RNA-dependent RNA polymerase
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RNA-dependent RNA polymerase (RdRp) or RNA replicase is an
enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
that catalyzes the replication of
RNA Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
from an RNA template. Specifically, it catalyzes synthesis of the RNA strand
complementary A complement is something that completes something else. Complement may refer specifically to: The arts * Complement (music), an interval that, when added to another, spans an octave ** Aggregate complementation, the separation of pitch-class ...
to a given RNA template. This is in contrast to typical
DNA-dependent RNA polymerase In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that synthesizes RNA from a DNA template. Using the enzyme helicase, RNAP locally opens the ...
s, which all organisms use to catalyze the
transcription Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including: Genetics * Transcription (biology), the copying of DNA into RNA, the fir ...
of RNA from a DNA template. RdRp is an essential protein encoded in the genomes of most RNA-containing
virus A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea. Since Dmitri Ivanovsky's 1 ...
es with no DNA stage including
SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) is a strain of coronavirus that causes COVID-19 (coronavirus disease 2019), the respiratory illness responsible for the ongoing COVID-19 pandemic. The virus previously had a ...
. Some eukaryotes also contain RdRps, which are involved in
RNA interference RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression. Historically, RNAi was known by o ...
and differ structurally from viral RdRps.


History

Viral RdRps were discovered in the early 1960s from studies on
mengovirus Mengovirus, also known as Columbia SK virus, mouse Elberfield virus, and Encephalomyocarditisvirus (EMCV), belongs to the genus ''Cardiovirus'' which is a member of the ''Picornaviridae''. Its genome is a single stranded positive-sense RNA molecu ...
and
polio virus A poliovirus, the causative agent of polio (also known as poliomyelitis), is a serotype of the species ''Enterovirus C'', in the family of ''Picornaviridae''. There are three poliovirus serotypes: types 1, 2, and 3. Poliovirus is composed of an ...
when it was observed that these viruses were not sensitive to
actinomycin D Dactinomycin, also known as actinomycin D, is a chemotherapy medication used to treat a number of types of cancer. This includes Wilms tumor, rhabdomyosarcoma, Ewing's sarcoma, trophoblastic neoplasm, testicular cancer, and certain types of ovari ...
, a drug that inhibits cellular DNA-directed RNA synthesis. This lack of sensitivity suggested that there is a virus-specific enzyme that could copy RNA from an RNA template and not from a DNA template.


Distribution

RdRps are highly conserved throughout viruses and are even related to
telomerase Telomerase, also called terminal transferase, is a ribonucleoprotein that adds a species-dependent telomere repeat sequence to the 3' end of telomeres. A telomere is a region of repetitive sequences at each end of the chromosomes of most euka ...
, though the reason for this is an ongoing question as of 2009. The similarity has led to speculation that viral RdRps are ancestral to human telomerase. The most famous example of RdRp is that of the
polio virus A poliovirus, the causative agent of polio (also known as poliomyelitis), is a serotype of the species ''Enterovirus C'', in the family of ''Picornaviridae''. There are three poliovirus serotypes: types 1, 2, and 3. Poliovirus is composed of an ...
. The viral genome is composed of RNA, which enters the cell through receptor-mediated
endocytosis Endocytosis is a cellular process in which substances are brought into the cell. The material to be internalized is surrounded by an area of cell membrane, which then buds off inside the cell to form a vesicle containing the ingested material. E ...
. From there, the RNA is able to act as a template for complementary RNA synthesis, immediately. The complementary strand is then, itself, able to act as a template for the production of new viral genomes that are further packaged and released from the cell ready to infect more host cells. The advantage of this method of replication is that there is no DNA stage; replication is quick and easy. The disadvantage is that there is no 'back-up' DNA copy. Many RdRps are associated tightly with membranes and are, therefore, difficult to study. The best-known RdRps are polioviral 3Dpol,
vesicular stomatitis virus ''Indiana vesiculovirus'', formerly ''Vesicular stomatitis Indiana virus'' (VSIV or VSV) is a virus in the family ''Rhabdoviridae''; the well-known ''Rabies lyssavirus'' belongs to the same family. VSIV can infect insects, cattle, horses and pigs ...
L, and
hepatitis C virus The hepatitis C virus (HCV) is a small (55–65 nm in size), enveloped, positive-sense single-stranded RNA virus of the family ''Flaviviridae''. The hepatitis C virus is the cause of hepatitis C and some cancers such as liver cancer ( hepato ...
NS5B Nonstructural protein 5B (NS5B) is a viral protein found in the hepatitis C virus (HCV). It is an RNA-dependent RNA polymerase, having the key function of replicating HCV's viral RNA by using the viral positive RNA strand as a template to catalyze ...
protein. Many
eukaryote Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bacte ...
s also have RdRps and these are involved in
RNA interference RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression. Historically, RNAi was known by o ...
: these amplify
microRNA MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miRN ...
s and
small temporal RNA Small temporal RNA (abbreviated stRNA) regulates gene expression during roundworm development by preventing the mRNAs they bind from being translated. In contrast to siRNA, stRNAs downregulate expression of target RNAs after translation initiation ...
s and produce double-stranded RNA using
small interfering RNA Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20-24 (normally 21) base pairs in length, similar to MicroRNA, miRNA, and op ...
s as primers. In fact these same RdRps that are used in the defense mechanisms can be usurped by RNA viruses for their benefit. Their evolutionary history has been reviewed.


