Reverse transcriptase (RNA-dependent DNA polymerase) | |||||||||
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Identifiers | |||||||||
Symbol | RVT_1 | ||||||||
Pfam | PF00078 | ||||||||
Pfam clan | CL0027 | ||||||||
InterPro | IPR000477 | ||||||||
PROSITE | PS50878 | ||||||||
SCOP2 | 1hmv / SCOPe / SUPFAM | ||||||||
CDD | cd00304 | ||||||||
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RNA-directed DNA polymerase | |||||||||
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Identifiers | |||||||||
EC no. | 2.7.7.49 | ||||||||
CAS no. | 9068-38-6 | ||||||||
Databases | |||||||||
IntEnz | IntEnz view | ||||||||
BRENDA | BRENDA entry | ||||||||
ExPASy | NiceZyme view | ||||||||
KEGG | KEGG entry | ||||||||
MetaCyc | metabolic pathway | ||||||||
PRIAM | profile | ||||||||
PDB structures | RCSB PDB PDBe PDBsum | ||||||||
Gene Ontology | AmiGO / QuickGO | ||||||||
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A reverse transcriptase (RT) is an enzyme used to convert RNA genome to DNA , a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, by retrotransposon mobile genetic elements to proliferate within the host genome, and by eukaryotic cells to extend the telomeres at the ends of their linear chromosomes. Contrary to a widely held belief, the process does not violate the flows of genetic information as described by the classical central dogma, as transfers of information from RNA to DNA are explicitly held possible. [2] [3] [4]
Retroviral RT has three sequential biochemical activities: RNA-dependent DNA polymerase activity, ribonuclease H (RNase H), and DNA-dependent DNA polymerase activity. Collectively, these activities enable the enzyme to convert single-stranded RNA into double-stranded cDNA. In retroviruses and retrotransposons, this cDNA can then integrate into the host genome, from which new RNA copies can be made via host-cell transcription. The same sequence of reactions is widely used in the laboratory to convert RNA to DNA for use in molecular cloning, RNA sequencing, polymerase chain reaction (PCR), or genome analysis.
Reverse transcriptases were discovered by Howard Temin at the University of Wisconsin–Madison in Rous sarcoma virions [5] and independently isolated by David Baltimore in 1970 at MIT from two RNA tumour viruses: murine leukemia virus and again Rous sarcoma virus. [6] For their achievements, they shared the 1975 Nobel Prize in Physiology or Medicine (with Renato Dulbecco).
Well-studied reverse transcriptases include:
The enzymes are encoded and used by viruses that use reverse transcription as a step in the process of replication. Reverse-transcribing RNA viruses, such as retroviruses, use the enzyme to reverse-transcribe their RNA genomes into DNA, which is then integrated into the host genome and replicated along with it. Reverse-transcribing DNA viruses, such as the hepadnaviruses, can allow RNA to serve as a template in assembling and making DNA strands. HIV infects humans with the use of this enzyme. Without reverse transcriptase, the viral genome would not be able to incorporate into the host cell, resulting in failure to replicate.[ citation needed ]
Reverse transcriptase creates double-stranded DNA from an RNA template.
In virus species with reverse transcriptase lacking DNA-dependent DNA polymerase activity, creation of double-stranded DNA can possibly be done by host-encoded DNA polymerase δ, mistaking the viral DNA-RNA for a primer and synthesizing a double-stranded DNA by a similar mechanism as in primer removal, where the newly synthesized DNA displaces the original RNA template.[ citation needed ]
The process of reverse transcription, also called retrotranscription or retrotras, is extremely error-prone, and it is during this step that mutations may occur. Such mutations may cause drug resistance.
