RNA Viruses and RNA Templates

Fundamental Concepts

• Concept 1: RNA virus genome conformation and polarity vary and determine replication strategies

  • Genomes can be unimolecular (single RNA strand) or segmented (like influenza).
  • RNA genomes can be single-stranded (ss) or double-stranded (ds), and can be circular.
  • Positive-sense genomes: can be directly translated into viral proteins; infectious even when deproteinized.
  • Negative-sense genomes: require immediate RdRp activity; cannot be translated directly. Their genomes are non-infectious if stripped of proteins.
  • Retroviruses are an exception to the general rule about RdRp requirements for replication.
  • Examples: influenza (segmented, negative-sense), picornaviruses (positive-sense, single segment), alphaviruses (positive-sense, sometimes with distinct replication steps).
  • Significance: genome conformation/polarity shapes how replication and transcription are organized and how RdRp is deployed.

• Concept 2: All RNA viruses must make mRNA and copies of their genome

  • Two essential outcomes for replication:
  • Generate messenger RNAs (mRNAs) that can be translated into viral proteins.
  • Make copies of the viral genome that will be packaged into progeny virions.
  • These requirements are universal across RNA virus families, regardless of genome conformation/polarity.

• Concept 3: An RNA-dependent RNA polymerase (RdRp) is essential

  • RdRp is required for RNA synthesis in all RNA viruses.
  • Polarity/conformation influences whether RdRp must be packaged in the virion:
  • Negative-sense and dsRNA viruses require RdRp at entry because their genomes cannot be directly copied or translated.
  • Positive-sense genomes are infectious on their own, but replication still requires RdRp expression to synthesize a negative-strand template for genome replication.
  • Retroviruses are a notable exception to the general rule about RdRp need.

• Concept 4: Similarities in genome conformation/polarity do not imply identical replication strategies

  • Even when two viruses share the same genome sense, they can differ in how they produce mRNA and replicate genomes.
  • Example: picornaviruses and alphaviruses are both positive-sense ssRNA viruses but differ in their mRNA and genome synthesis steps.

• Practical implications and connections

  • Understanding these fundamental concepts helps explain why different viruses have distinct replication complexes, genome packaging, and strategies for gene expression.
  • The concepts underpin the design of antiviral strategies that target RdRp, genome replication, and mRNA synthesis.

RNA Structure and Synthesis Strategies

• Overview: RNA viruses vary in genome conformation/polarity, which dictates how they replicate and synthesize mRNA and genomes. Some require nucleocapsid association to replicate due to genome polarity.

Genomic RNA and mRNA Synthesis Strategies

• (-) strand RNA Viruses

  • Single-stranded negative-sense genomes must first be converted to positive-sense RNA, which can serve as mRNA or as a template for synthesizing negative-strand genomes for progeny virions.
  • Strategies for mRNA and genome synthesis are shared with unimolecular or segmented genomes (e.g., influenza).

• (+) strand RNA Viruses

  • Positive-sense genomes do not immediately require RdRp activity because they can be translated directly.
  • To continue replication, expression of viral RdRp is required to synthesize a negative-strand template for producing additional copies of the positive-strand genome.
  • In alphaviruses, viral mRNAs can be synthesized separately from the negative strand; full-length complement to the original genome is produced differently from the negative strand.

• RNA Has Secondary Structure

  • RNA can fold into complex secondary structures, including stem-loops, hairpins, interior loops, bulges, and multibranched loops.
  • Pseudoknots: a loop base-pairs with sequences outside the stem-loop, adding complexity.
  • These structures can impact replication and transcription by influencing template accessibility and enzyme interactions.

• Ambisense and Double-Stranded RNA Genomes

  • Ambisense genomes contain both positive and negative sense segments; replication requires an intermediate antigenome RNA.
  • Ambisense viruses (e.g., bunyaviruses, arenaviruses) synthesize mRNA from genome and antigenome RNA due to dual sense regions.
  • Double-stranded RNA genomes (e.g., rotavirus, Reoviridae) begin replication by synthesizing a positive-strand RNA from the negative strand, which is then translated and used as a template for the complementary negative strand.