Replication process

RdRp differs from
RNA polymerase In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that synthesizes RNA from a DNA template. Using the enzyme helicase, RNAP locally opens the ...
as it works to catalyze the synthesis of an RNA strand complementary to a given RNA template, rather than using a DNA template. The RNA replication process is a four-step mechanism, as described. #
Nucleoside triphosphate A nucleoside triphosphate is a nucleoside containing a nitrogenous base bound to a 5-carbon sugar (either ribose or deoxyribose), with three phosphate groups bound to the sugar. They are the molecular precursors of both DNA and RNA, which are cha ...
(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 causes the RdRp to undergo a conformational change. # Active site closure – the conformational change, initiated by the correct NTP binding, results in the restriction of active site access and produces a catalytically competent state. #
Phosphodiester bond In chemistry, a phosphodiester bond occurs when exactly two of the hydroxyl groups () in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. The "bond" involves this linkage . Discussion of phosphodiesters is d ...
formation – two Mg2+ ions are present in the catalytically active state and arrange themselves in such a way around the newly synthesized RNA chain that the substrate NTP is able to undergo a phosphatidyl transfer and form a phosphodiester bond with the newly synthesized chain. Without the use of these Mg2+ ions, the active site is no longer catalytically stable and the RdRp complex changes to an open conformation. # Translocation – once the active site is open, the RNA template strand is able to move by one position through the RdRp protein complex and continue chain elongation by binding a new NTP, unless otherwise specified by the template. RNA synthesis can be performed by means of a
primer Primer may refer to: Arts, entertainment, and media Films * ''Primer'' (film), a 2004 feature film written and directed by Shane Carruth * ''Primer'' (video), a documentary about the funk band Living Colour Literature * Primer (textbook), a t ...
-independent (''de novo'') or a primer-dependent mechanism that utilizes a viral protein genome-linked (VPg) primer. The ''de novo'' initiation consists in the addition of a
nucleoside triphosphate A nucleoside triphosphate is a nucleoside containing a nitrogenous base bound to a 5-carbon sugar (either ribose or deoxyribose), with three phosphate groups bound to the sugar. They are the molecular precursors of both DNA and RNA, which are cha ...
(NTP) to the 3'-OH of the first initiating NTP. During the following so-called elongation phase, this nucleotidyl transfer reaction is repeated with subsequent NTPs to generate the complementary RNA product. Termination of the nascent RNA chain produced by RdRp is not completely known, however, it has been shown that RdRp termination is sequence-independent. One major drawback of RNA-dependent RNA polymerase replication is the immense error rate during transcription. RdRps are known to have a lack of fidelity on the order of 104 nucleotides, which is thought to be a direct result of its insufficient proofreading abilities. This high rate of variation is favored in viral genomes as it allows for the pathogen to overcome defenses developed by hosts trying to avoid infection allowing for evolutionary growth.


