the ts1 mutation are loosely linked and are located on two separate genes. PAA1rts1 showed a defect in viral DNA synthesis at 39.5 degrees C, which presumably can be attributed to the production of a PAA-resistant and thermolabile DNA polymerase. PAA1rts1 was also defective in the shutoff of host DNA synthesis at the restrictive temperature.     

A herpes simplex virus type 1 gamma34.5 second-site suppressor mutant that exhibits enhanced growth in cultured glioblastoma cells is severely attenuated in animals

We describe here the neurovirulence properties of a herpes simplex virus type 1 gamma34.5 second-site suppressor mutant. gamma34.5 mutants are nonneurovirulent in animals and fail to grow in a variety of cultured cells due to a block at the level of protein synthesis. Extragenic suppressors with restored capacity to replicate in cells that normally do not support the growth of the parental gamma34.5 deletion mutant have been isolated. Although the suppressor virus reacquires the ability to grow in nonpermissive cultured cells, it remains severely attenuated in mice and is indistinguishable from the mutant gamma34.5 parent virus at the doses investigated. Repairing the gamma34.5 mutation in the suppressor mutant restores neurovirulence to wild-type levels. These studies illustrate that (i) the protein synthesis and neurovirulence defects observed in gamma34.5 mutant viruses can be genetically separated by an extragenic mutation at another site in the viral chromosome; (ii) the extragenic suppressor mutation does not affect neurovirulence; and (iii) the attenuated gamma34.5 mutant, which replicates poorly in many cell types, can be modified by genetic selection to generate a nonpathogenic variant that regains the ability to grow robustly in a nonpermissive glioblastoma cell line. As this gamma34.5 second-site suppressor variant is attenuated and replicates vigorously in neoplastic cells, it may have potential as a replication-competent, viral antitumor agent.     

Isolation and primary identification of methylotrophic yeast Hansenula polymorpha mutants for peroxisome biogenesis

After exposure of cells of the methylotrophic yeast Hansenula polymorpha HF246 leu1-1 to N-nitro-N-nitrosoguanidine, a collection of 227 mutants unable to grow on methanol at elevated temperature (45 degrees C) was obtained. Ninety four ts mutants (35% of the total number of mutants), which were unable to grow on methanol only at 45 degrees C but could grow at optimal temperature (37 degrees C), were isolated. Complementation analysis of mutants using 12 deletion mutants for genes of peroxisome biogenesis (PEX) (available in this yeast species by the beginning of our work) allowed to assign 51 mutants (including 16 ts) to the separate group of mutants unable to complement deletion mutants with defects in eight PEX genes. These mutants were classified into three groups: group 1 contained 10 pex10 mutants (4 ts mutants among them); group 2 included 19 mutants that failed to complement other pex testers: 1 pex1; 2 pex4 (1 ts); 6 pex5 (5 ts); 3 pex8; 6 (3ts)- pex19; group 3 contained 22 "multiple" mutants. In mutants of group 3, hybrids with several testers do not grow on methanol. All mutants (51) carried recessive mutations, except for mutant 108, in which the mutation was dominant only at 30 degrees C, which suggests that it is ts-dominant. Recombination analysis of mutants belonging to group 2 revealed that only five mutants (two pex5 and three pex8) carried mutations for the corresponding PEX genes. The remaining 14 mutants yielded methanol-utilizing segregants in an arbitrarily chosen sample of hybrids with the pex tester, which indicates mutation location in other genes. In 19 mutants, random analysis of ascospores from hybrids obtained upon crossing mutants of group 3 with a strain lacking peroxisomal disorders (ade11) revealed a single mutation causing the appearance of a multiple phenotype. A more detailed study of two mutants from this group allowed the localization of this mutation in the only PEX gene (PEX or PEX2). The revealed disorder of complementation interactions between nonallelic genes is under debate.     