Retroviruses, also referred to as class VI ssRNA-RT viruses, are RNA reverse-transcribing viruses with a DNA intermediate. Their genomes consist of two molecules of positive-sense single-stranded RNA with a 5' cap and 3' polyadenylated tail. Examples of retroviruses include the human immunodeficiency virus (HIV) and the human T-lymphotropic virus (HTLV). Creation of double-stranded DNA occurs in the cytosol [10] as a series of these steps:
Creation of double-stranded DNA also involves strand transfer, in which there is a translocation of short DNA product from initial RNA-dependent DNA synthesis to acceptor template regions at the other end of the genome, which are later reached and processed by the reverse transcriptase for its DNA-dependent DNA activity. [11]
Retroviral RNA is arranged in 5’ terminus to 3’ terminus. The site where the primer is annealed to viral RNA is called the primer-binding site (PBS). The RNA 5’end to the PBS site is called U5, and the RNA 3’ end to the PBS is called the leader. The tRNA primer is unwound between 14 and 22 nucleotides and forms a base-paired duplex with the viral RNA at PBS. The fact that the PBS is located near the 5’ terminus of viral RNA is unusual because reverse transcriptase synthesize DNA from 3’ end of the primer in the 5’ to 3’ direction (with respect to the newly synthesized DNA strand). Therefore, the primer and reverse transcriptase must be relocated to 3’ end of viral RNA. In order to accomplish this reposition, multiple steps and various enzymes including DNA polymerase, ribonuclease H(RNase H) and polynucleotide unwinding are needed. [12] [13]
The HIV reverse transcriptase also has ribonuclease activity that degrades the viral RNA during the synthesis of cDNA, as well as DNA-dependent DNA polymerase activity that copies the sense cDNA strand into an antisense DNA to form a double-stranded viral DNA intermediate (vDNA). [14] The HIV viral RNA structural elements regulate the progression of reverse transcription. [15]
Self-replicating stretches of eukaryotic genomes known as retrotransposons utilize reverse transcriptase to move from one position in the genome to another via an RNA intermediate. They are found abundantly in the genomes of plants and animals. Telomerase is another reverse transcriptase found in many eukaryotes, including humans, which carries its own RNA template; this RNA is used as a template for DNA replication. [16]
Initial reports of reverse transcriptase in prokaryotes came as far back as 1971 in France (Beljanski et al., 1971a, 1972) and a few years later in the USSR (Romashchenko 1977 [17] ). These have since been broadly described as part of bacterial Retrons, distinct sequences that code for reverse transcriptase, and are used in the synthesis of msDNA. In order to initiate synthesis of DNA, a primer is needed. In bacteria, the primer is synthesized during replication. [18]
Valerian Dolja of Oregon State argues that viruses, due to their diversity, have played an evolutionary role in the development of cellular life, with reverse transcriptase playing a central role. [19]
The reverse transcriptase employs a "right hand" structure similar to that found in other viral nucleic acid polymerases. [20] [21] In addition to the transcription function, retroviral reverse transcriptases have a domain belonging to the RNase H family, which is vital to their replication. By degrading the RNA template, it allows the other strand of DNA to be synthesized. [22] Some fragments from the digestion also serve as the primer for the DNA polymerase (either the same enzyme or a host protein), responsible for making the other (plus) strand. [20]
There are three different replication systems during the life cycle of a retrovirus. The first process is the reverse transcriptase synthesis of viral DNA from viral RNA, which then forms newly made complementary DNA strands. The second replication process occurs when host cellular DNA polymerase replicates the integrated viral DNA. Lastly, RNA polymerase II transcribes the proviral DNA into RNA, which will be packed into virions. Mutation can occur during one or all of these replication steps. [23]
Reverse transcriptase has a high error rate when transcribing RNA into DNA since, unlike most other DNA polymerases, it has no proofreading ability. This high error rate allows mutations to accumulate at an accelerated rate relative to proofread forms of replication. The commercially available reverse transcriptases produced by Promega are quoted by their manuals as having error rates in the range of 1 in 17,000 bases for AMV and 1 in 30,000 bases for M-MLV. [24]
Other than creating single-nucleotide polymorphisms, reverse transcriptases have also been shown to be involved in processes such as transcript fusions, exon shuffling and creating artificial antisense transcripts. [25] [26] It has been speculated that this template switching activity of reverse transcriptase, which can be demonstrated completely in vivo, may have been one of the causes for finding several thousand unannotated transcripts in the genomes of model organisms. [27]
Two RNA genomes are packaged into each retrovirus particle, but, after an infection, each virus generates only one provirus. [28] After infection, reverse transcription is accompanied by template switching between the two genome copies (copy choice recombination). [28] There are two models that suggest why RNA transcriptase switches templates. The first, the forced copy-choice model, proposes that reverse transcriptase changes the RNA template when it encounters a nick, implying that recombination is obligatory to maintaining virus genome integrity. The second, the dynamic choice model, suggests that reverse transcriptase changes templates when the RNAse function and the polymerase function are not in sync rate-wise, implying that recombination occurs at random and is not in response to genomic damage. A study by Rawson et al. supported both models of recombination. [28] From 5 to 14 recombination events per genome occur at each replication cycle. [29] Template switching (recombination) appears to be necessary for maintaining genome integrity and as a repair mechanism for salvaging damaged genomes. [30] [28]
As HIV uses reverse transcriptase to copy its genetic material and generate new viruses (part of a retrovirus proliferation circle), specific drugs have been designed to disrupt the process and thereby suppress its growth. Collectively, these drugs are known as reverse-transcriptase inhibitors and include the nucleoside and nucleotide analogues zidovudine (trade name Retrovir), lamivudine (Epivir) and tenofovir (Viread), as well as non-nucleoside inhibitors, such as nevirapine (Viramune).[ citation needed ]
Reverse transcriptase is commonly used in research to apply the polymerase chain reaction technique to RNA in a technique called reverse transcription polymerase chain reaction (RT-PCR). The classical PCR technique can be applied only to DNA strands, but, with the help of reverse transcriptase, RNA can be transcribed into DNA, thus making PCR analysis of RNA molecules possible. Reverse transcriptase is used also to create cDNA libraries from mRNA. The commercial availability of reverse transcriptase greatly improved knowledge in the area of molecular biology, as, along with other enzymes, it allowed scientists to clone, sequence, and characterise RNA.
