For viral +RNAs being packaged and used as templates for genome replication in the viroplasm, the 3CS serves as a critical polymerase recognition element [31,36,37,47]. 6, reveal potential mechanisms of RV assortment and packaging. == Random versus selective genome segment packaging == Viruses that maintain their genomes as separate RNA molecules are faced with a daunting challenge during assemblyhow to package a full complement of genome segments. Some RNA viruses utilize a non-selective packaging mechanism in which segments are randomly encapsidated into virions. This random packaging mechanism creates a large number of particles that lack complete genomes and are thus unable to mediate subsequent infections [1]. Other RNA viruses, particularly those with three or more genome segments, have evolved a more sophisticated packaging mechanism whereby each viral RNA is explicitly recognized. Members of theReoviridaefamily are thought to utilize this gene-specific approach to package their genomes of 9, 10, 11, or 12 segments of double-stranded RNA (dsRNA). The strongest evidence in support of selective packaging, rather than random packaging, for theReovirdaecomes from the observation that the particle-to-plaque forming unit ratio can be quite low [2,3]. Moreover, aReoviridaemember has never been identified that contains more than one copy of each gene, suggesting a precise equimolar process of assortment [4]. While the exact manner by which theReoviridaeachieve assortment is unknown, these viruses share several features with the eight-segmented, negative-strand 2,2,2-Tribromoethanol RNA viruses of Rabbit polyclonal to CLOCK theOrthomyxoviridaefamily (e.g. influenza A virus) and the three-segmented, dsRNA bacteriophages of theCystoviridaefamily (e.g. 6) [5,6]. In particular, for all these viruses,cis-acting elements in single-stranded viral RNA are thought to determine segment selection, and viral proteins play critical roles in orchestrating the assortment and packaging processes. One of the remaining mysteries forReoviridaeis whether they (i) assort their genome segments prior to packaging, similar to influenza A virus, or (ii) package each segment individually into a pre-formed particle, similar to 6. In this review, we discuss the evidence supporting each of these assortment and packaging models for rotavirus (RV), aReoviridaefamily member and significant pediatric gastrointestinal pathogen [7]. == RV virion architecture and replication cycle == The mature RV virion is a triple-layered particle that encases eleven dsRNA genome segments (Figure 1) [812]. The outermost 2,2,2-Tribromoethanol layer of the virion has T=13 icosahedral symmetry and is composed of the VP7 glycoprotein with several embedded copies of the VP4 spike attachment protein [13,14]. The intermediate layer, also exhibiting T=13 symmetry, is made up of VP6 and surrounds a thin T=1 VP2 core shell [14]. Aqueous channels penetrate the VP6 and VP2 layers, allowing divalent cations and nucleotides to access the particle interior [14]. Viral polymerase complexes (PCs) consisting of a 2,2,2-Tribromoethanol single subunit each of the viral RNA-dependent RNA polymerase (VP1) and RNA capping enzyme (VP3) are attached to the inner surface of the VP2 shell, proximal to most if not all of the twelve fivefold axes [10,14,15]. These enzymes are linked to the core shell through interactions requiring the amino-terminal residues of VP2, which form inwardly protruding fivefold hubs [1418]. The RV dsRNA genome encodes six structural (VP1-4, VP6-7) and five or six nonstructural (NSP1-5/6) proteins and is predicted to be arranged as tubules that spool around the PCs [14,15]. == Figure 1. Architecture and protein composition of the RV virion. == The left panel shows a cryo-electron micrograph image reconstruction of a mature, RV triple-layered particle (TLP) at 9.5 resolution and was used with permission from B.V.V. Prasad (Baylor University). A portion of the particle has been computationally removed to reveal the internal virion layers. The smooth external surface is made up of the VP7 glycoprotein (yellow) 2,2,2-Tribromoethanol and is embedded 2,2,2-Tribromoethanol with the VP4 spike attachment protein (red). The intermediate VP6 layer is shown in blue and the thin VP2 core shell is shown in green. Ordered portions of viral dsRNA that line the VP2 shell are shown in gold. Polymerase complex (PC) components, VP1 (the viral polymerase) and VP3 (the viral capping.