A hamster temperature-sensitive alanyl-tRNA synthetase mutant causes degradation of cell-cycle related proteins and apoptosis

We have isolated a temperature-sensitive alanyl-tRNA synthetase mutant from hamster BHK21 cells, designated as ts ET12. It has a single nucleotide mutation, converting the 321st amino acid residue, 321Gly, to Arg. The mutation was localized between two RNA-binding domains of alanyl-tRNA synthetase. Thus far, we have isolated two temperature-sensitive aminoacyl-tRNA synthetase mutants from the BHK21 cell line: ts BN250 and ts BN269. They are defective in histidyl- and lysyl-tRNA synthetase respectively. Both mutants rapidly undergo apoptosis at the nonpermissive temperature, 39.5 degrees C. ts ET12 cells, however, did not undergo apoptosis until 48 h after a temperature-shift to 39.5 degrees C, while mutated alanyl-tRNA synthetase of ts ET12 cells was lost within 4 h. Loss of the mutated alanyl-tRNA synthetase was inhibited by a ubiquitin-dependent proteasome inhibitor, MG132, and by a protein-synthesis inhibitor, cycloheximide. Cell-cycle related proteins were also lost in ts ET12 cells at 39.5 degrees C, as shown in ts BN250. In contrast, the mutated aminoacyl-tRNA synthetases of ts BN250 and ts BN269 were stable at 39.5 degrees C. However, the defects of these mutants released EMAPII, an inducer of apoptosis at 39.5 degrees C. No release of EMAPII occurred in ts ET12 cells at 39.5 degrees C, consistent with the delay of apoptosis in these cells.     

Selective isolation of mutants of vesicular stomatitis virus defective in production of the viral glycoprotein.

We describe a procedure that enriches for temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV), Indiana serotype, which are conditionally defective in the biosynthesis of the viral glycoprotein. The selection procedure depends on the rescue of pseudotypes of known ts VSV mutants in complementation group V (corresponding to the viral G protein) by growth at 39.5 degrees C in cells preinfected with the avian retrovirus Rous-associated virus 1 (RAV-1). Seventeen nonleaky ts mutants were isolated from mutagenized stocks of VSV. Eight induced no synthesis of VSV proteins at the nonpermissive temperature and hence were not studied further. Four mutants belonged to complementation group V and resembled other ts (V) mutations in their thermolability, production at 39.5 degrees C of noninfectious particles specifically deficient in VSV G protein, synthesis at 39.5 degrees C of normal levels of viral RNA and protein, and ability to be rescued at 39.5 degrees C by preinfection of cells by avian retroviruses. Five new ts mutants were, unexpectedly, in complementation group IV, the putative structural gene for the viral nucleocapsid (N) protein. At 39.5 degrees C these mutants also induced formation of noninfectious particles relatively deficient in G protein, and production of infectious virus at 39.5 degrees C was also enhanced by preinfection with RAV-1, although not to the same extent as in the case of the group V mutants. We believe that the primary effect of the ts mutation is a reduced synthesis of the nucleocapsid and thus an inhibition of synthesis of all viral proteins; apparently, the accumulation of G protein at the surface is not sufficient to envelope all the viral nucleocapsids, or the mutation in the nucleocapsid prevents proper assembly of G into virions. The selection procedure, based on pseudotype formation with glycoproteins encoded by an unrelated virus, has potential use for the isolation of new glycoprotein mutants of diverse groups of enveloped viruses.     

Epidermal growth factor has a unique effect in combination with fetal bovine serum to bypass the ts-block of G0-specific ts mutant tsJT60

