25.3: RNA-Dependent Synthesis of RNA and DNA

RNA Viruses

Infections with RNA viruses place a constant burden on our healthcare systems and economy. Over the past century, this has been particularly true for infections with the Human immunodeficiency virus 1 (HIV-1), Influenza A virus (IAV), Rotavirus (RotaV), West Nile virus (WNV), Dengue virus (DV), Measles virus (MV), and the Porcine reproductive and respiratory syndrome virus (PRRSV). However, emergent RNA viruses can also have considerable consequences, including Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) in 2003, Middle East Respiratory Syndrome coronavirus (MERS-CoV), and, most recently, Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2) in December 2019.

Eukaryotes and bacteria can be infected with a wide variety of RNA viruses. On average, these pathogens exhibit little sequence similarity and employ distinct replication and transcription strategies. RNA virus genomes can consist of double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA), as shown in Figure \(\PageIndex{1}\) (Panel a). In turn, ssRNA viruses can be classified as positive-sense (+RNA) or negative-sense (−RNA), depending on whether their genetic material can be translated. As summarized for the four model RNA pathogens in Panel b, all RNA viruses use dedicated replication and transcription strategies to amplify their genetic material. The common denominator of these strategies is a conserved RNA-dependent polymerase domain. Figure \(\PageIndex{1}\) describes variations of the replication process in the different types of RNA viruses.

Panel A shows a simplified taxonomy of the genome architecture of representative RNA viruses.

Panel B (+RNA virus) shows infection with a +RNA virus, as exemplified here by a CoV-like virion, which releases a single-stranded RNA genome into the cytoplasm (1). (2) Translation of the 5′-terminal open-reading frame of the genome produces the viral replicase. (3) This multi-enzyme complex includes RNA-dependent RNA polymerase (RdRp) activity (orange) and associates with intracellular membranes before RNA synthesis commences. Newly synthesized RNAs are subsequently used to produce new +RNAs (4), which are typically capped (yellow) and polyadenylated (polyA). (Retrovirus) HIV-1 genomes are packaged as single-stranded RNA (ssRNA) in virions. When the ssRNA is released (1), a cDNA copy is synthesized by the reverse transcriptase enzyme (RT) (2). The RNA is then degraded by the RT's intrinsic RNase H activity (3), and the single-stranded cDNA is converted to dsDNA (4). The dsDNA is imported into the nucleus (5) for integration into the host’s genetic material. (−RNA virus) (1) As illustrated here with an IAV-like particle, infection with an RNA virus releases a viral RNA genome that is associated with a viral polymerase (orange) and nucleoprotein (green). (2) In the case of non-segmented RNA viruses, these complexes support transcription to produce viral mRNAs or cRNAs. (3) Viral mRNAs are next translated, and new viral proteins complex with cRNAs to synthesize new vRNAs. (5) The vRNA-containing complexes of some segmented-RNA viruses are imported into the nucleus of the host cell, where (6) the RdRp produces mRNAs or cRNAs. (7) mRNAs are transported to the cytoplasm, while new viral proteins bind cRNAs to form cRNPs for RNA synthesis. (dsRNA virus) Fully duplexed RNA genomes lack a cap and poly (A) elements. (1) The RdRp (orange), therefore, transcribes the viral genome inside the capsid of the virion (blue and red), so viral mRNAs can be (2) released into the cytoplasm as illustrated here with a rotavirus-like virion. In the cytoplasm, newly synthesized viral RdRps translate (3) or replicate the mRNA.

Viral RNA-dependent RNA Polymerases (RdRPs) were discovered in the early 1960s through studies on mengovirus and polioviruses, when researchers observed that these viruses were not sensitive to actinomycin D (a drug that inhibits cellular DNA-directed RNA synthesis). This lack of sensitivity suggested a virus-specific enzyme that can copy RNA from an RNA template but not from a DNA template. RdRps exhibit some structural similarity to telomerase enzymes, suggesting a potential ancestral relationship between telomerase and RdRps.

The most famous example of RdRp is the poliovirus. The viral genome is composed of RNA and enters the cell via receptor-mediated endocytosis. There, the RNA can act as a template for immediate synthesis of complementary RNA. The complementary strand can then serve as a template for producing new viral genomes, which are packaged and released from the cell, ready to infect more host cells. This replication method eliminates the need for a DNA stage, making replication quick and straightforward. The disadvantage is that there is no 'backup' DNA copy (Fig. 26.3.1b; + RNA virus).