Structure

Viral/prokaryotic RNA-directed RNA polymerases, along with many single-subunit DNA-directed polymerases, employ a fold whose organization has been linked to the shape of a right hand with three subdomains termed fingers, palm, and thumb. Only the palm subdomain, composed of a four-stranded antiparallel
beta sheet The beta sheet, (β-sheet) (also β-pleated sheet) is a common motif of the regular protein secondary structure. Beta sheets consist of beta strands (β-strands) connected laterally by at least two or three backbone hydrogen bonds, forming a g ...
with two
alpha helices The alpha helix (α-helix) is a common motif in the secondary structure of proteins and is a right hand-helix conformation in which every backbone N−H group hydrogen bonds to the backbone C=O group of the amino acid located four residues ear ...
, is well conserved among all of these enzymes. In RdRp, the palm subdomain comprises three well-conserved motifs (A, B, and C). Motif A (D-x(4,5)-D) and motif C (GDD) are spatially juxtaposed; the
aspartic acid Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. Like all other amino acids, it contains an amino group and a carboxylic acid. Its α-amino group is in the pro ...
residues of these motifs are implied in the binding of Mg2+ and/or Mn2+. The
asparagine Asparagine (symbol Asn or N) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form under biological conditions), an α-carboxylic acid group (which is in the depro ...
residue of motif B is involved in selection of ribonucleoside triphosphates over dNTPs and, thus, determines whether RNA rather than DNA is synthesized. The domain organization and the 3D structure of the catalytic centre of a wide range of RdRps, even those with a low overall sequence homology, are conserved. The catalytic center is formed by several motifs containing a number of conserved amino acid residues. Eukaryotic
RNA interference RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression. Historically, RNAi was known by o ...
requires a cellular RNA-dependent RNA polymerase (c RdRp). Unlike the "hand" polymerases, they resemble simplified multi-subunit
DNA-dependent RNA polymerase In molecular biology, RNA polymerase (abbreviated RNAP or RNApol), or more specifically DNA-directed/dependent RNA polymerase (DdRP), is an enzyme that synthesizes RNA from a DNA template. Using the enzyme helicase, RNAP locally opens the ...
s (DdRPs), specifically in the catalytic β/β' subunits, in that they use two sets of double-psi β-barrels in the active site. QDE1 () in ''
Neurospora crassa ''Neurospora crassa'' is a type of red bread mold of the phylum Ascomycota. The genus name, meaning "nerve spore" in Greek, refers to the characteristic striations on the spores. The first published account of this fungus was from an infestation ...
'', which has both barrels in the same chain, is an example of such an c RdRp enzyme.
Bacteriophage A bacteriophage (), also known informally as a ''phage'' (), is a duplodnaviria virus that infects and replicates within bacteria and archaea. The term was derived from "bacteria" and the Greek φαγεῖν ('), meaning "to devour". Bacteri ...
homologs of c RdRp, including the similarly single-chain DdRp yonO (), appear to be closer to c RdRps than DdRPs are.


