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Review
. 2015 Apr;3(2):MDNA3-0027-2014.
doi: 10.1128/microbiolspec.MDNA3-0027-2014.

Reverse Transcription of Retroviruses and LTR Retrotransposons

Stephen H Hughes  1
Affiliations
Review

Reverse Transcription of Retroviruses and LTR Retrotransposons

Stephen H Hughes. Microbiol Spectr. 2015 Apr.

Abstract

The enzyme reverse transcriptase (RT) was discovered in retroviruses almost 50 years ago. The demonstration that other types of viruses, and what are now called retrotransposons, also replicated using an enzyme that could copy RNA into DNA came a few years later. The intensity of the research in both the process of reverse transcription and the enzyme RT was greatly stimulated by the recognition, in the mid-1980s, that human immunodeficiency virus (HIV) was a retrovirus and by the fact that the first successful anti-HIV drug, azidothymidine (AZT), is a substrate for RT. Although AZT monotherapy is a thing of the past, the most commonly prescribed, and most successful, combination therapies still involve one or both of the two major classes of anti-RT drugs. Although the basic mechanics of reverse transcription were worked out many years ago, and the first high-resolution structures of HIV RT are now more than 20 years old, we still have much to learn, particularly about the roles played by the host and viral factors that make the process of reverse transcription much more efficient in the cell than in the test tube. Moreover, we are only now beginning to understand how various host factors that are part of the innate immunity system interact with the process of reverse transcription to protect the host-cell genome, the host cell, and the whole host, from retroviral infection, and from unwanted retrotransposition.

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Figures

FIGURE 1
FIGURE 1
Reverse transcription of the genome of HIV-1. This figure shows, in cartoon form, the steps that are involved in the conversion of the ssRNA genome found in virions into dsDNA. In the figure, RNA is shown in green and DNA in purple. For simplicity, the 5′ cap and the poly(A) tail, which are present on the viral genomic RNA, have been omitted. The various sequence elements in the viral genome, including the genes, are not drawn to scale. (A) The tRNA primer (green arrow on the left) is base paired to the primer-binding site (PBS). (B) RT has initiated minus-strand DNA synthesis from the tRNA primer, and has copied the U5 and R sequences at the 5′ end of the genome. This creates an RNA/DNA duplex, which allows RNase H to degrade the RNA that has been copied (dotted green line). (C) The minus-strand DNA (see text) has been transferred, using the R sequence found at both ends of the viral RNA, to the 3′ end of the viral RNA, and minus-strand DNA synthesis can resume. The HIV-1 genome has two purine-rich sequences (polypurine tracts, or PPTs, one immediately adjacent to U3; the other, the central PPT [cPPT] is in the pol gene). (D–G) The two PPTs are relatively resistant to RNase H (D) and they serve as primers for plus-strand DNA synthesis (E). Once plus-strand DNA synthesis has been initiated, RNase H removes the PPT from the plus-strand DNAs (F, G). The plus-strand that is initiated in U3 is extended until the first 18 bases to the tRNA primer are copied (see text); RNase H then removes the tRNA primer (E). It appears that the entire PPT primers are removed by RNase H; however, RNase H leaves a single riboA on the 5′ end of the minus-strand (E, F). Removing the tRNA primer sets the stage for the second-strand transfer (F). Both the plus- and minus-strand DNAs are then elongated. The plus-strand that was initiated at the U3 junction (on the left in the figure) displaces a segment of the plus-strand that was initiated from the cPPT, creating a small flap, called the central flap (cFLAP). doi:10.1128/microbiolspec.MDNA3–0027-2014.f1
FIGURE 2
FIGURE 2
HIV-1 RT in a complex with dsDNA and an incoming dNTP. RT is shown as a ribbon diagram; the DNA and the incoming dNTP are shown as space filling models. The p51 subunit (at the bottom) is shown in gray. The RNase H domain is shown in pink, and the four subdomains to the polymerase domain are shown in different colors: fingers, blue; thumb, green; palm, red; and connection, yellow. The DNA template strand (the strand that is being copied) is dark red and the primer strand (the strand that is being extended) is purple. The incoming dNTP is light blue. doi:10.1128/microbiolspec.MDNA3–0027-2014.f2
FIGURE 3
FIGURE 3
Movement of HIV-1 RT during polymerization. The colors used for the various subunits, domains, and subdomains of RT, and for the two DNA strands, are as in Fig. 2. The p51 subunit is gray. The RNase H domain is shown in pink, and the four subdomains to the polymerase domain are as follows: fingers, blue; thumb, green; palm, red; and connection, yellow. The DNA template is dark red and the primer strand is purple. The incoming dNTP is light blue. The structural changes in RT can be correlated with specific steps in the binding of the substrates and the incorporation of the incoming dNTP. In unliganded RT, the fingers and thumb are closed (A). Before the dsDNA (or other nucleic acid substrate) can be bound, the thumb must move (A → B); this allows the dsDNA to be bound (B). This sets the stage for the binding of the incoming dNTP. When the incoming dNTP binds, the fingers close, which allows the dNTP to be incorporated, with the release of pyrophosphate (PPi). The incorporation of the dNTP temporarily leaves the end of the primer stand in the N or nucleoside triphosphate-binding site (see text). Translocation moves the nucleic acid by 1 bp, which allows the next dNTP to be bound and incorporated. doi:10.1128/microbiolspec.MDNA3–0027-2014.f3
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