Reverse Transcriptase (RT) enzymes, common to retroviruses, convert the viral +RNA strand into a cDNA copy (Fig. 26.3.1b; retroviruses). The RT enzyme then degrades the RNA, and the single-stranded cDNA is converted into double-stranded DNA (dsDNA). The dsDNA is then integrated into the host genome, where it can remain dormant. Upon reactivation, the virus synthesizes +RNA, along with viral proteins used to assemble infectious viral particles.

Structure and Mechanism of RNA Viral Polymerases

The RNA-dependent polymerase domain is a member of the superfamily of template-dependent nucleic acid polymerases and typically consists of fewer than 400 amino acids. Its sequence is highly variable, with some regions showing less than ~10% conservation. Strong amino acid conservation is evident; however, in regions directly involved in nucleotide selection or catalysis, such as the strictly conserved glycine and aspartates at the domain center, this conservation is particularly notable. The prototypic RNA virus RNA polymerase domain contains seven such regions or motifs, arranged from amino to carboxy terminus as G, F1–3, A, B, C, D, and E. Each of the seven motifs in the RNA polymerase domain adopts a specific and conserved fold, as shown in Figure \(\PageIndex{2}\).

Panel A shows the structure of the FMDV RdRp. The motifs A, B, C, D, E, F, and G are color-coded yellow, gold, orange, red, light green, aquamarine, and blue. Overall, the polymerase structure resembles a cupped right hand. Herein, motifs A–E lie on the palm, while motifs F and G are part of the fingers. In the side view of the enzyme, the template and NTP channel locations are indicated.

Panel B shows conserved structural elements of the FMDV RdRp. Homomorphic A–G were mapped and color-coded yellow, gold, orange, red, light green, aquamarine, and blue, respectively. Images A and B are based on PDB accession 2E9R.

Figure \(\PageIndex{3}\) shows an interactive iCn3D model of the Foot-and-mouth disease virus RNA-polymerase in complex with a template-primer RNA, PPi, and UTP (2E9Z)

Active site region side chains are shown as sticks and labeled. The template strand is shown in yellow, and the primer is in green. Note the conserved amino acids Tyr-336, the catalytic Asp-338, and Lys-387 in motifs C and E, respectively. The primer 3'OH forms an H-bond with active site Asp 338 (in motif C). In some structures, Asp 338 binds a metal ion that interacts with the PPP of the incoming NTP. Two metal ions are involved in catalysis. Tyr 336 interacts with the primer nucleotide, while Lys 387 and Arg 388 interact with the primer backbone. These three residues are highly conserved. The acceptor base of the template strand is adjacent to the NTP-binding site. Viral RNA replication has no proofreading activity.

RNA-dependent RNA polymerases generally have a groove on top of the enzyme where RNA enters. It exists in the front. Nucleotides enter at a rear channel. This is illustrated in Figure \(\PageIndex{4}\) (Panel b). Following the convention for cellular DNA-dependent DNA polymerases (DdDp), the seven motifs and homomorphs are grouped into three subdomains. These subdomains are called fingers, palm, and thumb based on the polymerase domain’s likeness to a cupped right hand, as shown in Figure \(\PageIndex{4}\) (Panel A). The finger subdomain loops that interconnect the fingers with the thumb in the RNA-dependent RNA polymerases (RdRps) of +RNA and dsRNA viruses create an overall “closed-hand” conformation that is unique to RNA-dependent RNA polymerases (RdRps) and generally not seen in crystal structures of DNA-dependent DNA polymerases (DdDps) or reverse transcriptases (RTs).

The three subdomains of RNA-dependent polymerases work together to facilitate RNA and nucleotide (NTP) binding. The thumb subdomain contains residues involved in RNA binding, which generally pack into the minor groove of the template RNA. In some polymerases, the thumb also contains sequences that protrude into the template channel to help stabilize the initiating nucleotide triphosphates (NTPs) on the single-stranded RNA (ssRNA) template. Crucially, these protrusions can also undergo relatively large conformational rearrangements to facilitate template translocation after the first condensation reaction. The other residues of the thumb subdomain contribute to the formation of the NTP tunnel, which sits at an ~110° angle with the template channel as shown in Figure \(\PageIndex{4}\), Panel A. The cavity is lined with positively charged amino acids, although charge interactions are likely insufficient to guide NTPs into the interior. Indeed, molecular dynamics (MD) simulations have demonstrated that the residues of the NTP channel can also explore a relatively large volume of space, which may enable the RdRp to actively “pump” NTPs down the channel.

Panel (A) shows a ribbon representation of a typical picornaviral RdRP (model from the cardiovirus EMCV 3D^pol, PDB id. 4NZ0). The seven conserved motifs are indicated in different colors: motif A (red), motif B (green), motif C (yellow), motif D (sand), motif E (cyan), motif F (blue), and motif G (pink).

