RNA replication. Reverse transcription

RNA replication. Reverse transcription

How RNA viruses copy their genomes with a polymerase our cells do not make, why they change so fast, and how retroviruses, hepatitis B and our own cells copy RNA back into DNA.

Contents3 sections

At a glance

  1. Our cells copy DNA into DNA and DNA into RNA, but have no enzyme for copying RNA into RNA. Almost every RNA virus therefore brings the gene for its own, an RNA-dependent RNA polymerase (RdRp).
  2. A positive-strand RNA genome is itself an mRNA and is translated at once. Negative-strand and double-stranded RNA viruses carry their polymerase into the cell to make their mRNAs.
  3. RdRps do not proofread, so an RNA virus makes about one mistake in its genome each time it is copied. RNA viruses change fast, escaping antibodies and drugs; coronaviruses, with some of the largest RNA genomes, have a proofreading enzyme.
  4. Reverse transcriptase copies RNA into DNA. HIV starts from a cellular tRNA, and its integrase inserts the DNA into a chromosome as a provirus. Hepatitis B copies RNA into DNA inside its capsid.
  5. Our own cells reverse transcribe too: telomerase builds chromosome ends, and LINE-1 elements copy themselves through RNA. In the laboratory, reverse transcriptase makes DNA copies of mRNA.

Copying RNA into RNA

An RNA virus copies its genome through a complementary strand: each RNA is a template for its complement, which is a template for new genomes. Our cells have no polymerase made for copying RNA into RNA, so the virus brings the gene for one, the RNA-dependent RNA polymerase (RdRp); like other polymerases it builds its chain 5′ to 3′. Hepatitis D virus, which infects only people who also carry hepatitis B virus, is an exception: it makes no polymerase, and the cell's own RNA polymerases, chiefly RNA polymerase II, copy its RNA. Viruses are grouped by their genome and by how they make their mRNA, in the Baltimore classification, named after David Baltimore, one of the discoverers of reverse transcriptase.

GenomeWhat happens first in the cellExamples
Positive-strand (+) RNAthe RNA is read by ribosomes, making the viral proteins, the RdRp among thempoliovirus, hepatitis A and C viruses, dengue virus, coronaviruses
Negative-strand (−) RNAthe RdRp carried in the virion makes mRNAs from the genomeinfluenza, measles, mumps, rabies and Ebola viruses
Double-stranded RNAthe RdRp carried in the virion makes mRNAs from the negative strandrotavirus
(+) RNA copied through DNAreverse transcriptase carried in the virion makes DNAHIV and other retroviruses
DNA copied through RNAthe cell transcribes the viral DNA; the RNA is copied back into DNAhepatitis B virus

Positive-strand viruses. Because the genome is an mRNA, it can start an infection on its own. It is translated into long polyproteins, which viral proteases cut into the working proteins; the RdRp then copies the genome into negative strands, and each of these into many new positive strands.

Negative-strand viruses. Their RNA is the complement of an mRNA and cannot be translated, so the virion carries the RdRp in, bound to the genome. It first makes mRNAs, then full-length positive copies that are templates for new genomes. The influenza polymerase does not make its own caps. Its PB2 subunit grips the cap of a cell's pre-mRNA, its PA subunit cuts off the capped start, 10 to 13 nucleotides long, and the polymerase uses this piece to begin a viral mRNA: cap-snatching.

each time an RNA virus is copied, about
1mistake per genome
in a coronavirus genome, among the largest RNA genomes
25 to 32kilobases
comes from transposable elements, about
halfof our genome
living with HIV at the end of 2025
41.0 millionpeople

Copying without checking

RdRps have no proofreading, so on average they make about one mutation in the genome each time it is copied. An infected person therefore carries a swarm of slightly different viruses, and any variant that escapes antibodies or a drug can take over. Such error rates would destroy a long genome, which is thought to keep most RNA genomes under about 15 kilobases. Coronaviruses and their relatives with large genomes are the exception: coronavirus genomes, of 25 to 32 kilobases, are copied with the help of a proofreading exonuclease, nsp14.

