Two tort,erred sequence motifs, oc~:urrin$ in HIV-I r~verse transeriptase~ at re,,dues II0+A 16 trod 183~190. have been studied ufinB ~ite.direeted mutall,,,tesis of the cloned llene. In particular, asp, flares at positioni 185 and 11t6 have each been rout;tied to either asparalline or glutamate, The resulting mutant proteins wer~ catalytically inactive but still able to bind th~ template.primer complex, ooly rA.oli~o tiT. Other mutations in these re|lions resulted in reduced reverse transcriptase activity but the mutation of tyrosine-183 to serlne caused a stllnifit~mt increase in the g,~ for dTTP and the K+ f,~r inhibition by 3'-azi,lotltymidine.lriphosphatc, 2+.Y-dldeox.vthymidine-tripho~phatc and phosphonoformie acid,

The activity, metabolism, and mode of action of (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (H2G) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and varicella-zoster virus (VZV) were studied. Compared to acyclovir (ACV), H2G has superior activity against VZV (50% inhibitory concentration of 2.3 M) and Epstein-Barr virus (50% inhibitory concentration of 0.9 M), comparable activity against HSV-1, and weaker activity against HSV-2. The antiviral effect on HSV-1 showed persistence after removal of compound. H2G was metabolized to its mono-, di-and triphosphate derivatives in virus-infected cells, with H2G-triphosphate being the predominant product. Only small amounts of H2G-triphosphate were detected in uninfected cells (1 to 10 pmol/10 6 cells), whereas the level in HSV-1-infected cells reached 1,900 pmol/10 6 cells. H2G was a substrate for all three viral thymidine kinases and could also be phosphorylated by mitochondrial deoxyguanosine kinase. The intracellular half-life of H2G-triphosphate varied in uninfected (2.5 h) and infected (HSV-1, 14 h; VZV, 3.7 h) cells but was always longer than the half-life of ACV-triphosphate (1 to 2 h). H2G-triphosphate inhibited HSV-1, HSV-2, and VZV DNA polymerases competitively with dGTP (K i of 2.8, 2.2, and 0.3 M, respectively) but could not replace dGTP as a substrate in a polymerase assay. H2G was not an obligate chain terminator but would only support limited DNA chain extension. Only very small amounts of radioactivity, which were too low to be identified by high-performance liquid chromatography analysis of the digested DNA, could be detected in purified DNA from uninfected cells incubated with [ (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (H2G) is an acyclic guanosine analog with structural similarities to acyclovir (ACV){9-[(2-hydroxyethoxy)methyl]guanine}, the established treatment for herpesvirus infections (6), and penciclovir (PCV) [9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine], a compound recently licensed in some countries for the treatment of herpes zoster (29). The racemic mixture of (RS)H2G [previously abbreviated (Ϯ)2HM-HBG (1, 3, 17)] was originally shown to have good activity against herpes simplex virus (HSV) (18) and varicella-zoster virus (VZV) in vitro (3) and in vivo (17), and subsequent studies with the separated isomers showed that this activity resided predominantly with the R isomer (1, 25), now called H2G (25,27). H2G has activity against HSV type 1 (HSV-1) and type 2 (HSV-2) (1) and has been reported to have activity against human herpesvirus type 6 (4).Such a broad-spectrum anti-herpesvirus activity makes H2G an interesting candidate for clinical development, particularly for the treatment of VZV infection, where its impressive activity could lead to improved efficacy over current therapies.Strains of VZV that are deficient in thymidine kinase (TK) are resistant to H2G (1, 3), suggesting a mechanism of activation involving selective phosphorylation by herpesvirus-induced TKs, as is the case for ACV and PCV. This is support...