In viruses

There are 4 superfamilies of viruses that cover all RNA-containing viruses with no DNA stage: * Viruses containing positive-strand RNA or double-strand RNA, except
retrovirus A retrovirus is a type of virus that inserts a DNA copy of its RNA genome into the DNA of a host cell that it invades, thus changing the genome of that cell. Once inside the host cell's cytoplasm, the virus uses its own reverse transcriptase ...
es and ''
Birnaviridae ''Birnaviridae'' is a family of double-stranded RNA viruses. Salmonid fish, birds and insects serve as natural hosts. There are currently 11 species in this family, divided among seven genera. Diseases associated with this family include infec ...
'' ** All positive-strand RNA eukaryotic viruses with no DNA stage ** All RNA-containing
bacteriophage A bacteriophage (), also known informally as a ''phage'' (), is a duplodnaviria virus that infects and replicates within bacteria and archaea. The term was derived from "bacteria" and the Greek φαγεῖν ('), meaning "to devour". Bacteri ...
s; there are two families of RNA-containing bacteriophages: '' Leviviridae'' (positive ssRNA phages) and ''
Cystoviridae ''Cystovirus'' is a genus of double-stranded RNA viruses which infects bacteria. It is the only genus in the family ''Cystoviridae.'' The name of the group c''ysto'' derives from Greek ''kystis'' which means bladder or sack. There are seven spec ...
'' (dsRNA phages) ** dsRNA virus family ''
Reoviridae ''Reoviridae'' is a family of double-stranded RNA viruses. Member viruses have a wide host range, including vertebrates, invertebrates, plants, protists and fungi. They lack lipid envelopes and package their segmented genome within multi-layere ...
'', ''
Totiviridae ''Totiviridae'' is a family of double-stranded RNA viruses. Giardia lamblia, leishmania, trichomonas vaginalis, and fungi serve as natural hosts. The name of the group derives from Latin ''toti'' which means undivided or whole. There are 28 spec ...
'', ''
Hypoviridae ''Hypovirus'' is a genus of viruses, in the family ''Hypoviridae''. Fungi serve as natural hosts. There are four species in this genus. Infection reduces the virulence of its parasitic host, making it a hyperparasite useful for blight control. ...
'', ''
Partitiviridae ''Partitiviridae'' is a family of double-stranded RNA viruses. Plants, fungi, and protozoa serve as natural hosts. It has been suggested that they can also infect bacteria. The name comes from the Latin ''partitius,'' which means divided, and ...
'' * ''
Mononegavirales ''Mononegavirales'' is an order of negative-strand RNA viruses which have nonsegmented genomes. Some common members of the order are Ebola virus, human respiratory syncytial virus, measles virus, mumps virus, Nipah virus, and rabies virus. A ...
'' (negative-strand RNA viruses with non-segmented genomes; ) * Negative-strand RNA viruses with segmented genomes (), such as orthomyxoviruses and bunyaviruses * dsRNA virus family ''
Birnaviridae ''Birnaviridae'' is a family of double-stranded RNA viruses. Salmonid fish, birds and insects serve as natural hosts. There are currently 11 species in this family, divided among seven genera. Diseases associated with this family include infec ...
'' ()
Flavivirus ''Flavivirus'' is a genus of positive-strand RNA viruses in the family ''Flaviviridae''. The genus includes the West Nile virus, dengue virus, tick-borne encephalitis virus, yellow fever virus, Zika virus and several other viruses which may cau ...
es produce a polyprotein from the ssRNA genome. The polyprotein is cleaved to a number of products, one of which is NS5, an RNA-dependent RNA polymerase. This RNA-directed RNA polymerase possesses a number of short regions and motifs homologous to other RNA-directed RNA polymerases. RNA replicase found in positive-strand ssRNA viruses are related to each other, forming three large superfamilies. Birnaviral RNA replicase is unique in that it lacks motif C (GDD) in the palm. Mononegaviral RdRp (PDB 5A22) has been automatically classified as similar to (+)−ssRNA RdRps, specifically one from ''Pestivirus'' and one from ''Leviviridae''. Bunyaviral RdRp monomer (PDB 5AMQ) resembles the heterotrimeric complex of Orthomyxoviral (Influenza; PDB 4WSB) RdRp. Since it is a protein universal to RNA-containing viruses, RdRp is a useful marker for understanding their evolution. The overall structural evolution of viral RdRps has been reviewed.


Recombination

When replicating its (+)ssRNA genome, the
poliovirus A poliovirus, the causative agent of polio (also known as poliomyelitis), is a serotype of the species ''Enterovirus C'', in the family of ''Picornaviridae''. There are three poliovirus serotypes: types 1, 2, and 3. Poliovirus is composed of an ...
RdRp is able to carry out recombination. Recombination appears to occur by a copy choice mechanism in which the RdRp switches (+)ssRNA templates during negative strand synthesis. Recombination frequency is determined in part by the fidelity of RdRp replication. RdRp variants with high replication fidelity show reduced recombination, and low fidelity RdRps exhibit increased recombination. Recombination by RdRp strand switching also occurs frequently during replication in the (+)ssRNA plant
carmovirus ''Carmovirus'' was a genus of viruses. The genus was split in 2015 into three genera, each retaining -''carmovirus'' as part of their name: * '' Alphacarmovirus'' * '' Betacarmovirus'' * '' Gammacarmovirus'' These genera are in the same family, ...
es and
tombusvirus ''Tombusvirus'' is a genus of viruses, in the family '' Tombusviridae''. Plants serve as natural hosts. There are 17 species in this genus. Symptoms associated with this genus include mosaic. The name of the genus comes from ''Tomato bushy stunt ...
es.