• Nucleocapsid Organization in (-) Strand Genomes

  • Negative-strand genomes are packaged with nucleoprotein (N) into nucleocapsids, often with RdRp and accessory proteins.
  • Nucleoprotein complexes keep RNA in a single-stranded form, prevent RNA secondary structure formation, and shield RNA from RNases.
  • Replication occurs in the context of the nucleocapsid; RNA synthesis is intimately tied to nucleoprotein interactions

• Summary of Section 2 concepts

  • Genome conformation/polarity dictate replication strategies and need for nucleocapsid association in some viruses.
  • RNA secondary structures and nucleocapsid interactions influence replication and transcription fidelity and efficiency.

Mechanisms of RNA Synthesis

• Historical context: The first evidence for an RdRp that can synthesize RNA from an RNA template came from mengovirus and poliovirus studies in the 1960s (RdRp activity observed even with actinomycin D, which blocks DNA-templated transcription).

  • RdRps exist across RNA virus families; replication depends on viral RdRps and sometimes host factors.

• Rules that govern RdRp activity

  • RNA synthesis begins and ends at defined sites on templates; RdRp often requires virus- and/or host-encoded accessory proteins.
  • Some RdRps require a primer with a free 3'-OH; others can initiate de novo without a primer.
  • Primers can be protein-linked or capped on the 5' end.
  • Elongation is typically template-dependent and proceeds 5' to 3' on the nascent RNA.
  • Elongation requires coordination of two divalent metal ions at the active site (often coordinated by conserved aspartic acids).
  • Across RdRps, there is a universal set of motifs (A–E) that underpin polymerase function and fidelity.

• Nucleic acid polymerases share common motifs

  • Four conserved motifs A–D are found in RdRps; motif C contains a glycine-aspartic acid-aspartic acid sequence and signals RdRp identity in many positive-sense RNA viruses.
  • Motifs A and B: nucleotide recognition/binding; motifs A and C: phosphate transfer; motif D: palm structure; motif E: nucleotide primer binding (unique to RdRps).
  • All RdRps from diverse viruses share a common ancestral origin based on these motifs.

• Poliovirus 3Dpol structure and active site

  • RdRp enzymes often adopt a hand-like architecture with fingers, thumb, and a central palm active site containing motifs A–D.
  • The active site is a closed tunnel through which the template and NTPs pass during elongation.
  • Motif F forms the entry point for incoming NTPs.
  • Structural studies show conserved geometry around the catalytic site across RdRps.

• Active-site residues and fidelity checkpoints

  • Specific residues in the active site contribute to fidelity checkpoints that limit misincorporation.
  • Example: In poliovirus 3Dpol, Aspartic acid at position 238 (Asp-238) participates in fidelity by hydrogen bonding with the 2'-OH of the ribose in the incoming NTP, biasing incorporation toward NTPs over dNTPs; a corresponding residue in DNA polymerases (tyrosine) biases toward dNTPs.

• Classified polymerases: open vs closed structures

  • RdRps generally adopt a closed “hand” structure centered on the active site tunnel.
  • Other polymerases (e.g., Klenow fragment of DNA pol I, HIV-1 RT) are more open and cleft-like, lacking a closed tunnel.

• Process of RNA synthesis: template selection to poly(A) tail synthesis

  • Template selection and preparation: identify viral RNA template among many cellular RNAs; followed by template unwinding by helicases and viral/cellular factors.
  • Initiation: some RdRps initiate de novo; others require a primer (protein-linked or capped). Initiation can occur at template 3' end or internally at specific nucleotides.
  • Elongation: NTPs are added in a 5' to 3' direction; polymerase rearrangements position the NTP into the catalytic site.
  • Poly(A) tail synthesis: many RNA viruses add poly(A) tails to mRNAs; poly(A) addition can be achieved by reiterative copying (slippage) or by template-driven processes that yield a tail.
  • In influenza, poly(A) tails are generated by a moving template model where the 5' end is anchored and the 3' end is threaded, promoting reiterative copying of a poly(U) and adding poly(A).

• Template specificity mechanisms

  • Initiating nucleotide affinity: RdRps recognize initiating nucleotides depending on the virus (e.g., reovirus RdRp prefers G at position 2; bovine viral diarrhea virus RdRp prefers C at position 1).
  • 3'/5' end sequences or structures: cap-snatching in influenza shows selective initiation via conserved sequences in segments; poliovirus uses a cis-acting replication element (cre) as a priming site; internal RNA sequences like cre help recruit RdRp for priming.
  • Exclusion of viral RNA sequences: some viruses ensure host RNA transcription is used (cap-snatching) to prime viral mRNA, while others avoid competitions by selective primer usage.
  • Protein-protein interactions: specific viral proteins bridge the RNA template to the polymerase (e.g., VSV L–P complex bridging to template RNA via N protein).

