EXPERIENCIA NACIONAL

Reverse transcription can be divided into three stages - initiation, RNA-dependent DNA synthesis, and DNA-dependent DNA synthesis. The initiation step marks the assembly o f the polymerase complex, the tRNA primer, and the genomic RNA template. In the second phase, RT catalyses the incorporation o f nucleotides onto the genomic RNA template. The resultant R NA-DNA duplex is transient, as the RNAse H component o f RT degrades the genome template shortly after incorporation. The single-stranded cD N A copy acts as a template for second strand synthesis. The DNA-dependent DNA polymerase generates a complementary strand. Strand transfer events, described in more detail below, enable the regeneration o f two LTRs from a single template copy. The mechanics o f this process are detailed below, with FIGURE 1-5 providing an accompaniment to the text by charting the nucleic acid manoeuvres involved in reverse transcription.

In it ia tio n

The isoacceptor tRNA^^®^ is incorporated into HIV-1 virions and specifically primes reverse transcription. Modified nucleotides within the tRNA^^®^ anticodon loop specifically interact with a hairpin structure found in the U5 region o f the genome. This facilitates complementary base-pairing between the 3 ’-terminal 18 nucleotides o f tRNA^^®^ and the HIV-1 primer-binding site (PBS) - a highly structured and highly conserved ISbp region located almost immediately downstream o f the U5 hairpin (Marquet et al., 1995).

The nucleocapsid protein makes essential contributions to this duplex formation. It partially melts the tRNA and genomic RNA secondary structures and physically blocks R NA se H cleavage o f the genomic template (Druillennec et al., 1999a). Completion o f this ‘initiation com plex’ stimulates RT to incorporate five nucleotides complementary to the RNA template immediately downstream o f the 3 ’ terminus o f the tRNA^^®^ primer (Lanchy et al., 1996a).

R N A -De p e n d e n t D N A Sy n t h e s is

After the initial incorporation o f five nucleotides, a transition to quasi-processive elongation occurs, i.e. unlike the five initial incorporations, RT incorporates more than one nucleotide before dissociating from the primer-template heteroduplex. This transition is probably dependent upon interactions between RT, tRNA^^®^, the upstream hairpin structure, and PBS sequences (Lanchy et al., 1998). Starting at the PBS, and continuing for approximately 180 nucleotides to the 5 ’ end o f the template, reverse transcriptase synthesises ‘minus-strand strong-stop D N A ’ (step 2 in FIGURE 1-5). Approximately 10-15 bases behind the point o f nucleotide incorporation, the RNAse H domain completely degrades the RNA constituents o f the new ly synthesised RNA/DNA duplex (step 3 in FIGURE 1-5). However, the tRNA^^®^/PBS RNA /R N A homoduplex is resistant to this cleavage, allowing later copying o f the PBS (Gotte et al., 1995).

Continuation o f reverse transcription is marked by the annealing o f the minus-strand strong-stop D N A to the complementary bases o f the 3 ’ R region belonging either to the same template or to that o f the co-packaged RNA genome m olecule (Panganiban and Fiore, 1988). Homologous and heterologous acceptor genomes seem to be selected with equal frequency (van Wamel and Berkhout, 1998). This is the first ‘strand transfer’ event o f reverse transcription, and is greatly assisted by three properties o f nucleocapsid protein - strand-annealing, inhibition o f self-priming by the minus-strand D NA , and effecting changes in RNAse H action (Peliska et al., 1994; Guo et al., 1997). Synthesis o f minus-strand D N A restarts and extends across the genome to the PBS in the 5 ’ LTR. Again, the RNA template is degraded by the trailing R NAse H domain, with the exception o f the two polypurine tracts (PPTs, step 4 in FIGURE 1-5).

D N A -De p e n d e n t D N A Sy n t h e s is

Co-incident with minus-strand synthesis, the retained PPT RNA sequences prime synthesis o f two positive-sense D N A strands labelled the upstream and downstream segments (labelled U+ and D + respectively, step 5 in FIGURE 1-5). D+ synthesis proceeds as far as the 3 ’PPT primer. Here, the strand displacement facility o f RT separates the D N A /D N A duplex allowing elongation to the end o f the U3-R-U5 LTR

(step 6 in FIGURE 1-5). The displaced U + positive-strand D N A acts as template for a further minus-strand synthesis extending to the 3 ’PPT, and so two LTRs are generated.

U + synthesis halts at a methylated adenosine 19 bases inside the preserved tRNA^^®^ primer, thus copying the entire PBS (Ben Artzi et al., 1996). Eventually, RNAse H degrades the RNA at the two PPT sites and the tRNA primer is released. This allows the second strand transfer event to take place, facilitated by a dimeric integrase ‘bridge’ maintaining the homologous 5 ’ and 3 ’ termini in close proximity (Miller et al., 1997). The single-stranded regenerated U + PBS (primer binding site, see Initiation above) anneals to the complementary minus-strand PBS (Panganiban and Fiore, 1988). Transcription is resumed and proceeds until the growing D N A strand reaches the first deoxy-nucleotide o f the D+ segment. A second strand displacement event takes place, allowing the U + strand to be extended by a further 99 nucleotides until the ‘central termination sequence’. Here, the minor groove o f the D N A is compressed, ejecting the RT enzyme and terminating reverse transcription (Chameau et al., 1994). The three- strand ‘central D N A flap’ thus created is unique to lentiviral reverse transcription and was recently shown to be essential for the post-transcription nuclear import o f PICs (Zennou et al., 2000).

FIGURE 1-5: Schematic diagram o f the process o f reverse transcription (after Gotte et a/., R U5 PBS Gag 1. C=M K > I________ Pol Digested by RNAse H cPPT Env U3 R 3 ’PPT 2

.

RNAse H digestion retarded

RNA DNA % U+ 5. D Digested by RNAse H D+ U+ Pol Gag R U5 Env 7. PBS cPPT 3’PPT