Intragenic complementation

Sendai virus ''Murine respirovirus'', formerly ''Sendai virus'' (SeV) and previously also known as murine parainfluenza virus type 1 or hemagglutinating virus of Japan (HVJ), is an enveloped,150-200 nm in diameter, a negative sense, single-stranded RN ...
(family ''Paramyxoviridae'') has a linear, single stranded, negative-sense, nonsegmented RNA genome. The viral RdRp consists of two virus-encoded subunits, a smaller one P and a larger one L. When different inactive RdRp mutants with defects throughout the length of the L subunit where tested in pairwise combinations, restoration of viral RNA synthesis was observed in some combinations. This positive L–L interaction is referred to as
intragenic complementation Epistasis is a phenomenon in genetics in which the effect of a gene mutation is dependent on the presence or absence of mutations in one or more other genes, respectively termed modifier genes. In other words, the effect of the mutation is dep ...
and indicates that the L protein is an oligomer in the viral RNA polymerase complex.


Drug therapies

* RdRps can be used as drug targets for viral pathogens as their function is not necessary for eukaryotic survival. By inhibiting RNA-dependent RNA polymerase function, new RNAs cannot be replicated from an RNA template strand, however, DNA-dependent RNA polymerase will remain functional. * There are currently antiviral drugs against Hepatitis C and COVID-19 that specifically target RdRp. These include Sofosbuvir and
Ribavirin Ribavirin, also known as tribavirin, is an antiviral medication used to treat RSV infection, hepatitis C and some viral hemorrhagic fevers. For hepatitis C, it is used in combination with other medications such as simeprevir, sofosbuvir, pe ...
against Hepatitis C and
Remdesivir Remdesivir, sold under the brand name Veklury, Text was copied from this source which is © European Medicines Agency. Reproduction is authorized provided the source is acknowledged. is a broad-spectrum antiviral medication developed by the ...
, the only
FDA The United States Food and Drug Administration (FDA or US FDA) is a federal agency of the Department of Health and Human Services. The FDA is responsible for protecting and promoting public health through the control and supervision of food ...
approved drug against COVID-19. *
GS-441524 GS-441524 is a nucleoside analogue antiviral drug which was developed by Gilead Sciences. It is the main plasma metabolite of the antiviral prodrug remdesivir, and has a half-life of around 24 hours in human patients. Remdesivir and GS-441524 ...
triphosphate, is a substrate for RdRp, but not mammalian polymerases. It results in premature chain termination and inhibition of viral replication. GS-441524 triphosphate is the biologically active form of the phosphate pro-drug,
Remdesivir Remdesivir, sold under the brand name Veklury, Text was copied from this source which is © European Medicines Agency. Reproduction is authorized provided the source is acknowledged. is a broad-spectrum antiviral medication developed by the ...
. Remdesivir is classified as a nucleotide analog in which it works to inhibit the function of RdRp by covalently binding to and interrupting termination of the nascent RNA through early or delayed termination or preventing further elongation of the RNA polynucleotide. This early termination leads to nonfunctional RNA that will be degraded through normal cellular processes.


RNA interference

The use of RNA-dependent RNA polymerase plays a major role in
RNA interference RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression. Historically, RNAi was known by o ...
in eukaryotes, a process used to silence gene expression via small interfering RNAs ( siRNAs) binding to mRNA rendering them inactive. Eukaryotic RdRp becomes active in the presence of dsRNA, and is a less widely distributed compared to other RNAi components as it lost in some animals, though still found in ''
C. elegans ''Caenorhabditis elegans'' () is a free-living transparent nematode about 1 mm in length that lives in temperate soil environments. It is the type species of its genus. The name is a blend of the Greek ''caeno-'' (recent), ''rhabditis'' (r ...
'' and '' P. tetraurelia'' and
plants Plants are predominantly Photosynthesis, photosynthetic eukaryotes of the Kingdom (biology), kingdom Plantae. Historically, the plant kingdom encompassed all living things that were not animals, and included algae and fungi; however, all curr ...
. This presence of dsRNA triggers the activation of RdRp and RNAi processes by priming the initiation of RNA transcription through the introduction of siRNAs into the system. In ''C. elegans'', siRNAs are integrated into the RNA-induced silencing complex,
RISC In computer engineering, a reduced instruction set computer (RISC) is a computer designed to simplify the individual instructions given to the computer to accomplish tasks. Compared to the instructions given to a complex instruction set comput ...
, which works alongside mRNAs targeted for interference to recruit more RdRps to synthesize more secondary siRNAs and repress gene expression.


See also

* Spiegelman's Monster * NS5B inhibitor


Notes


References


External links

* * {{DEFAULTSORT:Rna-Dependent Rna Polymerase Gene expression RNA EC 2.7.7