• Helicases and cellular proteins in RNA synthesis

  • Viral helicases unwind RNA structures to expose templates for synthesis.
  • Cellular proteins can participate in RNA synthesis (e.g., poliovirus requires poly(rC)-binding protein PCbp and poly(A)-binding protein PAbp1 to initiate negative-strand synthesis; cytoskeletal interactions can also modulate replication efficiency in some viruses).

• Initiation strategies: de novo vs primer-dependent

  • De novo initiation: RdRps start synthesis without a primer; often less accessible due to lack of primer stabilization.
  • Primer-dependent initiation: can use protein-primed primers (e.g., VPg in poliovirus) or capped RNA primers in some viruses (e.g., influenza and bunyaviruses).
  • Poliovirus example: VPg is covalently linked to the 5' end of RNA and acts as a primer after uridylylation by the RdRp on the cre template.

• Poliovirus: protein priming and VPg chemistry

  • VPg becomes uridylylated (VPg-pUpU) to serve as a primer for RNA synthesis.
  • A ribonucleoprotein complex involving cloverleaf structure, PCbp, and 3CDpro initiates negative-strand synthesis; VPg is recruited and extended with uridines.

• Cellular proteins in poliovirus RNA synthesis

  • PCbp and PAbp1 are essential for initiating negative-strand synthesis; they link cloverleaf structure to the poly(A) tail to promote circular RNA topology for replication.
  • The replication complex includes interactions with 3CDpro and other host factors.

• Cap-snatching and initiation in influenza and bunyaviruses

  • Influenza uses cap-snatching to prime viral mRNA synthesis, stealing 5' caps from cellular mRNAs and using them as primers.
  • This process occurs with a conserved 11-nucleotide sequence at the 5' end of each segment that activates cap-binding and endonuclease activities to generate primers.

• Elongation and conflicts with translation

  • Upon initiation, elongation proceeds with NTPs entering the catalytic site and base-pairing with the template.
  • For positive-sense RNAs, a potential conflict exists between translation by ribosomes and RNA synthesis by RdRp, since transcription proceeds 3'→5' on the template while translation proceeds 5'→3' on the mRNA.
  • Two deconfliction strategies: separation in time (temporarily suppress translation to allow RNA synthesis) or physical compartmentalization (e.g., replication in a capsid or nucleus), reducing ribosome-RdRp collisions.

• Poly(A) synthesis strategies

  • Most RNA viruses terminate mRNA with a 3' poly(A) tail; except arenaviruses and reoviruses, which have different tail features.
  • Reiterative copying at a U-rich region can generate poly(A) tails (e.g., VSV).
  • The Influenza moving-template model explains how the RdRp can stall and slip on a poly(U) tract, generating a poly(A) tail at the 3' end of the nascent mRNA.

• Summary of Section 3 concepts

  • RdRps follow common mechanistic rules but with virus-specific adaptations (primer requirements, initiation location, and accessory factors).
  • Structural motifs and active-site residues govern fidelity and nucleotide selection.
  • RNA synthesis is a multi-step process requiring template selection, initiation, elongation, and poly(A) tail formation, with host factors and viral proteins shaping each step.

Making mRNA and RNA Genomes

• Goal of replication: switch from producing mRNAs to genome replication for progeny virions

  • The switch can occur at initiation or termination of RNA synthesis and is achieved via multiple strategies across virus families.

• RNA polymerase specificity switching (positive-sense viruses like coronaviruses, caliciviruses, and alphaviruses)

  • Sindbis virus (an alphavirus) example:
  • Early in replication, genome yields two non-structural polyproteins: P123 and P1234 from the same message via termination/read-through at a stop codon in P123 ORF.
  • P1234 is cleaved into nsP1–nsP4; nsP4 cleavage alters polymerase specificity.
  • After cleavage, polymerase can complete negative-strand synthesis, then synthesize full-length positive strand, then negative strand again