tsJT60 cells are G0-specific temperature-sensitive mutants of the cell cycle from Fischer rats i.e., they grow exponentially at both 34 degrees and 39.5 degrees C, but when stimulated with fetal bovine serum (FBS) from the resting state (G0) they enter S phase at 34 degrees C but not at 39.5 degrees C. Epidermal growth factor (EGF) also induced DNA synthesis, although weakly, in G0-arrested tsJT60 cells at 34 degrees C but failed at 39.5 degrees C. When G0-arrested tsJT60 cells were stimulated at 39.5 degrees C with FBS plus EGF, they entered S phase and divided. Somatomedin C, insulin, or transferrin had a weak effect in inducing DNA synthesis in G0-arrested cells when applied at 34 degrees C or with FBS at 39.5 degrees C. Fibroblast growth factor, platelet-derived growth factor, or 12-O-tetradecanoylphorbol 13-acetate had no such stimulatory effect at 39.5 degrees C. Binding of 125I-somatomedin C was not temperature-sensitive. Several other ts mutant cells that were blocked at 39.5 degrees C from entering S phase from the resting state following FBS addition were stimulated by FBS plus EGF at 34 degrees C but not at 39.5 degrees C.     

Preliminary characterization of coxsackievirus B3 temperature-sensitive mutants.

Prototype temperature-sensitive (ts) mutants of a coxsackievirus B3 parent virus capable of replication to similar levels at 34 or 39.5 degrees C were examined for the nature of the temperature-sensitive event restricting replication in HeLa cells at 39.5 degrees C. The ts mutant prototypes represented three different non-overlapping complementation groups. The ts1 mutant (complementation group III) synthesized less than 1% of the infectious genomic RNA synthesized by the coxsackievirus B3 parent virus at 39.5 degrees C and was designated an RNA- mutant. Agarose gel analysis of glyoxal-treated RNA from cells inoculated with ts1 virus revealed that cell RNA synthesis continued in the presence of synthesis of the small amount of viral RNA. This mutant was comparatively ineffective in inducing cell cytopathology and in directing synthesis of viral polypeptides, likely due to the paucity of nascent genomes for translation. The ts5 mutant (complementation group II) directed synthesis of appreciable quantities of both viral genomes (RNA+) and capsid polypeptides; however, assembly of these products into virions occurred at a low frequency, and virions assembled at 39.5 degrees C were highly unstable at that temperature. Shift-down experiments with ts5-inoculated cells showed that capsid precursor materials synthesized at 39.5 degrees C can, after shift to 34 degrees C, be incorporated into ts5 virions. We suggest that the temperature-sensitive defect in this prototype is in the synthesis of one of the capsid polypeptides that cannot renature into the correct configuration required for stability in the capsid at 39.5 degrees C. The ts11 mutant (complementation group I) also synthesized appreciable amounts of viral genomes (RNA+) and viral polypeptides at 39.5 degrees C. Assembly of ts11 virions at 39.5 degrees C occurred at a low frequency, and the stability of these virions at 39.5 degrees C was similar to that of the parent coxsackievirus B3 virions. The temperature-sensitive defect in the ts11 prototype is apparently in assembly. The differences in biochemical properties of the three prototype ts mutants at temperatures above 34 degrees C may ultimately offer insight into the differences in pathogenicity observed in neonatal mice for the three prototype ts mutants.     

Retroviral transduction of alveolar bone cells with a temperature-sensitive SV40 large T antigen

We have transduced adult human alveolar bone (AB) cells with a gene construct encoding a temperature-sensitive mutation of the SV40 large T antigen (tsT). Such cells divided rapidly, for more than 50 passages thus far, at a permissive low temperature (34.5 degrees C), comparable to the non-transduced parental cells at 37 degrees C. However, the tsT-transduced AB cells failed to grow at a non-permissive high temperature (39 degrees C) at which the T antigen is inactivated. Nevertheless, the cells formed mineralised nodules in vitro at both the low and high temperatures. Flow cytometry analysis showed that the transduced cells cultured at 34.5 degrees C, like the parental cells at 37 degrees C, were smaller and less granular than the transduced cells incubated at 39 degrees C. Moreover, the transduced cells grown at 34.5 degrees C were also found to express bone sialoprotein, osteopontin and type I collagen at levels similar to those of the parental cells at 37 degrees C, although osteonectin and fibronectin were down-regulated. When the transduced cells were incubated at 39 degrees C, the expression of all antigens was up-regulated, particularly osteonectin. Thus, we have obtained long-term cultures of tsT-transduced AB cells whose growth is temperature-dependent and which express certain features characteristic of bone-derived cells.     

