Chapter 6: Synthesis of RNA from RNA Templates

Synthesis of RNA from RNA Templates Introduction The genomes of RNA viruses may be uni molecular or segmented; single stranded of (+), (-), or ambisense polarity; double stranded; or circular These structurally diverse viral RNA genomes share a common requirement: they must be copied efficiently within the infected cell to provide both genomes for assembly into progeny virus particles and messenger RNAs for the synthesis of viral proteins The genomes of all RNA viruses except retroviruses and hepatitis delta virus encode an RNA-dependent RNA polymerase (RdRP) to catalyze the synthesis of new genomes and mRNAs Virus particles that contain (-) strand or double-stranded RNA genomes must contain the RdRp, because the incoming viral RNA can be neither translated nor copied by the cellular machinery In contrast, viral particles containing a (+) strand RNA genome lack a viral polymerase; the deproteinized RNAs of these viruses are infectious because they are translated in cells to produce, among other viral proteins, the viral RNA polymerase Nevertheless, each mechanism of viral RNA synthesis meets two essential requirements common to all infectious cycles: (i) during replication, the RNA genome must be copied from one end to the other with no loss of nucleotide sequence; and (ii) viral mRNAs that can be translated efficiently by the cellular protein synthetic machinery must be made The nature of the RNA template Secondary structures in viral RNA RNA molecules are not simple linear chains but can form secondary structures that are important for RNA synthesis, translation, and assembly Direct evidence for specific RNA structures comes from experiments in which RNAs are treated with enzymes or chemicals that attack single- Or double-stranded regions specifically Naked or nucleocapsid RNA The genomes of (-) strand viruses are organized into nucleocapsids in which protein molecules, including the RdRP and accessory proteins, are bound to the genomic to the genomic RNAs at regular intervals The RdRPs of (-)strand viruses copy viral RNAs only when they are present in the nucleocapsid, such as that formed by the N protein of vesicular stomatitis virus bound to genomic RNA. in contrast, the genomes of many (+) strand RNA viruses are not coated with proteins in the virus particle This difference is consistent with the fact that mRNAs are produced from the genomes of (-) strand RNA viruses upon cell entry, whereas the genomes of (+) strand RNA viruses are translated directly The viral nucleoproteins (NP) are cooperative, single-stranded-RNA-binding proteins, as are the single-stranded nucleic acid-binding proteins required during DNA-directed DNA synthesis Their function during replication is to keep the RNA single stranded and prevent base pairing between the template and product, so that additional rounds of RNA synthesis can occur The nucleoproteins of non-segmented (-) strand RNA viruses have a two-lobe architecture that forms a positively charged groove that birds and shields the genomic RNA The RdRP of segmented (-) strand RNA viruses can bind the NP-RNA template directly, whereas those of non-segmented (-) strand RNA viruses cannot: a phosphoprotein (P) is required to recruit the RdRP to the NP-RNA The genomes of many (+) strand RNA viruses encode helicase that serve functions similar to that of the nucleoproteins of (-) strand RNA viruses. The RNA synthesis machinery Identification of RNA-dependent RNA polymerases In these experiments, extracts were prepared from virus-infected cells and incubated with the four ribonucleoside triphosphates, one of which was radioactively labeled. The incorporation of nucleoside monophosphate into RNA was then measured Similar assays later demonstrated that the particles of (-) strand viruses and of double-stranded RNA viruses contain an RdRP that synthesizes mRNAs from the (-) strand RNA present in the particles Because polioviral genomic RNA contains a 3' poly(A) sequence, polymerase activity was measured with a poly (A)template and an oligo (u)primer Such assays for RdRP activity have been used to detect the presence of virus-specific enzymes in virus particles or in extracts of cells infected with a wide variety of RNA viruses RNA-directed RHA synthesis obeys a set of universal rules. RNA synthesis is catalyzed by virus-encoded polymerases and initiates and terminates at specific sites in the template, but viral accessory proteins and even host cell proteins may also be required A comparison of The structures and sequences of polynucleotide polymerases has led to the generality that all DNA and RNA polymerases catalyze synthesis by a mechanism that requires two metals RNA is usually synthesized by template-directed,stepwise incorporation of ribodeoxynucleoside monophosphates (NMPs) into the 3’-OH end of the growing RNA chain, which undergoes elongation in the 