Influenza shows what this means for vaccines. Small changes keep building up in the genes for its surface proteins, haemagglutinin and neuraminidase, so antibodies against last year's viruses fit this year's less well: antigenic drift, which is why the vaccine's composition is reviewed every year. Influenza A can also change abruptly, in antigenic shift, when a virus with new surface proteins, often carrying genes from viruses of birds or pigs, reaches people; the 2009 H1N1 pandemic began this way. Such viruses often arise by reassortment: the genome is in eight segments, and two viruses infecting the same cell can swap them.

Copying RNA into DNA

In the 1960s Howard Temin proposed that some RNA tumour viruses copy their genetic information into DNA, against the accepted rule that information flows only from DNA to RNA. In 1970, Temin and David Baltimore independently found the enzyme that does it in the virions, reverse transcriptase, and they shared the 1975 Nobel Prize in Physiology or Medicine with Renato Dulbecco. Reverse transcriptase copies RNA into DNA and DNA into DNA, and its RNase H part destroys the RNA of an RNA–DNA hybrid.

  1. 1

    A tRNA primes the first strand

    In the virion, a cellular tRNA for lysine is paired with the primer-binding site near the 5′ end of HIV's RNA. Once the virus is inside a cell, reverse transcriptase extends it, copying the RNA into DNA.
  2. 2

    The new DNA jumps

    The short first DNA moves to the matching repeat at the 3′ end of the RNA and is extended along the whole genome.
  3. 3

    RNase H clears the RNA

    RNase H cuts away the RNA template, except two short purine-rich stretches, which prime the second DNA strand.
  4. 4

    A double-stranded copy

    Reverse transcriptase finishes both strands, giving a linear double-stranded DNA with long terminal repeats (LTRs) at both ends.
  5. 5

    Integrase inserts it

    In the nucleus, integrase joins the DNA's 3′ ends to a chromosome, and the cell repairs the gaps. The inserted DNA, the provirus, is transcribed by RNA polymerase II from a promoter in its LTR, making viral mRNAs and new genomes.
Reverse transcription of HIV
HIV's genome copied into DNA, drawn flat as a ladder across the figure. First, the viral RNA, a red strand, runs along the top from its 5′ end on the left to its 3′ end on the right; near its left end a deeper red tRNA, its body hanging below, is paired with the RNA, and from it a short blue DNA strand runs left along the bottom to the RNA's end, where a green enzyme, reverse transcriptase, sits with its paler RNase H domain behind it. Next, the RNA's left end has gone, and the short DNA, with its tRNA, has moved to the right end, where it pairs with the RNA's last stretch; the blue strand now runs all the way back along the RNA, the enzyme at its left end. Then the RNA has gone apart from two short red pieces, and a deeper blue strand grows from each: one from the middle, with the enzyme at its growing end, and one from the piece near the right, which runs on to the tRNA. Next, the tRNA has gone and the short deeper blue piece on the right has moved to the far left, and both strands now run the full length, longer than the RNA was at both ends. Last, drawn smaller, the double-stranded DNA sits in a paler blue chromosome that runs on to either edge, joined to it on each side with a short gap in one strand, a deeper green integrase at each join. Step 1 of 5: A tRNA primes the first strand. A tRNA for lysine is paired with the PBS near the RNA's 5′ end. Reverse transcriptase extends it, copying U5 and R, until it reaches the end of the RNA.5′3′3′

Step 1 of 5A tRNA primes the first strand. A tRNA for lysine is paired with the PBS near the RNA's 5′ end. Reverse transcriptase extends it, copying U5 and R, until it reaches the end of the RNA.

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Reverse transcriptase copies HIV's RNA into double-stranded DNA in two jumps between matching sequences, which give the DNA a long terminal repeat at each end; integrase then inserts the DNA into a chromosome.

