Temperature-sensitive mutants and revertants in the coronavirus nonstructural protein 5 protease (3CLpro) define residues involved in long-distance communication and regulation of protease activity

Positive-strand RNA virus genomes are translated into polyproteins that are processed by viral proteases to yield functional intermediate and mature proteins. Coronaviruses (CoVs) carry genes that encode an nsp5 protease (also known as 3CLpro or Mpro) responsible for 11 maturation cleavages. The nsp5 structure contains two chymotrypsin-like domains (D1 and D2) and a unique domain (D3), and forms functional dimers. However, little is known of interactions or communication across the structure of the protease during nsp5 activity. Using reverse genetic mutagenesis of the CoV murine hepatitis virus (MHV) nsp5, we identified a new temperature-sensitive (ts) mutation in D2 of nsp5 (Ser133Ala) and confirmed a ts residue in D3 (Phe219Leu). Both D2-tsS133A and D3-tsF219L were impaired for viral replication and nsp5-mediated polyprotein processing at the nonpermissive temperature. Passage of tsS133A and tsF219L at the nonpermissive temperature resulted in emergence of multiple second-site suppressor mutations, singly and in combinations. Among the second-site mutations, a D2 His134Tyr change suppressed the ts phenotype of D2-tsS133A and D3-tsF219L, as well as the previously reported D2-tsV148A. Analysis of multiple CoV nsp5 structures, and alignment of nonredundant nsp5 primary sequences, demonstrated that ts and suppressor residues are not conserved across CoVs and are physically distant (>10 Å) from each other, from catalytic and substrate-binding residues, and from the nsp5 dimer interface. These findings demonstrate that long-distance communication pathways between multiple residues and domains of nsp5 play a significant role in nsp5 activity and viral replication, suggesting possible novel targets for non-active site inhibitors of nsp5.     

Histocompatibility gene mutation rates in the mouse: a 25-year review

 Mutation rates of H2 and non-H2 histocompatibility genes in the mouse are examined over a 25-year period. Detected by skin graft rejections, the mutations were screened in inbred and hybrid mice from a continuously maintained and monitored colony and from a regularly supplied set of mice provided from the National Cancer Institute for monitoring of genetic integrity. Twenty-five H2 mutations were recovered, involving the K, D, L, and Ab loci, as well as over 80 mutations of non-H2 histocompatibility genes. Aside from a single allele at a single locus (H2-K ^b ), the spontaneous mutation rate of H2 class I genes appears to be equivalent to that found estimated for non-H2 histocompatibility genes, and comparable to rates reported for a variety of mouse genes. This is in contrast with previous suggestions that H2 genes mutate at orders of magnitude greater than do “average” mammalian genes. The discrepancy is attributed to the H2-K ^b gene which accounts for over half of all reported H2 mutations and which mutates spontaneously at a rate of 1–2×10^–4 per gene per generation. Furthermore, over half of the spontaneous H2-K ^b mutations result in a single mutant phenotype (the “bg” group) which involve similar changes at amino acid residues 116 and 121. Thus, the high spontaneous mutation rate for H2-K ^b appears to be the exception among major histocompatibility genes, rather than the rule. Received: 18 April 1997 / Revised: 22 May 1997     

Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene

The vaccinia virus D5 gene encodes a 90 kDa early protein that is essential for viral DNA replication. In this report we map and explore the phenotypes of the temperature sensitive mutants bearing lesions in this gene:ts17,ts24,ts69, (WR strain) andts6389 (IHD strain). Viral DNA synthesis was virtually undetectable during non-permissive infections performed withts17, and incorporation of³H-thymidine ceased rapidly when cultures were shifted to the non-permissive temperature in the midst of replication. The D5 protein may therefore be involved in DNA synthesis at the replication fork. The lesions of the four mutants were localized within the D5orf by marker rescue, and the single nucleotide changes responsible for thets phenotype of the three WR mutants were identified. Unexpectedly, the three alleles with N-terminal mutations were impaired in marker rescue when homologous recombination with small (<2 kb), intragenic DNA fragments at 39.5°C was required. This deficiency was not due to degradation of transfected DNA under non-permissive conditions. Efficient marker rescue could be restored by incubation at the permissive temperature for a brief period after transfection, suggesting a requirement for functional D5 in genome/plasmid recombination. Marker rescue under non-permissive conditions could alternatively be restored by co-transfection of unlinked but contiguous DNA sequences.     