5' —> 3' direction Three-dimensional structures of RNA-dependent RNA polymerases Viral RdRPs show the highest degree of conservation of all viral proteins and are the only proteins common to all RNA viruses Seven conserved sequence motifs (A to G) that contain amino acid residues crucial for enzymatic function have been identified in all RdRPs The crystal structures of the four types of nucleic acid polymerases reveal that the enzymes resemble a right hand consisting of a palm, fingers, and a thumb, with the active site of the enzyme located in the palm The fingers and thumb subdomains of RdRPs show extensive interactions that encircle the active site and form a channel in which the template binds The closed structure creates a nucleoside triphosphate (NTP) entry tunnel on one face of the enzyme and a template-binding site on the other Residues within motif F, a conserved region unique to RdRPs form the NTP entry tunnel, while motif G is in a loop that outlines the template entry channel Three channels can be observed in the structures of RdRPs from (+) strand RNA and some double-stranded RNA viruses, which comprise the entry and exit paths for template and an NTP channel Mechanisms of RNA synthesis Initiation As polymerases synthesize nucleic acid in a 5' to 3' direction, the nucleotidyl transfer reaction is initiated at the 3' end of the template strand Most DNA polymerases are primer-dependent enzymes, while DNA - dependent RNA polymerases initiate RNA synthesis de novo In these cases, RNA polymerization is initiated by a protein-linked primer (picornaviruses) or an oligonucleotide cleaved from the 5' end of cellular pre-mRNA (influenza viruses) De novo initiation In this process, the first phosphodiester bond is made between the 3’-OH of the initiating NTP and the second NTP In these cases, initiation takes place at the exact 3’ end of the template, except during replication of the genomes of some (-) strand RNA viruses Initiation begins at an internal C, and after extension of a few nucleotides, the daughter strand is shifted in the 3' direction so that the 5’-terminal G residue is not base paired with the template strand Structural comparisons of viral RdRPs that catalyze de novo initiation reveal larger thumb subdomains with structural elements that fill most of the active-site cavity and provide a platform for initiating nucleotides After the product reaches a certain length, the polymerase undergoes a conformational change that moves the priming platform out of the way and allows the newly synthesized complementary RNA to exit as the enzyme moves along the template strand A protein platform also appears to participate in de novo priming by the reovirus RdRP, a cube-like structure with a catalytic site in the center that is accessible by four tunnels One tunnel allows template entry, one serves for the exit of newly synthesized double-stranded RNA, a third permits exit of mRNA, and a fourth is for substrate entry The loop supports the initiating NTP, then retracts into the palm and fits into the minor groove of the doublestranded RNA product Primer-dependent initiation Protein priming A protein-linked oligonucleotide serves as a primer for RNA synthesis by RdRPs of members of the picornaviridae and caliciviridae. Protein priming also occurs during DNA replication of adenoviruses, certain DNA-containing bacteriophages A terminal protein provides a hydroxyl group (in a tyrosine or serine residue) to which the first (priming) oligonucleotide can be linked by viral polymerases, via a phosphodiester bond Polioviral genomic RNA, as well as newly synthesized (+) and (-) strand RNAs, are covalently linked at their 5' ends to the 22-amino-acid protein VPg, initially suggesting that VPg might function as a primer for RNA synthesis Priming by capped RNA fragments Inhibition by alpha-amanitin is explained by a requirement for newly synthesized cellular transcripts made by this enzyme to provide primers for influenza viral mRNA synthesis Presumably, these cellular transcripts must be made continuously because they are exported rapidly from the nucleus once processed. Such transcripts are cleaved in the nucleus by an influenza virus-encoded, cap-dependent endonuclease that is part of the RdRP The resulting 10- to 13-nucleotide capped fragments serve as primers for the initiation of viral mRNA synthesis The structures of endonuclease domains from these viruses reveal the presence of a common nuclease fold Capping Three mechanisms can be distinguished: acquisition of preformed 5’ cap structures from cellular pre-mRNAs or mRNAs as described earlier, de novo synthesis by cellular enzymes, or synthesis by viral enzymes Elongation After an RdRP has associated stably with the nucleic acid template, the enzyme then adds nucleotides without dissociating from the template. Most RdRPs are highly processive; that is, they can add thousands of nucleotides