The key steps in the replication of the poliovirus genome, initiation of (‐) and (+) strands, require two different cis‐acting elements, oriR and oriL, respectively. It has been proposed that the spatial organization of these elements is maintained by tertiary (‘kissing’) interactions between the loops of two constituent hairpins. Here, the putative partners of the kissing interaction within the oriR of the full‐length poliovirus RNA were modified by site‐directed mutagenesis. The destabilization of this interaction resulted in a severe suppression of the viral RNA synthesis, but the mutant transcripts proved to be infectious. With a single exception, the potential for the kissing interaction within the oriR of the recovered viruses was partially or completely restored due to either true reversions or second‐site compensatory mutations. There was a good correlation between the restoration of this potential and the phenotypic properties of the viruses. It was concluded that the kissing interaction in the poliovirus oriR is functionally important. Using the above experimental data, a three‐dimensional structure was derived by molecular modeling techniques, which demonstrated the overall feasibility of the proposed interactions and displayed the poliovirus oriR as a quasi‐globular multi‐domain structure.

Higher-order RNA structures in the 3 untranslated region (3UTR) of enteroviruses are thought to play a pivotal role in viral negative-strand RNA synthesis. The structure of the 3UTR was predicted by thermodynamic calculations using the STAR (structural analysis of RNA) computer program and experimentally verified using chemical and enzymatic probing of in vitro-synthesized RNA. A possible pseudoknot interaction between the 3D polymerase coding sequence and domain Y and a "kissing" interaction between domains X and Y was further studied by mutational analysis, using an infectious coxsackie B3 virus cDNA clone (domain designation as proposed by E. V. Pilipenko, S. V. Maslova, A. N. Sinyakov, and V. I. Agol (Nucleic Acids Res. 20:1739-1745, 1992). The higher-order RNA structure of the 3UTR appeared to be maintained by an intramolecular kissing interaction between the loops of the two predominant hairpin structures (X and Y) within the 3UTR. Disturbing this interaction had no effect on viral translation and processing of the polyprotein but exerted a primary effect on viral replication, as was demonstrated in a subgenomic coxsackie B3 viral replicon, in which the capsid P1 region was replaced by the luciferase gene. Mutational analysis did not support the existence of the pseudoknot interaction between hairpin loop Y and the 3D polymerase coding sequence. Based on these experiments, we constructed a three-dimensional model of the 3UTR of coxsackie B virus that shows the kissing interaction as the essential structural feature of the origin of replication required for its functional competence.

The origin of replication ( oriR ) involved in the initiation of (-) strand enterovirus RNA synthesis is a quasi-globular multi-domain RNA structure which is maintained by a tertiary kissing interaction. The kissing interaction is formed by base pairing of complementary sequences within the predominant hairpin-loop structures of the enteroviral 3' untranslated region. In this report, we have fully characterised the kissing interaction. Site-directed mutations which affected the different base pairs involved in the kissing interaction were generated in an infectious coxsackie B3 virus cDNA clone. The kissing interaction appeared to consist of 6 bp. Distortion of the interaction by mispairing of each of the base pairs involved in this higher order RNA structure resulted in either temperature sensitive or lethal phenotypes. The nucleotide constitution of the base which gaps the major groove of the kissing domain was not relevant for virus growth. The reciprocal exchange of the complete sequence involved in the kissing resulted in a mutant virus with wild type virus growth characteristics arguing that the base pair constitution is of less importance for the initiation of (-) strand RNA synthesis than the existence of the tertiary structure itself.

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The coxsackie B3 virus ori R is an element of viral RNA thought to promote the assembly of a ribonucleoprotein complex involved in the initiation of genome replication. The mutual orientation of its two helical domains X and Y is determined by a kissing interaction between the loops of these domains. Here, a genetic approach was worked out to identify spatial orientation-dependent recognition signals in these helices. Spatial orientation changes (due to linear and rotational shifts) were introduced by appropriate insertions/deletions of a single base pair into one or both of the domains, and phenotypic consequences caused by these mutations were studied. The insertion of a base pair into domain Y caused a defect in viral reproduction that could be suppressed by a base-pair insertion into domain X. Similarly, a defect in viral replication caused by a base-pair deletion from domain X could be suppressed by a base-pair deletion from domain Y. Thus, certain areas of the two domains should cross-talk to one another in the sense that a change of space position of one of them required an adequate reply (change of space position) from the other. Phenotypic effects of the local rotation of one or more base pairs (and of some other mutations) in either domain X or domain Y suggested that the two most distal base pairs of these domains served as orientationdependent recognizable signals. The results were also consistent with the notion that the recognition of the distal base pair of domain Y involved a mechanism similar to the intercalation of an amino acid residue.

The application of Patterson methods to the solution of molecular structures with approximately equal atoms is usually limited by the translation function. The literature on translation functions for the positioning of a correctly oriented molecular fragment is surveyed and summarized. Similarities and differences in the published functions are discussed. Variations of these functions have been tested, with the goal of finding the function that will give the best results for small search fragments and that can be used in routine X-ray analyses of small and medium sized structures. We find that well known reciprocal-space correlation functions (which are shown to be closely related to the residual R 2 ), which make use of Fast Fourier Transform techniques, are fast and reliable. Best results are obtained when all symmetry elements are used simultaneously, and geometrically unacceptable positions are rejected. Test results show that for small fragments (containing less than 10% of the scattering matter of the asymmetric unit) one should use all observed reflections (including weak reflections) and the correlation function to be used should take care of the reciprocal space equivalent of sharpening, origin removal, and subtraction of intramolecular vectors. A computer program for the automatic positioning of a correctly oriented molecular fragment is described. § 1. IntroductionIn structure determination processes using direct methods to solve the phase problem, it often occurs that the resulting structural information is

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