Orsay virus CP-δ adopts a novel β-bracelet structural fold and incorporates into virions as a head fiber

Fiber proteins are commonly found in eukaryotic and prokaryotic viruses, where they play important roles in mediating viral attachment and host cell entry. They typically form trimeric structures and are incorporated into virions via noncovalent interactions. Orsay virus, a small RNA virus which specifically infects the laboratory model nematod

Initiation of RNA Polymerization and Polymerase Encapsidation by a Small dsRNA Virus

During the replication cycle of double-stranded (ds) RNA viruses, the viral RNA-dependent RNA polymerase (RdRP) replicates and transcribes the viral genome from within the viral capsid. How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families. In this study, we have determined a 2.4 angstrom resolution structure of an RdRP from the human picobirnavirus (hPBV). In addition to the conserved polymerase fold, the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays and site-directed mutagenesis showed that: (1) the hPBV RdRP is fully active using both ssRNA and dsRNA templates; (2) the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; (3) RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and (4) the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences. Co-expression of the hPBV RdRP and the capsid protein (CP) indicated that, under the conditions used, the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome. Additionally, the hPBV RdRP exhibited higher affinity towards the conserved 5'-terminal sequence of the viral RNA, suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly.Peer reviewe

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

Crystal Structure of the Human Astrovirus Capsid Protein

Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. HAstV is a nonenveloped virus with a T=3 capsid and a positive-sense RNA genome. The capsid protein (CP) of HAstV is synthesized as a 90-kDa precursor (VP90) that can be divided into three linear domains: a conserved N-terminal domain, a hypervariable domain, and an acidic C-terminal domain. Maturation of HAstV requires proteolytic processing of the astrovirus CP both inside and outside the host cell, resulting in the removal of the C-terminal domain and the breakdown of the rest of the CP into three predominant protein species with molecular masses of ∼34, 27/29, and 25/26 kDa, respectively. We have now solved the crystal structure of VP9071–415 (amino acids [aa] 71 to 415 of VP90) of human astrovirus serotype 8 at a 2.15-Å resolution. VP9071–415 encompasses the conserved N-terminal domain of VP90 but lacks the hypervariable domain, which forms the capsid surface spikes. The structure of VP9071–415 is comprised of two domains: an S domain, which adopts the typical jelly-roll β-barrel fold, and a P1 domain, which forms a squashed β-barrel consisting of six antiparallel β-strands similar to what was observed in the hepatitis E virus (HEV) capsid structure. Fitting of the VP9071–415 structure into the cryo-electron microscopy (EM) maps of HAstV produced an atomic model for a continuous, T=3 icosahedral capsid shell. Our pseudoatomic model of the human HAstV capsid shell provides valuable insights into intermolecular interactions required for capsid assembly and trypsin-mediated proteolytic maturation needed for virus infectivity. Such information has potential applications in the development of a virus-like particle (VLP) vaccine as well as small-molecule drugs targeting astrovirus assembly/maturation

Orsay δ protein is required for non-lytic viral egress

Nonenveloped gastrointestinal viruses, such as human rotavirus, can exit infected cells from the apical surface without cell lysis. The mechanism of such nonlytic exit is poorly understood. The nonenveloped Orsay virus is an RNA virus infecting the intestine cells of the nematode Caenorhabditis elegans Dye staining results suggested that Orsay virus exits from the intestine of infected worms in a nonlytic manner. Therefore, the Orsay virus-C. elegans system provides an excellent in vivo model to study viral exit. The Orsay virus genome encodes three proteins: RNA-dependent RNA polymerase, capsid protein (CP), and a nonstructural protein, δ. δ can also be expressed as a structural CP-δ fusion. We generated an ATG-to-CTG mutant virus that had a normal CP-δ fusion but could not produce free δ due to the lack of the start codon. This mutant virus showed a viral exit defect without obvious phenotypes in other steps of viral infection, suggesting that δ is involved in viral exit. Ectopically expressed free δ localized near the apical membrane of intestine cells in C. elegans and colocalized with ACT-5, an intestine-specific actin that is a component of the terminal web. Orsay virus infection rearranged ACT-5 apical localization. Reduction of the ACT-5 level via RNA interference (RNAi) significantly exacerbated the viral exit defect of the δ mutant virus, suggesting that δ and ACT-5 functionally interact to promote Orsay virus exit. Together, these data support a model in which the viral δ protein interacts with the actin network at the apical side of host intestine cells to mediate the polarized, nonlytic egress of Orsay virus.IMPORTANCE: An important step of the viral life cycle is how viruses exit from host cells to spread to other cells. Certain nonenveloped viruses can exit cultured cells in nonlytic ways; however, such nonlytic exit has not been demonstrated in vivo In addition, it is not clear how such nonlytic exit is achieved mechanistically in vivo Orsay virus is a nonenveloped RNA virus that infects the intestine cells of the nematode C. elegans It is currently the only virus known to naturally infect C. elegans Using this in vivo model, we show that the δ protein encoded by Orsay virus facilitates the nonlytic exit of the virus, possibly by interacting with host actin on the apical side of worm intestine cells

Two Distinct Size Classes of Immature and Mature Subviral Particles from Tick-Borne Encephalitis Virus

Flaviviruses assemble in the endoplasmic reticulum by a mechanism that appears to be driven by lateral interactions between heterodimers of the envelope glycoproteins E and prM. Immature intracellular virus particles are then transported through the secretory pathway and converted to their mature form by cleavage of the prM protein by the cellular protease furin. Earlier studies showed that when the prM and E proteins of tick-borne encephalitis virus are expressed together in mammalian cells, they assemble into membrane-containing, icosahedrally symmetrical recombinant subviral particles (RSPs), which are smaller than whole virions but retain functional properties and undergo cleavage maturation, yielding a mature form in which the E proteins are arranged in a regular T = 1 icosahedral lattice. In this study, we generated immature subviral particles by mutation of the furin recognition site in prM. The mutation resulted in the secretion of two distinct size classes of particles that could be separated by sucrose gradient centrifugation. Electron microscopy showed that the smaller particles were approximately the same size as the previously described mature RSPs, whereas the larger particles were approximately the same size as the virus. Particles of the larger size class were also detected with a wild-type construct that allowed prM cleavage, although in this case the smaller size class was far more prevalent. Subtle differences in endoglycosidase sensitivity patterns suggested that, in contrast to the small particles, the E glycoproteins in the large subviral particles and whole virions might be in nonequivalent structural environments during intracellular transport, with a portion of them inaccessible to cellular glycan processing enzymes. These proteins thus appear to have the intrinsic ability to form alternative assembly products that could provide important clues about the role of lateral envelope protein interactions in flavivirus assembly