In genetics, complementary DNA (cDNA) is DNA that was reverse transcribed from an RNA. cDNA exists in both single-stranded and double-stranded forms and in both natural and engineered forms.
A DNA virus is a virus that has a genome made of deoxyribonucleic acid (DNA) that is replicated by a DNA polymerase. They can be divided between those that have two strands of DNA in their genome, called double-stranded DNA (dsDNA) viruses, and those that have one strand of DNA in their genome, called single-stranded DNA (ssDNA) viruses. dsDNA viruses primarily belong to two realms: Duplodnaviria and Varidnaviria, and ssDNA viruses are almost exclusively assigned to the realm Monodnaviria, which also includes some dsDNA viruses. Additionally, many DNA viruses are unassigned to higher taxa. Reverse transcribing viruses, which have a DNA genome that is replicated through an RNA intermediate by a reverse transcriptase, are classified into the kingdom Pararnavirae in the realm Riboviria.
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. After invading a host cell's cytoplasm, the virus uses its own reverse transcriptase enzyme to produce DNA from its RNA genome, the reverse of the usual pattern, thus retro (backwards). The new DNA is then incorporated into the host cell genome by an integrase enzyme, at which point the retroviral DNA is referred to as a provirus. The host cell then treats the viral DNA as part of its own genome, transcribing and translating the viral genes along with the cell's own genes, producing the proteins required to assemble new copies of the virus. Many retroviruses cause serious diseases in humans, other mammals, and birds.
Transcription is the process of copying a segment of DNA into RNA. The segments of DNA transcribed into RNA molecules that can encode proteins produce messenger RNA (mRNA). Other segments of DNA are transcribed into RNA molecules called non-coding RNAs (ncRNAs).
A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create two identical DNA duplexes from a single original DNA duplex. During this process, DNA polymerase "reads" the existing DNA strands to create two new strands that match the existing ones. These enzymes catalyze the chemical reaction
Hepadnaviridae is a family of viruses. Humans, apes, and birds serve as natural hosts. There are currently 18 species in this family, divided among 5 genera. Its best-known member is hepatitis B virus. Diseases associated with this family include: liver infections, such as hepatitis, hepatocellular carcinomas, and cirrhosis. It is the sole accepted family in the order Blubervirales.
Ribonuclease H is a family of non-sequence-specific endonuclease enzymes that catalyze the cleavage of RNA in an RNA/DNA substrate via a hydrolytic mechanism. Members of the RNase H family can be found in nearly all organisms, from bacteria to archaea to eukaryotes.
Cauliflower mosaic virus (CaMV) is a member of the genus Caulimovirus, one of the six genera in the family Caulimoviridae, which are pararetroviruses that infect plants. Pararetroviruses replicate through reverse transcription just like retroviruses, but the viral particles contain DNA instead of RNA.
Viral replication is the formation of biological viruses during the infection process in the target host cells. Viruses must first get into the cell before viral replication can occur. Through the generation of abundant copies of its genome and packaging these copies, the virus continues infecting new hosts. Replication between viruses is greatly varied and depends on the type of genes involved in them. Most DNA viruses assemble in the nucleus while most RNA viruses develop solely in cytoplasm.
