Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation mechanism that is widespread in eukaryotic organisms. It is often associated with methylation of the transcribed region of the silenced gene and with accumulation of small RNAs (21 to 25 nucleotides) homologous to the silenced gene. In plants, PTGS can be triggered locally and then spread throughout the organism via a mobile signal that can cross a graft junction. Previously, we showed that the helper component-proteinase (HC-Pro) of plant potyviruses suppresses PTGS. Here, we report that plants in which PTGS has been suppressed by HC-Pro fail to accumulate the small RNAs associated with silencing. However, the transgene locus of these plants remains methylated. Grafting experiments indicate that HC-Pro prevents the plant from responding to the mobile silencing signal but does not eliminate its ability to produce or send the signal. These results demonstrate that HC-Pro functions downstream of transgene methylation and the mobile signal at a step preceding accumulation of the small RNAs. INTRODUCTIONPost-transcriptional gene silencing (PTGS) is a sequencespecific RNA degradation mechanism first discovered in transgenic plants (Napoli et al., 1990;Smith et al., 1990;van der Krol et al., 1990). Related processes have been found in diverse eukaryotic organisms including Neurospora , in which it is called quelling, and a variety of animal systems, in which it is referred to as RNA interference or RNAi Fire, 1999;Grant, 1999;Kooter et al., 1999;Ding, 2000;Matzke et al., 2001). Sequence-specific RNA degradation is triggered by double stranded RNA (dsRNA) in a variety of organisms Waterhouse et al., 1998;Sharp, 1999;Bass, 2000;Matzke et al., 2001). In plants, PTGS can be induced by RNA viruses, many of which replicate via dsRNA intermediates. Finally, in both plants and Caenorhabditis elegans , the process can be triggered locally and then spread to distant parts of the organism (Palauqui et al., 1997;Voinnet and Baulcombe, 1997;Fire et al., 1998;Jorgensen et al., 1998;Palauqui and Vaucheret, 1998;Voinnet et al., 1998). The relatedness of these sequence-specific RNA degradation processes in different organisms is evidenced by their requirement for a conserved set of gene products Matzke et al., 2001), including a protein with homology to translation factor eIF2C (Tabara et al., 1999;Catalanotto et al., 2000;Fagard et al., 2000), an RNA-dependent RNA polymerase (RdRp) (Cogoni and Macino, 1999a;Dalmay et al., 2000;Mourrain et al., 2000;Smardon et al., 2000), and proteins with homology to DNA helicases and RNase D (Cogoni and Macino, 1999b;Ketting et al., 1999). However, at this point, neither the roles of these various gene products nor the mechanisms for induction, maintenance, and spread of sequence-specific RNA degradation are clearly understood.Several molecular features characterize the sequencespecific RNA degradation processes found in diverse organisms. Studies in both plants and Drosophila have shown that silencing is accompanied b...

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A requisite step in reovirus infection of the murine intestine is proteolysis of outer-capsid proteins to yield infectious subvirion particles (ISVPs). When converted to ISVPs by intestinal proteases, virions of reovirus strain type 3 Dearing (T3D) lose 90% of their original infectivity due to cleavage of viral attachment protein ς1. In an analysis of eight field isolate strains of type 3 reovirus, we identified one additional strain, type 3 clone 31 (T3C31), that loses infectivity and undergoes ς1 cleavage upon conversion of virions to ISVPs. We examined the ς1 deduced amino acid sequences of T3D and the eight field isolate strains for a correlation between sequence variability and ς1 cleavage. The ς1 proteins of T3D and T3C31 contain a threonine at amino acid position 249, whereas an isoleucine occurs at this position in the ς1 proteins of the remaining strains. Thr249 occupies the d position of a heptad repeat motif predicted to stabilize ς1 oligomers through α-helical coiled-coil interactions. This region of sequence comprises a portion of the fibrous tail domain of ς1 known as the neck. Substitution of Thr249 with isoleucine or leucine resulted in resistance to cleavage by trypsin, whereas replacement with asparagine did not affect cleavage susceptibility. These results demonstrate that amino acid position 249 is an independent determinant of T3D ς1 cleavage susceptibility and that an intact heptad repeat is required to confer cleavage resistance. We performed amino-terminal sequence analysis on the ς1 cleavage product released during trypsin treatment of T3D virions to generate ISVPs and found that trypsin cleaves ς1 after Arg245. Thus, the sequence polymorphism at position 249 controls cleavage at a nearby site in the neck region. The relevance of these results to reovirus infection in vivo was assessed by treating virions with the contents of a murine intestinal wash under conditions that result in generation of ISVPs. The pattern of ς1 cleavage susceptibility generated by using purified protease was reproduced in assays using the intestinal wash. These results provide a mechanistic explanation for ς1 cleavage during exposure of virions to intestinal proteases and may account for certain strain-dependent patterns of reovirus pathogenesis.

Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation mechanism that is widespread in eukaryotic organisms. It is often associated with methylation of the transcribed region of the silenced gene and with accumulation of small RNAs (21 to 25 nucleotides) homologous to the silenced gene. In plants, PTGS can be triggered locally and then spread throughout the organism via a mobile signal that can cross a graft junction. Previously, we showed that the helper component-proteinase (HC-Pro) of plant potyviruses suppresses PTGS. Here, we report that plants in which PTGS has been suppressed by HC-Pro fail to accumulate the small RNAs associated with silencing. However, the transgene locus of these plants remains methylated. Grafting experiments indicate that HC-Pro prevents the plant from responding to the mobile silencing signal but does not eliminate its ability to produce or send the signal. These results demonstrate that HC-Pro functions downstream of transgene methylation and the mobile signal at a step preceding accumulation of the small RNAs.

A circular permuted variant of the potent human immunodeficiency virus (HIV)-inactivating protein cyanovirin-N (CV-N) was constructed. New N- and C-termini were introduced into an exposed helical loop, and the original termini were linked using residues of the original loop. Since the three-dimensional structure of wild-type cyanovirin-N is a pseudodimer, the mutant essentially exhibits a swap between the two pseudo-symmetrically related halves. The expressed protein, which accumulates in the insoluble fraction, was purified, and conditions for in vitro refolding were established. During refolding, a transient dimeric species is also formed that converts to a monomer. Similar to the wild-type CV-N, the monomeric circular permuted protein exhibits reversible thermal unfolding and urea denaturation. The mutant is moderately less stable than the wild-type protein, but it displays significantly reduced anti-HIV activity. Using nuclear magnetic resonance spectroscopy, we demonstrate that this circular permuted monomeric molecule adopts the same fold as the wild-type protein. Characterization of these two architecturally very similar molecules allows us to embark, for the first time, on a structure guided focused mutational study, aimed at delineating crucial features for the extraordinary difference in the activity of these molecules.