The Experts below are selected from a list of 243 Experts worldwide ranked by ideXlab platform
Paul Ahlquist - One of the best experts on this subject based on the ideXlab platform.
-
brome mosaic virus 1a nucleoside triphosphatase helicase domain plays crucial roles in recruiting rna replication templates
Journal of Virology, 2005Co-Authors: Xiaofeng Wang, Waiming Lee, Tokiko Watanabe, Michael Janda, Michael Schwartz, Paul AhlquistAbstract:Positive-strand RNA viruses are a large class of viral pathogens causing numerous clinically and economically important diseases of humans, animals, and plants. Although such viruses encompass substantial variation in morphology, genetic organization, host range, and other properties, they all share fundamental similarities in their basic replication mechanisms. For example, genome replication by positive-strand RNA viruses is universally associated with intracellular membranes, which usually are induced by viral replication proteins to form invaginations or vesicles (51). In the early steps of positive-strand RNA virus replication, the viral genomic RNAs first serve as templates for translating these replication proteins and often other viral proteins. Once such proteins induce formation of the membrane-associated replication complexes, the incoming viral genomic RNA must be recruited away from translation to serve as a template for RNA replication. This genomic RNA transition from translation to RNA replication is a crucial step in early infection and must be tightly regulated to effectively balance translation and replication (44). Nevertheless, the mechanisms of selecting and recruiting viral RNAs for replication are not well understood. One positive-strand RNA virus for which such processes have been studied is brome mosaic virus (BMV), a member of the alphavirus-like superfamily of human, animal, and plant viruses. The BMV genome is composed of three RNAs. RNA1 and RNA2 encode replication proteins 1a (109 kDa) and 2apol (94 kDa), respectively. 1a has an N-terminal domain with enzymatic activities required for capping viral RNA and a C-terminal superfamily I nucleoside triphosphatase (NTPase)/helicase-like (NTPase/hel) domain (2, 3, 30, 34). 2apol possesses a central polymerase-like domain and an N-terminal region that binds the 1a NTPase/hel domain (7, 27, 45). RNA3 encodes the 3a protein, required for cell-to-cell movement in plants (4, 42), and coat protein. Yeast (Saccharomyces cerevisiae) cells expressing 1a and 2apol support BMV RNA replication, in which negative- and positive-strand RNA3 and subgenomic RNA4 are produced and amplified using DNA-transcribed RNA3 as the original template (24). This yeast system duplicates the features of BMV RNA replication in natural host plants, including proper intracellular localization; dependence on 1a, 2apol, and specific cis-acting signals; and production of excess positive-strand over negative-strand RNA (52, 54). In addition, yeast support selective encapsidation of BMV RNAs (33). 1a is a key player in BMV RNA replication (7, 25, 52). In yeast expressing 1a alone, 1a is associated with perinuclear endoplasmic reticulum (ER) membranes and induces formation of compartments, or spherules, in which BMV RNA replication occurs (9, 49, 52). These spherules are 50- to 70-nm invaginations of the outer perinuclear ER membrane into the ER lumen, with interiors that are connected to the cytoplasm through a neck (52). Similar membrane invaginations are associated with RNA replication in natural infections by bromoviruses, alphaviruses, nodaviruses, and many other positive-stranded RNA viruses (15, 21, 28, 35, 51, 59). 1a also recruits 2apol to spherules by interacting with the 2apol N terminus (7, 27, 52). In the absence of 2apol, 1a recruits RNA3 to a membrane-associated, nuclease-resistant, and detergent-Susceptible State in which RNA3 half-life and accumulation increase by 20 to 50 fold and RNA3 translation is inhibited (25, 52). This State appears to correspond to the interior of the 1a-induced spherules, since in yeast cells expressing 1a and 2apol and replicating RNA3, positive- and negative-strand RNA3 templates and nascent RNA are retained in an indistinguishable, membrane-associated, nuclease-resistant State, and immunogold electron microscopy (EM) localizes bromo-UTP-labeled nascent RNA to spherules (52). Although helicases have traditionally been viewed as NTP-dependent double-stranded (ds) nucleic acid unwinding enzymes, recent data suggest that helicases may also be involved in RNA translocation, modulating RNA-protein interactions, etc. (39, 53, 56). 1a has a C-terminal superfamily I NTPase/hel domain (amino acids [aa] 562 to 961) containing seven helicase signature motifs, denoted I, Ia, and II to VI, with motifs I and II comprising a putative NTPase domain (Fig. (Fig.1)1) (3, 17, 31, 34). Multiple results show that the 1a NTPase/hel domain contributes to the RNA synthesis functions of the assembled replication complex. When preformed RNA replication complexes are shifted to a nonpermissive temperature, a strong temperature-sensitive insertion mutation near the 1a NTPase domain blocks further synthesis of positive- and negative-strand genomic RNAs and subgenomic RNA4 (34). Moreover, studies with other animal and plant-infecting members of the alphavirus superfamily show that conserved NTPase/hel domains paralleling that of BMV 1a have an RNA triphosphatase activity contributing to capping of viral RNA products by removing 5′ γ-phosphates (37, 57). In addition to these roles in RNA synthesis, the 1a NTPase/hel domain has a role(s) in earlier steps of RNA replication complex assembly. In particular, mutations in three of seven 1a helicase motifs block in vivo RNA3 stabilization (2). However, the nature of these contributions is not clear. FIG. 1. (A) Schematic of BMV 1a protein. 1a contains an N-terminal capping domain (aa 1 to 515) with m7G-methyltransferase and m7GMP binding activities and a C-terminal NTPase/helicase-like domain (aa 562 to 961) containing seven conserved signature helicase ... To gain more insight into the functions of the 1a NTPase/hel domain and the mechanisms by which 1a induces RNA3 in vivo to become membrane associated and stabilized, we made and tested additional mutations in six of the seven helicase signature motifs. We show here that mutations in each of these signature helicase motifs blocked BMV RNA replication. Most replication-defective mutations allowed spherule formation and readily detectable RNA3 recruitment to membranes but blocked RNA3 from achieving the nuclease-resistant State induced by wild-type (wt) 1a. The data show that the 1a NTPase/hel domain plays crucial roles in recruiting BMV RNA templates into replication complexes.