Panel (B) shows a lateral view of a surface representation of the enzyme (grey) that has been cut to expose the three channels that serve as entry and exit sites for the different substrates and reaction products. The structural elements that support motifs A–G are also shown as ribbons. This panel also shows the organization of the palm subdomain, with motif A displayed in two alternative conformations: the standard conformation (PDB ID 4NZ0), found in the apo form of most crystallized 3Dpol proteins, and the altered conformation found in the tetragonal crystal form of the EMCV enzyme (PDB ID 4NYZ). The alterations affect mainly Asp240, the amino acid in charge of incoming ribonucleotide triphosphate (rNTP) selection, and the neighboring Phe239 that moves ~10 Å away from its position in the enzyme catalytic cavity, directed towards the entrance of the nucleotide channel, approaching motif F;

Panel (C) shows a close-up of the structural superimposition of the two alternative conformations of the EMCV motif A;

Panel (D) shows the PV replication-elongation complexes. Sequential structures illustrating the movement of the different palm residues from a binary PV 3D^pol-RNA open complex (left) to an open 3D^pol-RNA-rNTP ternary complex (middle) where the incoming rNTP is positioned in the active site for catalysis and, a closed ternary complex (right) after nucleotide incorporation and pyrophosphate (PPi) release. The residues D_A (involved in rNTP selection through an interaction with the 2′ hydroxyl group), D_C (the catalytic aspartate of motif C), K_D (the general acid residue of motif D that can coordinate the export of the PPi group), and N_B (a conserved Asn of motif B, interacting with D_A) have been highlighted as sticks. The different structures correspond to the 3D^pol-RNA (PDB id. 3OL6), 3D^pol-RNA-CTP open complex (PDB id. 3OLB) and 3D^pol-RNA-CTP closed complex (PDB id. 3OL7) structures of PV elongation complexes, respectively

The finger subdomain residues mainly pack into the major groove of the RNA template. Furthermore, upon entry of the template, they can unstack the single strand at position +3, as shown in Figure \(\PageIndex{5}\) (Panel A). The non-conserved structural elements of the finger subdomain also contribute to RNA binding. In particular, the fingertips of dsRNA and some +RNA virus RdRps allow the polymerase to “hold” the template without extensive conformational changes. The variations and extensions in the fingers subdomain have also been shown to play roles in protein–protein interactions, phosphorylation, oligomerization, and nuclear import. In contrast, the HIV-1 RT lacks such extensions and adopts a more “open-hand” or U-shaped binding cleft. As a consequence, the RT structure must rearrange its subdomains to coordinate the binding of the template, nascent strand, and incoming dNTPs.

Panel a shows the structure of the PV active site as it moves from a native state or elongation complex (i) to an open complex (ii), and a closed complex (iii). The closed complex depicted here shows the active site after catalysis. Highlighted are the metal-binding aspartates of motifs A and C, and the lysine of motif D that acts as a general acid. Color coding by motif as in Fig. 26.3.2. Image based on PDB accessions 3Ol6, 3OLB, and 3OL7.

Panel b shows a schematic presentation of the RdRp active site. The aspartates (Asp) of motif A (yellow) and C (orange) bind divalent metal ions (marked Mg and shaded grey), which are used to coordinate the formation of a new phosphodiester bond at the 3′-OH (red in panel ii) of the nascent strand (yellow). The general acid (red Lys/His in panel ii) is positioned near the β-phosphate of the incoming NTP to protonate the PP_i leaving group.

Panel c shows a simplified schematic of the kinetic steps of RNA polymerases. Asterisk indicates a closed complex.

The NTP and template entry channels meet at the palm subdomain - Figure \(\PageIndex{5}\), Panel A. This structure comprises a central, partially formed three-stranded β-sheet, which is also present in RNA-recognition motifs (RRMs). The relative movement of these strands within the RRM is crucial for catalysis and depends on NTP binding. Only when the correct NTP binds can motif A and motif C align and complete the RRM fold. This catalytically competent conformation of the active site is often referred to as the closed complex (not to be confused with the “closed-hand”, which refers to the overall structure of the RdRp) - Figure \(\PageIndex{5}\), Panel d.

