Graham Darby

High resolution structures of HIV-1 RT from four RT-inhibitor complexes.

We have determined the structures of four complexes of HIV-1 reverse transcriptase with non-nucleoside inhibitors, three fully refined at high resolution. The highest resolution structure is of the RT-nevirapine complex which has an R-factor of 0.186 and a root-mean-square bond length deviation of 0.015 Å for all data to 2.2 Å. The structures reveal a common mode of binding for these chemically diverse compounds. The common features of binding are largely hydrophobic interactions and arise from induced shape complementarity achieved by conformational rearrangement of the enzyme and conformational/ conf igurational rearrangement of the compounds.

The Role of Herpes Simplex Virus Type 1 Thymidine Kinase in Pathogenesis

A genetically engineered herpes simplex virus type 1 (HSV-1)thymidine kinase (TK) deletion mutant has been constructed and used to investigate the role of this gene in pathogenesis. Inoculation of mice with the HSV TK deletion mutant resulted in the establishment of latent ganglionic infection as demonstrated by superinfection of explanted ganglia with wild-type (wt) virus but not by routine explant culture suggesting that the virus-encoded TK is not essential for the establishment of latent infection but may be necessary for either reactivation or virus replication following reactivation. In addition, Southern blot hybridization has been used to demonstrate in vivo complementation of this mutant by wt virus in both peripheral and central nervous system tissues of mice during acute infection and to show that such complementation can result in the establishment and reactivation of latent TK- infection.

Zidovudine sensitivity of human immunodeficiency viruses from high-risk, symptom-free individuals during therapy

Human immunodeficiency type 1 isolates from 18 initially symptom-free men who were treated with zidovudine for 2 years were investigated for drug sensitivity. At the start all the men had persistent core antigenaemia; the acquired immunodeficiency syndrome developed in 6 during the study. The polymerase chain reaction was used to detect mutations at residue 215 of reverse transcriptase, a mutation associated with reduced drug sensitivity. After 2 years 16/18 isolates were mutant. However, after about 6 months of treatment the mutation was detected in only 7 isolates, 4 from individuals who subsequently had AIDS. Limited direct virus sensitivity data correlated with the genetic data. The rate of appearance of the 215 mutation seemed to correlate with CD4 counts and viral virulence.

Isolation and characterization of acyclovir-resistant mutants of herpes simplex virus

Mutants of HSV which are resistant to acyclovir (acycloguanosine) have been isolated following serial passages of several herpes simplex virus (HSV) strains in the presence of the drug. The majority of the mutants isolated are defective in induction of thymidine kinase (TK) and this is consistent with the observation that independently isolated TK- viruses are naturally resistant to ACV. One mutant is described (SC16 R9C2) which is resistant in biochemically transformed cells which express HSV TK. This suggests that its resistance resides at a level other than TK. It is also resistant to phosphonoacetic acid, suggesting that the DNA polymerase locus may be involved. A further mutant is described [Cl (101) P2C5] which induces normal levels of TK, although the nature of resistance of this virus is not yet elucidated.

Evidence that the 'active centre' of the herpes simplex virus thymidine kinase involves an interaction between three distinct regions of the polypeptide.

The nucleotide sequence of the coding region of the thymidine kinase gene from each of three mutant strains of herpes simplex virus type 1 and from the parental strain, SC16, has been determined. The mutants were known to express thymidine kinase enzymes with distinct substrate binding properties. Consideration of the lesions in the genes responsible for these altered biochemical properties. Consideration of the lesions in the genes responsible for these altered biochemical properties has led us to postulate a preliminary model for the active centre of the enzyme, involving the cooperation of three distinct regions of the polypeptide.

Herpes simplex virus latency: the cellular location of virus in dorsal root ganglia and the fate of the infected cell following virus activation.

Latent infections have been established in cervical dorsal root ganglia of Balb/c mice following peripheral inoculation of ts mutants of HSV-1. Activation of the mutant LS2 at the non-permissive temperature in vitro by culture of excised ganglia, or in vivo by neurosurgery has allowed the unambiguous identification of the neuron as the site of latent infection. Evidence is presented which shows that activation of virus infection in latently infected ganglia by neurosurgery results in a reduction of the number of latent foci in that ganglion. One interpretation of this observation is that the productive infection which follows activation of latent virus in the neuron leads to the destruction of that cell.