The isolation and characterization of temperature-dependent ricin A chain molecules in Saccharomyces cerevisiae

Ricin is a heterodimeric plant protein that is potently toxic to mammalian cells. Toxicity results from the catalytic depurination of eukaryotic ribosomes by ricin toxin A chain (RTA) that follows toxin endocytosis to, and translocation across, the endoplasmic reticulum membrane. To ultimately identify proteins required for these later steps in the entry process, it will be useful to express the catalytic subunit within the endoplasmic reticulum of yeast cells in a manner that initially permits cell growth. A subsequent switch in conditions to provoke innate toxin action would permit only those strains containing defects in genes normally essential for toxin retro-translocation, refolding or degradation to survive. As a route to such a screen, several RTA mutants with reduced catalytic activity have previously been isolated. Here we report the use of Saccharomyces cerevisiae to isolate temperature-dependent mutants of endoplasmic reticulum-targeted RTA. Two such toxin mutants with opposing phenotypes were isolated. One mutant RTA (RTAF108L/L151P) allowed the yeast cells that express it to grow at 37 degrees C, whereas the same cells did not grow at 23 degrees C. Both mutations were required for temperature-dependent growth. The second toxin mutant (RTAE177D) allowed cells to grow at 23 degrees C but not at 37 degrees C. Interestingly, RTAE177D has been previously reported to have reduced catalytic activity, but this is the first demonstration of a temperature-sensitive phenotype. To provide a more detailed characterization of these mutants we have investigated their N-glycosylation, stability, catalytic activity and, where appropriate, a three-dimensional structure. The potential utility of these mutants is discussed.     

Control of ribonucleotide reductase in heat- and cold-sensitive mammalian cell-cycle mutants

Two heat-sensitive (reversibly arrested in G1 phase at 39.5 degrees C, multiplying at 33 degrees C) and two cold-sensitive (reversibly arrested in G1 phase at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were tested for ribonucleotide reductase activity, using cells made permeable to nucleotides. After transfer of the heat-sensitive mutant cells to 39.5 degrees C, ribonucleotide reductase activity, similar to thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77-85), but unlike DNA polymerase alpha (Schneider, E., Müller, B. and Schindler, R. (1985) Biochim. Biophys. Acta 825, 375-383), decreased rapidly and in parallel with numbers of cells in S phase, whereas in the cold-sensitive mutant cells brought to 33 degrees C, ribonucleotide reductase activity decreased approx. 8 h later than numbers of DNA-synthesizing cells. When arrested heat- or cold-sensitive mutant cells were returned to the permissive temperature, ribonucleotide reductase activities, similar to DNA polymerase alpha and to thymidine kinase in heat-sensitive mutants, increased essentially in parallel with reentry of cells into S phase, whereas the increase in thymidine kinase activity in the cold-sensitive mutants was previously shown to occur approx. one cell-cycle time later. This indicates that ribonucleotide reductase and thymidine kinase are coordinately expressed in the heat-sensitive, but independently regulated in the cold-sensitive mutants.     

Apurinic sites cause mutations in simian virus 40

SV40 has been used as a molecular probe to study the mutagenicity of apurinic sites (Ap) in mammalian cells. Untreated or UV-irradiated monkey kidney cells were transfected with depurinated DNA from the temperature-sensitive tsB201 SV40 late mutant which grows normally at the permissive temperature of 33 degrees C but which is unable to grow at 41 degrees C. Phenotypic revertants were screened at 41 degrees C for their ability to grow at the restrictive temperature and the mutation frequency was calculated in the viral progeny. Ap sites were introduced into DNA by heating at 70 degrees C under acid conditions (pH 4.8). This treatment induces one Ap site per SV40 genome per 15 min of heating as measured by alkaline denaturation or by treatment with the T4-encoded UV-specific endonuclease which possesses Ap-endonuclease activity. The experiments reported here show that Ap sites strongly decrease virus survival with a lethal hit corresponding roughly to 3 Ap lesions per SV40 genome, and indicate for the first time that apurinic sites produced by heating are highly mutagenic in animal cells. UV irradiation of the host cells 24 h prior to transfection with depurinated DNA did not modify the mutation frequency in the virus progeny.     