RNase H-defective mutants of Escherichia coli: a possible discriminatory role of RNase H in initiation of DNA replication

Summary Mutants of Escherichia coli completely deficient in RNase H activity were isolated by inserting transposon Tn3 into the structural gene for RNase H, rnh, and its promoter. These rnh ^- mutants exhibited the following phenotypes; (1) the mutants grew fairly normally, (2) rnh ^- cells could be transformed with ColE1 derivative plasmids, pBR322 and pML21, though the plasmids were relatively unstable, under non selective conditions, (3) rnh ^- mutations partially suppressed the temperature-sensitive phenotype of plasmid pSC301, a DNA replication initiation mutant derived from pSC101, (4) rnh ^- mutations suppressed the temperature-sensitive growth character of dnaA ^ts mutant, (5) rnh ^- cells showed continued DNA synthesis in the presence of chloramphenicol (stable DNA replication). Based on these findings we propose a model for a role of RNase H in the initiation of chromosomal DNA replication. We suggest that two types of RNA primers for initiation of DNA replication are synthesized in a dnaA/oriC-dependent and-independent manner and that only the dnaA/oriC-dependent primer is involved in the normal DNA replication since the dnaA/oriC independent primer is selectively degraded by RNase H.Abbreviations AP^r ampicillin-resistant - kb kilobase pair(s) - NEM N-ethyl maleimide - Ts temperature-sensitive     

Clonal analysis of two mutations in the large subunit of RNA polymerase II of Drosophila

Summary Two mutations in the gene, RpII215, were analyzed to determine their effects on cell differentiation and proliferation. The mutations differ in that one, RpII215 ^ts(ts), only displays a conditional recessive lethality, while the other, RpII215 ^Ubl (Ubl), is a recessive lethal mutation that also displays a dominant mutant phenotype similar to that caused by the mutation Ultrabithorax (Ubx). Ubl causes a partial transformation of the haltere into a wing; however, this transformation is more complete in flies carrying both Ubl and Ubx. The present study shows that patches of Ubl/- tissue in gynandromorphs are morphologically normal. Cuticle that has lost the wild-type copy of the RpII215 locus fails to show a haltere to wing transformation, nor does it show the synergistic enhancement of Ubx by Ubl. We conclude that an interaction between the two RpII215 alleles, Ubl and RpII215 ⁺, is responsible for the mutant phenotype. Gynandromorphs carrying the ts allele, when raised at permissive temperature, display larger patches of ts/- cuticle than expected, possibly indicating that the proliferation of ts/+ cells is reduced. This might result from an antagonistic interaction between different RpII215 alleles. Classical negative complementation does not appear to be the cause of the antagonistic interaction described above, as only one RpII215 subunit is thought to be present in an active multimeric polymerase enzyme. We have therefore coined the term negative heterosis to describe the aforementioned interactions.We also observed that the effects of mutationally altered RNA polymerase II on somatic cells are different from its effects on germ cells. Mutant somatic cells (either Ubl/- or ts/-, the latter shifted to restrictive temperature) reduce cell proliferation, but otherwise do not appear to disrupt cell differentiation. However, mutant germ cells often differentiate into morphologically abnormal oocytes.     

