Stephen H. Hughes

Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA.

We have determined the 3.0 Å resolution structure of wild‐type HIV‐1 reverse transcriptase in complex with an RNA:DNA oligonucleotide whose sequence includes a purine‐rich segment from the HIV‐1 genome called the polypurine tract (PPT). The PPT is resistant to ribonuclease H (RNase H) cleavage and is used as a primer for second DNA strand synthesis. The ‘RNase H primer grip’, consisting of amino acids that interact with the DNA primer strand, may contribute to RNase H catalysis and cleavage specificity. Cleavage specificity is also controlled by the width of the minor groove and the trajectory of the RNA:DNA, both of which are sequence dependent. An unusual ‘unzipping’ of 7 bp occurs in the adenine stretch of the PPT: an unpaired base on the template strand takes the base pairing out of register and then, following two offset base pairs, an unpaired base on the primer strand re‐establishes the normal register. The structural aberration extends to the RNase H active site and may play a role in the resistance of PPT to RNase H cleavage.

Structure and Function of HIV-1 Reverse Transcriptase: Molecular Mechanisms of Polymerization and Inhibition

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.

Selective Excision of AZTMP by Drug-Resistant Human Immunodeficiency Virus Reverse Transcriptase

ABSTRACT Two distinct mechanisms can be envisioned for resistance of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to nucleoside analogs: one in which the mutations interfere with the ability of HIV-1 RT to incorporate the analog, and the other in which the mutations enhance the excision of the analog after it has been incorporated. It has been clear for some time that there are mutations that selectively interfere with the incorporation of nucleoside analogs; however, it has only recently been proposed that zidovudine (AZT) resistance can involve the excision of the nucleoside analog after it has been incorporated into viral DNA. Although this proposal resolves some important issues, it leaves some questions unanswered. In particular, how do the AZT resistance mutations enhance excision, and what mechanism(s) causes the excision reaction to be relatively specific for AZT? We have used both structural and biochemical data to develop a model. In this model, several of the mutations associated with AZT resistance act primarily to enhance the binding of ATP, which is the most likely pyrophosphate donor in the in vivo excision reaction. The AZT resistance mutations serve to increase the affinity of RT for ATP so that, at physiological ATP concentrations, excision is reasonably efficient. So far as we can determine, the specificity of the excision reaction for an AZT-terminated primer is not due to the mutations that confer resistance, but depends instead on the structure of the region around the HIV-1 RT polymerase active site and on its interactions with the azido group of AZT. Steric constraints involving the azido group cause the end of an AZT 5′-monophosphate-terminated primer to preferentially reside at the nucleotide binding site, which favors excision.

Flexible Use of Nuclear Import Pathways by HIV-1

HIV-1 replication requires transport of nascent viral DNA and associated virion proteins, the retroviral preintegration complex (PIC), into the nucleus. Too large for passive diffusion through nuclear pore complexes (NPCs), PICs use cellular nuclear transport mechanisms and nucleoporins (NUPs), the NPC components that permit selective nuclear-cytoplasmic exchange, but the details remain unclear. Here we identify a fragment of the cleavage and polyadenylation factor 6, CPSF6, as a potent inhibitor of HIV-1 infection. When enriched in the cytoplasm, CPSF6 prevents HIV-1 nuclear entry by targeting the viral capsid (CA). HIV-1 harboring the N74D mutation in CA fails to interact with CPSF6 and evades the nuclear import restriction. Interestingly, whereas wild-type HIV-1 requires NUP153, N74D HIV-1 mimics feline immunodeficiency virus nuclear import requirements and is more sensitive to NUP155 depletion. These findings reveal a remarkable flexibility in HIV-1 nuclear transport and highlight a single residue in CA as essential in regulating interactions with NUPs.

High-resolution structures of HIV-1 reverse transcriptase/TMC278 complexes: Strategic flexibility explains potency against resistance mutations.

TMC278 is a diarylpyrimidine (DAPY) nonnucleoside reverse transcriptase inhibitor (NNRTI) that is highly effective in treating wild-type and drug-resistant HIV-1 infections in clinical trials at relatively low doses (∼25–75 mg/day). We have determined the structure of wild-type HIV-1 RT complexed with TMC278 at 1.8 Å resolution, using an RT crystal form engineered by systematic RT mutagenesis. This high-resolution structure reveals that the cyanovinyl group of TMC278 is positioned in a hydrophobic tunnel connecting the NNRTI-binding pocket to the nucleic acid-binding cleft. The crystal structures of TMC278 in complexes with the double mutant K103N/Y181C (2.1 Å) and L100I/K103N HIV-1 RTs (2.9 Å) demonstrated that TMC278 adapts to bind mutant RTs. In the K103N/Y181C RT/TMC278 structure, loss of the aromatic ring interaction caused by the Y181C mutation is counterbalanced by interactions between the cyanovinyl group of TMC278 and the aromatic side chain of Y183, which is facilitated by an ∼1.5 Å shift of the conserved Y183MDD motif. In the L100I/K103N RT/TMC278 structure, the binding mode of TMC278 is significantly altered so that the drug conforms to changes in the binding pocket primarily caused by the L100I mutation. The flexible binding pocket acts as a molecular “shrink wrap” that makes a shape complementary to the optimized TMC278 in wild-type and drug-resistant forms of HIV-1 RT. The crystal structures provide a better understanding of how the flexibility of an inhibitor can compensate for drug-resistance mutations.

