Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies, which establish this eubacterial Ago as a bona fide guide DNA strand-mediated site-specific RNA endonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the 5' phosphate of the guide strand can be anchored in a basic pocket, and the mRNA can be positioned for site-specific cleavage by RNase H-type divalent cation-coordinated catalytic Asp residues of the PIWI domain. Domain swap experiments involving chimeras of human Ago (hAgo1) and cleavage-competent hAgo2 reinforce the role of the PIWI domain in "slicer" activity. We propose a four-step Ago-mediated catalytic cleavage cycle model, which provides distinct perspectives into the mechanism of guide strand-mediated mRNA cleavage within the RISC.

The subcellular localization of transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV) (group I and group II coronaviruses, respectively) nucleoproteins (N proteins) were examined by confocal microscopy. The proteins were shown to localize either to the cytoplasm alone or to the cytoplasm and a structure in the nucleus. This feature was confirmed to be the nucleolus by using specific antibodies to nucleolin, a major component of the nucleolus, and by confocal microscopy to image sections through a cell expressing N protein. These findings are consistent with our previous report for infectious bronchitis virus Coronaviruses are enveloped RNA viruses with nonsegmented, single-stranded, positive-sense RNA genomes of 27 to 32 kb that are 5Ј capped and 3Ј polyadenylated (26). The 5Ј two-thirds of the coronavirus genome encodes the virus contribution to the replicase-transcription complex, Rep1a and Rep1b, the latter resulting from a Ϫ1 frameshift (8). During coronavirus replication, a 3Ј-coterminal nested set of subgenomic mRNAs, which encode other viral proteins, including nucleoprotein (N protein), are synthesized. In part, based on similar genome replication strategies (17, 61), the coronavirus family, Coronaviridae, has been grouped together with the arterivirus family, Arteriviridae, into the order Nidovirales (11). While gene functions and distributions for the two families are similar, there are some differences that might lead to subtle differences in replication strategies. Recently, we have reported that the coronavirus infectious bronchitis virus (IBV) N protein localizes to the cytoplasm and a structure in the nucleus proposed to be the nucleolus in both IBV-infected cells and cells transfected with a plasmid expressing IBV N protein under the control of a PolII promoter (23). A similar result was reported with the arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) N protein (54), suggesting that localization of N protein to the nucleolus was probably common to these two virus families and potentially common to all Nidovirales.Coronavirus replication is generally accepted to occur in the cytoplasm of infected cells (66), although for IBV an intact cell nucleus has been proposed to be necessary for virus replication (20). In addition, proteins normally associated with the nucleus have been implicated in the replication of the murine coronavirus mouse hepatitis virus (MHV) (30). The nucleolus is a structure found within the nucleus and is only present during interphase (1). It is the site where rRNA is synthesized and where biogenesis of ribosomal subunits and polymerase III transcripts occurs (10, 57). The nucleolus also sequesters regulatory complexes and has been implicated in the regulation of the cell cycle (10). The possible involvement of the nucleolus in coronavirus replication is not exclusive to coronaviruses. As a consequence of infection or a deliberate process, a number of viruses, including adenoviruses (37) and poliovirus (65), redistribute nucleolin, ...

The 2b proteins encoded by cucumovirus act as post-transcriptional gene silencing suppressors to counter host defence during infection. Here we report the crystal structure of Tomato aspermy virus 2b (TAV2b) protein bound to a 19 bp small interfering RNA (siRNA) duplex. TAV2b adopts an all a-helix structure and forms a homodimer to measure siRNA duplex in a length-preference mode. TAV2b has a pair of hook-like structures to recognize simultaneously two a-helical turns of A-form RNA duplex by fitting its a-helix backbone into two adjacent major grooves of siRNA duplex. The conserved p-stackings between tryptophan and the 5 0 -terminal base of siRNA duplex from both ends enhance the recognition. TAV2b further oligomerizes to form a dimer of dimers through the conserved leucine-zipper-like motif at its amino-terminal a-helix. Biochemical experiments suggest that TAV2b might interfere with the post-transcriptional gene silencing pathway by directly binding to siRNA duplex.