An autoselection system in recombinant Kluyveromyces lactis enhances cloned gene stability and provides freedom in medium selection

An autoselection system for increasing plasmid stability in Kluyveromyces lactis, based on the blockage of the pyrimidine de novo and salvage pathways, was investigated. In a manner analogous to that used in Saccharomyces cerevisiae, a putative “fur1” mutation was selected in a uraA K. lactis strain using 5-fluorouracil and 5-fluorocytosine plates. Survival of the mutant required expression of a plasmid-borne URA3 gene regardless of the culture medium employed, verifying the efficacy of this autoselection system in K. lactis. The expression of heterologous invertase, encoded by the S. cerevisiae SUC2 gene, was studied during long-term sequential batch cultures (70 generations) in complex yeast/peptone/glucose medium. The fur1 mutant successfully retained the plasmid; invertase specific activity remained above 90% of the initial level. Furthermore, no mutation reversion was observed. In contrast, for the control non-fur1 strain, only 4% of the cells retained the plasmid after 70 generations, and invertase specific activity dropped to less than 10% of the initial level. Experiments comparing growth and activity in different media indicated the potential for improving productivity through medium enrichment using this autoselection system. Received: 1 April 1997 / Received revision: 16 August 1997 / Accepted: 11 September 1997     

Involvement of Gene 49 in Recombination of Bacteriophage T4

The role of T4 gene 49 in recombination was investigated using its conditional-lethal amber (am) and temperature-sensitive (ts) mutants. When measured in genetic tests, defects in gene 49 produced a recombination-deficient phenotype. However, DNA synthesized in cells infected with a ts mutant (tsC9) at a nonpermissive temperature appeared to be in a recombinogenic state: after restitution of gene function by shifting to a permissive temperature, the recombinant frequency among progeny increased rapidly even when DNA replication was blocked by an inhibitor. Growth of a gene 49-defective mutant was suppressed by an additional mutation in gene uvsX, but recombination between rII markers was not.     

A mutant ofLactobacillus acidophilus with temperature-sensitive regulation of DNA synthesis

Among other temperature-sensitive mutants ofLactobacillus acidophilus the mutant “ts 9” with temperature-sensitive initiation of DNA synthesis was isolated. In this mutant, the course of DNA synthesis under non-permissive conditions proceeds in two phases. During the first 90–120 min, a slight increase (20–50%) of DNA content takes place. Then during further incubation at 40°C, the capacity for initiation of further DNA synthesis increases and a second round of DNA synthesis starts after 3–4h of incubation. The initiation of DNA synthesis is prevented by chloramphenicol and the preceding lag is temperature-dependent. It is concluded that an accumulation of an initiation factor is required for the onset of a new cycle of DNA synthesis and that in thets 9 mutant this accumulation is inhibited at non-permissive temperature.     

Analysis of mutagenesis induced by a thermolabile T4 phage deoxycytidylate hydroxymethylase suggests localized deoxyribonucleotide pool imbalance

Summary To understand the molecular basis of mutation stimulated by deoxyribonucleotide pool imbalance, we studied a temperature-sensitive T4 phage gene 42 mutant (LB3), which specifies a thermolabile deoxycytidylate hydroxymethylase. Analysis of rII mutations, revertible to wild type along either GC-to-AT or AT-to-GC transition pathways, showed 8- to 80-fold stimulation of GC-to-AT mutations at a semi-permissive temperature (34° C). One such marker, rII SN103, which showed the highest stimulation at 34° C, was sequenced after amplification of the template by polymerase chain reaction. The mutant site in rII SN103 was identified at nucleotide position 265 from the rII B translational start as an AT-to-GC transition, which changes TCA to CCA. Sequence analysis of revertants and pseudorevertants generated at 34° C showed that both cytosines within this triplet can undergo change to either thymine or adenine, consistent with the hypothesis that hydroxymethyldeoxycytidine triphosphate pools are depleted at replication sites. However, dNTP pool measurements in extracts of 34° C cultures showed no significant deviations from values obtained at 30° C, suggesting that pool imbalances occur only locally, close to replication forks. Our studies support the hypothesis that the imitator phenotype displayed by ts LB3 at semi-permissive temperature is a consequence of perturbation of the flow of nucleotide precursors into the DNA replication machinery. A putative localized depletion of hm-dCTP presumably enlarges effective dTTP/hm-dCTP and dATP/hm-dCTP pool ratios, resulting in the observed C-to-T transition and C-to-A transversion mutations.     

