Poliovirus RNA-dependent RNA polymerase (3D(pol)). Assembly of stable, elongation-competent complexes by using a symmetrical primer-template substrate (sym/sub)

Detailed studies of the kinetics and mechanism of nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus, 3D(pol), have been limited by the inability to assemble elongation complexes that permit activity to be monitored by extension of end-labeled primers. We have solved this problem by employing a short, symmetrical, heteropolymeric RNA primer-template that we refer to as 'sym/sub'. Formation of 3D(pol)- sym/sub complexes is slow owing to a slow rate of association (0.1 μM-1 s-1) of 3D(pol) and sym/sub and a slow isomerization (0.076 s-1) of the 3D(pol)-sym/sub complex that is a prerequisite for catalytic competence of this complex. Complex assembly is stoichiometric under conditions in which competing reactions, such as enzyme inactivation, are eliminated. Inactivation of 3D(pol) occurs at a maximal rate of 0.051 s-1 at 22 °C in reaction buffer lacking nucleotide. At this temperature, ATP protects 3D(pol) against inactivation with a K0.5 of 37 μM. Once formed, 3D(pol)-sym/sub elongation complexes are stable (t( 1/2 ) = 2 h at 22 °C) and appear to contain only a single polymerase monomer. In the presence of Mg2+, AMP, 2'-dAMP, and 3'-dAMP are incorporated into sym/sub by 3D(pol) at rates of 72, 0.6, and 1 s-1, respectively. After incorporation of AMP, 3D(pol)-sym/sub product complexes have a half-life of 8 h at 22 °C. The stability of 3D(pol)-sym/sub complexes is temperature-dependent. At 30 °C, there is a 2-8-fold decrease in complex stability. Complex dissociation is the rate-limiting step for primer utilization. 3D(pol) dissociates from the end of template at a rate 10-fold faster than from internal positions. The sym/sub system will facilitate mechanistic analysis of 3D(pol) and permit a direct kinetic and thermodynamic comparison of the RNA-dependent RNA polymerase to the other classes of nucleic acid polymerases

Structure-function relationships of the viral RNA-dependent RNA polymerase: Fidelity, replication speed, and initiation mechanism determined by a residue in the ribose-binding pocket

Studies of the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV), 3Dpol, have shown that Asn-297 permits this enzyme to distinguish ribose from 2′-deoxyribose. All animal RNA viruses have Asn at the structurally homologous position of their polymerases, suggesting a conserved function for this residue. However, all prokaryotic RNA viruses have Glu at this position. In the presence of Mg2+, the apparent affinity of Glu-297 3Dpol for 2′-deoxyribonucleotides was decreased by 6-fold relative to wild type without a substantial difference in the fidelity of 2′-dNMP incorporation. The fidelity of ribonucleotide misincorporation for Glu-297 3Dpol was reduced by 14-fold relative to wild type. A 4- to 11-fold reduction in the rate of ribonucleotide incorporation was observed. Glu-297 PV was unable to grow in HeLa cells due to a replication defect equivalent to that observed for a mutant PV encoding an inactive polymerase. Evaluation of the protein-(VPg)-primed initiation reaction showed that only half of the Glu-297 3Dpol initiation complexes were capable of producing VPg-pUpU product and that the overall yield of uridylylated VPg products was reduced by 20-fold relative to wild-type enzyme, a circumstance attributable to a reduced affinity for UTP. These studies identify the first RdRp derivative with a mutator phenotype and provide a mechanistic basis for the elevated mutation frequency of RNA phage relative to animal RNA viruses observed in culture. Although protein-primed initiation and RNA-primed elongation complexes employ the same polymerase active site, the functional differences reported here imply significant structural differences between these complexes

Poliovirus RNA-dependent RNA polymerase (3D(pol)) is sufficient for template switching in vitro