A Chinese hamster ovary cell mutant with a heat-sensitive, conditional- lethal defect in vacuolar function

We describe a mutant derived from Chinese hamster ovary cells that is offt-sensitive for viability and for resistance to certain protein toxins. This mutant, termed G.7.1, grows normally at 34 degrees C but does not grow in Dulbecco's modified Eagle's medium at 39.5 degrees C. However, when this medium is supplemented with FeSO4, the mutant cells will grow at the elevated temperature. At 39.5 degrees C, G.7.1 cells acquire resistance to diphtheria toxin, modeccin, and Pseudomonas aeruginosa exotoxin A, all of which are protein toxins that require endocytosis and exposure to a low pH within vesicles before they can invade the cytosol and kill cells. The properties of mutant G.7.1 could result from a heat-sensitive lesion that impairs vacuolar acidification. We assayed the ATP-stimulated generation of pH gradients across the membrane of vesicles in cell-free preparations from mutant and parental cells by the partitioning of acridine orange into acidic compartments and found that the acidification response of the mutant cells was heat-labile. Altogether the evidence suggests that G.7.1 cells contain a heat-sensitive lesion that impairs vacuolar acidification and that they fail to grow in normal medium at 39.5 degrees C because they cannot extract Fe+3 from transferrin, a process that normally requires exposing transferrin to a low pH within endosomal vesicles.     

Effects of elevated temperature on growth, respiratory-deficient mutation, respiratory activity, and ethanol production in yeast

Some mesophilic yeasts and a thermotolerant strain of Saccharomyces cerevisiae were found to grow at 40 degrees C in complex media containing 1% yeast extract when an inoculum of 10(6) or more cells.mL-1 was used. Yeast extract (6%) permitted Saccharomyces cerevisiae to grow at 40 degrees C even with a smaller inoculum size (10(5) cells.mL-1). The fraction of respiratory-deficient (petite) mutants in 40 degrees C grown culture was less than 10% except for the thermotolerant strain, which showed greatly increased levels depending on culture conditions. Seven of eight yeast strains exhibited extremely reduced cytochrome oxidase activity when grown at 40 degrees C irrespective of the frequency of the petite mutation. In contrast, the accumulation of ethanol in the medium and the ethanol-producing activity of the cells were not affected by growth at 40 degrees C.     

tsJT60, a cell cycle G0-ts mutant, becomes lethal at non-permissive temperature by transformation with adenovirus 5 when the expression of E1B gene is lacking

tsJT60, a temperature-sensitive (ts) mutant cell line of Fischer rat, is viable at both permissive (34 degrees C) and non-permissive (39.5 degrees C) temperatures. The cells grow normally in exponential growth phase at both temperatures, but when stimulated with fetal bovine serum (FBS) from G0 phase they re-enter S phase at 34 degrees C but not at 39.5 degrees. When tsJT60 cells were transformed with adenovirus (Ad) 5 wild type, they grew well at both temperatures, expressed E1A and E1B genes, and formed colonies in soft agar. When tsJT60 cells were transformed with Ad5 dl313, that lacks E1B gene, the transformed cells grew well at 34 degrees C but failed to form colony in soft agar. They died very soon at 39.5 degrees C. 3Y1 cells (a parental line of tsJT60) transformed with dl313 grew well at both temperatures, although neither expressed E1B gene nor formed colonies in soft agar. The phenotype of being lethal at 39.5 degrees C of dl313-transformed tsJT60 cells was complemented by cell fusion with 3Y1BUr cells (5-BrdU-resistant 3Y1), but not with tsJT60TGr cells (6-thioguanine resistant tsJT60). These results indicate that the lethal phenotype is related to the ts mutation of tsJT60 cells and also to the deletion of E1B gene of Ad5.     