Analysis of HCF, the cellular cofactor of VP16, in herpes simplex virus-infected cells

Herpes simplex virus (HSV) immediate-early (IE) gene expression is initiated via the recruitment of the structural protein VP16 onto specific sites upstream of each IE gene promoter in a multicomponent complex (TRF.C) that also includes the cellular proteins Oct-1 and HCF. In vitro results have shown that HCF binds directly to VP16 and stabilizes TRF.C. Results from transfection assays have also indicated that HCF is involved in the nuclear import of VP16. However, there have been no reports on the role or the fate of HCF during HSV type 1 (HSV-1) infection. Here we show that the intracellular distribution of HCF is dramatically altered during HSV-1 infection and that the protein interacts with and colocalizes with VP16. Moreover, viral protein synthesis and replication were significantly reduced after infection of a BHK-21-derived temperature-sensitive cell line (tsBN67) which contains a mutant HCF unable to associate with VP16 at the nonpermissive temperature. Intracellular distribution of HCF and of newly synthesized VP16 in tsBN67-infected cells was similar to that observed in Vero cells, suggesting that late in infection the trafficking of both proteins was not dependent on their association. We constructed a stable cell line (tsBN67r) in which the temperature-sensitive phenotype was rescued by using an epitope-tagged wild-type HCF. In HSV-1-infected tsBN67r cells at the nonpermissive temperature, direct binding of HCF to VP16 was observed, but virus protein synthesis and replication were not restored to levels observed at the permissive temperature or in wild-type BHK cells. Together these results indicate that the factors involved in compartmentalization of VP16 alter during infection and that late in infection, VP16 and HCF may have additional roles reflected in their colocalization in replication compartments.     

Production of infectious particles at the nonpermissive temperature by a temperature-sensitive mutant of bacteriophage SH-133 specific forPseudomonas facilis

The preliminary characterization of a unique temperature-sensitive (ts) mutant of bacteriophage SH-133, designatedts18, is reported. The mutant showed a substantial reduction in the ability to form plaques at the nonpermissive temperature (32°C) when compared with its plaqueforming ability at the permissive temperature (27°C). However, the supernatant fromts18-infected cells grown at 32°C exhibited significant infectivity when assayed at 27°C, which indicates that the reduced titer ofts18 at 32°C is not due to its inability to form phage particles at that temperature. Phage particles produced at 32°C, but not at 27°C, were thermolabile when tested at 32°C. The thermolability of phage yields from cells mixedly infected at 32°C with increasing wild-type/ts18 input ratios was independent of the quantity of wild-type gene product per cell. Thermostable phage particles were yielded byts18-infected cells that received short pulses of permissive temperature during the latter part of the latent period. These data indicate that the defect of the mutant is due to the production of a nonstructural assembly protein that misfunctions when viral maturation proceeds at the nonpermissive temperature.     

Transforming NRK cells with avian sarcoma virus reduces the extracellular Ca requirement without affecting the calcicalmodulin requirement for the G1/S transition

NRK rat cells infected with a transformation-defective, temperature-sensitive (ts) mutant of the avian sarcoma virus could not proliferate in Ca²⁺-deficient medium at a nonpermissive temperature (40 °C) that inactivated the viral pp60^v-scr-transforming product and rendered the cells phenotypically untransformed. However, these arrested cells were stimulated to initiate DNA replication with little or no delay while still in the Ca²⁺-deficient medium, either by adding Ca²⁺ or calmodulin at 40 °C or by reducing the temperature to 36 °C which restored the transformed phenotype by rapidly reactivating pp60^v-src. The G1/S transition triggered by restoring the transformed phenotype was suppressed by three different anticalmodulin drugs (R24571, trifluoperazine, W7). The suppression by one of these drugs, trifluoperazine, was overcome by adding calmodulin. Thus, neoplastic transformation by the avian sarcoma virus sharply reduces the extracellular Ca²⁺ requirement for the initiation of DNA replication without bypassing a calcical-modulin-dependent mechanism also needed for the G1/S transition.     