The polymerase reaction creates new phosphodiester bonds between nucleotide triphosphates (NTPs) using RNA as a template. The NTP substrates involved in this reaction are coordinated by two metal ions, which are bound by the conserved aspartates of motifs A and C - Figure \(\PageIndex{5}\) - panel a, i. They also position the NTP’s triphosphate optimally for attack by the sugar moiety of the nascent strand once its 3′ carbon has lost a proton- Figure \(\PageIndex{5}\), panel b. The N-terminal aspartate of motif C thus uses a metal ion to fix the α-phosphate of the incoming NTP and reduce the pK_a of the nascent RNA’s 3′-OH to facilitate the attack - Figure \(\PageIndex{5}\), panel b, ii. The amino-terminal carboxylate of motif A, on the other hand, stabilizes the β- and γ-phosphates with its divalent metal as well as the pentavalent (phosphorane) intermediate that forms during catalysis - Figure \(\PageIndex{5}\), panel b, ii. Structural and biochemical analyses have shown that the formation of this transient structure is dependent on the attack of the NTP’s α-phosphate by the 3′-OH, which is often the rate-limiting catalytic step in NTP condensation - Figure \(\PageIndex{5}\), panel b.

Motif D’s lysine or histidine assists the N-terminal aspartate of motif A in coordinating the β-phosphate of the incoming NTP, analogous to the trigger loop in DdDps. However, when the positively charged side chain of motif D approaches the β-phosphate, it can also protonate the PPi leaving group - Figure \(\PageIndex{5}\), panel b, ii. This second protonation step in the active site is not essential for the polymerase reaction, since catalysis can still occur when motif D’s lysine is mutated to a residue with a different pK_a. The polymerase reaction rate will nevertheless be 50- to 1,000-fold higher when a lysine or histidine is present. Recent data even suggest that motif D can coordinate the export of the PPi group from the active site once catalysis has occurred, thereby triggering RNA translocation.

Overall, the RNA replication process can be summarized with this four-step mechanism:

1. Nucleoside triphosphate (NTP) binding - initially, the RdRp presents with a vacant active site in which an NTP binds, complementary to the corresponding nucleotide on the template strand. Correct NTP binding induces a conformational change in the RdRp.
2. Active site closure - the conformational change, initiated by the correct NTP binding, restricts active site access and produces a catalytically competent state.
3. Phosphodiester bond formation - two Mg²⁺ ions in the catalytically active state arrange themselves around the newly synthesized RNA chain so the substrate NTP can undergo phosphoryl transfer and form a phosphodiester bond with the chain. With these Mg²⁺ ions, the active site is no longer catalytically stable, and the RdRp complex shifts to an open conformation.
4. Translocation - once the active site opens, the RNA template strand can move one position through the RdRp complex and continue chain elongation by binding a new NTP, unless the template specifies otherwise.

Figure \(\PageIndex{6}\) shows a more detailed representation of the elongation catalytic cycle.

Figure \(\PageIndex{6}\): Elongation catalytic cycle of RNA-dependent RNA polymerases

RNA synthesis can occur via a primer-independent (de novo) or primer-dependent mechanism that uses a viral protein genome-linked (VPg) primer. De novo initiation consists of adding a nucleoside triphosphate (NTP) to the 3'-OH of the first initiating NTP. During the subsequent elongation phase, this nucleotidyl transfer reaction repeats with subsequent NTPs to generate the complementary RNA product. The termination of the nascent RNA chain produced by RdRp is not completely known. However, RdRp termination has been shown to be sequence-independent.

One feature of RNA-dependent RNA polymerase replication is the immense error rate during transcription. RdRps and RTs are known to lack fidelity by about 10⁴ nucleotides, which is thought to result from insufficient proofreading. This high rate of variation is favored in viral genomes because it enables the pathogen to overcome host defenses, thereby facilitating evolutionary growth.

Let's look at another RdRp from the poliovirus. The virus has a single-stranded, positive-sense RNA genome, which makes it infectious on its own but much more infectious when replicated. It is translated into a polyprotein that is cleaved into 12 distinct proteins. One protein, 3Dpol, is an RNA-dependent RNA polymerase that transcribes the infecting +RNA strand into a -RNA strand, which then serves as a template for synthesizing additional +RNA strands.

Figure \(\PageIndex{7}\) shows an interactive iCn3D model of the Poliovirus polymerase elongation complex with 2',3'-dideoxy-CTP (3OLB). The model shows the palm, finger, and thumb domains.