Structures of HIV‐1 reverse transcriptase with pre‐ and post‐translocation AZTMP‐terminated DNA

AZT (3′‐azido‐3′‐deoxythymidine) resistance involves the enhanced excision of AZTMP from the end of the primer strand by HIV‐1 reverse transcriptase. This reaction can occur when an AZTMP‐terminated primer is bound at the nucleotide‐binding site (pre‐translocation complex N) but not at the ‘priming’ site (post‐translocation complex P). We determined the crystal structures of N and P complexes at 3.0 and 3.1 Å resolution. These structures provide insight into the structural basis of AZTMP excision and the mechanism of translocation. Docking of a dNTP in the P complex structure suggests steric crowding in forming a stable ternary complex that should increase the relative amount of the N complex, which is the substrate for excision. Structural differences between complexes N and P suggest that the conserved YMDD loop is involved in translocation, acting as a springboard that helps to propel the primer terminus from the N to the P site after dNMP incorporation.

H3K4me3 Interactions with TAF3 Regulate Preinitiation Complex Assembly and Selective Gene Activation

Histone modifications regulate chromatin-dependent processes, yet the mechanisms by which they contribute to specific outcomes remain unclear. H3K4me3 is a prominent histone mark that is associated with active genes and promotes transcription through interactions with effector proteins that include initiation factor TFIID. We demonstrate that H3K4me3-TAF3 interactions direct global TFIID recruitment to active genes, some of which are p53 targets. Further analyses show that (1) H3K4me3 enhances p53-dependent transcription by stimulating preinitiation complex (PIC) formation; (2) H3K4me3, through TAF3 interactions, can act either independently or cooperatively with the TATA box to direct PIC formation and transcription; and (3) H3K4me3-TAF3/TFIID interactions regulate gene-selective functions of p53 in response to genotoxic stress. Our findings indicate a mechanism by which H3K4me3 directs PIC assembly for the rapid induction of specific p53 target genes.

Structural and Functional Analyses of the Second-Generation Integrase Strand Transfer Inhibitor Dolutegravir (S/GSK1349572)

Raltegravir (RAL) and related HIV-1 integrase (IN) strand transfer inhibitors (INSTIs) efficiently block viral replication in vitro and suppress viremia in patients. These small molecules bind to the IN active site, causing it to disengage from the deoxyadenosine at the 3′ end of viral DNA. The emergence of viral strains that are highly resistant to RAL underscores the pressing need to develop INSTIs with improved resistance profiles. Herein, we show that the candidate second-generation drug dolutegravir (DTG, S/GSK1349572) effectively inhibits a panel of HIV-1 IN variants resistant to first-generation INSTIs. To elucidate the structural basis for the increased potency of DTG against RAL-resistant INs, we determined crystal structures of wild-type and mutant prototype foamy virus intasomes bound to this compound. The overall IN binding mode of DTG is strikingly similar to that of the tricyclic hydroxypyrrole MK-2048. Both second-generation INSTIs occupy almost the same physical space within the IN active site and make contacts with the β4–α2 loop of the catalytic core domain. The extended linker region connecting the metal chelating core and the halobenzyl group of DTG allows it to enter farther into the pocket vacated by the displaced viral DNA base and to make more intimate contacts with viral DNA, compared with those made by RAL and other INSTIs. In addition, our structures suggest that DTG has the ability to subtly readjust its position and conformation in response to structural changes in the active sites of RAL-resistant INs.

Transgenic chickens: Insertion of retroviral genes into the chicken germ line

We infected early chicken embryos by injection of wild-type and recombinant avian leukosis viruses into the yolk of unincubated, fertile eggs. The viremic males (designated generation 0 (G-0] were tested for transmission of proviral DNA to their G-1 progeny. Nine of 37 G-0 viremic males were mosiac and proviral DNA was transmitted to their progeny at frequencies varying from 1 to 11%. All of the G-1 progeny examined by restriction enzyme analysis for clonality of proviral junction fragments had one to three simple but different fragments. The proviral DNA was transmitted from G-1 to the G-2 progeny in a Mendelian fashion thus proving that retroviral genes have been inserted into the chicken germ line. One of the viruses is a candidate vector for insertion of foreign genes into the chicken germ line.

Avian bic, a gene isolated from a common retroviral site in avian leukosis virus-induced lymphomas that encodes a noncoding RNA, cooperates with c-myc in lymphomagenesis and erythroleukemogenesis.

ABSTRACT bic is a novel gene identified at a common retroviral integration site in avian leukosis virus-induced lymphomas and has been implicated as a collaborator with c-myc in B lymphomagenesis. It lacks an extensive open reading frame and is believed to function as an untranslated RNA (W. Tam, Gene 274:157-167, 2001; W. Tam, D. Ben-Yehuda, and W. S. Hayward, Mol. Cell. Biol. 17:1490-1502, 1997). The oncogenic potential of bic, particularly its ability to cooperate with c-myc in oncogenesis, was tested directly by expressing c-myc and bic, either singly or in pairwise combination, in cultured chicken embryo fibroblasts (CEFs) and in chickens using replication-competent retrovirus vectors. Coexpression of c-myc and bic in CEFs caused growth enhancement of cells. Most importantly, chick oncogenicity assays demonstrated that bic can cooperate with c-myc in lymphomagenesis and erythroleukemogenesis. The present study provides direct evidence for the involvement of untranslated RNAs in oncogenesis and provides further support for the role of noncoding RNAs as riboregulators.