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We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses defective in virus-associated (VA) RNA expression. The results show that the virus-associated RNAs, VA RNAI and VA RNAII, function as suppressors of RNAi by interfering with the activity of Dicer. The VA RNAs bind Dicer and function as competitive substrates squelching Dicer. Further, we show that VA RNAI and VA RNAII are processed by Dicer, both in vitro and during a lytic infection, and that the resulting short interfering RNAs (siRNAs) are incorporated into active RISC. Dicer cleaves the terminal stem of both VA RNAI and VA RNAII. However, whereas both strands of the VA RNAI-specific siRNA are incorporated into RISC, the 3' strand of the VA RNAII-specific siRNA is selectively incorporated during a lytic infection. In summary, our work shows that adenovirus suppresses RNAi during a lytic infection and gives insight into the mechanisms of RNAi suppression by VA RNA.

The untranslated leader RNA is the most conserved part of the human immunodeficiency virus type I (HIV-1) genome. It contains many regulatory motifs that mediate a variety of steps in the viral life cycle. Previous work showed that the full-length leader RNA can adopt two alternative structures: a long distance interaction (LDI) and a branched multiple-hairpin (BMH) structure. The BMH structure exposes the dimer initiation site (DIS) hairpin, whereas this motif is occluded in the LDI structure. Consequently, these structures differ in their capacity to form RNA dimers in vitro. The BMH structure is dimerization-competent, due to DIS hairpin formation, but also presents the splice donor (SD) and RNA packaging (Psi) hairpins. In the LDI structure, an extended RNA packaging (Psi(E)) hairpin is folded, which includes the splice donor site and gag coding sequences. The gag initiation codon is engaged in a long distance base pairing interaction with sequences in the upstream U5 region in the BMH structure, thus forming the evolutionarily conserved U5-AUG duplex. Therefore, the LDI-BMH equilibrium may affect not only the process of RNA dimer formation but also translation initiation. In this study, we designed mutations in the 3'-terminal region of the leader RNA that alter the equilibrium of the LDI-BMH structures. The mutant leader RNAs are affected in RNA dimer formation, but not in their translation efficiency. These results indicate that the LDI-BMH status does not regulate HIV-1 RNA translation, despite the differential presentation of the gag initiation codon in both leader RNA structures.

A pseudotriloop is formed by transloop base pairing between the first (5') and the fifth nucleotide in a hexanucleotide RNA loop ("hexaloop") to subtend a triloop of nucleotides 2-4. This structure has been found in hairpins involved in the regulation of iron metabolism in mammalian cells and in transcription of plant virus subgenomic RNA. Several hexaloop hairpins, including HIV-transactivation-responsive element and hepatitis B virus , potentially adopt a pseudotriloop conformation. Here we show that an RNA plant virus whose replication depends on a conventional triloop hairpin can be used to verify the existence of pseudotriloop structures in vivo. Our data suggest that the pseudotriloop may represent a common motif in RNA-protein recognition.

In the Bromoviridae family of plant viruses, trinucleotide hairpin loops play an important role in RNA transcription. Recently, we reported that Brome mosaic virus (BMV) subgenomic (sg) transcription depended on the formation of an unusual triloop hairpin. By native gel electrophoresis, enzymatic structure probing, and NMR spectroscopy it is shown here that in the absence of viral replicase the hexanucleotide loop 5'C1AUAG5A3' of this RNA structure can adopt a pseudo trinucleotide loop conformation by transloop base pairing between C1 and G5. By means of in vitro replication assays using partially purified BMV RNA-dependent RNA polymerase (RdRp) it was found that other base pairs contribute to sg transcription, probably by stabilizing the formation of this pseudo triloop, which is proposed to be the primary element recognized by the viral replicase. The BMV pseudo triloop structure strongly resembles iron-responsive elements (IREs) in cellular messenger RNAs and may represent a general protein-binding motif. In addition, in vitro replication assays showed that the BMV sg hairpin is functionally equivalent to the minus-strand core promoter hairpin stem-loop C at the 3' end of BMV RNAs. Replacement of the sg hairpin by stem-loop C yielded increased sg promoter activity whereas replacement of stem-loop C by the sg hairpin resulted in reduced minus-strand promoter activity. We conclude that AUA triloops represent the common motif in the BMV sg and minus-strand promoters required for recruitment of the viral replicase. Additional sequence elements of the minus-strand promoter are proposed to direct the RdRp to the initiation site at the 3' end of the genomic RNA.