Mechanisms of inhibition of HIV replication by non-nucleoside reverse transcriptase inhibitors

Abstract

The non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are a therapeutic class of compounds that are routinely used, in combination with other antiretroviral drugs, to treat HIV-1 infection. NNRTIs primarily block HIV-1 replication by preventing RT from completing reverse transcription of the viral single-stranded RNA genome into DNA. However, some NNRTIs, such as efavirenz, have been shown to inhibit the late stages of HIV-1 replication by interfering with HIV-1 Gag-Pol polyprotein processing, while others, such as the pyrimidinediones, have been shown to inhibit both HIV-1 RT-mediated reverse transcription and HIV-1/HIV-2 viral entry. Accordingly, in this review we describe the multiple mechanisms by which NNRTIs inhibit HIV-1 reverse transcription (and in some cases HIV-2 reverse transcription) and other key steps involved in HIV-1/HIV-2 replication.

Fig. 1

Diagram of HIV reverse transcription mediated by the DNA polymerase, RNase H and strand-transfer activities of the viral RT. Each step is described in more detail in the text. RDDP and DDDP refer to the RNA- and DNA-dependent DNA polymerase activities of RT, respectively.

Fig. 2

Chemical structures of the NNRTIs described in this review.

Fig. 3

Ribbon representation of the HIV-1 RT in complex with efavirenz (PDB coordinates 1FK9). The p66 and p51 subunits of HIV-1 RT are colored red and blue, respectively. Residues located in the DNA polymerase active site (Asp110, Asp185, Asp186) and the RNase H active site (Glu478, Asp443, Asp498) are indicated with yellow spheres.

Fig. 4

The HIV life-cycle. (1) HIV binds to a CD4 receptor and one of two co-receptors (CXCR4 or CCR5) on the surface of the CD4+ T-lymphocyte. (2) The viral envelope then fuses with the host cell membrane. After fusion, the viral capsid is released into the host cell cytoplasm. (3) The HIV RT converts the single-stranded HIV RNA into double-stranded DNA (dsDNA). (4) The proviral dsDNA integrates into the host cell's genome in a process mediated by the viral IN. (5) The replication and transcription machinery of the host cell is involved in provirus replication, and in viral mRNA synthesis. (6) Viral mRNAs are used in the synthesis of the viral proteins Gag, Gag-Pol and Env, as well as accessory proteins such as Nef, Vif, Vpr, and Vpu. (7) Sets of viral protein chains come together with 2 copies of the viral RNA to generate an immature virus particle that pushes out (or buds) from the cell, taking some of the cell membrane with it. (8) The virus then matures, which involves the processing of viral proteins by the HIV PR.

Fig. 5

Diagram of the temporal events involved in the initiation of plus-strand DNA synthesis.

Fig. 6

Superimposition of the HIV-1 RT in complex with MSK-076 (blue; PDB co-ordinates 1DTT) with the apo-form of HIV-2 RT (orange; PDB co-ordinates 1MU2), illustrating location of the NNRTI-binding pocket (in HIV-1) and conserved residues in the DNA polymerase active site (Asp110, Asp185, Asp186). Amino acid residues that were mutated in HIV-1 (Lys101, Tyr181 and Gly190) and HIV-2 (Ala101 and Gly112) during selection experiments with MSK-076 are also depicted.

Fig. 7

Western blot analysis of sequential processing products from a model Pol construct, pol-pET28a(+), expressed in E. coli strain BL21(DE3)pLysS and grown in the absence or presence of 5 μM efavirenz. Samples were taken at 60, 75, 90, 105, 120, 150 and 180 min post-induction, respectively. Western blots were probed with mAB 5B2 (anti-RT). As is clearly evident, more p66/p51 RT is generated in the efavirenz reaction than in the absence of drugs.