Regulation of polypeptide chain initiation and activity of initiation factor eIF-2 in Chinese-hamster-ovary cell mutants containing temperature-sensitive aminoacyl-tRNA synthetases

The regulation of polypeptide chain initiation has been investigated in extracts from a number of well-characterized Chinese hamster ovary (CHO) cell mutants containing different temperature-sensitive aminoacyl-tRNA synthetases. These cells exhibit a large decline in the rate of initiation when cultures are shifted from the permissive temperature of 34 degrees C to the non-permissive temperature of 39.5 degrees C. During a brief incubation with [35S]Met-tRNAMetf or [35S]methionine, formation of initiation complexes on native 40S ribosomal subunits and 80S ribosomes is severely impaired in extracts from the mutant cell lines exposed to 39.5 degrees C. Wild-type cells exposed to 39.5 degrees C do not show any inhibition of protein synthesis or initiation complex formation. Inhibition of formation of 40S initiation complexes in the extracts from mutant cells, incubated at the non-permissive temperature, is shown to be independent of possible changes in mRNA binding or the rate of polypeptide chain elongation and is not due to any decrease in the total amount of initiation factor eIF-2 present. However, assays of eIF-2 X GTP X Met-tRNAMetf ternary complex formation in postribosomal supernatants from the temperature-sensitive mutants reveal a marked defect in the activity of eIF-2 after exposure of the cells to 39.5 degrees C and addition of exogenous eIF-2 to cell-free protein-synthesizing systems from cells incubated at 34 degrees C and 39.5 degrees C eliminates the difference in activity between them. The activity of the initiation factor itself is not directly temperature-sensitive in the mutant CHO cells. The results suggest that the activity of aminoacyl-tRNA synthetases can affect the ability of eIF-2 to bind Met-tRNAMetf and form 40S initiation complexes in intact cells, indicating a regulatory link between polypeptide chain elongation and chain initiation.     

In vivo replication of an ICP34.5 second-site suppressor mutant following corneal infection correlates with in vitro regulation of eIF2 alpha phosphorylation

In animal models of herpes simplex virus type 1 (HSV-1) infection, ICP34.5-null viruses are avirulent and also fail to grow in a variety of cultured cells due to their inability to prevent RNA-dependent protein kinase (PKR)-mediated inhibition of protein synthesis. We show here that the inability of ICP34.5 mutants to grow in vitro is due specifically to the accumulation of phosphorylated eIF2 alpha. Mutations suppressing the in vitro phenotype of ICP34.5-null mutants have been described which map to the unique short region of the HSV-1 genome, resulting in dysregulated expression of the US11 gene. Despite the inability of the suppressor mutation to suppress the avirulent phenotype of the ICP34.5-null parental virus following intracranial inoculation, the suppressor mutation enhanced virus growth in the cornea, trigeminal ganglia, and periocular skin following corneal infection compared to that with the ICP34.5-null virus. The phosphorylation state of eIF2 alpha following in vitro infection with the suppressor virus was examined to determine if in vivo differences could be attributed to differential regulation of eIF2 alpha phosphorylation. The suppressor virus prevented accumulation of phosphorylated eIF2 alpha, while the wild-type virus substantially reduced eIF2 alpha phosphorylation levels. These data suggest that US11 functions as a PKR antagonist in vivo, although its activity may be modulated by tissue-specific differences in translation regulation.     

Mutations which alter the level or structure of nsP4 can affect the efficiency of Sindbis virus replication in a host-dependent manner.