Stamenless, a tomato mutant with homeotic conversions in petals and stamens

A tomato (Lycopersicon esculentum Mill.) monogenic semidominant mutation, stamenless (sl), which results in homeotic conversions in two adjacent floral whorls, was studied. When grown at standard temperature, flowers of sl/sl plants showed sepaloid petals in the second whorl and strong transformation of stamens to carpels in whorl three. These transformed carpels were fused with each other and with the genuine carpels in the fourth whorl to form a unique gynoecium. The mutation is semidominant since heterozygous plants showed a phenotype intermediate between that of the wild type (WT) and that of homozygous mutant plants, with nearly WT petals but with feminized stamens bearing naked ovules on the base of their adaxial face. The initiation and position of organ primordia in sl/sl flowers were not altered when compared with WT primordia although development of organ primordia in the second and third whorls deviated from WT at an early stage as observed by scanning electron microscopy. The mutant phenotype is temperature sensitive and when sl/sl plants were cultured at low temperature, the morphology of some flowers resembled that of the WT. This reversion of the mutant phenotype is also induced by treatment of young sl/sl plants with gibberellic acid, providing evidence that gibberellin synthesis or sensitivity could mediate the effect of low temperature on the mutant phenotype. Southern blot analyses using a Deficiens-homologous gene from Solanum tuberosum as a probe showed a restriction-fragment-length polymorphism (RFLP) linked to the sl mutation. This result indicates that the mutation affects a Deficiens-like gene that controls the identity of petals and stamens. Received: 10 December 1998 / Accepted: 29 March 1999     

Physiological suppression of the temperature-sensitive sporulation defect in a Bacillus subtilis RNA polymerase mutant

Summary Five hundred putative RNA polymerase mutants of Bacillus subtilis were isolated by selecting for resistance to the RNA polymerase inhibitors rifampin (Rif^r), streptovaricin (Str^r) or streptolydigan (Std^r). This collection was screened for mutants that were unable to sporulate at the non-permissive temperature of 46°C, yet which sporulated well at 37°C and had normal vegetative growth (Spo^ts phenotype). Nearly one half of the Rif^r and one quarter of the Stv^r mutants were Spo^ts, whereas none of the Std^r mutants had this phenotype.The streptovaricin resistant strain stv84 was studied in detail. The stv84 mutation maps between cysA14 and strA39 on the B. subtilis chromosome, and the Stv^r and Spo^ts phenotypes cotransform at a frequency of 100%. The Spo^ts phenotype of stv84 could be physiologically corrected by supplementing the growth medium with inhibitors of RNA synthesis such as rifampin or azauracil, with carbohydrates such as ribose, mannose or glycerol, or with lipids such as Tween 40 or fatty acids native to Bacillus subtilis membranes. A Spo^ts phenotype resembling that of stv84 was produced in wild type B. subtilis by adding cerulenin, an inhibitor of fatty acid biosynthesis, to the growth medium. This cerulenin-induced sporulation defect was reversed by the same treatments that correct the temperature-sensitive genetic defect of stv84. These data indicate that the Spo^ts phenotype of strain stv84 is not due to an intrinsic inability of the mutant RNA polymerase to transcribe developmentally-specific genes at the nonpermissive temperature. Rather, the data suggest that the stv84 lesion causes a physiological imbalance which disrupts membrane structure or function in sporulating cells.     