Human Immunodeficiency Virus MUTATIONS IN THE VIRAL PROTEASE THAT CONFER RESISTANCE TO SAQUINAVIR INCREASE THE DISSOCIATION RATE CONSTANT OF THE PROTEASE-SAQUINAVIR COMPLEX

Mutations in the human immunodeficiency virus (HIV) protease (L90M, G48V, and L90M/G48V) arise when HIV is passaged in the presence of the HIV protease inhibitor saquinavir. These mutations yield a virus with less sensitivity to the drug (L90M > G48V ≫ L90M/G48V). L90M, G48V, and L90M/G48V proteases have 1/20, 1/160, and 1/1000 the affinity for saquinavir compared to WT protease, respectively. Therefore, the affinity of mutant protease for saquinavir decreased as the sensitivity of the virus to saquinavir decreased. Association rate constants for WT and mutant proteases with saquinavir were similar, ranging from 2 to 4 × 107 M−1 s−1. In contrast, the dissociation rate constants for WT, L90M, G48V, and L90M/G48V proteases complexed with saquinavir were 0.0014, 0.019, 0.128, and 0.54 s−1, respectively. This indicated that the reduced affinity for mutant proteases and saquinavir is primarily the result of larger dissociation rate constants. The increased dissociation rate constants may be the result of a decrease in the internal equilibrium between the bound inhibitor with the protease flaps up and the bound inhibitor with the flaps down. Interestingly, the affinity of these mutant proteases for VX-478, ABT-538, AG-1343, or L-735,524 was not reduced as much as that for saquinavir. Finally, the catalytic constants of WT and mutant proteases were determined for eight small peptide substrates that mimic the viral cleavage sites in vivo. WT and L90M proteases had similar catalytic constants for these substrates. In contrast, G48V and L90M/G48V proteases had catalytic efficiency (kcat/Km) values with TLNF-PISP, RKIL-FLDG, and AETF-YVDG that were 1/10 to 1/20 the value of WT protease. The decreased catalytic efficiencies were primarily the result of increased Km values. Thus, mutations in the protease decrease the affinity of the enzyme for saquinavir and the catalytic efficiency with peptide substrates.

Characterization of a DNA polymerase mutant of herpes simplex virus from a severely immunocompromised patient receiving acyclovir

A series of herpes simplex virus isolates were recovered from a bone marrow transplant patient who received prolonged acyclovir therapy for indolent herpes simplex mouth and throat ulceration. Of 14 isolates received 10 were resistant to acyclovir and partially resistant to phosphonoacetic acid. Biochemical characterization revealed that resistance was due to an alteration in the virus DNA polymerase. DNA sequence analysis of the polymerase gene of a plaque-purified resistant virus isolate revealed a single nucleotide change when compared with the sequence of the gene of a plaque-purified sensitive isolate. This single base change resulted in a predicted amino acid substitution of Gly to Ser at residue number 841, a putative functional region of the polymerase.

Characterization of Abnormal Thymidine Kinases Induced by Drug-resistant Strains of Herpes Simplex Virus Type 1

Two TK+ acyclovir-resistant variants of herpes simplex virus (HSV) (S1 and Tr7) and one TK+ BVdU-resistant variant (B3) induce abnormal thymidine kinases with impaired ability to phosphorylate the drugs used in their isolation. These enzymes have been purified and their properties compared with those of the wild-type (wt) parent, SC16. The enzyme induced by S1 differed markedly from the other three in both its responses to salt and to pH. B3 TK recognized the enzymes natural substrates, thymidine, deoxycytidine, dTMP and ATP as well as the wt enzyme. In contrast, Tr7 and S1 TKs failed to bind deoxycytidine and bind thymidine less well than wt. Tr7 and S1 TKs had affinities for dTMP similar to those of B3 and the wt enzymes. ATP binding to wt, Tr7 and B3 enzymes was similar but this substrate bound only weakly to S1 TK. Each mutant displayed a characteristically distinct pattern of affinities for a range of nucleoside analogue substrates, suggesting that they will show some cross-resistance to drugs which have a similar mechanism of action to acyclovir and BVdU.

The multifunctional folic acid sythesis fas gene of Pneumocystis carinii appears to encode dihydropteroate synthase and hydroxymethyldihydropterin pyrophosphokinase

We describe the cloning of a multifunctional folic acid synthesis (fas) gene from Pneumocystis carinii. The nucleotide sequence contains an open reading frame interrupted by three introns, that encodes a protein of 740 amino acids with an Mr of 97,278. The predicted Fas protein has homology to two enzyme domains, dihydropteroate synthase and 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase, both of which are involved in folate synthesis, and at least one other region of unknown function.