Sandbox Reserved 1684
From Proteopedia
DiseasePolio or poliomyelitis is a disabling and life-threatening diseased caused by the poliovirus. This virus spreads from person to person and can infect a person’s spinal cord, resulting in paralysis. Most people that get infected with the poliovirus will not develop any visible symptoms. However, about one in four people with poliovirus infection will have flu-like symptoms that include: sore throat, fever, tiredness, nausea, headache, and stomach pain. These symptoms usually last from two to five days before disappearing. A smaller fraction of people infected with virus will develop more serious symptoms that affect the brain and spinal cord such as: paresthesia, meningitis, and paralysis. This last symptom is the most associated with polio because it can lead to permanent disability and death[1]. Between 2 and 10% of people who develop paralysis will die because the virus affects the diaphragm and other muscles that aid in respiration. Even young children who seem to completely recover from this virus can develop muscle pain or weakness and paralysis as adults, usually 15 to 40 years later. This is called post-polio syndrome. The U.S has been polio-free since 1979, poliovirus 2 was eradicated globally in 1999, and there has not been a case of poliovirus 3 since 2012. However, according to the WHO (World Health Organization) poliovirus 1 only affects two countries as of 2020, Pakistan and Afghanistan[2]. There is a vaccine for this virus called the Inactivated polio vaccine (IPV), for best protection children should get four doses of this vaccine. Viral RNA ClassificationThe poliovirus comes in three different serotypes: poliovirus 1 (PV1), poliovirus 2 (PV2), or poliovirus 3 (PV3). These viruses are non-enveloped, single-stranded positive-sense RNA. The poliovirus is a member of the picornavirus family which includes a significant number of pathogens for humans and livestock. This virus is very small and consists of an icosahedral protein coat. The 7500 nucleotide single-strand RNA genome of poliovirus contains one long open reading frame which is translated into a 247 kDa polyprotein. RNA-Dependent RNA Polymerase FunctionRNA-dependent RNA polymerases(RdRps)are one of the most versatile enzymes of RNA viruses that are vital for genome replication as well as for carrying out transcription. They have such a name due to their function where they use RNA template to synthesize mRNA which will later be translated into proteins and spread virus among the host. The core structural features of these polymerases are conserved, however, there is some divergence among their sequences[3]. The structure of the RNA-dependent RNA polymerases resembles a cupped right hand which also consists of fingers, palm and thumb subdomains. In most cases catalysis involves several conserved aspartate residues together with These RdRps are such a great target for antiviral drugs because they are in charge of viral genome replication as well as viral genome transcription, meaning these proteins allow viruses to grow in number and spread to other cells or parts of the body. Therefore, if there are drugs that target these enzymes, viruses would not be able to survive inside a host. Since it has a positive-sense, single stranded RNA genome, this can be translated right away by the ribosome and synthesize the many structural and non structural proteins that will form the Replication and Transcription Complex (RTC), and most importantly it will synthesize the RdRp that is encoded in the genome [4]. Structural FeaturesThe Poliovirus RNA-Dependent RNA polymerase is a 53kDa polymerase which together with other host proteins carries out viral RNA replication on the host cell cytoplasm. The poliovirus RdRp’s shape is common to that of other polymerases, with a palm subdomain which contains a core structure very similar to other polymerases, and different structures of the fingers and thumb from those of other polymerases. The palm subdomain contains four amino acid sequence of RNA-dependent RNA polymerases, referred to as A, B, C, and D. These fold into a structure that forms the core of the palm subdomain. This core structure consists of two α helices that pack beneath a four-stranded antiparallel β sheet. This same core structure is present in the palm subdomains of all four categories of polymerases. There is a fifth motif, motif E, unique to RNA-dependent polymerases, that pack between the palm and thumb subdomains[5]. Motif A of the poliovirus polymerase forms one of the four β strands (β1) of the core structure followed by a short helical turn (αE) at the C-terminal end of the motif. Near the end of the β strand of motif A just preceding the helix is the completely conserved aspartate that has been aligned in all previous sequence and structure comparisons; this residue is expected to coordinate catalytically essential metal ions. There is a highly conserved Asp238 residue in poliovirus polymerase, an aspartate at this position in RNA-dependent RNA polymerases, could favor NTPs over dNTPs, perhaps by interacting directly with the 2′hydroxyl group of an incoming NTP[6]. Motif B of poliovirus polymerase forms one of two α helices that pack beneath the four-stranded antiparallel β sheet of the polymerase core structure. However, the C-terminal portion of motif B, forms part of a long α helix. A portion of this helix is similarly positioned in all four categories of polymerases: it is in this region that all four motifs come together to form the ‘heart’ of the core structure of the polymerase palm subdomains. In motif B, residue Asn297 hydrogen bonds with the conserved Asp238 of motif A, helping to discriminate between NTPs and dNTPs[6]. Motif C of poliovirus polymerase forms a β-turn-β structure, which is part of the antiparallel β sheet of the polymerase core. The turn region of motif C contains two aspartates (Asp328 and Asp329) that are highly conserved in RNA-dependent polymerases. The two adjacent aspartates of motif C are quite close to the conserved aspartate of motif A, and these clustered aspartates are proposed to coordinate catalytically essential metals. Indeed, for poliovirus polymerase, mutating the conserved aspartate of motif A (Asp233) or the first conserved aspartate of motif C (Asp328), results in an inactive polymerase. Changing the second aspartate of motif C (Asp329) to asparagine results in a change in metal specificity. In the crystal structure of poliovirus polymerase, strong electron density is observed between the aspartate of motif A and the second aspartate of motif C. Motif D of poliovirus polymerase forms an α helix-turn-β strand structure. The α helix packs beneath the β sheet of the core structure.The β strand of motif D makes limited antiparallel β sheet interactions with the outside of motif A to complete the