-
Brome Mosaic Virus 1a Nucleoside Triphosphatase/Helicase Domain Plays Crucial Roles in Recruiting RNA Replication Templates
Journal of virology, 2005Co-Authors: Xiaofeng Wang, Waiming Lee, Tokiko Watanabe, Michael Janda, Michael Schwartz, Paul AhlquistAbstract:Positive-strand RNA viruses are a large class of viral pathogens causing numerous clinically and economically important diseases of humans, animals, and plants. Although such viruses encompass substantial variation in morphology, genetic organization, host range, and other properties, they all share fundamental similarities in their basic replication mechanisms. For example, genome replication by positive-strand RNA viruses is universally associated with intracellular membranes, which usually are induced by viral replication proteins to form invaginations or vesicles (51). In the early steps of positive-strand RNA virus replication, the viral genomic RNAs first serve as templates for translating these replication proteins and often other viral proteins. Once such proteins induce formation of the membrane-associated replication complexes, the incoming viral genomic RNA must be recruited away from translation to serve as a template for RNA replication. This genomic RNA transition from translation to RNA replication is a crucial step in early infection and must be tightly regulated to effectively balance translation and replication (44). Nevertheless, the mechanisms of selecting and recruiting viral RNAs for replication are not well understood. One positive-strand RNA virus for which such processes have been studied is brome mosaic virus (BMV), a member of the alphavirus-like superfamily of human, animal, and plant viruses. The BMV genome is composed of three RNAs. RNA1 and RNA2 encode replication proteins 1a (109 kDa) and 2apol (94 kDa), respectively. 1a has an N-terminal domain with enzymatic activities required for capping viral RNA and a C-terminal superfamily I nucleoside triphosphatase (NTPase)/helicase-like (NTPase/hel) domain (2, 3, 30, 34). 2apol possesses a central polymerase-like domain and an N-terminal region that binds the 1a NTPase/hel domain (7, 27, 45). RNA3 encodes the 3a protein, required for cell-to-cell movement in plants (4, 42), and coat protein. Yeast (Saccharomyces cerevisiae) cells expressing 1a and 2apol support BMV RNA replication, in which negative- and positive-strand RNA3 and subgenomic RNA4 are produced and amplified using DNA-transcribed RNA3 as the original template (24). This yeast system duplicates the features of BMV RNA replication in natural host plants, including proper intracellular localization; dependence on 1a, 2apol, and specific cis-acting signals; and production of excess positive-strand over negative-strand RNA (52, 54). In addition, yeast support selective encapsidation of BMV RNAs (33). 1a is a key player in BMV RNA replication (7, 25, 52). In yeast expressing 1a alone, 1a is associated with perinuclear endoplasmic reticulum (ER) membranes and induces formation of compartments, or spherules, in which BMV RNA replication occurs (9, 49, 52). These spherules are 50- to 70-nm invaginations of the outer perinuclear ER membrane into the ER lumen, with interiors that are connected to the cytoplasm through a neck (52). Similar membrane invaginations are associated with RNA replication in natural infections by bromoviruses, alphaviruses, nodaviruses, and many other positive-stranded RNA viruses (15, 21, 28, 35, 51, 59). 1a also recruits 2apol to spherules by interacting with the 2apol N terminus (7, 27, 52). In the absence of 2apol, 1a recruits RNA3 to a membrane-associated, nuclease-resistant, and detergent-Susceptible State in which RNA3 half-life and accumulation increase by 20 to 50 fold and RNA3 translation is inhibited (25, 52). This State appears to correspond to the interior of the 1a-induced spherules, since in yeast cells expressing 1a and 2apol and replicating RNA3, positive- and negative-strand RNA3 templates and nascent RNA are retained in an indistinguishable, membrane-associated, nuclease-resistant State, and immunogold electron microscopy (EM) localizes bromo-UTP-labeled nascent RNA to spherules (52). Although helicases have traditionally been viewed as NTP-dependent double-stranded (ds) nucleic acid unwinding enzymes, recent data suggest that helicases may also be involved in RNA translocation, modulating RNA-protein interactions, etc. (39, 53, 56). 1a has a C-terminal superfamily I NTPase/hel domain (amino acids [aa] 562 to 961) containing seven helicase signature motifs, denoted I, Ia, and II to VI, with motifs I and II comprising a putative NTPase domain (Fig. (Fig.1)1) (3, 17, 31, 34). Multiple results show that the 1a NTPase/hel domain contributes to the RNA synthesis functions of the assembled replication complex. When preformed RNA replication complexes are shifted to a nonpermissive temperature, a strong temperature-sensitive insertion mutation near the 1a NTPase domain blocks further synthesis of positive- and negative-strand genomic RNAs and subgenomic RNA4 (34). Moreover, studies with other animal and plant-infecting members of the alphavirus superfamily show that conserved NTPase/hel domains paralleling that of BMV 1a have an RNA triphosphatase activity contributing to capping of viral RNA products by removing 5′ γ-phosphates (37, 57). In addition to these roles in RNA synthesis, the 1a NTPase/hel domain has a role(s) in earlier steps of RNA replication complex assembly. In particular, mutations in three of seven 1a helicase motifs block in vivo RNA3 stabilization (2). However, the nature of these contributions is not clear. FIG. 1. (A) Schematic of BMV 1a protein. 1a contains an N-terminal capping domain (aa 1 to 515) with m7G-methyltransferase and m7GMP binding activities and a C-terminal NTPase/helicase-like domain (aa 562 to 961) containing seven conserved signature helicase ... To gain more insight into the functions of the 1a NTPase/hel domain and the mechanisms by which 1a induces RNA3 in vivo to become membrane associated and stabilized, we made and tested additional mutations in six of the seven helicase signature motifs. We show here that mutations in each of these signature helicase motifs blocked BMV RNA replication. Most replication-defective mutations allowed spherule formation and readily detectable RNA3 recruitment to membranes but blocked RNA3 from achieving the nuclease-resistant State induced by wild-type (wt) 1a. The data show that the 1a NTPase/hel domain plays crucial roles in recruiting BMV RNA templates into replication complexes.
-
a positive strand rna virus replication complex parallels form and function of retrovirus capsids
Molecular Cell, 2002Co-Authors: Michael Janda, Paul Ahlquist, Michael P Schwartz, Jianbo Chen, Michael L Sullivan, Johan Den BoonAbstract:We show that brome mosaic virus (BMV) RNA replication protein 1a, 2a polymerase, and a cis-acting replication signal recapitulate the functions of Gag, Pol, and RNA packaging signals in conventional retrovirus and foamy virus cores. Prior to RNA replication, 1a forms spherules budding into the endoplasmic reticulum membrane, sequestering viral positive-strand RNA templates in a nuclease-resistant, detergent-Susceptible State. When expressed, 2a polymerase colocalizes in these spherules, which become the sites of viral RNA synthesis and retain negative-strand templates for positive-strand RNA synthesis. These results explain many features of replication by numerous positive strand RNA viruses and reveal that these viruses, reverse transcribing viruses, and dsRNA viruses share fundamental similarities in replication and may have common evolutionary origins.
