Structure and function of HIV-1 reverse transcriptase: molecular mechanisms of polymerization and inhibition

Abstract

The rapid replication of HIV-1 and the errors made during viral replication cause the virus to evolve rapidly in patients, making the problems of vaccine development and drug therapy particularly challenging. In the absence of an effective vaccine, drugs are the only useful treatment. Anti-HIV drugs work; so far drug therapy has saved more than three million years of life. Unfortunately, HIV-1 develops resistance to all of the available drugs. Although a number of useful anti-HIV drugs have been approved for use in patients, the problems associated with drug toxicity and the development of resistance means that the search for new drugs is an ongoing process. The three viral enzymes, reverse transcriptase (RT), integrase (IN), and protease (PR) are all good drug targets. Two distinct types of RT inhibitors, both of which block the polymerase activity of RT, have been approved to treat HIV-1 infections, nucleoside analogs (NRTIs) and nonnucleosides (NNRTIs), and there are promising leads for compounds that either block the RNase H activity or block the polymerase in other ways. A better understanding of the structure and function(s) of RT and of the mechanism(s) of inhibition can be used to generate better drugs; in particular, drugs that are effective against the current drug-resistant strains of HIV-1.

Figure 1

Reverse transcription of the HIV-1 genome (with permission from Annual Review of Biochemistry). Each retroviral particle contains two copies of the RNA genome. Minus-strand DNA synthesis starts near the 5’ end of the plus-stand RNA genome using as a primer a host tRNA_lys3 that anneals at the primer-binding site (PBS). Step 1: Synthesis proceeds to the 5’ end of the RNA genome through the U5 region, ending at the R region at the 5’end, forming the “minus-strand strong stop DNA.” Step 2: DNA synthesis is accompanied by RNase H digestion of the RNA portion of the RNA-DNA hybrid product, thus exposing the single-strand DNA product. Step 3: This exposure facilitates hybridization with the R region at the 3’ end of the same, or the second RNA genome, a strand-transfer reaction known as the “first jump”. Step 4: When minus-strand elongation passes a polypurine rich region called the polypurine tract (PPT) region, a unique plus-strand RNA primer is formed by RNase H cleavage at its borders. Plus-strand synthesis then continues back to the U5 region using the minus-strand DNA as a template. Step 5: Meanwhile, minus-strand synthesis continues through the genome using the plus-strand RNA as a template, and removing the RNA template in its wake via RNase H activity. Step 6: The RNase H digestion products formed are presumed to provide additional primers for plus-strand synthesis at a number of internal locations along the minus-strand DNA. Step 7: PPT-initiated plus-strand DNA synthesis stops after copying the annealed portion of the tRNA to generate the plus-strand DNA form o f the PBS, forming the “plus-strand strong stop” product. The tRNA is then removed by the RNase H activity of RT. Step 8: This may facilitate annealing to the PBS complement on the minus-strand DNA, providing the complementarity for the “second jump.” DNA synthesis then continues. Step 9: Strand displacement synthesis by RT to the PBS and PPT ends, and/or repair and ligation of a circular intermediate produces a linear duplex with long terminal repeats (LTRs) at both ends.

Figure 2

Ribbon representation of HIV-1 RT in a complex with nucleic acid. The fingers, palm, thumb, connection, and RNase H subdomains of the p66 subunit are shown in blue, red, green, yellow, and orange, respectively. The p51 subunit is shown in dark brown. The template and primer DNA strands are shown in light and dark gray, respectively.

Figure 3

Metal chelation in the polymerase active site of an enzyme/DNA/dNTP ternary complex. The nucleotide binding site (N site or pre-translocation site) and the priming site (P site or post-translocation site) are shown.

Figure 4

Reaction steps during the mechanism of DNA polymerization by HIV-1 RT.

Figure 5

Chemical structures of NRTIs.

Figure 6

Chemical structures of NNRTIs.

Figure 7

Ribbon representation of the NNRTI-binding pocket, showing the residues where NNRTI-resistance mutations occur.

Figure 8

Chemical structure of RNase H inhibitors (8A): 1, N-(4-t-butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone (BBNH); 2, dihydroxy benzoyl naphthyl hydrazone (DHBNH); 3, 4-chlorophenylhydrazone of mesoxalic acid (CPHM); 4, diketo acid; 5, N-hydroxyimide; 6, hydroxyl tropolone (2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one, or β-thujaplicinol); excision inhibitors (8B), and 4’-substituted nucleoside analogs (8C): 1, 4’ethynyl-2Fluoro deoxyadenosine (EFdA); 2, 4’methyl-deoxythymidine (MdT), 4’ethynyl-deoxythymidine (EdT).

Figure 9

Ribbon representation of HIV-1 RT with DHBNH bound (with permission from ACS Chem Biol). In the crystal structure of DHBNH, an RNase H inhibitor binds >50 Å away from the RNase H subdomain, at a site that partially overlaps the NNIBP. The subdomains of the p66 subunit are color-coded (fingers in blue, palm in red, thumb in green, connection in yellow, and RNase H in gold). Upper left inset: a close-up of the DHBNH binding site. The inhibitor is shown in magenta. The pocket that is occupied by NNRTIs is shown in gray (the NNRTI pocket is not occupied in this structure).

Figure 10

Potentiation of AZT antiviral activity against AZT-resistant HIV-1 by BPH218.