Speciation via interspecific or intergeneric hybridization and polyploidization triggers genomic responses involving genetic and epigenetic alterations. Such modifications may be induced by small RNAs, which affect key cellular processes, including gene expression, chromatin structure, cytosine methylation and transposable element (TE) activity. To date, the role of small RNAs in the context of wide hybridization and polyploidization has received little attention. In this work, we performed high-throughput sequencing of small RNAs of parental, intergeneric hybrid, and allopolyploid plants that mimic the genomic changes occurring during bread wheat speciation. We found that the percentage of small RNAs corresponding to miRNAs increased with ploidy level, while the percentage of siRNAs corresponding to TEs decreased. The abundance of most miRNA species was similar to midparent values in the hybrid, with some deviations, as seen in overrepresentation of miR168, in the allopolyploid. In contrast, the number of siRNAs corresponding to TEs strongly decreased upon allopolyploidization, but not upon hybridization. The reduction in corresponding siRNAs, together with decreased CpG methylation, as shown here for the Veju element, represent hallmarks of TE activation. TE-siRNA downregulation in the allopolyploid may contribute to genome destabilization at the initial stages of speciation. This phenomenon is reminiscent of hybrid dysgenesis in Drosophila.

The retroviral Gag precursor plays an important role in the assembly of virion particles. The capsid (CA) protein of the Gag molecule makes a major contribution to this process. In the crystal structure of the free CA protein of the human immunodeficiency virus type 1 (HIV-1), 11 residues of the C terminus were found to be unstructured, and to date no information exists on the structure of these residues in the context of the Gag precursor molecule. We performed phylogenetic analysis and demonstrated a high degree of conservation of these 11 amino acids. Deletion of this cluster or introduction of various point mutations into these residues resulted in significant impairment of particle infectivity. In this cluster, two putative structural regions were identified, residues that form a hinge region (353-VGGP-356) and those that contribute to an ␣-helix (357-GHKARVL-363). Overall, mutations in these regions resulted in inhibition of virion production, but mutations in the hinge region demonstrated the most significant reduction. Although all the Gag mutants appeared to have normal Gag-Gag and Gag-RNA interactions, the hinge mutants were characterized by abnormal formation of cytoplasmic Gag complexes. Gag proteins with mutations in the hinge region demonstrated normal membrane association but aberrant rod-like membrane structures. More detailed analysis of these structures in one of the mutants demonstrated abnormal trapped Gag assemblies. These data suggest that the conserved CA C terminus is important for HIV-1 virion assembly and release and define a putative target for drug design geared to inhibit the HIV-1 assembly process.

A specific interaction between the nucleocapsid (NC) domain of the Gag polyprotein and the RNA encapsidation signal (⌿) is required for preferential incorporation of the retroviral genomic RNA into the assembled virion. Using the yeast three-hybrid system, we developed a genetic screen to detect human immunodeficiency virus type 1 (HIV-1) Gag mutants with altered RNA binding specificities. Specifically, we randomly mutated full-length HIV-1 Gag or its NC portion and screened the mutants for an increase in affinity for the Harvey murine sarcoma virus encapsidation signal. These screens identified several NC zinc finger mutants with altered RNA binding specificities. Furthermore, additional zinc finger mutants that also demonstrated this phenotype were made by site-directed mutagenesis. The majority of these mutants were able to produce normal virion-like particles; however, when tested in a single-cycle infection assay, some of the mutants demonstrated higher transduction efficiencies than that of wild-type Gag. In particular, the N17K mutant showed a seven-to ninefold increase in transduction, which correlated with enhanced vector RNA packaging. This mutant also packaged larger amounts of foreign RNA. Our results emphasize the importance of the NC zinc fingers, and not other Gag sequences, in achieving specificity in the genome encapsidation process. In addition, the described mutations may contribute to our understanding of HIV diversity resulting from recombination events between copackaged viral genomes and foreign RNA.The assembly process of retroviruses includes the encapsidation of the genomic RNA inside the nascent virion by Gag proteins in the cytoplasm. Although this RNA accounts for Ͻ1% of the total cytoplasmic RNA, it reaches Ͼ50% of the virion nucleic acids by weight (reviewed in reference 7). This selective enrichment is the result of specific interactions between cis-acting packaging elements in the genomic RNA and trans-acting RNA binding sites in the Gag precursor.For human immunodeficiency virus type 1 (HIV-1), many studies have demonstrated that the major encapsidation signal (⌿) is a structured RNA sequence that lies downstream of the primer binding site and extends into the 5Ј portion of the gag gene (29,30,34,44,45). Within this region, four adjacent stem-loop structures contribute to the encapsidation of the viral RNA, with each stem-loop binding the nucleocapsid (NC) domain in the HIV-1 Gag precursor with a different affinity (2,3,8,16,17,21,46,47). Two copies of a Cys-His box, containing the consensus sequence Cys-X2-Cys-X4-His-X4-Cys, in the HIV-1 NC are crucial for an interaction with RNA. These form two "zinc finger" or "zinc knuckle" motifs (also designated F1 for the N-terminal motif and F2 for the C-terminal motif), since the conserved cysteine and histidine residues bind a zinc ion, which leads to a rigid conformation. The compact structure of each of the zinc fingers forms a direct contact with the RNA (21, 61). This structure is important for the encapsidation of the viral RNA, a...