Michael Janda - One of the best experts on this subject based on the ideXlab platform.
-
brome mosaic virus 1a nucleoside triphosphatase helicase domain plays crucial roles in recruiting rna replication templates
Journal of Virology, 2005Co-Authors: Xiaofeng Wang, Waiming Lee, Tokiko Watanabe, Michael Janda, Michael Schwartz, Paul AhlquistAbstract:Positive-strand RNA viruses are a large class of viral pathogens causing numerous clinically and economically important diseases of humans, animals, and plants. Although such viruses encompass substantial variation in morphology, genetic organization, host range, and other properties, they all share fundamental similarities in their basic replication mechanisms. For example, genome replication by positive-strand RNA viruses is universally associated with intracellular membranes, which usually are induced by viral replication proteins to form invaginations or vesicles (51). In the early steps of positive-strand RNA virus replication, the viral genomic RNAs first serve as templates for translating these replication proteins and often other viral proteins. Once such proteins induce formation of the membrane-associated replication complexes, the incoming viral genomic RNA must be recruited away from translation to serve as a template for RNA replication. This genomic RNA transition from translation to RNA replication is a crucial step in early infection and must be tightly regulated to effectively balance translation and replication (44). Nevertheless, the mechanisms of selecting and recruiting viral RNAs for replication are not well understood. One positive-strand RNA virus for which such processes have been studied is brome mosaic virus (BMV), a member of the alphavirus-like superfamily of human, animal, and plant viruses. The BMV genome is composed of three RNAs. RNA1 and RNA2 encode replication proteins 1a (109 kDa) and 2apol (94 kDa), respectively. 1a has an N-terminal domain with enzymatic activities required for capping viral RNA and a C-terminal superfamily I nucleoside triphosphatase (NTPase)/helicase-like (NTPase/hel) domain (2, 3, 30, 34). 2apol possesses a central polymerase-like domain and an N-terminal region that binds the 1a NTPase/hel domain (7, 27, 45). RNA3 encodes the 3a protein, required for cell-to-cell movement in plants (4, 42), and coat protein. Yeast (Saccharomyces cerevisiae) cells expressing 1a and 2apol support BMV RNA replication, in which negative- and positive-strand RNA3 and subgenomic RNA4 are produced and amplified using DNA-transcribed RNA3 as the original template (24). This yeast system duplicates the features of BMV RNA replication in natural host plants, including proper intracellular localization; dependence on 1a, 2apol, and specific cis-acting signals; and production of excess positive-strand over negative-strand RNA (52, 54). In addition, yeast support selective encapsidation of BMV RNAs (33). 1a is a key player in BMV RNA replication (7, 25, 52). In yeast expressing 1a alone, 1a is associated with perinuclear endoplasmic reticulum (ER) membranes and induces formation of compartments, or spherules, in which BMV RNA replication occurs (9, 49, 52). These spherules are 50- to 70-nm invaginations of the outer perinuclear ER membrane into the ER lumen, with interiors that are connected to the cytoplasm through a neck (52). Similar membrane invaginations are associated with RNA replication in natural infections by bromoviruses, alphaviruses, nodaviruses, and many other positive-stranded RNA viruses (15, 21, 28, 35, 51, 59). 1a also recruits 2apol to spherules by interacting with the 2apol N terminus (7, 27, 52). In the absence of 2apol, 1a recruits RNA3 to a membrane-associated, nuclease-resistant, and detergent-Susceptible State in which RNA3 half-life and accumulation increase by 20 to 50 fold and RNA3 translation is inhibited (25, 52). This State appears to correspond to the interior of the 1a-induced spherules, since in yeast cells expressing 1a and 2apol and replicating RNA3, positive- and negative-strand RNA3 templates and nascent RNA are retained in an indistinguishable, membrane-associated, nuclease-resistant State, and immunogold electron microscopy (EM) localizes bromo-UTP-labeled nascent RNA to spherules (52). Although helicases have traditionally been viewed as NTP-dependent double-stranded (ds) nucleic acid unwinding enzymes, recent data suggest that helicases may also be involved in RNA translocation, modulating RNA-protein interactions, etc. (39, 53, 56). 1a has a C-terminal superfamily I NTPase/hel domain (amino acids [aa] 562 to 961) containing seven helicase signature motifs, denoted I, Ia, and II to VI, with motifs I and II comprising a putative NTPase domain (Fig. (Fig.1)1) (3, 17, 31, 34). Multiple results show that the 1a NTPase/hel domain contributes to the RNA synthesis functions of the assembled replication complex. When preformed RNA replication complexes are shifted to a nonpermissive temperature, a strong temperature-sensitive insertion mutation near the 1a NTPase domain blocks further synthesis of positive- and negative-strand genomic RNAs and subgenomic RNA4 (34). Moreover, studies with other animal and plant-infecting members of the alphavirus superfamily show that conserved NTPase/hel domains paralleling that of BMV 1a have an RNA triphosphatase activity contributing to capping of viral RNA products by removing 5′ γ-phosphates (37, 57). In addition to these roles in RNA synthesis, the 1a NTPase/hel domain has a role(s) in earlier steps of RNA replication complex assembly. In particular, mutations in three of seven 1a helicase motifs block in vivo RNA3 stabilization (2). However, the nature of these contributions is not clear. FIG. 1. (A) Schematic of BMV 1a protein. 1a contains an N-terminal capping domain (aa 1 to 515) with m7G-methyltransferase and m7GMP binding activities and a C-terminal NTPase/helicase-like domain (aa 562 to 961) containing seven conserved signature helicase ... To gain more insight into the functions of the 1a NTPase/hel domain and the mechanisms by which 1a induces RNA3 in vivo to become membrane associated and stabilized, we made and tested additional mutations in six of the seven helicase signature motifs. We show here that mutations in each of these signature helicase motifs blocked BMV RNA replication. Most replication-defective mutations allowed spherule formation and readily detectable RNA3 recruitment to membranes but blocked RNA3 from achieving the nuclease-resistant State induced by wild-type (wt) 1a. The data show that the 1a NTPase/hel domain plays crucial roles in recruiting BMV RNA templates into replication complexes.