before dissociating The vesicular stomatitis virus P protein enhances the processivity of the RdRP (L protein), possibly as a result of conformational changes that occur upon binding of P The increased processivity induced by p protein is enhanced in the presence of N, perhaps because the template must be kept unstructured so as not to impede the progress of L in general, nucleic acid synthesis begins with the formation of a complex of RdRP, template-primer, and initiating NTP. The NTP alpha-phosphate undergoes nucleophilic attack by the 3'-OH of the primer strand The nucleotidyl transfer reaction then takes place, pyrophosphate is released, and the template-primer moves by one base Based on these structures, it has been proposed that the catalytic cycle comprises six structural states: templateprimer binding, NTP binding, active-site closure, catalysis, opening of the active site, and translocation and pyrophosphate release In most cases, the RdRP first binds the RNA template-primer such that the templating base is above the active site. In this state, the RdRP conformation is the same as in the unbound form NTP selection is via interactions between the ribose 2' and 3' hydroxyl groups and three conserved residues on motifs B and A. These interactions cause a subtle restructuring of the Palm domain, closing the active site After catalysis, the active site is opened by movement of motif A, and the template moves one base to place the next base in the active site Closure of the active site by movements of the palm domain appears to be a feature of the RdRPs of all (+)RNA viruses but not (-)strand or double-stranded RNA viruses; their palm domains are already structured in the unbound form Functions of additional polymerase domains Additional N- and C-terminal domains that surround the RdRP cores are often encoded in the genomes of larger RNA viruses The core RdRPs of double-stranded RNA viruses are flanked by large N- and C-terminal domains. The former surrounds the fingers and thumb subdomains, closing the enzyme in a cage-like structure The c-terminal domains are shaped like bracelets and resemble the sliding clamps that contribute to the efficiency of DNA polymerases The minimal RdRP of vesicular stomatitis virus is surrounded by three globular domains with three enzymatic activities required for mRNA 5'-cap synthesis: 2’-O-methyltransferase, guanine-N7-methyltransferase, and polyribonucleotidyl transferase RNA polymerase oligomerization RNA polymerases of multiple (+) and (-) strand RNA viruses have been reported to form dimers and higher-order oligomers In many cases, deletions of amino acids that prevent oligomerization also inhibit or cause complete loss of enzyme activity The presence of a second interface, formed by N-terminal polypeptide segments, may lead to a network of polymerase fibers. These interacting N-terminal polypeptide segments may originate from different polymerase molecules and are required for enzyme activity Template specificity Viral RdRPs must select viral templates from among a vast excess of cellular mRNAs and then initiate correctly to ensure accurate RNA synthesis Template specificity may also be conferred by the recognition of RNA sequences or structures at the 5' and 3'ends of viral RNAs by viral proteins During mRNA synthesis by influenza virus polymerase sequences at the RNA termini ensure that the 5' ends of newly synthesized viral mRNAs are not cleaved and used as primers Unwinding the RNA template Base-paired regions In viral RNA must be disrupted to permit copying by RdRP. RNA helicases, which are encoded in the genomes of many RNA viruses, are thought to unwind the genomes of double-stranded RNA viruses, as well as secondary structures in template RNAs These molecules comprise three domains that mediate hydrolysis of NTPs and RNA binding. Between the domains is a cleft that is large enough to accommodate single-stranded but not double-stranded RNA Unwinding of double-stranded RNA probably occurs as one strand of RNA passes through the cleft and the other is excluded This RdRP has a plow-like protuberance around the entrance to the template channel that is thought to separate the two strands, allowing only one to enter the channel Role of Cellular Proteins Host cell components required for viral RNA synthesis were initially called "host factors", because nothing was known about their chemical composition This viral RdRP [bacteriophage Q-beta enzyme] is a multisubunit enzyme, consisting of a 65 kDa virus-encoded protein and four host proteins: ribosomal protein S1, translation elongation proteins (EF-Tu and EF-Ts), and an RNA binding protein The 65-kDa viral protein has sequence and structural similarity to known RdRPs, but exhibits no RNA polymerase activity in the absence of the host proteins When purified polioviral RNA is incubated with a cytoplasmic extract prepared from uninfected permissive