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We have found that human and ape ribosomal genes undergo concerted evolution involving genetic exchanges among nucleolus organizers on nonhomologous chromosomes. This conclusion is based upon restriction enzyme analysis of the ribosomal gene families in man and five ape species. Certain structural features were found to differ among (but not within) species even though the ribosomal genes have a multichromosomal distribution. Genetic exchanges among nucleolus organizer regions may be related to the well-known phenomenon of acrocentric chromosome associations observed in man and apes. Length variation in a region of the nontranscribed spacer was found in both chimpanzee species we examined. The nature of this length variation was found to be identical to that previously described in man. The origin of the length variation and its polymorphism within these three species might be explained by unequal alignment and unequal crossing-over among the ribosomal genes. An especially surprising finding was a nucleotide sequence polymorphism present in each individual human and ape we examined. Some ribosomal genes of each individual have a HindIl site in the 28S gene about 800 base pairs from the EcoRI site in this gene. The remaining 28S genes lack this HindII site. The presence of this polymorphism within individuals of every species we examined suggests that it has been maintained by natural selection.

The viral RNA segments in influenza virions were shown to be circular in conformation by using psoralen crosslinking methods. Electron microscopy of purified RNA following treatment of virus with the psoralen reagent 4'-aminomethyltrioxsalen (AMT) revealed circles with lengths corresponding to the individual segments. RNA blot analysis using polyacrylamide gels demonstrated that RNA from AMTtreated virus had a slowed migration, consistent with it being a single-stranded circle. Furthermore, nuclease S1 protection assays indicated that the termini of the RNA segments form an approximately 15-base-pair-long panhandle. This structure is consistent with the partial sequence complementarity that has been observed for the termini of all influenza virus RNAs. By RNA blot analysis, circular structures of viral sense RNA were also found in influenza virus-infected cells at early and late time points. The circular RNA was the predominant species at the time when the major transcription product is message RNA. This finding and the observation that the termination signal for mRNA synthesis directly abuts the panhandle suggest that a panhandle in the template viral RNA is a cis regulatory signal promoting the synthesis of mRNA instead of plus-sense template. Also, since the panhandle is present in high concentration in virions, we suggest that it is required for packaging and that the input RNA after infection is in the proper conformation for synthesis of primary transcripts.Influenza A virus contains eight negatively stranded RNA segments that encode at least 10 distinct proteins (for review, see ref. 1). The core of the virus consists of eight individual nucleocapsids, which are made up of the RNA segments, the three polymerase (P) proteins, and nucleoprotein molecules (NP). Early analyses revealed that these nucleocapsids have different molecular weights (2) and that they appear as strands (3) that show loops at one end. The tertiary structure is formed by folding back on itself and coiling, which results in a large double helix (3, 4). The lengths of the nucleocapsid strands can be correlated with the molecular weights of the viral RNA segments. Also, the presence of multiple nucleocapsid segments correlates with the finding that the mRNAs are transcribed independently (5, 6). Each of the nucleocapsids directs the synthesis of complementary RNA starting from the 3' ends of the virion RNAs (7). Although sequencing studies of the 3' and 5' termini of influenza A, B, and C virus RNAs revealed the presence of inverted complementary sequences (8-10), it was not known whether the termini actually basepair in ribonucleoprotein complexes.In the present study, we show that UV crosslinking of virion RNAs in the presence of 4'-substituted psoralen leads to circular RNA structures. This finding supports the proposition that stable hairpin structures involving the RNA termini are present in infectious influenza virus particles. In addition, we find that the viral RNA in infected cells is predominantly circular during the pha...

Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higherorder NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.antiinfluenza | oligomerization | polymerase inhibitor | protein-protein interaction | cooperative inhibition

We succeeded in rescuing infectious influenza virus by transfecting cells with RNAs derived from specific recombinant DNAs. RNA corresponding to the neuraminidase (NA) gene of influenza A/WSN/33 (WSN) virus was transcribed in vitro from plasmid DNA and, following the addition of purified influenza virus RNA polymerase complex, was transfected into MDBK cells. Superinfection with helper virus lacking the WSN NA gene resulted in the release of virus containing the WSN NA gene. We then introduced five point mutations into the WSN NA gene by cassette mutagenesis of the plasmid DNA. Sequence analysis of the rescued virus revealed that the genome contained all five mutations present in the mutated plasmid. The ability to create viruses with site-specific mutations will allow the engineering of influenza viruses with dermed biological properties.

Variation in influenza A viruses was examined by comparison of nucleotide sequences of the NS gene (890 bases) of 15 human viruses isolated over 53 years (1933 to 1985). Changes in the genes accumulate with time, and an evolutionary tree based on the maximum parsimony method can be constructed. The evolutionary rate is approximately 2 X 10(-3) substitution per site per year in the NS genes, which is about 10(6) times the evolutionary rate of germline genes in mammals. This uniform and rapid rate of evolution in the NS gene is a good molecular clock and is compatible with the hypothesis that positive selection is operating on the hemagglutinin (or perhaps some other viral genes) to preserve random mutations in the NS gene.

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