-
Brome Mosaic Virus 1a Nucleoside Triphosphatase/Helicase Domain Plays Crucial Roles in Recruiting RNA Replication Templates
Journal of virology, 2005Co-Authors: Xiaofeng Wang, Waiming Lee, Tokiko Watanabe, Michael Janda, Michael Schwartz, Paul AhlquistAbstract:Positive-strand RNA viruses are a large class of viral pathogens causing numerous clinically and economically important diseases of humans, animals, and plants. Although such viruses encompass substantial variation in morphology, genetic organization, host range, and other properties, they all share fundamental similarities in their basic replication mechanisms. For example, genome replication by positive-strand RNA viruses is universally associated with intracellular membranes, which usually are induced by viral replication proteins to form invaginations or vesicles (51). In the early steps of positive-strand RNA virus replication, the viral genomic RNAs first serve as templates for translating these replication proteins and often other viral proteins. Once such proteins induce formation of the membrane-associated replication complexes, the incoming viral genomic RNA must be recruited away from translation to serve as a template for RNA replication. This genomic RNA transition from translation to RNA replication is a crucial step in early infection and must be tightly regulated to effectively balance translation and replication (44). Nevertheless, the mechanisms of selecting and recruiting viral RNAs for replication are not well understood. One positive-strand RNA virus for which such processes have been studied is brome mosaic virus (BMV), a member of the alphavirus-like superfamily of human, animal, and plant viruses. The BMV genome is composed of three RNAs. RNA1 and RNA2 encode replication proteins 1a (109 kDa) and 2apol (94 kDa), respectively. 1a has an N-terminal domain with enzymatic activities required for capping viral RNA and a C-terminal superfamily I nucleoside triphosphatase (NTPase)/helicase-like (NTPase/hel) domain (2, 3, 30, 34). 2apol possesses a central polymerase-like domain and an N-terminal region that binds the 1a NTPase/hel domain (7, 27, 45). RNA3 encodes the 3a protein, required for cell-to-cell movement in plants (4, 42), and coat protein. Yeast (Saccharomyces cerevisiae) cells expressing 1a and 2apol support BMV RNA replication, in which negative- and positive-strand RNA3 and subgenomic RNA4 are produced and amplified using DNA-transcribed RNA3 as the original template (24). This yeast system duplicates the features of BMV RNA replication in natural host plants, including proper intracellular localization; dependence on 1a, 2apol, and specific cis-acting signals; and production of excess positive-strand over negative-strand RNA (52, 54). In addition, yeast support selective encapsidation of BMV RNAs (33). 1a is a key player in BMV RNA replication (7, 25, 52). In yeast expressing 1a alone, 1a is associated with perinuclear endoplasmic reticulum (ER) membranes and induces formation of compartments, or spherules, in which BMV RNA replication occurs (9, 49, 52). These spherules are 50- to 70-nm invaginations of the outer perinuclear ER membrane into the ER lumen, with interiors that are connected to the cytoplasm through a neck (52). Similar membrane invaginations are associated with RNA replication in natural infections by bromoviruses, alphaviruses, nodaviruses, and many other positive-stranded RNA viruses (15, 21, 28, 35, 51, 59). 1a also recruits 2apol to spherules by interacting with the 2apol N terminus (7, 27, 52). In the absence of 2apol, 1a recruits RNA3 to a membrane-associated, nuclease-resistant, and detergent-Susceptible State in which RNA3 half-life and accumulation increase by 20 to 50 fold and RNA3 translation is inhibited (25, 52). This State appears to correspond to the interior of the 1a-induced spherules, since in yeast cells expressing 1a and 2apol and replicating RNA3, positive- and negative-strand RNA3 templates and nascent RNA are retained in an indistinguishable, membrane-associated, nuclease-resistant State, and immunogold electron microscopy (EM) localizes bromo-UTP-labeled nascent RNA to spherules (52). Although helicases have traditionally been viewed as NTP-dependent double-stranded (ds) nucleic acid unwinding enzymes, recent data suggest that helicases may also be involved in RNA translocation, modulating RNA-protein interactions, etc. (39, 53, 56). 1a has a C-terminal superfamily I NTPase/hel domain (amino acids [aa] 562 to 961) containing seven helicase signature motifs, denoted I, Ia, and II to VI, with motifs I and II comprising a putative NTPase domain (Fig. (Fig.1)1) (3, 17, 31, 34). Multiple results show that the 1a NTPase/hel domain contributes to the RNA synthesis functions of the assembled replication complex. When preformed RNA replication complexes are shifted to a nonpermissive temperature, a strong temperature-sensitive insertion mutation near the 1a NTPase domain blocks further synthesis of positive- and negative-strand genomic RNAs and subgenomic RNA4 (34). Moreover, studies with other animal and plant-infecting members of the alphavirus superfamily show that conserved NTPase/hel domains paralleling that of BMV 1a have an RNA triphosphatase activity contributing to capping of viral RNA products by removing 5′ γ-phosphates (37, 57). In addition to these roles in RNA synthesis, the 1a NTPase/hel domain has a role(s) in earlier steps of RNA replication complex assembly. In particular, mutations in three of seven 1a helicase motifs block in vivo RNA3 stabilization (2). However, the nature of these contributions is not clear. FIG. 1. (A) Schematic of BMV 1a protein. 1a contains an N-terminal capping domain (aa 1 to 515) with m7G-methyltransferase and m7GMP binding activities and a C-terminal NTPase/helicase-like domain (aa 562 to 961) containing seven conserved signature helicase ... To gain more insight into the functions of the 1a NTPase/hel domain and the mechanisms by which 1a induces RNA3 in vivo to become membrane associated and stabilized, we made and tested additional mutations in six of the seven helicase signature motifs. We show here that mutations in each of these signature helicase motifs blocked BMV RNA replication. Most replication-defective mutations allowed spherule formation and readily detectable RNA3 recruitment to membranes but blocked RNA3 from achieving the nuclease-resistant State induced by wild-type (wt) 1a. The data show that the 1a NTPase/hel domain plays crucial roles in recruiting BMV RNA templates into replication complexes.
-
a positive strand rna virus replication complex parallels form and function of retrovirus capsids
Molecular Cell, 2002Co-Authors: Michael Janda, Paul Ahlquist, Michael P Schwartz, Jianbo Chen, Michael L Sullivan, Johan Den BoonAbstract:We show that brome mosaic virus (BMV) RNA replication protein 1a, 2a polymerase, and a cis-acting replication signal recapitulate the functions of Gag, Pol, and RNA packaging signals in conventional retrovirus and foamy virus cores. Prior to RNA replication, 1a forms spherules budding into the endoplasmic reticulum membrane, sequestering viral positive-strand RNA templates in a nuclease-resistant, detergent-Susceptible State. When expressed, 2a polymerase colocalizes in these spherules, which become the sites of viral RNA synthesis and retain negative-strand templates for positive-strand RNA synthesis. These results explain many features of replication by numerous positive strand RNA viruses and reveal that these viruses, reverse transcribing viruses, and dsRNA viruses share fundamental similarities in replication and may have common evolutionary origins.