Avian infectious bronchitis virus (IBV) is a member of theCoronaviridae (order Nidovirales) (9), members of which are enveloped viruses with single-stranded, positive-sense RNA genomes that are 5Ј capped and 3Ј polyadenylated (30, 63). The 5Ј two-thirds of the coronavirus genome encodes the replicase gene producing two polyproteins, Rep1a and Rep1ab, the latter resulting from a Ϫ1 frameshift (7). The remaining proteins, which include the nucleoprotein (N), are expressed from a nested set of subgenomic mRNAs (sgRNAs) that are produced via a discontinuous transcription mechanism (6, 30). Each of these sgRNAs has a short nontranslated leader sequence (64 nucleotides for IBV) derived from the 5Ј end of the genome. Present within the leader sequence is a consensus sequence, which we have termed the transcription-associated sequence (TAS) (24). The TAS contains a conserved core motif, which in the case of IBV is CUUAACAA, which is also located in the genome, proximal to the start site for each sgRNA. For different coronaviruses, the core sequence varies and can be present more than once per TAS.N protein, the virus RNA binding protein, is one of the most abundant viral proteins in an infected cell (31). Several functions have been postulated for the coronavirus N protein throughout the virus life cycle (31); primarily, it complexes with the genomic RNA to form a ribonucleocapsid structure (17) and associates with the M protein (19, 39) to form the viral core (48). While N protein is required in trans to rescue the full-length clone of IBV (8) and a porcine coronavirus transmissible gastroenteritis virus clone (82), it is not required for others (1,71,72). Certainly, in the case of the rescue of the full-length clone of severe acute respiratory syndrome coronavirus, the presence of N protein increases viral titers compared to rescue performed in the absence of N transcript (83), suggesting that N protein may be involved in the efficiency of replication but that it is not essential.Based on amino acid sequence comparisons, three domains have been identified in the murine coronavirus, mouse hepatitis virus (MHV) N protein (46), of which the central domain (domain II) was identified as a potential RNA binding site (35, 40) capable of binding both coronavirus-and non-coronavirusderived RNA sequences (35,68). However, whether this binding occurs with equal or different affinity is uncertain (14,35,49). N protein has been shown to associate with several motifs on viral RNA, including the leader RNA sequence, with particular affinity for the core sequence of the TAS (2, 41), sequences at the 3Ј end of the genomic RNA (84), and the packaging signal (37). How these sequences promote N binding is unknown.Several coronavirus N proteins have been shown to be phosphorylated, including IBV, MHV, and transmissible gastroenteritis virus N proteins, although the precise sites were not identified (31). The role of phosphorylation in the virus life cycle is unknown, although the phosphorylation state of N protein has been predicted to pl...

Coronavirus nucleoproteins (N proteins) Infectious bronchitis virus (IBV), a member of the Coronavirus genus of the Coronaviridae family, order Nidovirales (13), is an enveloped virus with a single-stranded, positive-sense RNA genome of 27,608 nucleotides (9) that is 5Ј capped and 3Ј polyadenylated which replicates in the cytoplasm of infected cells. The 5Ј two-thirds of the IBV genome encodes the replicase-transcription complex, Rep1a and Rep1ab, the latter resulting from a Ϫ1 frameshift (10). During IBV replication, a 3Ј-coterminal nested set of six subgenomic mRNAs are synthesized that encode other viral proteins, including nucleoprotein (N protein). Recently, we have reported that IBV N protein localizes to the cytoplasm and a structure in the nucleus proposed to be the nucleolus both in IBV-infected cells and in cells transfected with a plasmid expressing IBV N protein under the control of a PolII promoter (26), a result subsequently confirmed in species-specific and -nonspecific cells expressing the mouse hepatitis virus (MHV) and porcine transmissible gastroenteritis virus (TGEV) N proteins (64).The nucleolus is only present during interphase in mammalian cells (1) and is formed around ribosomal DNA repeats, which cluster at chromosomal loci called nucleolar organizer regions. It is the site where 5.8S, 18S, and 28S rRNAs are transcribed, processed, and assembled into ribosome subunits (11, 51). The nucleolus also sequesters regulatory complexes and has been implicated in the regulation of the cell cycle, telomerase activity, signal recognition particle biogenesis, small RNA processing, and mRNA transport (40, 41). The nucleolus is a dynamic structure composed of (or contains) at least 271 proteins (4), including nucleolin, fibrillarin, spectrin, B23, and the ribosomal proteins S5 and L9 (12, 51). Nucleolin (also called C23) represents 10% of the total nucleolar protein content, is highly phosphorylated and methylated, and also can be ADP-ribosylated (21). One of the main functions of nucleolin is processing the first cleavage step of rRNA in the presence of U3 snoRNP (21). Nucleolin may also function as a chaperone for correct folding in pre-rRNA processing (2). Fibrillarin is highly conserved in sequence, structure, and function in eukaryotes (5) and is directly involved in many posttranscriptional processes, including pre-rRNA processing, pre-rRNA methylation, and ribosome assembly (60).As a consequence of infection, a number of viral proteins interact with the nucleolus and can reorganize nucleolar antigens (25), with examples from retroviruses, DNA viruses, and RNA viruses. These include human immunodeficiency virus type 1 (HIV-1) Rev (16) and tat (56), Newcastle disease virus matrix protein (42), adenovirus IVa2 gene product (37) and V protein (39), Marek's disease virus MEQ protein (36), hepatitis D virus large-delta antigen (54), and porcine reproductive and respiratory syndrome virus nucleocapsid protein (49). The nucleolus is also the site of Borna disease virus replication and transcription (47...