Color coding is as follows:

• red: finger domain
• pink: pinky finger part of the finger domain
• blue: thumb domain
• light blue: primer grip at the beginning of the thumb domain
• cyan: RNA template
• green: RNA product
• gray: palm domain

Structural studies suggest that the pinky finger is involved in the initiation, while nucleotide binding and catalysis use the palm domain. The thumb domain appears to affect translocation. In most nucleases, the finger binds and positions NTPs in the active site. After catalysis, the reverse motion opens the active site, allowing translocation by ratcheting the template by one base pair, driven by PP_i release. These conformational changes are not as easy to achieve in +RNA-stranded viral RdRps.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

RNA viruses represent a major global health burden encompassing pathogens as diverse as HIV-1, influenza A, poliovirus, SARS-CoV-2, rotavirus, dengue, and measles. Despite their enormous sequence diversity, all RNA viruses share a common mechanistic requirement: an RNA-dependent polymerase to replicate and transcribe their RNA genomes, a function not provided by host cell machinery. RNA virus genomes are organized as double-stranded RNA (dsRNA), positive-sense single-stranded RNA (+ssRNA), negative-sense single-stranded RNA (−ssRNA), or retroviral +ssRNA, each employing a distinct replication strategy.

Positive-sense ssRNA viruses (exemplified by poliovirus and coronaviruses) deliver an immediately translatable genome that directly encodes the viral replicase; the RdRp then synthesizes a complementary RNA strand as template for the production of new +RNA genomes. Negative-sense ssRNA viruses (exemplified by influenza A) must first use their genome-associated RdRp to transcribe mRNAs before translation can begin; in segmented viruses such as influenza, the ribonucleoprotein complexes are imported into the nucleus for transcription. dsRNA viruses (such as rotavirus) carry out transcription inside the intact capsid, releasing capped +RNA mRNAs into the cytoplasm. Retroviruses (HIV-1) represent the most indirect strategy: the +RNA genome is reverse-transcribed into a −strand cDNA, the RNA template is degraded by the intrinsic RNase H activity of reverse transcriptase, the second cDNA strand is synthesized to produce dsDNA, and this dsDNA is nuclear-imported and integrated into the host genome by integrase, where it can remain latent before being activated to produce new viral proteins and genomes.

The structural heart of all RNA virus replication is the RdRp domain, comprising fewer than ~400 amino acids organized around seven conserved sequence motifs (G, F1-3, A, B, C, D, E). These motifs adopt conserved folds organized into three functional subdomains that together resemble a cupped right hand: the fingers (motifs F, G) that pack into the major groove of the RNA template and unstack the single strand; the thumb (motif E and associated regions) that binds the minor groove, stabilizes initiating NTPs, and helps form the NTP entry channel; and the palm (motifs A-D) that contains the catalytic center. A defining structural feature of RdRps from +RNA and dsRNA viruses is their "closed-hand" conformation, in which fingertip loops bridge the fingers and thumb to create a fully enclosed template channel—unlike the open U-shaped cleft of HIV-1 reverse transcriptase, which requires larger conformational rearrangements during catalysis.

Catalysis by RdRp proceeds through a conserved two-metal-ion mechanism. Two Mg²⁺ ions are coordinated by conserved Asp residues in motifs A and C: the motif C Asp positions the incoming NTP's α-phosphate and reduces the pKa of the nascent RNA 3′-OH to facilitate nucleophilic attack; the motif A Asp stabilizes the β- and γ-phosphates and the pentavalent transition state. A conserved Lys or His in motif D acts as a general acid to protonate the pyrophosphate leaving group, accelerating catalysis 50- to 1000-fold and subsequently facilitating PPi export to trigger translocation. The four-step elongation cycle—NTP binding, active site closure (only achieved with the correct NTP through motif A-C alignment to complete the palm β-sheet), phosphodiester bond formation, and template translocation—operates without proofreading. This lack of fidelity (~1 error per 10⁴ nucleotides) generates the genetic variation that enables RNA viruses to rapidly evolve resistance to antiviral drugs and evade host immune responses, while simultaneously providing the mechanistic rationale for nucleoside analog chain terminators (such as 2′,3′-dideoxy analogs lacking the 3′-OH required for chain extension) as antiviral therapeutics targeting these polymerases.

References

1. te Velthuis, A.J.W. (2014) Common and unique features of viral RNA-dependent polymerases. Cell. & Mol Life Sci 71:4403-4420. Available at: https://link.springer.com/article/10.1007/s00018-014-1695-z#Sec1

2. Wikipedia contributors. (2021, August 2). RNA-dependent RNA polymerase. In Wikipedia, The Free Encyclopedia. Retrieved 02:47, August 8, 2021, from https://en.Wikipedia.org/w/index.php?title=RNA-dependent_RNA_polymerase&oldid=1036813791

3. Ferrer-Orta, C., Ferrero, D., and Verdaguer, N. (2015) RNA-Dependent RNA Polymerases of Picornaviruses: From the Structure to Regulatory Mechanisms (2015 Viruses 7(8):4438-4460. Available at: https://www.mdpi.com/1999-4915/7/8/2829/htm