Xiaofeng Wang - One of the best experts on this subject based on the ideXlab platform.
-
brome mosaic virus 1a nucleoside triphosphatase helicase domain plays crucial roles in recruiting rna replication templates
Journal of Virology, 2005Co-Authors: Xiaofeng Wang, Waiming Lee, Tokiko Watanabe, Michael Janda, Michael Schwartz, Paul AhlquistAbstract:Positive-strand RNA viruses are a large class of viral pathogens causing numerous clinically and economically important diseases of humans, animals, and plants. Although such viruses encompass substantial variation in morphology, genetic organization, host range, and other properties, they all share fundamental similarities in their basic replication mechanisms. For example, genome replication by positive-strand RNA viruses is universally associated with intracellular membranes, which usually are induced by viral replication proteins to form invaginations or vesicles (51). In the early steps of positive-strand RNA virus replication, the viral genomic RNAs first serve as templates for translating these replication proteins and often other viral proteins. Once such proteins induce formation of the membrane-associated replication complexes, the incoming viral genomic RNA must be recruited away from translation to serve as a template for RNA replication. This genomic RNA transition from translation to RNA replication is a crucial step in early infection and must be tightly regulated to effectively balance translation and replication (44). Nevertheless, the mechanisms of selecting and recruiting viral RNAs for replication are not well understood. One positive-strand RNA virus for which such processes have been studied is brome mosaic virus (BMV), a member of the alphavirus-like superfamily of human, animal, and plant viruses. The BMV genome is composed of three RNAs. RNA1 and RNA2 encode replication proteins 1a (109 kDa) and 2apol (94 kDa), respectively. 1a has an N-terminal domain with enzymatic activities required for capping viral RNA and a C-terminal superfamily I nucleoside triphosphatase (NTPase)/helicase-like (NTPase/hel) domain (2, 3, 30, 34). 2apol possesses a central polymerase-like domain and an N-terminal region that binds the 1a NTPase/hel domain (7, 27, 45). RNA3 encodes the 3a protein, required for cell-to-cell movement in plants (4, 42), and coat protein. Yeast (Saccharomyces cerevisiae) cells expressing 1a and 2apol support BMV RNA replication, in which negative- and positive-strand RNA3 and subgenomic RNA4 are produced and amplified using DNA-transcribed RNA3 as the original template (24). This yeast system duplicates the features of BMV RNA replication in natural host plants, including proper intracellular localization; dependence on 1a, 2apol, and specific cis-acting signals; and production of excess positive-strand over negative-strand RNA (52, 54). In addition, yeast support selective encapsidation of BMV RNAs (33). 1a is a key player in BMV RNA replication (7, 25, 52). In yeast expressing 1a alone, 1a is associated with perinuclear endoplasmic reticulum (ER) membranes and induces formation of compartments, or spherules, in which BMV RNA replication occurs (9, 49, 52). These spherules are 50- to 70-nm invaginations of the outer perinuclear ER membrane into the ER lumen, with interiors that are connected to the cytoplasm through a neck (52). Similar membrane invaginations are associated with RNA replication in natural infections by bromoviruses, alphaviruses, nodaviruses, and many other positive-stranded RNA viruses (15, 21, 28, 35, 51, 59). 1a also recruits 2apol to spherules by interacting with the 2apol N terminus (7, 27, 52). In the absence of 2apol, 1a recruits RNA3 to a membrane-associated, nuclease-resistant, and detergent-Susceptible State in which RNA3 half-life and accumulation increase by 20 to 50 fold and RNA3 translation is inhibited (25, 52). This State appears to correspond to the interior of the 1a-induced spherules, since in yeast cells expressing 1a and 2apol and replicating RNA3, positive- and negative-strand RNA3 templates and nascent RNA are retained in an indistinguishable, membrane-associated, nuclease-resistant State, and immunogold electron microscopy (EM) localizes bromo-UTP-labeled nascent RNA to spherules (52). Although helicases have traditionally been viewed as NTP-dependent double-stranded (ds) nucleic acid unwinding enzymes, recent data suggest that helicases may also be involved in RNA translocation, modulating RNA-protein interactions, etc. (39, 53, 56). 1a has a C-terminal superfamily I NTPase/hel domain (amino acids [aa] 562 to 961) containing seven helicase signature motifs, denoted I, Ia, and II to VI, with motifs I and II comprising a putative NTPase domain (Fig. (Fig.1)1) (3, 17, 31, 34). Multiple results show that the 1a NTPase/hel domain contributes to the RNA synthesis functions of the assembled replication complex. When preformed RNA replication complexes are shifted to a nonpermissive temperature, a strong temperature-sensitive insertion mutation near the 1a NTPase domain blocks further synthesis of positive- and negative-strand genomic RNAs and subgenomic RNA4 (34). Moreover, studies with other animal and plant-infecting members of the alphavirus superfamily show that conserved NTPase/hel domains paralleling that of BMV 1a have an RNA triphosphatase activity contributing to capping of viral RNA products by removing 5′ γ-phosphates (37, 57). In addition to these roles in RNA synthesis, the 1a NTPase/hel domain has a role(s) in earlier steps of RNA replication complex assembly. In particular, mutations in three of seven 1a helicase motifs block in vivo RNA3 stabilization (2). However, the nature of these contributions is not clear. FIG. 1. (A) Schematic of BMV 1a protein. 1a contains an N-terminal capping domain (aa 1 to 515) with m7G-methyltransferase and m7GMP binding activities and a C-terminal NTPase/helicase-like domain (aa 562 to 961) containing seven conserved signature helicase ... To gain more insight into the functions of the 1a NTPase/hel domain and the mechanisms by which 1a induces RNA3 in vivo to become membrane associated and stabilized, we made and tested additional mutations in six of the seven helicase signature motifs. We show here that mutations in each of these signature helicase motifs blocked BMV RNA replication. Most replication-defective mutations allowed spherule formation and readily detectable RNA3 recruitment to membranes but blocked RNA3 from achieving the nuclease-resistant State induced by wild-type (wt) 1a. The data show that the 1a NTPase/hel domain plays crucial roles in recruiting BMV RNA templates into replication complexes.