We have performed a systematic, quantitative analysis of the kinetics of nucleotide incorporation catalyzed by poliovirus RNA-dependent RNA polymerase, 3D(pol). Homopolymeric primer/templates of defined length were used to evaluate the contribution of primer and template length and sequence to the efficiency of nucleotide incorporation without the complication of RNA structure. Interestingly, thermodynamic stability of the duplex region of these primer/templates was more important for efficient nucleotide incorporation than either primer or template length. Surprisingly, products greater than unit length formed in all reactions regardless of length or sequence. Neither a distributive nor a processive slippage mechanism could be used to explain completely the formation of long products. Rather, the data were consistent with a template-switching mechanism. All of the nucleotide could be polymerized during the course of the reaction. However, very few primers could be extended, suggesting an inverse correlation between the efficiency of primer utilization and that of nucleotide incorporation. Therefore, the greatest fraction of incorporated nucleotide derives from a small fraction of enzyme when radioactive nucleotide and homopolymeric primer/template substrates are employed. The impact of these results on mechanistic studies of 3D(pol)-catalyzed nucleotide incorporation and RNA recombination are discussed

Poliovirus RNA-dependent RNA polymerase (3D(pol)). Divalent cation modulation of primer, template, and nucleotide selection

We have analyzed the divalent cation specificity of poliovirus RNA- dependent RNA polymerase, 3D(pol). The following preference was observed: Mn2+ > Co2+ > Ni2+ > Fe2+ > Mg2+ > Ca2+ > Cu2+, and Zn2+ was incapable of supporting 3D(pol)-catalyzed nucleotide incorporation. In the presence of Mn2+, 3D(pol) activity was increased by greater than 10-fold relative to that in the presence of Mg2+. Steady-state kinetic analysis revealed that the increased activity observed in the presence of Mn2+ was due, primarily, to a reduction in the K(M) value for 3D(pol) binding to primer/template, without any significant effect on the K(M) value for nucleotide. The ability of 3D(pol) to catalyze RNA synthesis de novo was also stimulated approximately 10-fold by using Mn2+, and the enzyme was now capable of also utilizing a DNA template for primer-independent RNA synthesis. Interestingly, the use of Mn2+ as divalent cation permitted 3D(pol) activity to be monitored by following extension of 5'-32P-end- labeled, heteropolymeric RNA primer/templates. The kinetics of primer extension were biphasic because of the enzyme binding to primer/template in both possible orientations. When bound in the incorrect orientation, 3D(pol) was capable of efficient addition of nucleotides to the blunt-ended duplex; this activity was also apparent in the presence of Mg2+. In the presence of Mn2+, 3D(pol) efficiently utilized dNTPs, ddNTPs, and incorrect NTPs. On average, three incorrect nucleotides could be incorporated by 3D(pol). The ability of 3D(pol) to incorporate the correct dNTP, but not the correct ddNTP, was also observed in the presence of Mg2+. Taken together, these results provide the first glimpse into the nucleotide specificity and fidelity of the poliovirus polymerase and suggest novel alternatives for the design of primer/templates to study the mechanism of 3D(pol)-catalyzed nucleotide incorporation

Single-nucleotide resolution of RNA strands in the presence of their RNA complements

Double-stranded (ds)RNA is important for a variety of biological systems. The discovery of the dsRNA-binding motif (dsRBM), coupled with the occurrence of this motif in a wide variety of functionally diverse proteins, has led to increased interest and study of - dsRNA (6,14). For example, the dsRNA- activated protein kinase (PKR), an enzyme involved in the cellular antiviral response, contains two tandem copies of the dsRBM. In addition, the dsRNA adenine deaminases (dsRADs) contain three tandem copies of this motif (7). Likewise, the study of the RNA-dependent RNA polymerase (RdRP) activity associated with RNA virus transcriptases and replicases also requires the use of dsRNA. In each of these systems, the length of the typical RNA used is in the 10–80 bp range (1,9)

