Structural comparison strengthens the higher-order classification of proteases related to chymotrypsin

Specific cleavage of proteins by proteases is essential for several cellular, physiological, and viral processes. Chymotrypsin-related proteases that form the PA clan in the MEROPS classification of proteases is one of the largest and most diverse group of proteases. The PA clan comprises serine proteases from bacteria, eukaryotes, archaea, and viruses and chymotrypsin-related cysteine proteases from positive-strand RNA viruses. Despite low amino acid sequence identity, all PA clan proteases share a conserved double beta-barrel structure. Using an automated structure-based hierarchical clustering method, we identified a common structural core of 72 amino acid residues for 143 PA clan proteases that represent 12 protein families and 11 subfamilies. The identified core is located around the catalytic site between the two beta-barrels and resembles the structures of the smallest PA clan proteases. We constructed a structure-based distance tree derived from the properties of the identified common core. Our structure-based analyses support the current classification of these proteases at the subfamily level and largely at the family level. Structural alignment and structure-based distance trees could thus be used for directing objective classification of PA clan proteases and to strengthen their higher order classification. Our results also indicate that the PA clan proteases of positive-strand RNA viruses are related to cellular heat-shock proteases, which suggests that the exchange of protease genes between viruses and cells might have occurred more than once.Peer reviewe

RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine

RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2′-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine

Native RNA Purification Method for Small RNA Molecules Based on Asymmetrical Flow Field-Flow Fractionation

RNA molecules provide promising new possibilities for the prevention and treatment of viral infections and diseases. The rapid development of RNA biology and medicine requires advanced methods for the purification of RNA molecules, which allow fast and efficient RNA processing, preferably under non-denaturing conditions. Asymmetrical flow field-flow fractionation (AF4) enables gentle separation and purification of macromolecules based on their diffusion coefficients. The aim of the study was to develop an AF4 method for efficient purification of enzymatically produced antiviral small interfering (si)RNA molecules and to evaluate the overall potential of AF4 in the separation of short single-stranded (ss) and double-stranded (ds) RNA molecules. We show that AF4 separates monomeric ssRNA from dsRNA molecules of the same size and monomeric ssRNA from multimeric forms of the same ssRNA. The developed AF4 method enabled the separation of enzymatically produced 27-nt siRNAs from partially digested substrate dsRNA, which is potentially toxic for mammalian cells. The recovery of AF4-purified enzymatically produced siRNA molecules was about 70%, which is about 20% higher than obtained using anion-exchange chromatography. The AF4-purified siRNAs were not toxic for mammalian cells and fully retained their biological activity as confirmed by efficient inhibition of herpes simplex virus 1 replication in cell culture. Our work is the first to develop AF4 methods for the separation of short RNA molecules

Evidence for a Non-Catalytic Ion-Binding Site in Multiple RNA-Dependent RNA Polymerases

Peer reviewe

Native RNA Purification Method for Small RNA Molecules Based on Asymmetrical Flow Field-Flow Fractionation

RNA molecules provide promising new possibilities for the prevention and treatment of viral infections and diseases. The rapid development of RNA biology and medicine requires advanced methods for the purification of RNA molecules, which allow fast and efficient RNA processing, preferably under non-denaturing conditions. Asymmetrical flow field-flow fractionation (AF4) enables gentle separation and purification of macromolecules based on their diffusion coefficients. The aim of the study was to develop an AF4 method for efficient purification of enzymatically produced antiviral small interfering (si)RNA molecules and to evaluate the overall potential of AF4 in the separation of short single-stranded (ss) and double-stranded (ds) RNA molecules. We show that AF4 separates monomeric ssRNA from dsRNA molecules of the same size and monomeric ssRNA from multimeric forms of the same ssRNA. The developed AF4 method enabled the separation of enzymatically produced 27-nt siRNAs from partially digested substrate dsRNA, which is potentially toxic for mammalian cells. The recovery of AF4-purified enzymatically produced siRNA molecules was about 70%, which is about 20% higher than obtained using anion-exchange chromatography. The AF4-purified siRNAs were not toxic for mammalian cells and fully retained their biological activity as confirmed by efficient inhibition of herpes simplex virus 1 replication in cell culture. Our work is the first to develop AF4 methods for the separation of short RNA molecules

Herpes Simplex Virus Type 1 Clinical Isolates Respond to UL29-Targeted siRNA Swarm Treatment Independent of Their Acyclovir Sensitivity

Acyclovir is the drug of choice for the treatment of herpes simplex virus (HSV) infections. Acyclovir-resistant HSV strains may emerge, especially during long-term drug use, and subsequently cause difficult-to-treat exacerbations. Previously, we set up a novel treatment approach, based on enzymatically synthesized pools of siRNAs, or siRNA swarms. These swarms can cover kilobases-long target sequences, reducing the likelihood of resistance to treatment. Swarms targeting the UL29 essential gene of HSV-1 have demonstrated high efficacy against HSV-1 in vitro and in vivo. Here, we assessed the antiviral potential of a UL29 siRNA swarm against circulating strains of HSV-1, in comparison with acyclovir. All circulating strains were sensitive to both antivirals, with the half-maximal inhibitory concentrations (IC50) in the range of 350–1911 nM for acyclovir and 0.5–3 nM for the UL29 siRNA swarm. Additionally, we showed that an acyclovir-resistant HSV-1, devoid of thymidine kinase, is highly sensitive to UL29 siRNA treatment (IC50 1.0 nM; Imax 97%). Moreover, the detected minor variations in the RNAi target of the HSV strains had no effect on the potency or efficacy of UL29 siRNA swarm treatment. Our findings support the development of siRNA swarms for the treatment of HSV-1 infections, in order to circumvent any potential acyclovir resistance

Herpes Simplex Virus Type 1 Clinical Isolates Respond to UL29-Targeted siRNA Swarm Treatment Independent of Their Acyclovir Sensitivity

Acyclovir is the drug of choice for the treatment of herpes simplex virus (HSV) infections. Acyclovir-resistant HSV strains may emerge, especially during long-term drug use, and subsequently cause difficult-to-treat exacerbations. Previously, we set up a novel treatment approach, based on enzymatically synthesized pools of siRNAs, or siRNA swarms. These swarms can cover kilobases-long target sequences, reducing the likelihood of resistance to treatment. Swarms targeting the UL29 essential gene of HSV-1 have demonstrated high efficacy against HSV-1 in vitro and in vivo. Here, we assessed the antiviral potential of a UL29 siRNA swarm against circulating strains of HSV-1, in comparison with acyclovir. All circulating strains were sensitive to both antivirals, with the half-maximal inhibitory concentrations (IC50) in the range of 350–1911 nM for acyclovir and 0.5–3 nM for the UL29 siRNA swarm. Additionally, we showed that an acyclovir-resistant HSV-1, devoid of thymidine kinase, is highly sensitive to UL29 siRNA treatment (IC50 1.0 nM; Imax 97%). Moreover, the detected minor variations in the RNAi target of the HSV strains had no effect on the potency or efficacy of UL29 siRNA swarm treatment. Our findings support the development of siRNA swarms for the treatment of HSV-1 infections, in order to circumvent any potential acyclovir resistance

Insights into the pre-initiation events of bacteriophage phi6 RNA-dependent RNA polymerase : towards the assembly of a productive binary complex

The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3 terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the 6 RdRP can be greatly enhanced.The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3 terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the 6 RdRP can be greatly enhanced.The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3 terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the 6 RdRP can be greatly enhanced.Peer reviewe

Backtracking behavior in viral RNA-dependent RNA polymerase provides the basis for a second initiation site

Peer reviewe