Gammaretrovirus is a genus in the Retroviridae family. Example species are the murine leukemia virus and the feline leukemia virus. They cause various sarcomas, leukemias and immune deficiencies in mammals, reptiles and birds.
Baltimore classification is a system used to classify viruses based on their manner of messenger RNA (mRNA) synthesis. By organizing viruses based on their manner of mRNA production, it is possible to study viruses that behave similarly as a distinct group. Seven Baltimore groups are described that take into consideration whether the viral genome is made of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), whether the genome is single- or double-stranded, and whether the sense of a single-stranded RNA genome is positive or negative.
In molecular biology and genetics, the sense of a nucleic acid molecule, particularly of a strand of DNA or RNA, refers to the nature of the roles of the strand and its complement in specifying a sequence of amino acids. Depending on the context, sense may have slightly different meanings. For example, the negative-sense strand of DNA is equivalent to the template strand, whereas the positive-sense strand is the non-template strand whose nucleotide sequence is equivalent to the sequence of the mRNA transcript.
Multicopy single-stranded DNA (msDNA) is a type of extrachromosomal satellite DNA that consists of a single-stranded DNA molecule covalently linked via a 2'-5'phosphodiester bond to an internal guanosine of an RNA molecule. The resultant DNA/RNA chimera possesses two stem-loops joined by a branch similar to the branches found in RNA splicing intermediates. The coding region for msDNA, called a "retron", also encodes a type of reverse transcriptase, which is essential for msDNA synthesis.
RNA-dependent RNA polymerase (RdRp) or RNA replicase is an enzyme that catalyzes the replication of RNA from an RNA template. Specifically, it catalyzes synthesis of the RNA strand complementary to a given RNA template. This is in contrast to typical DNA-dependent RNA polymerases, which all organisms use to catalyze the transcription of RNA from a DNA template.
Nucleic acid sequence-based amplification, commonly referred to as NASBA, is a method in molecular biology which is used to produce multiple copies of single stranded RNA. NASBA is a two-step process that takes RNA and anneals specially designed primers, then utilizes an enzyme cocktail to amplify it.
Hepatitis B virus DNA polymerase is a hepatitis B viral protein. It is a DNA polymerase that can use either DNA or RNA templates and a ribonuclease H that cuts RNA in the duplex. Both functions are supplied by the reverse transcriptase (RT) domain.
The retroviral ribonuclease H is a catalytic domain of the retroviral reverse transcriptase (RT) enzyme. The RT enzyme is used to generate complementary DNA (cDNA) from the retroviral RNA genome. This process is called reverse transcription. To complete this complex process, the retroviral RT enzymes need to adopt a multifunctional nature. They therefore possess 3 of the following biochemical activities: RNA-dependent DNA polymerase, ribonuclease H, and DNA-dependent DNA polymerase activities. Like all RNase H enzymes, the retroviral RNase H domain cleaves DNA/RNA duplexes and will not degrade DNA or unhybridized RNA.
Positive-strand RNA viruses are a group of related viruses that have positive-sense, single-stranded genomes made of ribonucleic acid. The positive-sense genome can act as messenger RNA (mRNA) and can be directly translated into viral proteins by the host cell's ribosomes. Positive-strand RNA viruses encode an RNA-dependent RNA polymerase (RdRp) which is used during replication of the genome to synthesize a negative-sense antigenome that is then used as a template to create a new positive-sense viral genome.
Riboviria is a realm of viruses that includes all viruses that use a homologous RNA-dependent polymerase for replication. It includes RNA viruses that encode an RNA-dependent RNA polymerase, as well as reverse-transcribing viruses that encode an RNA-dependent DNA polymerase. RNA-dependent RNA polymerase (RdRp), also called RNA replicase, produces RNA from RNA. RNA-dependent DNA polymerase (RdDp), also called reverse transcriptase (RT), produces DNA from RNA. These enzymes are essential for replicating the viral genome and transcribing viral genes into messenger RNA (mRNA) for translation of viral proteins.
Orthornavirae is a kingdom of viruses that have genomes made of ribonucleic acid (RNA), including genes which encode an RNA-dependent RNA polymerase (RdRp). The RdRp is used to transcribe the viral RNA genome into messenger RNA (mRNA) and to replicate the genome. Viruses in this kingdom share a number of characteristics which promote rapid evolution, including high rates of genetic mutation, recombination, and reassortment.