Two mutants of Sindbis virus have been isolated which grow inefficiently at 34.5 degrees C in mosquito cells yet replicate normally in chicken embryo fibroblast cells at the same temperature. In addition, these mutants exhibit temperature-sensitive growth in both cell types and are RNA- at the nonpermissive temperatures (K.J. Kowal and V. Stollar, Virology 114:140-148, 1981). To clarify the basis of this host restriction, we have mapped the causal mutations for these temperature-dependent, host-restricted mutants. Functional mapping and sequence analysis of the mutant cDNAs revealed several mutations which mapped to the amino terminus of nsP4, the putative polymerase subunit of the viral RNA replicase. These mutations resulted in the following amino acid changes in nsP4: leucine to valine at residue 48, aspartate to glycine at residue 142, and proline to arginine at residue 187. Virus containing any of these mutations was restricted in its ability to replicate in mosquito but not chicken embryo fibroblast cells at 34.5 degrees C. In addition to its temperature-dependent, host-restricted phenotype, virus derived from one cDNA clone also exhibited decreased levels of nsP34 and nsP4 yet contained only a silent change in its genome. This C-to-U mutation occurred at nucleotide 5751, the first nucleotide after the opal termination codon separating nsP3 and nsP4. Our results suggest that this substitution decreases readthrough of the opal codon and diminishes production of nsP34 and nsP4. Such a decrease in synthesis rates might lead to levels of these products which are insufficient for viral RNA replication in mosquito cells at the higher temperature. This work provides the first evidence that nsP4 function can be strongly influenced by the host environment.     

Disruptions in Golgi structure and membrane traffic in a conditional lethal mammalian cell mutant are corrected by epsilon-COP

The CHO cell temperature-sensitive mutant ldlF exhibits two defects in membrane traffic at the nonpermissive temperature (39.5 degrees C): rapid degradation of LDL receptors, possibly caused by endocytic missorting, and disruption of ER-through-Golgi transport. Here, we show that at 39.5 degrees C, the Golgi in ldlF cells dissociated into vesicles and tubules. This dissociation was inhibited by AlF4-, suggesting trimeric G proteins are involved in the dissociation mechanism. This resembled the effects of brefeldin A on wild-type cells. We isolated a hamster cDNA that specifically corrected the ts defects of ldlF cells, but not those of other similar ts mutants (ldlE, ldlG, ldlH, and End4). Its predicted protein sequence is conserved in humans, rice, Arabidopsis, and Caenorhabditis elegans, and is virtually identical to that of bovine epsilon-COP, a component of the coatomer complex implicated in membrane transport. This provides the first genetic evidence that coatomers in animal cells can play a role both in maintaining Golgi structure and in mediating ER-through-Golgi transport, and can influence normal endocytic recycling of LDL receptors. Thus, along with biochemical and yeast genetics methods, mammalian somatic cell mutants can provide powerful tools for the elucidation of the mechanisms underlying intracellular membrane traffic.     

Isolation and characterization of temperature-sensitive mutants of adenovirus type 7.

Fifty temperature-sensitive mutants, which replicate at 32 degrees C but not at 39.5 degrees C, were isolated after mutagenesis of the vaccine strain of adenovirus type 7 with hydroxylamine (mutation frequency of 9.0%) or nitrous acid (mutation frequency of 3.8%). Intratypic complementation analyses separated 46 of these mutants into seven groups. Intertypic complementation tests with temperature-sensitive mutants of adenovirus type 5 showed that the mutant in complementation group A failed to complement H5ts125 (a DNA-binding protein mutant), that mutants in group B and C did not complement adenovirus type 5 hexon mutants, and that none of the mutants was defective in fiber production. Further phenotypic characterization showed that at the nonpermissive temperature the mutant in group A failed to make immunologically reactive DNA-binding protein, mutants in groups B and C were defective in transport of trimeric hexons to the nucleus, mutants in groups D, E, and F assembled empty capsids, and mutants in group G assembled DNA-containing capsids as well as empty capsids. The mutants of the complementation groups were physically mapped by marker rescue, and the mutations were localized between the following map coordinates: groups B and C between 50.4 and 60.2 map units (m.u.), groups D and E between 29.6 and 36.7 m.u., and group G between 36.7 and 42.0 m.u. or 44.0 and 47.0 m.u. The mutant in group A proved to be a double mutant.