four-stranded antiparallel β sheet of the core structure. The turn region packs against the base of the fingers subdomain[5]. Motif E is positioned between the palm and thumb subdomains and is not integral to the conserved core structure. Motif E forms a short β-turn-β structure that interacts extensively with the face of the β sheet of the core structure. These interactions are distinctly hydrophobic and account for the conservation of several hydrophobic residues in motifs A, C, and D of RNA-dependent polymerases[5]. The of the poliovirus RdRp is composed of two polypeptide segments, a larger segment that precedes motif A and a smaller segment composed of residues between motifs A and B of the palm subdomain. This fingers subdomain is composed of two polypeptide segments, an N-terminal of the palm subdomain and the second between motifs A and B. The thumb subdomain is composed primarily ofthe C-terminal-most 80 amino acid residues. The thumb subdomain of this polymerase begins with a beta strand that interacts with the edge of the beta strands of motif E to from a short three-stranded antiparallel beta sheet. The remainder of the thumb is composed of a series of five alpha helices. The first three from a three-helix bundle, the fourth is positioned at the top of the thumb subdomain and the fifth is positioned along the front edge of the beta strand of the thumb subdomain. The is composed of mostly residues C-terminal of the palm subdomain and is largely alpha helical. The core structure comprises motifs A-D, and it consists of two alpha helices that pack beneath a four-stranded antiparallel beta sheet. The strands of the antiparallel beta sheet are composed of residues from motifs A, C, and part of D, while the alpha helices are composed of residues from motif B and the remainder of motif D. Motif E packs between the pal and thumb subdomains. Near the end of the beta strand of motif A just before the helix is a completely conserved aspartate residue that is expected to coordinate catalytically essential metal ions[5].
Catalytic site and Elongation of TranscriptionDifferent from other RdRps, the poliovirus RdRp does not involve a major nucleotide repositioning step where the nascent template-NTP base pair is moved from a pre-insertion site into the active by a swinging motion of the fingers. Rather active site closure is achieved via initial NTP base-pairing to a fully prepositioned templating nucleotide followed by recognition of the ribose hydroxyl that dives structural changes within the palm domain to enable metal binding and subsequent catalysis. Before transcription, the RdRp adopts a where extensive interactions between the thumb and fingers domain completely encircle the active site and create the at the back of the polymerase[6]. This is held in place by a clamping structure composed of an extended loop from the pinky finger inserting into the major groove and an α-helix from the thumb domain packing into the opposite minor groove. The complete structure of the RdRp reveals that the very N-terminus of this protein must be buried in a pocket on the back of the fingers domain. This buried terminus stabilizes a structure that directly positions Asp238 for binding with the 2' hydroxyl group of the incoming nucleotide in the active site. When this Asp 238 was mutated to an alanine it abolishes poliovirus polymerase activity and all viral viability. The significance of having an aspartate in this place is that is important for the the . The phosphate groups of the NTP are trailing out through the entry tunnel where they interact with conserved basic residues in a structure that appears to select for a complete triphosphate. These are ionic interactions. . There is a proline residue (119) which is 100% conserved and is in the middle of Gly 117 and Gly[6]. It was shown that this Pro119 residue is essential for elongation activity in the poliovirus RdRp. This residue is a key conformational change in the pinky finger that locks the enzyme-substrate complex into the stable elongation-competent mode. As the ribose is pulled down into the active site it collides with Asp 238which repositions that residue to interact with Lys61 and Ser288. The NTP 2' hydroxyl group now forms a hydrogen bond with Ser288 and with Asn 297. The net result of this process is a tight network of ribose hydroxyl interactions that positions the NTP for catalysis and also causes a rearrangement of the motif A backbone to form a 3-stranded β-sheet with motif C in the palm domain. more importantly, this realignment causes Asp233 to swing toward the RNA and it now coordinates the Mg2+ ions necessary to complete the active site and enable catalysis. There is a suggested 6 step process for the catalytic cycle of the poliovirus RdRp. The first state is composed of the initial polymerase structure in the absence of a bound NTP. In the second state the loading of the NTP binding site takes place where the nucleotide is bound in the open conformation active site but catalysis has not taken place. In the third state there is a closure of the active site to generate a pre-catalysis state, followed by a post-catalysis fourth state. After catalysis the active site is reset to the open conformation in a post-catalysis and pre-translocation fifth state. Finally a yet not characterized sixth state takes place where its conformation remains unknown and serves as a translocation intermediate[7]. Additionally, nucleotide incorporation can be also be summarized as a two step process composed of NTP binding followed by recognition of hydroxyl groups at both the 2' and 3' in the ribose ring to trigger closure of the active site and subsequent catalysis[7]. Due to the low fidelity of RdRps in positive strand RNA viruses there are high mutation rates that allows viral genomes to rapidly evolve and thrive in different host cell environments, ensuring the efficient propagation of the virus and improving its fitness for survival. Conserved sites/residuesOnce the template strand is bound and NTP entry tunnel is formed, we can find several conserved basic residues (Arg 163, Lys 167, Arg 174) that were previously described binding the RdRp to the template strand. There is a kink at the top of the NTP entry tunnel which is stabilized by hydrogen bonding interactions involving Pro40, Glu47, and Arg49. these residues are highly conserved across picornaviruses , suggesting that the kink is common to all these viral polymerases[6]. One side of the binding pocket for the N-terminus is almost entirely made up of glycines (Gly284, Gly285) which provide some torsional flexibility. These glycines are 100% conserved among picornaviral polymerase sequences. There is a between index and ring fingers that lines the NTP entry tunnel, Lys61 and Glu177 form this bridge and are highly conserved among picornaviruses. References
| ||||||||||||