-
Brome Mosaic Virus 1a Nucleoside Triphosphatase/Helicase Domain Plays Crucial Roles in Recruiting RNA Replication Templates
Journal of virology, 2005Co-Authors: Xiaofeng Wang, Waiming Lee, Tokiko Watanabe, Michael Janda, Michael Schwartz, Paul AhlquistAbstract:Positive-strand RNA viruses are a large class of viral pathogens causing numerous clinically and economically important diseases of humans, animals, and plants. Although such viruses encompass substantial variation in morphology, genetic organization, host range, and other properties, they all share fundamental similarities in their basic replication mechanisms. For example, genome replication by positive-strand RNA viruses is universally associated with intracellular membranes, which usually are induced by viral replication proteins to form invaginations or vesicles (51). In the early steps of positive-strand RNA virus replication, the viral genomic RNAs first serve as templates for translating these replication proteins and often other viral proteins. Once such proteins induce formation of the membrane-associated replication complexes, the incoming viral genomic RNA must be recruited away from translation to serve as a template for RNA replication. This genomic RNA transition from translation to RNA replication is a crucial step in early infection and must be tightly regulated to effectively balance translation and replication (44). Nevertheless, the mechanisms of selecting and recruiting viral RNAs for replication are not well understood. One positive-strand RNA virus for which such processes have been studied is brome mosaic virus (BMV), a member of the alphavirus-like superfamily of human, animal, and plant viruses. The BMV genome is composed of three RNAs. RNA1 and RNA2 encode replication proteins 1a (109 kDa) and 2apol (94 kDa), respectively. 1a has an N-terminal domain with enzymatic activities required for capping viral RNA and a C-terminal superfamily I nucleoside triphosphatase (NTPase)/helicase-like (NTPase/hel) domain (2, 3, 30, 34). 2apol possesses a central polymerase-like domain and an N-terminal region that binds the 1a NTPase/hel domain (7, 27, 45). RNA3 encodes the 3a protein, required for cell-to-cell movement in plants (4, 42), and coat protein. Yeast (Saccharomyces cerevisiae) cells expressing 1a and 2apol support BMV RNA replication, in which negative- and positive-strand RNA3 and subgenomic RNA4 are produced and amplified using DNA-transcribed RNA3 as the original template (24). This yeast system duplicates the features of BMV RNA replication in natural host plants, including proper intracellular localization; dependence on 1a, 2apol, and specific cis-acting signals; and production of excess positive-strand over negative-strand RNA (52, 54). In addition, yeast support selective encapsidation of BMV RNAs (33). 1a is a key player in BMV RNA replication (7, 25, 52). In yeast expressing 1a alone, 1a is associated with perinuclear endoplasmic reticulum (ER) membranes and induces formation of compartments, or spherules, in which BMV RNA replication occurs (9, 49, 52). These spherules are 50- to 70-nm invaginations of the outer perinuclear ER membrane into the ER lumen, with interiors that are connected to the cytoplasm through a neck (52). Similar membrane invaginations are associated with RNA replication in natural infections by bromoviruses, alphaviruses, nodaviruses, and many other positive-stranded RNA viruses (15, 21, 28, 35, 51, 59). 1a also recruits 2apol to spherules by interacting with the 2apol N terminus (7, 27, 52). In the absence of 2apol, 1a recruits RNA3 to a membrane-associated, nuclease-resistant, and detergent-Susceptible State in which RNA3 half-life and accumulation increase by 20 to 50 fold and RNA3 translation is inhibited (25, 52). This State appears to correspond to the interior of the 1a-induced spherules, since in yeast cells expressing 1a and 2apol and replicating RNA3, positive- and negative-strand RNA3 templates and nascent RNA are retained in an indistinguishable, membrane-associated, nuclease-resistant State, and immunogold electron microscopy (EM) localizes bromo-UTP-labeled nascent RNA to spherules (52). Although helicases have traditionally been viewed as NTP-dependent double-stranded (ds) nucleic acid unwinding enzymes, recent data suggest that helicases may also be involved in RNA translocation, modulating RNA-protein interactions, etc. (39, 53, 56). 1a has a C-terminal superfamily I NTPase/hel domain (amino acids [aa] 562 to 961) containing seven helicase signature motifs, denoted I, Ia, and II to VI, with motifs I and II comprising a putative NTPase domain (Fig. (Fig.1)1) (3, 17, 31, 34). Multiple results show that the 1a NTPase/hel domain contributes to the RNA synthesis functions of the assembled replication complex. When preformed RNA replication complexes are shifted to a nonpermissive temperature, a strong temperature-sensitive insertion mutation near the 1a NTPase domain blocks further synthesis of positive- and negative-strand genomic RNAs and subgenomic RNA4 (34). Moreover, studies with other animal and plant-infecting members of the alphavirus superfamily show that conserved NTPase/hel domains paralleling that of BMV 1a have an RNA triphosphatase activity contributing to capping of viral RNA products by removing 5′ γ-phosphates (37, 57). In addition to these roles in RNA synthesis, the 1a NTPase/hel domain has a role(s) in earlier steps of RNA replication complex assembly. In particular, mutations in three of seven 1a helicase motifs block in vivo RNA3 stabilization (2). However, the nature of these contributions is not clear. FIG. 1. (A) Schematic of BMV 1a protein. 1a contains an N-terminal capping domain (aa 1 to 515) with m7G-methyltransferase and m7GMP binding activities and a C-terminal NTPase/helicase-like domain (aa 562 to 961) containing seven conserved signature helicase ... To gain more insight into the functions of the 1a NTPase/hel domain and the mechanisms by which 1a induces RNA3 in vivo to become membrane associated and stabilized, we made and tested additional mutations in six of the seven helicase signature motifs. We show here that mutations in each of these signature helicase motifs blocked BMV RNA replication. Most replication-defective mutations allowed spherule formation and readily detectable RNA3 recruitment to membranes but blocked RNA3 from achieving the nuclease-resistant State induced by wild-type (wt) 1a. The data show that the 1a NTPase/hel domain plays crucial roles in recruiting BMV RNA templates into replication complexes.
Robert Powers - One of the best experts on this subject based on the ideXlab platform.