Simplified: the strands drawn flat, as a ladder, not as helices; not to scale: every region is far longer than drawn, and the genome's middle is much shortened; one RNA drawn; each virion carries two copies, and the first jump can land on either; the tRNA drawn whole; to pair with the PBS, its 3′ end unfolds from the cloverleaf; one reverse transcriptase drawn, and the RNA cleared in one step; in fact RNase H cuts the RNA while the minus strand is being made; the two plus strands shown meeting in the middle; in fact the one from the left displaces the start of the one from the central PPT for a short way; integrase drawn at each end; it holds both ends together, and first cuts one strand at each end to leave a 3′-OH.

5 sources
  1. Coffin JM, Hughes SH, Varmus HE, editors. Retroviruses. Cold Spring Harbor Laboratory Press, 1997. Overview of reverse transcription.
  2. Coffin JM, Hughes SH, Varmus HE, editors. Retroviruses. Cold Spring Harbor Laboratory Press, 1997. Reverse transcription of the viral genome in vivo.
  3. Coffin JM, Hughes SH, Varmus HE, editors. Retroviruses. Cold Spring Harbor Laboratory Press, 1997. Transcription.
  4. Piekna-Przybylska D, Dykes C, Demeter LM, Bambara RA. Sequences in the U3 region of human immunodeficiency virus 1 improve efficiency of minus strand transfer in infected cells. Virology, 2011.
  5. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell, 4th edition. Garland Science, 2002. Site-specific recombination.

The provirus stays for the life of the cell, and in resting cells it can lie silent for years. Reverse transcriptase does not proofread either, and each virion carries two copies of the RNA, which the enzyme can switch between, recombining them. HIV therefore changes as it multiplies, and a virus resistant to one drug arises easily; this is why at least two drugs from different classes are used together.

Hepatitis B goes the other way round. Its small DNA genome, a partly double-stranded circle, is completed in the nucleus into a closed circle, cccDNA, which RNA polymerase II transcribes. One of its transcripts, the pregenomic RNA, is packed into a new capsid with the viral polymerase, which copies it back into DNA there, priming the first strand with the polymerase protein itself. The cccDNA stays in the nucleus, and drugs that block the polymerase do not remove it.

Reverse transcription in our own cells

Telomerase is a reverse transcriptase that carries its own RNA template and adds repeats to the ends of chromosomes (DNA replication). Many of our transposable elements also move through RNA: a LINE-1 element encodes a protein that nicks the chromosome and copies LINE-1 RNA into DNA at the nick, and Alu elements, short sequences with no protein of their own, are copied by LINE-1's enzymes. About half of our genome comes from transposable elements, most of them long since unable to move. Occasionally one still does: a new LINE-1 copy inserted into the factor VIII gene has caused haemophilia A.

In the laboratory, reverse transcriptase copies mRNA into complementary DNA (cDNA), which can be cloned, sequenced or amplified. In RT-PCR the cDNA is then amplified by PCR, and the quantitative form of the test measures how much HIV RNA is in a patient's blood, the viral load (Molecular biology methods).

Check yourselfAnswer, then say how sure you were: a lucky guess still counts as a weak point.

1The purified RNA of poliovirus, put into a cell, starts an infection; the purified RNA of measles virus does not. Why?

2What primes the first DNA strand that HIV's reverse transcriptase makes?

3Why can antiretroviral drugs control HIV but not cure it?

4Why is the composition of the influenza vaccine reviewed every year?

5How does zidovudine stop HIV reverse transcription?

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Reverse transcription

RNA replication and reverse transcription.

  • Antiviral drugsPharmacologyNot written yetReverse transcriptase inhibitors such as tenofovir, a nucleotide analogue, block it.
  • Chronic viral hepatitisInternal medicineNot written yetHepatitis B copies itself through an RNA intermediate, so tenofovir and entecavir suppress it.