Enzymatic and nonenzymatic functions of viral RNA-dependent RNA polymerases within oligomeric arrays

Abstract

Few antivirals are effective against positive-strand RNA viruses, primarily because the high error rate during replication of these viruses leads to the rapid development of drug resistance. One of the favored current targets for the development of antiviral compounds is the active site of viral RNA-dependent RNA polymerases. However, like many subcellular processes, replication of the genomes of all positive-strand RNA viruses occurs in highly oligomeric complexes on the cytosolic surfaces of the intracellular membranes of infected host cells. In this study, catalytically inactive polymerases were shown to participate productively in functional oligomer formation and catalysis, as assayed by RNA template elongation. Direct protein transduction to introduce either active or inactive polymerases into cells infected with mutant virus confirmed the structural role for polymerase molecules during infection. Therefore, we suggest that targeting the active sites of polymerase molecules is not likely to be the best antiviral strategy, as inactivated polymerases do not inhibit replication of other viruses in the same cell and can, in fact, be useful in RNA replication complexes. On the other hand, polymerases that could not participate in functional RNA replication complexes were those that contained mutations in the amino terminus, leading to altered contacts in the folded polymerase and mutations in a known polymerase-polymerase interaction in the two-dimensional protein lattice. Thus, the functional nature of multimeric arrays of RNA-dependent RNA polymerase supplies a novel target for antiviral compounds and provides a new appreciation for enzymatic catalysis on membranous surfaces within cells.

Surface for catalysis by poliovirus RNA dependent RNA polymerase

“…To assess the role of 3Dpol oligomerization during RNA replication, the structure of a macromolecular array of catalytically inactive 3Dpol YGAA (D328A, D329A) was analyzed by electron microscopy. 3Dpol YGAA oligomerized after incubation at 30 °C formed a 100Å diameter narrow filament 15 as shown in Fig. 2B, surprisingly similar to the wild type cold filament ( Fig.…”

“…The binding site for the RNA synthesis primer VPg 9; 31; 32; 33; 34 and the catalytic centers of the enzymes hang above the RNA channel, so that another round of RNA synthesis could be initiated immediately after an RNA template passes by. Interestingly, the 3Dpol YGAA oligomer itself is inactive, but is capable of enhancing RNA elongation in vitro when a concentration of wild type 3Dpol which is too low for observable replication is present 15 . Interface-II contacts may be maintained by 3Dpol YGAA , keeping adjacent 3Dpols in register to facilitate RNA binding.…”

Three dimensional imaging of the intracellular assembly of a functional viral RNA replicase complex

“…Our data indicate that the compartment is divided into domains in which the protein is active, regardless of molecule aggregation. Previous studies have suggested that close packing or oligomeric association of RNA-dependent RNA polymerase molecules is important for viral RNA replication in cells (Lyle et al, 2002;Spagnolo et al, 2010;Risco et al, 2012). This fundamental step in viral multiplication nonetheless depends on the specific virus and cell host.…”

Intracellular peptide delivery using amphiphilic lipid‐based formulations

“…Interestingly, the delivered proteins retain their activity as demonstrated in functional studies (Cassinelli et al, 2006(Cassinelli et al, , 2009Chiu et al, 2010;Duerr et al, 2009;Giordano et al, 2010;Leshchyns'ka et al, 2006;Moreno et al, 2009;Ohnuma et al, 2010;Okamoto et al, 2011;Rivas et al, 2009;Schafer et al, 2009;Spagnolo et al, 2010;Ying et al, 2007;Zhang et al, 2008).…”

“…In earlier studies, several groups successfully delivered proteins (Ohnuma et al, 2010;Spagnolo et al, 2010;Weill et al, 2008a;Zhang et al, 2008) including antibodies (Cassinelli et al, 2006(Cassinelli et al, , 2009Cortes et al, 2007;Duerr et al, 2009;Okamoto et al, 2011;Weill et al, 2008b;Ying et al, 2007), peptides (Chiu et al, 2010;Giordano et al, 2010;Leshchyns'ka et al, 2006;Moreno et al, 2009;Rivas et al, 2009) as well as quantum-dots (Delehanty et al, 2009) using the proprietary cationic lipid-mediated delivery reagent PULSin. Interestingly, the delivered proteins retain their activity as demonstrated in functional studies (Cassinelli et al, 2006(Cassinelli et al, , 2009Chiu et al, 2010;Duerr et al, 2009;Giordano et al, 2010;Leshchyns'ka et al, 2006;Moreno et al, 2009;Ohnuma et al, 2010;Okamoto et al, 2011;Rivas et al, 2009;Schafer et al, 2009;Spagnolo et al, 2010;Ying et al, 2007;Zhang et al, 2008).…”