Mechanism for Coordinated RNA Packaging and Genome Replication by Rotavirus Polymerase VP1

Rotavirus RNA-dependent RNA polymerase, VP1, catalyzes RNA synthesis within a subviral particle. This activity depends on core shell protein VP2. A conserved sequence at the 3′ end of plusstrand RNA templates is important for polymerase association and genome replication. We have determined the structure of VP1 at 2.9 Å resolution, as apoenzyme and in complex with RNA. The cage-like enzyme is similar to reovirus λ3, with four tunnels leading to or from a central, catalytic cavity. A distinguishing characteristic of VP1 is specific recognition, by conserved features of the template-entry channel, of four bases, UGUG, in the conserved 3′ sequence. Well-defined interactions with these bases position the RNA so that its 3′ end overshoots the initiating register, producing a stable but catalytically inactive complex. We propose that specific 3′ end recognition selects rotavirus RNA for packaging and that VP2 activates the auto-inhibited VP1/RNA complex to coordinate packaging and genome replication.Molecular and Cellular Biolog

Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing

Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids. Cohesion is thought to occur through the entrapment of DNA within the tripartite ring (Smc1, Smc3 and Rad21) with enforcement from a fourth subunit (SA1/SA2). Surprisingly, cohesin rings do not play a major role in sister telomere cohesion. Instead, this role is replaced by SA1 and telomere binding proteins (TRF1 and TIN2). Neither the DNA binding property of SA1 nor this unique telomere cohesion mechanism is understood. Here, using single-molecule fluorescence imaging, we discover that SA1 displays two-state binding on DNA: searching by one-dimensional (1D) free diffusion versus recognition through subdiffusive sliding at telomeric regions. The AT-hook motif in SA1 plays dual roles in modulating non-specific DNA binding and subdiffusive dynamics over telomeric regions. TRF1 tethers SA1 within telomeric regions that SA1 transiently interacts with. SA1 and TRF1 together form longer DNA-DNA pairing tracts than with TRF1 alone, as revealed by atomic force microscopy imaging. These results suggest that at telomeres cohesion relies on the molecular interplay between TRF1 and SA1 to promote DNA-DNA pairing, while along chromosomal arms the core cohesin assembly might also depend on SA1 1D diffusion on DNA and sequence-specific DNA binding

Characterization of the Interaction between the Cohesin Subunits Rad21 and SA1/2

The cohesin complex is responsible for the fidelity of chromosomal segregation during mitosis. It consists of four core subunits, namely Rad21/Mcd1/Scc1, Smc1, Smc3, and one of the yeast Scc3 orthologs SA1 or SA2. Sister chromatid cohesion is generated during DNA replication and maintained until the onset of anaphase. Among the many proposed models of the cohesin complex, the ﾒcoreﾒ cohesin subunits Smc1, Smc3, and Rad21 are almost universally displayed as tripartite ring. However, other than its supportive role in the cohesin ring, little is known about the fourth core subunit SA1/SA2. To gain deeper insight into the function of SA1/SA2 in the cohesin complex, we have mapped the interactive regions of SA2 and Rad21 in vitro and ex vivo. Whereas SA2 interacts with Rad21 through a broad region (301ﾖ750 aa), Rad21 binds to SA proteins through two SA-binding motifs on Rad21, namely N-terminal (NT) and middle part (MP) SA-binding motif, located At 60-81 aa of the N-terminus and 383ﾖ392 aa of the MP of Rad21, respectively. The MP SA-binding motif is a 10 amino acid, a-helical motif. Deletion of these 10 amino acids or mutation of three conserved amino acids (L385, F389, and T390) in this ahelical motif significantly hinders Rad21 from physically interacting with SA1/2. Besides the MP SA-binding motif, the NT SAbinding motif is also important for SA1/2 interaction. Although mutations on both SA-binding motifs disrupt Rad21-SA1/2 interaction, they had no apparent effect on the Smc1-Smc3-Rad21 interaction. However, the Rad21-Rad21 dimerization was reduced by the mutations, indicating potential involvement of the two SA-binding motifs in the formation of the two-ring handcuff for chromosomal cohesion. Furthermore, mutant Rad21 proteins failed to significantly rescue precocious chromosome separation caused by depletion of endogenous Rad21 in mitotic cells, further indicating the physiological significance of the two SA-binding motifs of Rad21

The Crystal Structure and RNA-Binding of an Orthomyxovirus Nucleoprotein

Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses, whose genome consists of multiple negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes. To better understand the structural features of orthomyxovirus RNPs that allow them to be packaged, we determined the crystal structure of the nucleoprotein (NP) of a fish orthomyxovirus, the infectious salmon anemia virus (ISAV) (genus Isavirus). As the major protein component of the RNPs, ISAV-NP possesses a bi-lobular structure similar to the influenza virus NP. Because both RNA-free and RNA-bound ISAV NP forms stable dimers in solution, we were able to measure the NP RNA binding affinity as well as the stoichiometry using recombinant proteins and synthetic oligos. Our RNA binding analysis revealed that each ISAV-NP binds ,12 nts of RNA, shorter than the 24ﾖ28 nts originally estimated for the influenza A virus NP based on population average. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. Considering that RNPs of ISAV and the influenza viruses have similar morphologies and dimensions, our findings suggest that NP-free RNA may exist on orthomyxovirus RNPs, and selective RNP packaging may be accomplished through direct RNA-RNA interactions

Structure and assembly of the influenza A virus ribonucleoprotein complex

The genome of influenza A viruses consists of eight segments of single-stranded, negative-sense RNA that are encapsidated as individual rod-shaped ribonucleoprotein complexes (RNPs). Each RNP contains a viral RNA, a viral polymerase and multiple copies of the viral nucleoprotein (NP). Influenza A virus RNPs play important roles during virus infection by directing viral RNA replication and transcription, intracellular transport of the viral RNA, gene reassortment as well as viral genome packaging into progeny particles. As a unique genomic entity, the influenza A virus RNP has been extensively studied since the 1960s. Recently, exciting progress has been made in studying the RNP structure and its assembly, leading to a better understanding of the structural basis of various RNP functions

RNA virus replication machineries.

<p>(A) RdRps of hepatitis C virus and reovirus. Hepatitis C virus is a (+)RNA virus from the Flaviviridae family, while reovirus is a dsRNA virus belonging to the Reoviridae family. In both structures, the fingers, palm, and the thumb of the polymerase are colored in blue, red, and green, respectively. Yellow and magenta highlight the N- and C-terminal domains, respectively. The three aspartic acid residues in the polymerase catalytic active site are shown in cyan. Reovirus RdRP has large N- and C-terminal domains with distinct functions: the N-terminal domain maintains the closed conformation of the active site, and the C-terminal domain serves as a processivity factor like a sliding clamp. (B) The yeast L-A virus (Totiviridae family). The two independent sets of capsid protein molecules, 60 copies each, are shown in red and yellow, respectively. The viral RdRP is likely to be tethered to the inner capsid near the 5-fold symmetry axis, and viral transcripts made inside the core are released through channels on or near the 5-fold axes. (C) An influenza A virus (Orthomyxoviridae family) RNP model. Blue spheres represent NP monomers, and the green line shows vRNA. A short duplex formed between the 5′ and the 3′ ends provides the binding site for the heterotrimeric RdRp. Overall, the RNP assumes a rod-shaped, double-helical structure that remains intact even after the vRNA is removed.</p