RNA-dependent RNA polymerase

About: RNA-dependent RNA polymerase is a research topic. Over the lifetime, 13904 publications have been published within this topic receiving 767954 citations. The topic is also known as: RdRp & RNA replicase.

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

Abstract: The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation.

Flavivirus Capsid Is a Dimeric Alpha-Helical Protein

Abstract: The Flaviviridae family of enveloped RNA viruses causes significant disease in both humans and agriculturally important animals. Flavivirus, the largest of the three genera of Flaviviridae, comprises over 70 viruses, mostly arthropod transmitted, including yellow fever virus (YF), dengue virus (DEN), West Nile virus, and tick-borne encephalitis virus (TBE) (15). The mature flavivirus particle is spherical with a diameter of 50 nm and contains multiple copies of three different structural proteins (C, M, and E), a host-derived membrane bilayer, and a single copy of a positive-sense RNA genome of approximately 11,000 nucleotides. The RNA genome is translated from a single open reading frame generating a polyprotein that is processed by viral and host proteases to yield the three structural proteins located at the N terminus followed by at least seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (23). The nonstructural proteins associate to form the viral replicase complex, for which several enzymatic functions have been identified, including protease, helicase, methyltransferase, and RNA-dependent RNA polymerase (15). The replicase complex is associated with intracellular membranes of the infected host and induces discrete membrane structures (16). Recent evidence suggests that the process of particle assembly may be coupled to genome replication (6, 12). This coupling of replication and virus assembly has made analysis of flavivirus assembly difficult, and thus, little is known about this important aspect of the virus life cycle. The structure of DEN, recently determined by cryoelectron microscopy (cryo-EM) and three-dimensional image reconstruction, has elucidated the molecular organization of the end product of the flavivirus assembly pathway (11). The organization of the E protein within the DEN particle was determined by modeling the atomic resolution structure of TBE E protein (22) into the outer density of the cryo-EM reconstruction. The model consists of a herringbone arrangement of E proteins, with three E protein monomers in the asymmetric unit of the virus, but lacking the anticipated T=3 icosahedral symmetry. The M protein is thought to reside just below E with both M and E being associated with the host-derived lipid bilayer. Internal to the lipid bilayer is the nucleocapsid core (NC) of the virus, consisting of multiple copies of capsid (C) protein that surround a single copy of the viral RNA genome. The density observed for the NC is roughly half of that observed for the outer density and suggests disorder or movement of the NC within the virus particle. The location of the NC within the virus particle indicates that the C protein directly contacts the genome RNA. A common phenomenon during flavivirus infection is the production and release into the extracellular medium of virus-like particles (VLPs) (24). NC is not present in VLPs, rendering them noninfectious. VLPs have also been produced in several heterologous expression systems in which only prM and E are present (1, 4, 9, 20, 21). Thus, the ability of E, in the presence of prM, to form a closed spherical protein shell is an intrinsic property of these two proteins and is independent of NC formation. However, the production of infectious particles requires the formation of NC, suggesting that an early stage of virus assembly involves the interaction of C protein with the genome RNA (7). The NC presumably then interacts with prM and/or E, resulting in the formation of a virus particle upon concomitant budding into the lumen of the endoplasmic reticulum (15). Unfortunately, details concerning the interactions of C protein with either the RNA genome or prM and E are not discernible in the cryo-EM reconstruction of DEN. Given the role of C in promoting encapsidation of the viral RNA and subsequent assembly of infectious virus particles, it is important to understand the structural and biophysical properties of the C protein that allow it to function in the flavivirus life cycle.

RNA mimics of green fluorescent protein

Abstract: A major class of molecules in cells is ribonucleic acid (RNA). Cellular RNAs comprise ribosomal RNA …