cells, the genomic RNA is translated and the viral RNA polymerase is made One of these host cell proteins required for poliovirus RNA synthesis is poly(rC)-binding protein, which binds to a cloverleaf structure that forms in the first 108 nucleotides of the viral (+) strand RNA Interaction of poly(rC)-binding protein with the cloverleaf facilitates the binding of polioviral protein 3CD to the opposite side of the same cloverleaf Paradigms for Viral RNA synthesis Exact replicas of the RNA genome must be made for assembly of infectious viral particles. However, the mRNAs Of most RNA viruses are not complete copies of the viral genome (+) strand RNA Newly synthesized (+) strand RNA molecules can serve as templates for further genomic replication, as mRNAs for the synthesis of viral proteins, or as genomic RNAs to be packaged into progeny virions Such simplicity comes at a price, however, because synthesis of individual viral proteins cannot be regulated. However, polioviral gene expression can be controlled by the rate and extent of polyprotein processing The mechanisms of mRNA synthesis of other (+) strand RNA viruses allow structural and nonstructural proteins (generally needed in greater and lesser quantities, respectively) to be made separately The nonstructural proteins are synthesized from full-length (+) strand (genomic) RNA, while structural proteins are translated from subgenomic mRNAs The mRNAs synthesized during infection by most RNA viruses contain a 3' poly(A) sequence, as do the vast majority of cellular mRNAs. The poly(A) sequence is encoded in the genome of (+) strand viruses Synthesis of nested subgenomic mRNAs An unusual pattern of mRNA synthesis occurs in cells infected with members of the families Coronaviridae and Arteriviridae, in which subgenomic mRNAs that form a 3'-coterminal nested set with the viral genome are synthesized The subgenomic mRNAs of these viruses comprise a leader and a body that are synthesized from noncontiguous sequences at the 5' and 3' ends, respectively, of the viral (+) strand genome Subgenome-length (-) strands are produced when the template loops out as the polymerase completes synthesis of the leader RNA (-) strand RNA The genes of RNA viruses with a nonsegmented (-) strand RNA genome are expressed by the production of subgenomic mRNAs In infected cells Individual mRNAs are produced by a series of initiation and termination reactions as the RdRP moves down the viral genome This start-stop mechanism accounts for the observation that 3'-proximal genes must be copied before downstream genes Vesicular stomatitis virus mRNA synthesis illustrates a second mechanism for poly(A) addition: reiterative copying of, or "stuttering" at, a short u sequence in the (-) strand template to produce a full length (+) strand RNA, the stop-start reactions at intergenic regions most be suppressed, a process that depends on the synthesis of the N and P proteins The number of N-P protein assembles in infected cells therefore regulates the relative efficiencies of mRNA synthesis and genome RNA replication The (-) strand RNA genome of paramyxoviruSES is copied efficiently only when its length in nucleotides is a multiple of 6. This requirement, called the rule of six, is probably a consequence of the association of each N monomer with six nucleotides Assembly of the nucleocapsid begins with the first nucleotide at the 5' end of the RNA and continues until the 3' end is reached. If the genome length is not a multiple of 6, then the 3' end of the genome will not be precisely aligned with the last N monomer Intracellular concentrations of NP protein are therefore an important determinant of whether mRNAs or full-length (+) strands are synthesized Ambisense RNA Although arenaviruses are considered (-) strand RNA viruses, their genomic RNA is in fact ambisense: mRNAs are produced both from (-) strand genomic RNA and from complementary full-length (+) strands Later in infection, the block imposed by the stem-loop structure is overcome, permitting the synthesis of full-length S and L (+) strand RNAs It now seems more likely that two different configurations of the RNA polymerases are made in infected cells: one for synthesis of mRNA and a second for synthesis of full-length copies of the genome Double-stranded RNA A distinctive feature of the infectious cycle of double-stranded RNA viruses is the production of mRNAs and genomic RNAs from distinct templates In different viral particles Viral mRNAs are synthesized by the polymerase inside the subviral parental core and then extruded into the cytoplasm through this pore. Attachment of the polymerase molecules to the pores ensures that mRNAs are actively threaded out of the particle, without depending upon diffusion, which would be very inefficient Viral (+) strand RNAs that will serve as templates for (-) strand RNA synthesis are first packaged into newly assembled subviral