Human Kv1.6 current displays a C-type-like inactivation when re-expressed in cos-7 cells.

The human Kv1.6K(+) channel was functionally re-expressed in COS-7 cells at different levels. Voltage-activated K(+) currents are recorded upon cell membrane depolarization independently of the level of Kv1.6 expression. The current acquires a fast inactivation when Kv1.6 expression is increased. Inactivation was not affected by divalent cations or by extracellular tetraethylammonium. We have characterized the inactivation properties in biophysical terms. The fraction of inactivated current and the kinetics of inactivation are increased as the cell becomes more depolarized. Inactivated current can be reactivated according to a bi-exponential function of time. Additional experiments indicate that Kv1.6 inactivation properties are close to those of a conventional C-type inactivation. This work suggests that the concentration of Kv1.6 channel in the cell membrane strongly modulates the kinetic properties of Kv1.6-induced K(+) current. The physiological implications of these modifications are discussed.

Phosphatidylinositol-4,5-bisphosphate, PIP2, controls KCNQ1/KCNE1 voltage-gated potassium channels: a functional homology between voltage-gated and inward rectifier K+ channels.

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is a major signaling molecule implicated in the regulation of various ion transporters and channels. Here we show that PIP(2) and intracellular MgATP control the activity of the KCNQ1/KCNE1 potassium channel complex. In excised patch-clamp recordings, the KCNQ1/KCNE1 current decreased spontaneously with time. This rundown was markedly slowed by cytosolic application of PIP(2) and fully prevented by application of PIP(2) plus MgATP. PIP(2)-dependent rundown was accompanied by acceleration in the current deactivation kinetics, whereas the MgATP-dependent rundown was not. Cytosolic application of PIP(2) slowed deactivation kinetics and also shifted the voltage dependency of the channel activation toward negative potentials. Complex changes in the current characteristics induced by membrane PIP(2) was fully restituted by a model originally elaborated for ATP-regulated two transmembrane-domain potassium channels. The model is consistent with stabilization by PIP(2) of KCNQ1/KCNE1 channels in the open state. Our data suggest a striking functional homology between a six transmembrane-domain voltage-gated channel and a two transmembrane-domain ATP-gated channel.

A role of Sep1 (= Kem1, Xrn1) as a microtubule-associated protein in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells lacking the SEP1 (also known as XRN1, KEM1, DST2, RAR5) gene function exhibit a number of phenotypes in cellular processes related to microtubule function. Mutant cells show increased sensitivity to the microtubule-destabilizing drug benomyl, increased chromosome loss, a karyogamy defect, impaired spindle pole body separation, and defective nuclear migration towards the bud neck. Analysis of the arrest morphology and of the survival during arrest strongly suggests a structural defect accounting for the benomyl hypersensitivity, rather than a regulatory defect in a checkpoint. Biochemical analysis of the purified Sep1 protein demonstrates its ability to promote the polymerization of procine brain and authentic S.cerevisiae tubulin into flexible microtubules in vitro. Furthermore, Sep1 co-sediments with these microtubules in sucrose cushion centrifugation. Genetic analysis of double mutant strains containing a mutation in SEP1 and in one of the genes coding for alpha- or beta-tubulin further suggests interaction between Sep1 and microtubules. Taken together these three lines of evidence constitute compelling evidence for a role of Sep1 as an accessory protein in microtubule function in the yeast S.cerevisiae.

Purification of assembly-competent tubulin from Saccharomyces cerevisiae.

We have developed a straightforward, two-step procedure to isolate highly purified yeast tubulin that reproducibly assembles into microtubules. The starting extracts are obtained from cells genetically engineered to overproduce both the alpha and beta subunits of tubulin, under control of the galactose promoter, to approximately 10-times wild-type levels. The first step of purification is carried out with the high-speed supernatant of lysed cells loaded onto a DEAE-Sephadex column; after this step the tubulin preparation is approximately 30% pure. In the second step, the tubulin fractions are loaded onto an immunoaffinity column prepared by coupling the anti-(alpha-tubulin) monoclonal antibody YL 1/2 to Sepharose-4B. Following elution with 0.8 M KCl, the tubulin present in the peak is 90% pure. Upon addition of porcine brain microtubule-associated proteins or DEAE-dextran, this tubulin preparation is functionally active for assembly into microtubules, as visualized by electron microscopy on negatively stained samples. Virtually identical microtubule structures are produced in parallel experiments on the assembly of yeast or porcine brain tubulin, with differences observed only at acidic pH values. Overall, this relatively simple procedure provides a useful tool for the production of functional tubulin suitable both for structural studies and for investigations of the assembly process.

Wide occurrence of measles virus subgenomic RNAs in attenuated live-virus vaccines.

Nine measles vaccine preparations, including four different viral strains, provided by eight different manufacturers were analysed by Northern blot for the nature of their nucleocapsid RNAs. Out of nine preparations, six were shown to contain subgenomic RNAs, along with the full length genomic RNA. Presence or absence of the subgenomic RNAs correlated strictly with the viral strains used. The role of the defective interfering particles in measles virus vaccine attenuation, and in its seroconversion efficacy upon vaccination, as well as the potential hazard of the presence of defective interfering particles in live-virus vaccine preparations, is discussed.

Translational requirement for La Crosse virus S-mRNA synthesis: a possible mechanism.

Ongoing protein synthesis is required for La Crosse S-mRNA synthesis in vivo, and complete S-mRNA can be made in vitro only in the presence of an active rabbit reticulocyte lysate. Using in vitro systems based on the polymerase activity of purified virions, we further support the notion that it is translation of the nascent mRNA that is required for complete transcription. Since replacement of guanosine with inosine in the nascent mRNA substitutes for the translational requirement, it appears that translation is required to prevent interactions of the nascent chain from taking place, which, if not prevented, lead to premature termination. These interactions appear to be between the nascent mRNA chain and its nucleocapsid template. A model for the translational requirement for complete S-mRNA synthesis is presented.