RNA virus error catastrophe: Direct molecular test by using ribavirin

RNA viruses evolve rapidly. One source of this ability to rapidly change is the apparently high mutation frequency in RNA virus populations. A high mutation frequency is a central tenet of the quasispecies theory. A corollary of the quasispecies theory postulates that, given their high mutation frequency, animal RNA viruses may be susceptible to error catastrophe, where they undergo a sharp drop in viability after a modest increase in mutation frequency. We recently showed that the important broad-spectrum antiviral drug ribavirin (currently used to treat hepatitis C virus infections, among others) is an RNA virus mutagen, and we proposed that ribavirin's antiviral effect is by forcing RNA viruses into error catastrophe. However, a direct demonstration of error catastrophe has not been made for ribavirin or any RNA virus mutagen. Here we describe a direct demonstration of error catastrophe by using ribavirin as the mutagen and poliovirus as a model RNA virus. We demonstrate that ribavirin's antiviral activity is exerted directly through lethal mutagenesis of the viral genetic material. A 99.3% loss in viral genome infectivity is observed after a single round of virus infection in ribavirin concentrations sufficient to cause a 9.7-fold increase in mutagenesis. Compiling data on both the mutation levels and the specific infectivities of poliovirus genomes produced in the presence of ribavirin, we have constructed a graph of error catastrophe showing that normal polio-virus indeed exists at the edge of viability. These data suggest that RNA virus mutagens may represent a promising new class of antiviral drugs

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Numerous cellular proteins interact with membrane surfaces to affect essential cellular processes. These interactions can be directed towards a specific lipid component within a membrane, as in the case of phosphoinositides (PIPs), to ensure specific subcellular localization and/or activation. PIPs and cellular PIP-binding domains have been studied extensively to better understand their role in cellular physiology. We applied a pH modulation assay on supported lipid bilayers (SLBs) as a tool to study protein-PIP interactions. In these studies, pH sensitive ortho-Sulforhodamine B conjugated phosphatidylethanolamine is used to detect protein-PIP interactions. Upon binding of a protein to a PIP-containing membrane surface, the interfacial potential is modulated (i.e. change in local pH), shifting the protonation state of the probe. A case study of the successful usage of the pH modulation assay is presented by using phospholipase C delta1 Pleckstrin Homology (PLC-δ1 PH) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) interaction as an example. The apparent dissociation constant (Kd,app) for this interaction was 0.39 ± 0.05 µM, similar to Kd,app values obtained by others. As previously observed, the PLC-δ1 PH domain is PI(4,5)P2 specific, shows weaker binding towards phosphatidylinositol 4-phosphate, and no binding to pure phosphatidylcholine SLBs. The PIP-on-a-chip assay is advantageous over traditional PIP-binding assays, including but not limited to low sample volume and no ligand/receptor labeling requirements, the ability to test high- and low-affinity membrane interactions with both small and large molecules, and improved signal to noise ratio. Accordingly, the usage of the PIP-on-a-chip approach will facilitate the elucidation of mechanisms of a wide range of membrane interactions. Furthermore, this method could potentially be used in identifying therapeutics that modulate protein's capacity to interact with membranes

Hepatitis C Virus RNA-dependent RNA Polymerase (NS5B) as a Mediator of the Antiviral Activity of Ribavirin

Ribavirin is administered in combination with interferon-α for treatment of hepatitis C virus (HCV) infection. Recently, we demonstrated that the antiviral activity of ribavirin can result from the ability of a viral RNA polymerase to utilize ribavirin triphosphate and to incorporate this nucleotide with reduced specificity, thereby mutagenizing the genome and decreasing the yield of infectious virus (Crotty, S., Maag, D., Arnold, J. J., Zhong, W., Lau, J. Y., Hong, Z., Andino, R., and Cameron, C. E. (2000) Nat. Med. 6, 1375-1379). In this study, we performed a quantitative analysis of a novel HCV RNA polymerase derivative that is capable of utilizing stably annealed primer-template substrates and exploited this derivative to evaluate whether lethal mutagenesis of the HCV genome is a possible mechanism for the anti-HCV activity of ribavirin. These studies demonstrate HCV RNA polymerase-catalyzed incorporation of ribavirin opposite cytidine and uridine. In addition, we demonstrate that templates containing ribavirin support CMP and UMP incorporation with equivalent efficiency. Surprisingly, templates containing ribavirin can also cause a significant block to RNA elongation. Together, these data suggest that ribavirin can exert a direct effect on HCV replication, which is mediated by the HCV RNA polymerase. We discuss the implications of this work on the development of nucleoside analogs for treatment of HCV infection

