What is an example of reverse transcriptase?

Avian myeloblastosis virus (AMV) reverse transcriptase is one of the most common RTs used in the lab. The 170kDa heterodimer requires 6–10mM Mg2+ or Mn2+ for activity, and reactions often include sodium pyrophosphate and spermidine to increase full-length cDNA production and decrease formation of hairpins during synthesis (3). AMV RT is less sensitive to inhibition by strong RNA secondary structure than Moloney murine leukemia virus (M-MLV) RT (4).

Optimal enzyme activity and maximum cDNA length occur at 42–48°C, but the reaction temperature can range from 25°C to 58°C (5). The higher reaction temperature helps denature regions of strong RNA secondary structure, which can cause RTs to stall and limit cDNA size (6–7). For this reason, AMV RT is often used to reverse transcribe RNAs with strong secondary structure. Like other RTs, AMV RT is compatible with gene-specific primers, oligo(dT)15 primers or random hexamers, although use of random hexamers requires a reduced reaction temperature of 37°C. Gene-specific RT primers with suitably high melting temperatures are recommended when the reaction temperature exceeds 42°C.

Although high reaction temperatures can effectively resolve regions of strong secondary structures, these temperatures are detrimental to RNA integrity. RNA is thermolabile and susceptible to metal-catalyzed degradation. Normally, hydrolysis occurs at a low frequency, but RNA hydrolysis becomes a concern under certain conditions (e.g., nonoptimal pH, high temperatures, the presence of divalent cations). Thus, cDNA synthesis—in particular cDNA synthesis of long RNAs—benefits from not exposing RNA to higher reaction temperatures. To minimize the amount of time that RNA spends at high temperatures, cDNA synthesis protocols using AMV and M-MLV RTs often incorporate an initial denaturation step, where the RNA and RT primer are combined, briefly heated to help denature any secondary structure then quickly cooled on ice to maintain the denatured state. The RT, reaction buffer and dNTPs are added, and the reaction is incubated at the desired temperature.

AMV RT possesses an intrinsic RNase H activity, which degrades the RNA strand of an RNA/DNA hybrid and can cleave the RNA template if the RT pauses during synthesis (8). This reduces total cDNA yield and the percentage of full-length cDNA, limiting the usefulness of AMV RT to reverse transcribe RNAs longer than ~5kb.

Typical RT-PCR conditions include the use of up to 5µg of total RNA or up to 100ng of polyA+ mRNA, 20–30 units of enzyme and a 60-minute incubation at 42°C. AMV RT is more processive than M-MLV RT (5–6), so fewer units are required to generate the same amount of cDNA; 25 units of AMV RT is equivalent to approximately 200 units of M-MLV RT. Prior to PCR, AMV must be inactivated because AMV RT, like M-MLV RT, can inhibit Taq DNA polymerase (9). The enzyme can be inactivated by heating at 70–100°C, followed by a 5-minute incubation on ice. The reverse transcription reaction is often diluted prior to PCR or the volume of cDNA added to the PCR is limited because spermidine can inhibit PCR (10). This limitation can negatively affect the ability to detect low-abundance RNAs.

AMV RT is recommended for one-step and two-step RT-PCR and RT-qPCR, reverse transcription of RNAs <5kb and primer extension, particularly if the template RNA has strong secondary structure.

Reverse transcription begins when the viral particle enters the cytoplasm of a target cell. The viral RNA genome enters the cytoplasm as part of a nucleoprotein complex that has not been well characterized. The process of reverse transcription generates, in the cytoplasm, a linear DNA duplex via an intricate series of steps. This DNA is colinear with its RNA template, but it contains terminal duplications known as the long terminal repeats (LTRs) that are not present in viral RNA (). Extant models for reverse transcription propose that two specialized template switches known as strand-transfer reactions or “jumps” are required to generate the LTRs.

Figure 1

Reverse transcription of the viral RNA genome generates a linear DNA duplex. The positions of the R, U5, and U3 regions, the polypurine tract (PPT), and the primer-binding site (PBS) are indicated. Reverse transcription creates duplications of the U5 (more...)

Retroviral DNA synthesis is absolutely dependent on the two distinct enzymatic activities of RT: a DNA polymerase that can use either RNA or DNA as a template, and a nuclease, termed ribonuclease H (RNase H), that is specific for the RNA strand of RNA:DNA duplexes. Although a role for other proteins cannot be ruled out, and it is likely that certain viral proteins (e.g., nucleocapsid, NC) increase the efficiency of reverse transcription, all of the enzymatic functions required to complete the series of steps involved in the generation of a retroviral DNA can be attributed to either the DNA polymerase or the RNase H of RT. The process of retroviral DNA synthesis is believed to follow the scheme outlined in :

Minus-strand DNA synthesis is initiated using the 3′end of a partially unwound transfer RNA which is annealed to the primer-binding site (PBS) in genomic RNA, as a primer. Minus-strand DNA synthesis proceeds until the 5′end of genomic RNA is reached, generating a DNA intermediate of discrete length termed minus-strand strong-stop DNA (–sssDNA). Since the binding site for the tRNA primer is near the 5′ end of viral RNA, –sssDNA is relatively short, on the order of 100–150 bases

Following RNase-H-mediated degradation of the RNA strand of the RNA:–sssDNA duplex, the first strand transfer causes –sssDNA to be annealed to the 3′end of a viral genomic RNA. This transfer is mediated by identical sequences known as the repeated (R) sequences, which are present at the 5′ and 3′ends of the RNA genome. The 3′end of –sssDNA was copied from the R sequences at the 5′end of the viral genome and therefore contains sequences complementary to R. After the RNA template has been removed, –sssDNA can anneal to the R sequences at the 3′end of the RNA genome. The annealing reaction appears to be facilitated by the NC.

Once the –sssDNA has been transferred to the 3′R segment on viral RNA, minus-strand DNA synthesis resumes, accompanied by RNase H digestion of the template strand. This degradation is not complete, however.

The RNA genome contains a short polypurine tract (PPT) that is relatively resistant to RNase H degradation. A defined RNA segment derived from the PPT primes plus-strand DNA synthesis. Plus-strand synthesis is halted after a portion of the primer tRNA is reverse-transcribed, yielding a DNA called plus-strand strong-stop DNA (+sssDNA). Although all strains of retroviruses generate a defined plus-strand primer from the PPT, some viruses generate additional plus-strand primers from the RNA genome.

RNase H removes the primer tRNA, exposing sequences in +sssDNA that are complementary to sequences at or near the 3′end of plus-strand DNA.

Annealing of the complementary PBS segments in +sssDNA and minus-strand DNA constitutes the second strand transfer.

Plus- and minus-strand syntheses are then completed, with the plus and minus strands of DNA each serving as a template for the other strand.

Figure 2

Process of reverse transcription of the retroviral genome. (Black line) RNA; (light color) minus-strand DNAs; (dark color) plus-strand DNA. See text for a description of this process.

What is an example of reverse transcription?

What types of viruses have reverse transcriptase?

What is reverse transcriptase also known as?

Where is reverse transcriptase found?