To determine if West Nile virus (WNV) infection of insect cells induces a protective RNAi response, Drosophila melanogaster S2 and Aedes albopictus C6/36 cells were infected with WNV, and the production of WNV-homologous small RNAs was assayed as an indicator of RNAi induction. A distinct population of approximately 25 nt WNV-homologous small RNAs was detected in infected S2 cells but not C6/36 cells. RNAi knockdown of Argonaute 2 in S2 cells resulted in slightly increased susceptibility to WNV infection, suggesting that some WNV-homologous small RNAs produced in infected S2 cells are functional small interfering RNAs. WNV was shown to infect adult D. melanogaster, and adult flies containing mutations in each of four different RNAi genes (Argonaute 2, spindle-E, piwi, and Dicer-2) were significantly more susceptible to WNV infection than wildtype flies. These results combined with the analysis of WNV infection of S2 and C6/36 cells support the conclusion that WNV infection of D. melanogaster, but perhaps not Ae. albopictus, induces a protective RNAi response.

Variant histones that differ in amino acid sequence from S-phase histones are widespread in eukaryotes, yet the structural changes they cause to nucleosomes and how those changes affect relevant cellular processes have not been determined. H2A.F/Z is a highly conserved family of H2A variants. H2Av, the H2A.F/Z variant of Drosophila melanogaster, was localized in polytene chromosomes by indirect immunofluorescence and in diploid chromosomes by chromatin immunoprecipitation. H2Av was widely distributed in the genome and not limited to sites of active transcription. H2Av was present in thousands of euchromatic bands and the heterochromatic chromocenter of polytene chromosomes, and the H2Av antibody precipitated both transcribed and nontranscribed genes as well as noncoding euchromatic and heterochromatic sequences. The distribution of H2Av was not uniform. The complex banding pattern of H2Av in polytene chromosomes did not parallel the concentration of DNA, as did the pattern of immunofluorescence using H2A antibodies, and the density of H2Av measured by immunoprecipitation varied between different sequences. Of the sequences assayed, H2Av was least abundant on 1.688 satellite sequences and most abundant on the hsp70 genes. Finally, transcription caused, to an equivalent extent, both H2Av and H2A to be less tightly associated with DNA.The basic unit of chromatin in eukaryotes is the nucleosome. A nucleosome consists of 146 base pairs of DNA wrapped around an octamer of histone proteins H2A, H2B, H3, and H4 (1). Although chromatin is a highly reiterative structure, cells create heterogeneity in the structure of nucleosomes to facilitate and regulate DNA-mediated processes such as transcription. Heterogeneity is created, in part, by posttranslational modifications of histone proteins, including acetylation, phosphorylation, methylation, ubiquitination, and ADP-ribosylation (2-4). Acetylation status of the amino termini of histones H3 and H4, in particular, plays a important role in transcriptional regulation (2, 5).Heterogeneity in nucleosome structure also results from incorporation of variant histone proteins into the nucleosome. In contrast to the canonical histones, which are multicopy genes expressed during S-phase of the cell cycle, variant histones are encoded by single copy genes that differ in amino acid sequence from S-phase histones and whose expression is not limited to S-phase (6, 7). Variant histones allow specialization of nucleosome structure for specific purposes. Sperm-specific variant histones, for example, facilitate the dramatic compaction of DNA that occurs during spermatogenesis (4,8). A specific variant of histone H3 is incorporated specifically at centromeres creating a specialized chromatin structure required for proper function of the kinetochore (9 -14), and macro-H2A, a histone H2A variant, localizes preferentially to the inactive X chromosome in mammals and may alter chromatin in a way that helps suppress transcription (15). These and other variants have been identified for histones H2A,...