particles During conservative replication, the double-stranded RNA that exits the polymerase must be melted, so that the newly synthesized (+) strand is released and the template (-) strand reanneals with the original (+) strand Attachment of the 5' cap to the polymerase facilitates insertion of the 3' end of the (-) strand into the template channel. This arrangement allows very efficient reinitiation of RNA synthesis in the crowded core of the particle Unique mechanisms of mRNA and genome synthesis of hepatitis delta virus The genome does not encode an RdRP: viral RNAs are produced by host cell RNA polymerase II, and the hepatitis delta virus RNAs are RNA catalysts, or ribozymes All hepatitis delta virus RNAs are synthesized in the nucleus by DNA-dependent RNA polymerase II. The ability of this cellular enzyme to copy an RNA template provides a missing link in molecular evolution During the course of evolution, enzymes with this property acquired the ability to copy DNA templates, facilitating the transition from RNA to DNA genomes The switch from mRNA synthesis to the production of full-length (+) strand RNA of hepatitis delta virus is controlled by suppression of a poly(A) signal Full-length (-) and (+) strand RNAs are copied by a rolling-circle mechanism, and ribozyme self-cleavage releases linear monomers. Subsequent ligation of the two termini by the same ribozyme produces a monomeric circular RNA The RNA downstream of the poly(A)site is not degraded, in contrast to that of other mRNA precursors made by RNA polymerase II, but is elongated until a complete full-length (+) strand is made The poly(A) addition site in this full-length (+) strand RNA is not used, perhaps because the delta antigen bound to the rod-like RNA blocks access of cellular enzymes to the poly(A) signal Do ribosomes and RNA polymerases collide? At a certain point in infection, the RNA polymerase copies the RNA in a 3’ —> 5’ direction while ribosomes traverse it in an opposite direction, raising the question of whether the viral polymerase avoids collisions with ribosomes These results suggest that ribosomes must be cleared from viral RNA before it can serve as a template for (-) strand RNA synthesis; in other words, replication and translation cannot occur simultaneously Origins of diversity in RNA virus genomes Misincorporation of nucleotides DNA directed DNA polymerases have proofreading capabilities in the form of exonuclease active sites that can correct such mistakes. Most RdRPs do not possess this capability Many of the polymerization errors cause lethal amino acid changes, but other mutations that are not lethal are retained in the genomes of infectious virus particles This phenomenon has led to the realization that RNA virus populations are quasispecies, or mixtures of many different genome sequences Nucleotide binding occurs in two steps: first, the NTP is bound in such a way that the ribose cannot interact properly with the Asp of motif A and the Asn of motif B If the NTP is correctly base paired with the template, then there is a conformational change in the enzyme, which reorients the triphosphate and allows phosphoryl transfer to occur The closed active-site polymerase structures reveal a network of hydrogen bonds from the 2' hydroxyl of the basepaired NTP to motif B in the fingers domain and the top of motif A Although this amino acid [G64S] is remote from the active site, it participates in hydrogen bonding to motif A, which is important in holding the NTP In an appropriate conformation for catalysis Subtle changes in the enzyme caused by this substitution make it more dependent on correct NTP base pairing in the active site, thereby increasing replication fidelity Segment reassortment and RNA recombination Reassortment is the exchange of entire RNA molecules between genetically related viruses with segmented genomes In contrast to reassortment, recombination is the exchange of nucleotide sequences among different genome RNA molecules Recombination can occur by two different mechanisms: non replicative, the non-homologous end joining of two different RNA molecules; or replicative, the switching of templates Non replicative recombination is highly inefficient and thought to influence virus evolution minimally. Replicative recombination mainly occurs between nucleotide sequences of two parental genome RNA strands that have a high percentage of nucleotide identity This mechanism of RNA recombination is coupled with the process of genome RNA replication: it occurs by template switching during (-) strand synthesis The RNA polymerase first copies the 3’ end of one parental (+) strand, then switches templates and continues synthesis at the corresponding position on a second parental (+) strand Because of their smaller size, subgenomic RNAs replicate more rapidly than full-length RNA, and ultimately compete for the components of the RNA synthesis machinery