HIV reverse rr#nscripcvaie; RNuLEW; Probin cnpinecrin$: s-Trrbulin epiropc; Immunaulfniry puriReatian I. INTRODUCTIONReverse transcriptase is an enzyme of central importancc in rhc replication of HIV, the causative agent of AIDS [l]. Part of the HIV-I polgenecodes for a p66RT subunit that is cleaved at the C-terminal region by the HIV protcase to give a p66/p51 heterodimcr, the form of RT observed in virions f2), An RNaseH domain has been identified within the last 120 amino acids of the Cterminal region of the p66 subunit by sequence analysis [3] and functional studies [4].We have previously described immunoaffinity chromatography of RT using immobilized monoclonal antibodies raised to HIV-I RT [5]. Elution of RT from these columns requires vigorous conditions which may have deleterious effects on enzyme structure. This together with the C-terminal heterogeneity observed in these preparations [6], may contribute to the disorder observed in three different crystal forms of RT when ex- amincd in an X-ray beam [7]. We thus wished to devise a mild isolation procedure which could produce protein with an intact C-terminus, The work of Wehland et al, [8] on the detailed mapping of the amino acid sequence requirement for the binding of m-tubulin to the monoclonal antibody YL1/2 [9] showed that an essentially linear epitope was located at the C-terminus OF the polypeptide chain. There was a requirement for a C-terminal aromatic residue (either tyrosine or phenylalanine) containing a free carboxyl group, preceded by an acidic residue (glutamic or aspartic acid). A further adjacent acidic residue greatly increased binding, It was also demonstrated that the loss of the C-terminal aromatic residue abolished the binding of cr-tubulin as well as related peptides to the YL1/2 antibody [8]. We therefore reasoned that incorporation of this epitope into HIV RT and RNaseH could afford a method of purifying these proteins with a homogeneous C-terminus using a simple peptide elution procedure from columns of immobilized YLl/:! antibody. Incorporation of this a-tubulin epitope into p66RT and RNaseH can be achieved by mutating the C-terminus from Lys-Val-Leu to Glu-Glu-Phe. The site of HIV protease cleavage of the p66RT subunit has been shown to be between a Phe and Tyr giving a ~51 subunit with a C-terminal sequence of Glu-Thr+he [IO]. Therefore only a single change of Thr to Glu is required to incorporate the C+ tubulin epitope at the C-terminus of the ~51 polypeptide. In changing the natural amino acid sequence of a protein there is the possibility of changing the biological characteristics of that protein. It has, however, previously been shown that the C-terminus of HIV-1 RT 298Published by Elsevier Sciettce Publishers B. V.

DNA plasmids encoding the open reading frames of canine oral papillomavirus (COPV) nonstructural early genes E1, E2, or E7 protein were delivered into both oral mucosal and cutaneous epithelial sites in beagle dogs using particle-mediated immunotherapeutic delivery (PMID) technology. Control dogs were vaccinated with plasmid encoding either hepatitis B virus surface antigen (HBVs) or COPV L1. Using a prophylactic immunisation protocol, a priming dose of plasmid DNA was followed by a booster dose 6 weeks later. Four weeks after boost, all dogs were challenged with infectious COPV particles. Following viral challenge, as shown previously (M. A. Stanley et al., 2001, Vaccine 19, 2783-2792), mucosal papillomas developed in the negative-control HBVs vaccinated dogs, but all animals in the COPV L1 group were fully protected from disease development. In the early gene-vaccinated groups five of six in the E1-vaccinated dogs, two of six in E2-vaccinated dogs, and three of six in the E7-vaccinated beagles developed oral papillomas. Compared to the HBVs negative-control group the oral papillomas that did develop in the early-gene vaccinated beagles were significantly smaller, shorter in duration, and fewer in number. Taken together the disease burden was markedly reduced and this was statistically significant. In a second experiment one group of animals was vaccinated with plasmid encoding the wild-type COPV E1 gene, and a separate group was vaccinated with plasmid encoding a synthetic codon-optimised COPV E1 gene sequence. None of the codon-optimised E1-vaccinated animals developed papillomas at any challenge site. However, all animals vaccinated with wild-type E1 had papillomas. These data suggest that immunisation by PMID with papillomavirus early genes can significantly impact upon subsequent disease development and that full protection can be achieved using improved vectors encoding codon-optimised gene sequences perhaps emphasizing the importance of antigen load in the generation of protective responses to papillomavirus proteins.