-
metabolic changes associated with adaptive resistance to daptomycin in streptococcus mitis oralis
BMC Microbiology, 2020Co-Authors: Allison Parrett, Joseph M Reed, Stewart G Gardner, Nagendra N Mishra, Arnold S Bayer, Robert PowersAbstract:Viridans group streptococci of the Streptococcus mitis-oralis subgroup are important endovascular pathogens. They can rapidly develop high-level and durable non-susceptibility to daptomycin both in vitro and in vivo upon exposure to daptomycin. Two consistent genetic adaptations associated with this phenotype (i.e., mutations in cdsA and pgsA) lead to the depletion of the phospholipids, phosphatidylglycerol and cardiolipin, from the bacterial membrane. Such alterations in phospholipid biosynthesis will modify carbon flow and change the bacterial metabolic status. To determine the metabolic differences between daptomycin-Susceptible and non-Susceptible bacteria, the physiology and metabolomes of S. mitis-oralis strains 351 (daptomycin-Susceptible) and 351-D10 (daptomycin non-Susceptible) were analyzed. S. mitis-oralis strain 351-D10 was made daptomycin non-Susceptible through serial passage in the presence of daptomycin. Daptomycin non-Susceptible S. mitis-oralis had significant alterations in glucose catabolism and a re-balancing of the redox status through amino acid biosynthesis relative to daptomycin Susceptible S. mitis-oralis. These changes were accompanied by a reduced capacity to generate biomass, creating a fitness cost in exchange for daptomycin non-susceptibility. S. mitis-oralis metabolism is altered in daptomycin non-Susceptible bacteria relative to the daptomycin Susceptible parent strain. As demonstrated in Staphylococcus aureus, inhibiting the metabolic changes that facilitate the transition from a daptomycin Susceptible State to a non-Susceptible one, inhibits daptomycin non-susceptibility. By preventing these metabolic adaptations in S. mitis-oralis, it should be possible to deter the formation of daptomycin non-susceptibility.
Michael Schwartz - One of the best experts on this subject based on the ideXlab platform.
-
brome mosaic virus 1a nucleoside triphosphatase helicase domain plays crucial roles in recruiting rna replication templates
Journal of Virology, 2005Co-Authors: Xiaofeng Wang, Waiming Lee, Tokiko Watanabe, Michael Janda, Michael Schwartz, Paul AhlquistAbstract:Positive-strand RNA viruses are a large class of viral pathogens causing numerous clinically and economically important diseases of humans, animals, and plants. Although such viruses encompass substantial variation in morphology, genetic organization, host range, and other properties, they all share fundamental similarities in their basic replication mechanisms. For example, genome replication by positive-strand RNA viruses is universally associated with intracellular membranes, which usually are induced by viral replication proteins to form invaginations or vesicles (51). In the early steps of positive-strand RNA virus replication, the viral genomic RNAs first serve as templates for translating these replication proteins and often other viral proteins. Once such proteins induce formation of the membrane-associated replication complexes, the incoming viral genomic RNA must be recruited away from translation to serve as a template for RNA replication. This genomic RNA transition from translation to RNA replication is a crucial step in early infection and must be tightly regulated to effectively balance translation and replication (44). Nevertheless, the mechanisms of selecting and recruiting viral RNAs for replication are not well understood. One positive-strand RNA virus for which such processes have been studied is brome mosaic virus (BMV), a member of the alphavirus-like superfamily of human, animal, and plant viruses. The BMV genome is composed of three RNAs. RNA1 and RNA2 encode replication proteins 1a (109 kDa) and 2apol (94 kDa), respectively. 1a has an N-terminal domain with enzymatic activities required for capping viral RNA and a C-terminal superfamily I nucleoside triphosphatase (NTPase)/helicase-like (NTPase/hel) domain (2, 3, 30, 34). 2apol possesses a central polymerase-like domain and an N-terminal region that binds the 1a NTPase/hel domain (7, 27, 45). RNA3 encodes the 3a protein, required for cell-to-cell movement in plants (4, 42), and coat protein. Yeast (Saccharomyces cerevisiae) cells expressing 1a and 2apol support BMV RNA replication, in which negative- and positive-strand RNA3 and subgenomic RNA4 are produced and amplified using DNA-transcribed RNA3 as the original template (24). This yeast system duplicates the features of BMV RNA replication in natural host plants, including proper intracellular localization; dependence on 1a, 2apol, and specific cis-acting signals; and production of excess positive-strand over negative-strand RNA (52, 54). In addition, yeast support selective encapsidation of BMV RNAs (33). 1a is a key player in BMV RNA replication (7, 25, 52). In yeast expressing 1a alone, 1a is associated with perinuclear endoplasmic reticulum (ER) membranes and induces formation of compartments, or spherules, in which BMV RNA replication occurs (9, 49, 52). These spherules are 50- to 70-nm invaginations of the outer perinuclear ER membrane into the ER lumen, with interiors that are connected to the cytoplasm through a neck (52). Similar membrane invaginations are associated with RNA replication in natural infections by bromoviruses, alphaviruses, nodaviruses, and many other positive-stranded RNA viruses (15, 21, 28, 35, 51, 59). 1a also recruits 2apol to spherules by interacting with the 2apol N terminus (7, 27, 52). In the absence of 2apol, 1a recruits RNA3 to a membrane-associated, nuclease-resistant, and detergent-Susceptible State in which RNA3 half-life and accumulation increase by 20 to 50 fold and RNA3 translation is inhibited (25, 52). This State appears to correspond to the interior of the 1a-induced spherules, since in yeast cells expressing 1a and 2apol and replicating RNA3, positive- and negative-strand RNA3 templates and nascent RNA are retained in an indistinguishable, membrane-associated, nuclease-resistant State, and immunogold electron microscopy (EM) localizes bromo-UTP-labeled nascent RNA to spherules (52). Although helicases have traditionally been viewed as NTP-dependent double-stranded (ds) nucleic acid unwinding enzymes, recent data suggest that helicases may also be involved in RNA translocation, modulating RNA-protein interactions, etc. (39, 53, 56). 1a has a C-terminal superfamily I NTPase/hel domain (amino acids [aa] 562 to 961) containing seven helicase signature motifs, denoted I, Ia, and II to VI, with motifs I and II comprising a putative NTPase domain (Fig. (Fig.1)1) (3, 17, 31, 34). Multiple results show that the 1a NTPase/hel domain contributes to the RNA synthesis functions of the assembled replication complex. When preformed RNA replication complexes are shifted to a nonpermissive temperature, a strong temperature-sensitive insertion mutation near the 1a NTPase domain blocks further synthesis of positive- and negative-strand genomic RNAs and subgenomic RNA4 (34). Moreover, studies with other animal and plant-infecting members of the alphavirus superfamily show that conserved NTPase/hel domains paralleling that of BMV 1a have an RNA triphosphatase activity contributing to capping of viral RNA products by removing 5′ γ-phosphates (37, 57). In addition to these roles in RNA synthesis, the 1a NTPase/hel domain has a role(s) in earlier steps of RNA replication complex assembly. In particular, mutations in three of seven 1a helicase motifs block in vivo RNA3 stabilization (2). However, the nature of these contributions is not clear. FIG. 1. (A) Schematic of BMV 1a protein. 1a contains an N-terminal capping domain (aa 1 to 515) with m7G-methyltransferase and m7GMP binding activities and a C-terminal NTPase/helicase-like domain (aa 562 to 961) containing seven conserved signature helicase ... To gain more insight into the functions of the 1a NTPase/hel domain and the mechanisms by which 1a induces RNA3 in vivo to become membrane associated and stabilized, we made and tested additional mutations in six of the seven helicase signature motifs. We show here that mutations in each of these signature helicase motifs blocked BMV RNA replication. Most replication-defective mutations allowed spherule formation and readily detectable RNA3 recruitment to membranes but blocked RNA3 from achieving the nuclease-resistant State induced by wild-type (wt) 1a. The data show that the 1a NTPase/hel domain plays crucial roles in recruiting BMV RNA templates into replication complexes.
