Ltr1

transcription reverse transcription

FIGURE 11 -23 Mechanism of retroviral integration and transposition of viral-like retrotransposons. The top panel shows integrated provirus, for a more detaifed view of the LTR sequences, see the figures in Box 11-2. The promoter for transcription of the viral RNA is embedded in the left tTR as shown. cDNA synthesis from this viral RNA is explained in Bo* 11-2. The integrase-catalyzed DNA cleavage and DNA st:and transfer steps are shown.

integrase-cataiyzed 3'end cleavage

I 5 SOU

released dinucleotide target DNA 3.

integrase-cataiyzed 3'end cleavage

I 5 SOU

released dinucleotide target DNA 3.

integrase-cataiyzed Dt\iA strand transfer

new integrated copy gap repair and ligation s I

tion. This reconstruction of the LTRs is essential for recognition of the cDNA by integrase and for subsequent recombination,

DNA Transposases and Retroviral Integrases Are Members of a Protein Superfamily

As we have seen, DNA cleavage of the 3' ends of the transposon DNA (or cDNA] and DNA strand transfer are common steps used for DNA transposition and the movement of viral-like retrotransposons and retroviruses. This conserved recombination mechanism is reflected in the structure of the transposasc/integrase proteins {Figure 11-24). High-resolution structures reveal that many different transposases

322 Site-Specific Recombination and Transposition o) DNA Box 11-2 The Pathway of Retroviral cDNA Formation

To understand the process of retroviral reverse transcription (or that of the viral-like retrotransposons), we first need to look in more detail at the structure of the LTR sequences. Each LTR is constructed of three sequence elements. These are called: U3 (for unique 3' end), R (for repeat), and U5 (for unique 5' end). Transcription from the integrated copy of the retroviral genome generates the viral RNA with the R sequence at each end (Box 11-2 Figure 1). Therefore, during the process of reverse transcription, one additional U3 and Ub region must be synthesized. As explained below, this duplication happens because priming of DNA synthesis occurs at internal sires within the RNA genome and the R sequence allows two "strand switches" to occur during the replication process.

It is the viral RNA that is packaged into virus partides, and this RNA enters the new cell during infection The viral RNA is packaged with a cellular tRNA molecule (see Chapter 14) that serves as the primer for synthesis of the first cDNA strand. This tRNA forms base pairs with a specific sequence near the U5 region, known as the primer-binding site (PBS) (Box 11-2 Figure 2a). DNA synthesis by the reverse transcriptase enzyme then copies the U5 region and the first R segment (Box 11-2 Figure 2b).

Reverse transcriptase has two enzymatic activities that are important for cDNA formation: a DNA polymerase activity and an RNAse H activity. RNAse H enzymes degrade RNA that is base-paired with DNA (as we discussed in Chapter 8). Dunng reverse transcription, RNAse H removes the template RNA strands. When this step occurs on the first RNA-DNA hybrid intermediate (see Box 11-2 Figures 2b and 2c), the U5-R DNA strand is released in a single-stranded form.

This U5-R DNA strand can then base-pair with the R region on the other end of the viral RNA molecule (Box 11-2 Figure 2d). This step is the first of the two strand switches. Once this switching occurs, reverse transcriptase continues DNA synthesis to copy the remainder of the RNA template (Box 11-2 Figure 2e). The resulting DNA strand ends with the PBS sequence at its 3' terminus. The RNA template strand is removed, as before, by RNAse H (Box 11-2 Figures 2d and 2e)

RNAse H mediated degradation of the viral RNA also generates an RNA fragment that serves as the primer for synthesis of the second cDNA strand I his region of RNA remains base-paired with a sequence celled the polypurine tract (PPT) at the edge of the U3 sequence (Box 11-2 Figures 2e and 2f). Elongation of this primer copies the U3, R, U5, and PBS sequences into DNA

Once the tRNA primer is removed from the first cDNA strand, the second strand switch occurs. The complementary sequence of the PBS on the 3' ends allows base-pairing interactions between the two DNA strands and formation of a circular intermediate. Elongation of each of the 3' DNA ends present in this intermediate to the end of the other strand generates the double-stranded cDNA with two complete LTR sequences, This DNA molecule is then ready to be integrated into the cell's genome by the integrase protein-

Reverse transcriptase is a virus-encoded (or retrotranspo-son encoded) enzyme end serves no essential cellular function. It is, however, absolutely essential for retrovirus replication. Thus, it is a common target of antiviral drugs, including many of the drugs that have been used to fight the AIDS epidemic.

R US gag pol env U3 RNA S'M "1 " I 1. I M3'

reverse transcription integration

DNA U3 R U5 gag pot em U3

LTR LTR

| transcription

BOX 11-2 FIGURE 1 Detailed view of the sequence elements near the ends of the retroviral RNA and cONA. Viral like retrotransposons have a very similar sequence organization. The pat gene encodes both reverse transcriptase (including the RNAse H activity) and mlegrase

Box 11-2 (Continued)

a rna

(RNA primer

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