Mutagenesis of the conserved 51-nucleotide region of Sindbis virus

We have constructed 25 site-specific mutations in a domain of 51 nucleotides in Sindbis virus that is highly conserved among all alphaviruses sequenced to date. These 51 nucleotides are capable of forming two hairpin structures and are found from nucleotides 155 to 205 in Sindbis virus within the region encoding nsP1. Of the mutations, 21 were silent and did not lead to a change in the amino acid sequence encoded. These silent mutations changed not only the linear sequence but also the stability of the hairpins in most cases. Two double mutants that were constructed led to the replacement of one base pair by another so that the linear sequence was altered but the nature of the hairpins was not. All of the mutants with silent mutations were viable, but 19 of the 21 mutants were severely impaired for growth in both chicken and mosquito cells. Compared with the parental virus, they grew slowly and produced virus at rates of 10(-1) to 10(-4) times the parental rate. Surprisingly, however, the plaques produced by these mutants were indistinguishable from those produced by the parental virus. Two of the silent mutations, found within the first hairpin structure, produced virus at a faster rate than the parental virus. It is clear that the exact sequence of this region is important for some aspect of virus replication. We suggest that one or more proteins, either virus encoded or cellular, bind to the hairpin structures in a sequence-specific fashion in a step that promotes replication of the viral RNA. Of the mutations that resulted in a change of coding, only one of four was viable, suggesting that the amino acid sequence encoded in this domain is essential for virus replication

Defined mutations in the 5' nontranslated sequence of Sindbis virus RNA

We have constructed 24 deletion mutants which contain deletions of from 1 to 15 nucleotides in the 5' nontranslated region of Sindbis virus RNA and tested the effect of these mutations on virus replication. The results showed that the first 44 nucleotides, which are capable of forming a hairpin structure, are important for virus replication, as all deletions tested in this region were either lethal or resulted in virus that grew poorly in comparison to the parental virus. Many of these deletions had different effects in mosquito cells than in chicken cells, suggesting that cellular factors, presumably proteins, bind to this region. This domain may function in at least two processes in viral replication. It seems likely that in the minus strand, this sequence element is bound by the viral replicase and promotes RNA replication. In the plus strand, this element may modulate initiation of translation of the nonstructural proteins. The results suggest that the hairpin structure itself is important. All deletions within it had deleterious effects on virus replication, and in particular, deletion of one of the G residues at nucleotide 7 or 8 or of one of the C residues at nucleotide 36 or 37 which are theoretically base-paired with these G's resulted in temperature-sensitive viruses that behaved very similarly. In contrast, large deletions between the 44-nucleotide hairpin and the translation start site at nucleotides 60 to 62 resulted in virus that grew as well as or better than the parental virus in both chicken and mosquito cells. The A residue at position 5 of the HRSP strain used was examined in more detail. Deletion of this A was lethal, whereas substitution by G resulted in a virus that grew poorly, despite the fact that G is present at position 5 in the AR339 parent of HRSP. U at position 5 resulted in a virus that grew less well than the A5 strain but better than the G5 mutant

Humphrey Center News: Spring 1988 v. 3, no. 1

Newsletter of the Hubert H. Humphrey Cancer Research Center at Boston University School of Medicine

Resonant and antiresonant bouncing droplets

When placed onto a vibrating liquid bath, a droplet may adopt a permanent bouncing behavior, depending on both the forcing frequency and the forcing amplitude. The relationship between the droplet deformations and the bouncing mechanism is studied experimentally and theoretically through an asymmetric and dissipative bouncing spring model. Antiresonance effects are evidenced. Experiments and theoretical predictions show that both resonance at specific frequencies and antiresonance at Rayleigh frequencies play crucial roles in the bouncing mechanism. In particular, we show that they can be exploited for droplet size selection.Comment: 4 pages, 4 figures and 1 vide

Entscheidungsfindungsprozesse im Vatikan. Beobachtungen zum Regierungsstil Pius’ XI. (1922-1939)

Seit die Akten der Jahre 1922 bis 1939 im Vatikanischen Geheimarchiv zugänglich sind, können die Verfahren der Entscheidungsfindung dieser Zeit detailliert nachgezeichnet werden. Es zeigt sich, dass damals Entwicklungen zu einem vorläufigen Abschluss kamen, die eine Kernfrage der Ekklesiologie berühren: das Spannungsfeld zwischen monarchischer und kollegialer Kirchenleitung. Pius XI. berief die Kongregation für die Außerordentlichen Kirchlichen Angelegenheiten kaum noch ein, das Konsistorium nutzte er vor allem noch, um Entscheidungen zu verkünden. Er entschied allein, wenn ihm die Präfekten der Kongregationen oder der Kardinalstaatssekretär in Privataudienzen Fragen vorlegten, während diese kaum miteinander sprachen. Wichtig waren neben den Nuntien informelle Netzwerke, zugleich stellte die steigende Flut einlaufender Informationen die Kurie vor große Herausforderungen. Diese Strukturen prägen den Vatikan bis heute – und haben in jüngster Zeit vermehrt zu Rufen nach Reformen geführt.Since all records from the pontificate of Pius XI (1922-1939) became accessible in the Vatican Secret Archives as of fall 2006, the procedures of decision-making of this period can be retraced in detail. It appears that during these years age-long developments came to a preliminary conclusion; these refer to a crucial question of ecclesiology: the area of conflict between monarchic and collegial church leadership. Pius XI rarely convoked the Congregation for Extraordinary Ecclesiastical Affairs, in which important issues of church policy had been discussed before, and used the consistory mainly as a forum to announce his decisions and to let them be received. Solely the pope decided when the prefects of the congregations and particularly the Cardinal Secretary of State were to submit questions to him, while the latter hardly spoke with each other. Consequently, the private audiences emerged as the main place of decision. Besides the nuncios, informal networks of informants played an important role; at the same time, the increasing flood of daily arriving information confronted the Curia with considerable challenges. The structures from the time of Pius XI shape the Vatican until today – and have increasingly led to demands for reforms and especially for a periodically sitting cabinet consisting of leading representatives of the congregations and the curial councils chaired by the pope