-
Brome Mosaic Virus 1a Nucleoside Triphosphatase/Helicase Domain Plays Crucial Roles in Recruiting RNA Replication Templates
Journal of virology, 2005Co-Authors: Xiaofeng Wang, Waiming Lee, Tokiko Watanabe, Michael Janda, Michael Schwartz, Paul AhlquistAbstract:Positive-strand RNA viruses are a large class of viral pathogens causing numerous clinically and economically important diseases of humans, animals, and plants. Although such viruses encompass substantial variation in morphology, genetic organization, host range, and other properties, they all share fundamental similarities in their basic replication mechanisms. For example, genome replication by positive-strand RNA viruses is universally associated with intracellular membranes, which usually are induced by viral replication proteins to form invaginations or vesicles (51). In the early steps of positive-strand RNA virus replication, the viral genomic RNAs first serve as templates for translating these replication proteins and often other viral proteins. Once such proteins induce formation of the membrane-associated replication complexes, the incoming viral genomic RNA must be recruited away from translation to serve as a template for RNA replication. This genomic RNA transition from translation to RNA replication is a crucial step in early infection and must be tightly regulated to effectively balance translation and replication (44). Nevertheless, the mechanisms of selecting and recruiting viral RNAs for replication are not well understood. One positive-strand RNA virus for which such processes have been studied is brome mosaic virus (BMV), a member of the alphavirus-like superfamily of human, animal, and plant viruses. The BMV genome is composed of three RNAs. RNA1 and RNA2 encode replication proteins 1a (109 kDa) and 2apol (94 kDa), respectively. 1a has an N-terminal domain with enzymatic activities required for capping viral RNA and a C-terminal superfamily I nucleoside triphosphatase (NTPase)/helicase-like (NTPase/hel) domain (2, 3, 30, 34). 2apol possesses a central polymerase-like domain and an N-terminal region that binds the 1a NTPase/hel domain (7, 27, 45). RNA3 encodes the 3a protein, required for cell-to-cell movement in plants (4, 42), and coat protein. Yeast (Saccharomyces cerevisiae) cells expressing 1a and 2apol support BMV RNA replication, in which negative- and positive-strand RNA3 and subgenomic RNA4 are produced and amplified using DNA-transcribed RNA3 as the original template (24). This yeast system duplicates the features of BMV RNA replication in natural host plants, including proper intracellular localization; dependence on 1a, 2apol, and specific cis-acting signals; and production of excess positive-strand over negative-strand RNA (52, 54). In addition, yeast support selective encapsidation of BMV RNAs (33). 1a is a key player in BMV RNA replication (7, 25, 52). In yeast expressing 1a alone, 1a is associated with perinuclear endoplasmic reticulum (ER) membranes and induces formation of compartments, or spherules, in which BMV RNA replication occurs (9, 49, 52). These spherules are 50- to 70-nm invaginations of the outer perinuclear ER membrane into the ER lumen, with interiors that are connected to the cytoplasm through a neck (52). Similar membrane invaginations are associated with RNA replication in natural infections by bromoviruses, alphaviruses, nodaviruses, and many other positive-stranded RNA viruses (15, 21, 28, 35, 51, 59). 1a also recruits 2apol to spherules by interacting with the 2apol N terminus (7, 27, 52). In the absence of 2apol, 1a recruits RNA3 to a membrane-associated, nuclease-resistant, and detergent-Susceptible State in which RNA3 half-life and accumulation increase by 20 to 50 fold and RNA3 translation is inhibited (25, 52). This State appears to correspond to the interior of the 1a-induced spherules, since in yeast cells expressing 1a and 2apol and replicating RNA3, positive- and negative-strand RNA3 templates and nascent RNA are retained in an indistinguishable, membrane-associated, nuclease-resistant State, and immunogold electron microscopy (EM) localizes bromo-UTP-labeled nascent RNA to spherules (52). Although helicases have traditionally been viewed as NTP-dependent double-stranded (ds) nucleic acid unwinding enzymes, recent data suggest that helicases may also be involved in RNA translocation, modulating RNA-protein interactions, etc. (39, 53, 56). 1a has a C-terminal superfamily I NTPase/hel domain (amino acids [aa] 562 to 961) containing seven helicase signature motifs, denoted I, Ia, and II to VI, with motifs I and II comprising a putative NTPase domain (Fig. (Fig.1)1) (3, 17, 31, 34). Multiple results show that the 1a NTPase/hel domain contributes to the RNA synthesis functions of the assembled replication complex. When preformed RNA replication complexes are shifted to a nonpermissive temperature, a strong temperature-sensitive insertion mutation near the 1a NTPase domain blocks further synthesis of positive- and negative-strand genomic RNAs and subgenomic RNA4 (34). Moreover, studies with other animal and plant-infecting members of the alphavirus superfamily show that conserved NTPase/hel domains paralleling that of BMV 1a have an RNA triphosphatase activity contributing to capping of viral RNA products by removing 5′ γ-phosphates (37, 57). In addition to these roles in RNA synthesis, the 1a NTPase/hel domain has a role(s) in earlier steps of RNA replication complex assembly. In particular, mutations in three of seven 1a helicase motifs block in vivo RNA3 stabilization (2). However, the nature of these contributions is not clear. FIG. 1. (A) Schematic of BMV 1a protein. 1a contains an N-terminal capping domain (aa 1 to 515) with m7G-methyltransferase and m7GMP binding activities and a C-terminal NTPase/helicase-like domain (aa 562 to 961) containing seven conserved signature helicase ... To gain more insight into the functions of the 1a NTPase/hel domain and the mechanisms by which 1a induces RNA3 in vivo to become membrane associated and stabilized, we made and tested additional mutations in six of the seven helicase signature motifs. We show here that mutations in each of these signature helicase motifs blocked BMV RNA replication. Most replication-defective mutations allowed spherule formation and readily detectable RNA3 recruitment to membranes but blocked RNA3 from achieving the nuclease-resistant State induced by wild-type (wt) 1a. The data show that the 1a NTPase/hel domain plays crucial roles in recruiting BMV RNA templates into replication complexes.