Multiple full-length NS3 molecules are required for optimal unwinding of oligonucleotide DNA in vitro

NS3 (nonstructural protein 3) from the hepatitis C virus is a 3′ → 5′ helicase classified in helicase superfamily 2. The optimally active form of this helicase remains uncertain. We have used unwinding assays in the presence of a protein trap to investigate the first cycle of unwinding by full-length NS3. When the enzyme was in excess of the substrate, NS3 (500 nM) unwound >80% of a DNA substrate containing a 15-nucleotide overhang and a 30-bp duplex (45:30-mer; 1 nM). This result indicated that the active form of NS3 that was bound to the DNA prior to initiation of the reaction was capable of processive DNA unwinding. Unwinding with varying ratios of NS3 to 45:30-mer allowed us to investigate the active form of NS3 during the first unwinding cycle. When the substrate concentration slightly exceeded that of the enzyme, little or no unwinding was observed, indicating that if a monomeric form of the protein is active, then it exhibits very low processivity. Binding of NS3 to the 45:30-mer was measured by electrophoretic mobility shift assays, resulting in KD = 2.7 ± 0.4 nM. Binding to individual regions of the substrate was investigated by measuring the KD for a 15-mer oligonucleotide as well as a 30-mer duplex. NS3 bound tightly to the 15-mer (KD = 1.3 ± 0.2 nM) and, surprisingly, fairly tightly to the double-stranded 30-mer (KD = 11.3 ± 1.3 nM). However, NS3 was not able to rapidly unwind a blunt-end duplex. Thus, under conditions of optimal unwinding, the 45:30-mer is initially saturated with the enzyme, including the duplex region. The unwinding data are discussed in terms of a model whereby multiple molecules of NS3 bound to the single-stranded DNA portion of the substrate are required for optimal unwinding

Mutations in HIV reverse transcriptase which alter RNase H activity and decrease strand transfer efficiency are suppressed by HIV nucleocapsid protein

Structural studies of authentic HIV reverse transcriptase (RT) suggest a role for the p51 carboxyl terminus in forming an active RNase H conformation [Rodgers, D. W., Gamblin, S. J., Harris, B. A., Ray, S., Culp, J. S., Hellmig, B., Woolf, D. J., Debouck, C. and Harrison, S.C. (1995) Proc. Natl. Acad. Sci. USA 92, 1222-1226]. We have purified mutant RT heterodimers containing deletion of 5, 9, or 13 amino acids from the p51 carboxyl terminus. These 'selectively deleted' heterodimers have been analyzed for changes in RNA-dependent DNA polymerase activity, RNase H activity, and the ability to catalyze DNA strand transfer. As deletions extended into the p51 subunit, a decrease in the stability of the RT-DNA complex was apparent. The largest effect was observed for p66/p51Δ13 RT, which showed a 3-fold decrease relative to wild-type RT. RNase H activity was measured by digestion of the RNA in a 5' 32P-labeled RNA/DNA hybrid. Deletion of 5 or 9 amino acids from pSI had little effect on synthesis-dependent and synthesis- independent RNase H activities. In contrast, deletion of 13 amino acids from p51 increased the length of the hydrolysis products of both RNase H activities by 8-10 bp, thus changing the spatial relationship between the polymerase and RNase H active sites from a distance of 17-18 bp to 26-27 bp. The Δ13 derivative was also incapable of efficient DNA strand transfer. This defect in strand transfer could be suppressed by the 71-amino acid form of HIV nucleocapsid protein (NC) but not by the 55-amino acid form (NC55) or by equine infectious anemia virus NC. These results provide evidence for the existence of a specific complex between RT and NC and are discussed in terms of the role of this complex in proviral DNA synthesis