Phenotypic Characterization of Adenovirus Type 12 Temperature-Sensitive Mutants in Productive Infection and Transformation

Eleven temperature-sensitive mutants of adenovirus type 12, capable of forming plaques in human cells at 33 C but not at 39.5 C, were isolated from a stock of a wild-type strain after treatment with either nitrous acid or hydroxyl-amine. Complementation tests in doubly infected human cells permitted a tentative assignment of eight of these mutants to six complementation groups. Temperature-shift experiments revealed that one mutant is affected early and most of the other mutants are affected late. Only the early mutant, H12ts505, was temperature sensitive in viral DNA replication. Infectious virions of all the mutants except H12ts505 and two of the late mutants produced at 33 C, appeared to be more heat labile than those of the wild type. Only H12ts505 was temperature sensitive for the establishment of transformation of rat 3Y1 cells. One of the late mutants (H12ts504) had an increased transforming ability at the permissive temperature. Results of temperature-shift transformation experiments suggest that a viral function affected in H12ts505 is required for “initiation” of transformation. Some of the growth properties of H12ts505-transformed cells were also temperature dependent, suggesting that a functional expression of a gene mutated in H12ts505 is required to maintain at least some aspects of the transformed state.     

The Temperature Sensitivity of a Mutation in the Essential tRNA Modification Enzyme tRNA Methyltransferase D (TrmD)

Conditional temperature-sensitive (ts) mutations are important reagents to study essential genes. Although it is commonly assumed that the ts phenotype of a specific mutation arises from thermal denaturation of the mutant enzyme, the possibility also exists that the mutation decreases the enzyme activity to a certain level at the permissive temperature and aggravates the negative effect further upon temperature upshifts. Resolving these possibilities is important for exploiting the ts mutation for studying the essential gene. The trmD gene is essential for growth in bacteria, encoding the enzyme for converting G37 to m¹G37 on the 3′ side of the tRNA anticodon. This conversion involves methyl transfer from S-adenosyl methionine and is critical to minimize tRNA frameshift errors on the ribosome. Using the ts-S88L mutation of Escherichia coli trmD as an example, we show that although the mutation confers thermal lability to the enzyme, the effect is relatively minor. In contrast, the mutation decreases the catalytic efficiency of the enzyme to 1% at the permissive temperature, and at the nonpermissive temperature, it renders further deterioration of activity to 0.1%. These changes are accompanied by losses of both the quantity and quality of tRNA methylation, leading to the potential of cellular pleiotropic effects. This work illustrates the principle that the ts phenotype of an essential gene mutation can be closely linked to the catalytic defect of the gene product and that such a mutation can provide a useful tool to study the mechanism of catalytic inactivation.     

Genetic Variation During Persistent Reovirus Infection: Presence of Extragenically Suppressed Temperature-Sensitive Lesions in Wild-Type Virus Isolated from Persistently Infected L Cells

Persistent reovirus infection of L cells was established with a serially passaged stock of temperature-sensitive (ts) mutant C(447) containing greater than 90% defective interfering particles. Within a month after establishment of the carrier culture, the ts mutant was replaced by virus that expressed the wild-type (ts⁺) temperature phenotype (R. Ahmed and A. F. Graham, J. Virol. 23:250-262, 1977). To determine whether the ts⁺ phenotype of the virus was due to intragenic reversion or to the presence of an extragenic mutation suppressing the original ts defect, several clones were backcrossed to wild-type reovirus, and the progeny of each cross were screened for temperature sensitivity. The results indicated that the original tsC lesion had reverted. However, in two of the seven clones examined, new ts lesions were found. These new ts lesions appeared phenotypically as ts⁺ due to the presence of extragenic suppressor mutations. Temperature-sensitive mutants representing three different groups were rescued from one suppressed clone, indicating that this ts⁺ clone contained multiple ts lesions. Among the ts mutants rescued were the initial isolates of a new recombination group which we have designated H. Some of the ts mutants rescued from the suppressed clones are capable of interfering with the growth of wild-type reovirus and may play a role in maintaining the carrier state. The results of this study show that persistently infected L cells contain a genetically heterogeneous population of reovirus even though all virus clones express the ts⁺ phenotype. It is thus critical to distinguish between genotype and phenotype when analyzing viruses that emerge during persistent infection.