Intratypic recombination of polioviruses: evidence for multiple crossing-over sites on the viral genome.

Intratypic recombinant polioviruses were isolated from cells that were coinfected with two temperature-sensitive (ts) mutants of poliovirus type 1, ts035Gr and ts247. After phenotypic characterization of these recombinants, their proteins were studied by polyacrylamide gel electrophoresis, and their genomes were analyzed by RNase T1 fingerprinting and partial nucleotide sequencing. Segregation of specific phenotypic and biochemical characteristics inherited from the parental viruses demonstrated that crossing-over could occur in at least four distinct regions of the genome. Possible mechanisms for recombination are discussed.

Multiple mutations involved in the phenotype of a temperature-sensitive small-plaque mutant of poliovirus.

A temperature-sensitive small-plaque mutant of poliovirus type 1, ts247, has been analyzed previously. Several mutations were detected in the P3 region of the genome by analysis of proteins and by T1 oligonucleotide mapping of viral RNA. We have now studied spontaneous reversion of ts247 to the wild-type phenotype. This was found to be a two-step event, reversion to a ts+ phenotype (revertant R247-51) being distinct from acquisition of normal plaque size (revertant R247-12). The mutation responsible for the ts phenotype of ts247, implicated also in virus aggregation and heat lability, could not be detected by biochemical studies. Analysis of homotypic recombinants obtained by crossing ts247 with a guanidine-resistant derivative of a temperature-sensitive replicase mutant mapped this mutation to the P1 region or to the 5' end of the P2 region of the genome. The small-plaque phenotype of ts247 and R247-51 was correlated with an abnormality in polypeptide 3C (protease); direct sequencing of viral RNA revealed a U to C change at nucleotide 5658, which altered an isoleucine to threonine in the protease of ts247 and R247-51 but not of R247-12. Two other mutations were present in the region of the genome coding for polypeptide 3D of ts247 and of both classes of revertants. They thus seemed to play no role in the phenotype of ts247. One mutation, an A to G change at nucleotide 7135, was silent at the protein level, whereas the other, an A to G change at nucleotide 6264, determined a major amino acid change from glutamate to glycine in the viral replicase.

Translational requirement of La Crosse virus S-mRNA synthesis: in vitro studies.

The exceptional requirement of La Crosse virus mRNA synthesis for ongoing protein synthesis in vivo was examined in vitro by using purified virions and a reticulocyte lysate. Transcription from the S genome produced two incomplete transcripts (110 and 205 nucleotides [nt]) in the absence of the lysate, whereas S-mRNA (900 nt) was predominantly made when the lysate was present. The addition of drugs which inhibit protein synthesis also inhibited the synthesis of S-mRNA, and in some cases led to the reappearance of the 205-nt RNA. Reconstruction experiments demonstrated that the incomplete transcripts were not the result of rapid and selective degradation of S-mRNA but were due to premature termination of the polymerase at defined sites. The requirement for ongoing protein synthesis for productive transcription in vitro is not at the level of chain initiation but for elongation of the nascent RNA beyond these sites.

Recombination and rescue between temperature-sensitive mutants of poliovirus type 1.

Four different temperature-sensitive (ts) mutants derived from the Mahoney strain of poliovirus type 1, were crossed in an infectious center recombination test. Evidence for recombination was obtained in three crosses, with a different segregation of an unselected marker, resistance to guanidine, in each case. Evidence for genetic complementation between ts mutants was not found, except with one set of RNA- mutants, ts 221 and ts 035. The marked virus yield enhancement which was observed in cells mixedly infected by these two mutants resulted from a nonreciprocal rescue of ts 035 by ts 221. The effects of ts 221 input multiplicity and of guanidine inhibition of viral RNA replication on the rescue were analyzed. The results showed that yield enhancement of ts 035 in mixed infection could be correlated to the low level RNA replication of ts 221 at the nonpermissive temperature.

Biochemical characterization of poliovirus type 1 temperature-sensitive mutants.

Four temperature-sensitive mutants selected upon chemical mutagenesis of the poliovirus type 1 Mahoney strain [H. Agut, T. Matsukura, C. Bellocq, M. Dréano, J. C. Nicolas, and M. Girard, Ann. Virol. (Inst. Pasteur), 132E, 445-460 (1981)]. were analyzed by T1 oligonucleotide mapping. Three mutants (ts 203, ts 221, and ts 035) had T1 fingerprints identical to that of wild-type virus while mutant ts 247 exhibited two differences on oligonucleotides that mapped in the region of the genome coding for the replicase (polypeptide 4b) and for the protease (polypeptide 7c) of the virus. Cells were infected with each of the four mutants separately, labeled with [35S]methionine, and the labeled polypeptides were analyzed by SDS-polyacrylamide gel electrophoresis. Mutants and wild-type virus polypeptides showed a similar electrophoretic pattern except for the replicase and the protease of ts 247 which showed abnormal apparent molecular weights. The labeled proteins were subjected to two-dimensional isoelectrofocusing and SDS-polyacrylamide gel electrophoresis. Polypeptides 4b (replicase) and 2 (the common precursor to polypeptides 7c and 4b) of ts 247 and ts 035 exhibited distinct charge alterations when compared to the corresponding wild-type polypeptides. These alterations were also found on polypeptide 6a in the case of ts 247 and on polypeptide 6b in the case of ts 035, both polypeptides resulting from an alternate cleavage of polypeptide 2.