Full text loading...
Chapter 7 : Genome Replication I: the Players
Category: Viruses and Viral Pathogenesis
Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase
This chapter reviews the viral and host players in the viral RNA replication process and the forms of picornavirus RNA utilized, as well as the RNA/protein complexes that facilitate viral RNA synthesis. Cleavage of poly(rC)-binding protein 2 (PCBP2) by 3CD contributes to a switch from translation to RNA replication for poliovirus, as full-length PCBP2 functions in viral translation but the truncated PCBP2 cleavage product can only function in RNA replication. The cis- acting replication element (CRE) is an RNA structure required for picornavirus RNA replication and was first discovered in the HRV14 genome by McKnight and Lemon. The picornavirus polymerase will homodimerize, oligomerize, and interact with the viral proteins 3AB, VPg, and 3CD. Although research efforts designed to elucidate the mechanisms of RNA replication utilized by picornaviruses have been comprehensive during the past several decades, there is still much to understand about the protein players and viral RNA sequences involved in replication. It is still unknown how 3Dpol can bind to sequences as disparate as the 3’ NCR/poly(A) tract of picornavirus genomic RNAs and those found at the 3’ ends of negative-strand RNA intermediates. Importantly, additional inhibitors of picornavirus replication that target specific players involved in RNA replication complex assembly, initiation, and chain elongation need to be developed as potential therapeutics against this important class of human and animal viruses.
Cascade of poliovirus protein processing, with an emphasis on nonstructural proteins. The intact initial polyprotein, representing the complete long open reading frame of the viral genomic RNA, is shown below the depiction of the viral genome. This polyprotein is cleaved by viral proteolytic enzymes to generate intermediate precursors (P1, P2, and P3 or precursors that contain both P2 and P3 amino acid sequences). The triangles indicate cleavage sites recognized by viral proteinases 3C/3CD and 2A. Precursor polypeptides are further processed by viral proteinases to yield mature viral proteins. A brief description of the functions of the viral proteins is provided. As shown, precursors and mature protein cleavage products may have distinct roles in the viral replication cycle. The steps in processing the structural protein precursor (P1) are omitted from this figure for simplicity. (Modified from Encyclopedia of Virology, 3rd ed., vol. 4, 2008 [ 165 ], with permission from Elsevier.)
Forms of viral RNA in a picornavirus-infected cell. Following entry and uncoating, the picornavirus genomic RNA is altered by the cleavage of VPg from its 5′ terminus by an unidentified host cellular enzyme termed unlinkase. Genomic picorna-virus RNA molecules lacking VPg are the templates for translation. These templates for translation also serve as the templates for negative-strand RNA synthesis, which results in a duplex of template and newly synthesized product RNA termed the RF. Negative-strand RNA molecules (perhaps derived from the RF) act as template for positive-strand RNA synthesis in RI complexes. The RI complexes have multiple positive-strand RNAs synthesized from a single negative-strand template, resulting in asymmetric levels of positive- versus negative-strand viral RNAs in the infected cell. The positive-strand viral RNA molecules can then serve as templates for additional rounds of translation or negative-strand RNA synthesis, or they are packaged into virions for subsequent infection of other host cells.
Simplified scheme of ribonucleoprotein complexes formed on genomic RNAs and that have been demonstrated to play a role in enterovirus RNA replication. In this example, viral (i.e., 3CD, 3AB, 3Dpol, and VPg) or host cell (PCBP and PABP) proteins are shown interacting with stem-loop I (also known as the cloverleaf), the CRE, or the 3′ poly(A) tract. The a, b, c, and d subelements for stem-loop I are shown, as are RNA secondary structures representing the CRE and the 3′ NCR of enterovirus genomic RNA. The IRES and protein-coding region are also indicated (not drawn to scale).
Proposed closed loop model of poliovirus negative-strand RNA synthesis. Host cell protein PCBP2 and viral protein 3CD form a ternary complex with stem-loop I at the 5′ end of poliovirus genomic RNA. This complex interacts with the host cell protein PABP, which has been demonstrated to interact with the 3′ poly(A) tract of positive-strand viral RNAs. This interaction has been proposed to facilitate communication between the termini of the viral genome as a prerequisite for 3D polymerase binding and negative-strand RNA synthesis. Although several features of the model have been verified by using recombinant proteins and partial reactions, a fully functional cyclized complex has not yet been detected in virus-infected cells or in vitro. (Adapted from references 13 and 75 .)
Examples of RNA and protein complexes involved in picornavirus replication