A mevalonate-independent pathway of isoprenoid biosynthesis present in Plasmodium falciparum was shown to represent an effective target for chemotherapy of malaria. This pathway includes 1-deoxy-D-xylulose 5-phosphate (DOXP) as a key metabolite. The presence of two genes encoding the enzymes DOXP synthase and DOXP reductoisomerase suggests that isoprenoid biosynthesis in P. falciparum depends on the DOXP pathway. This pathway is probably located in the apicoplast. The recombinant P. falciparum DOXP reductoisomerase was inhibited by fosmidomycin and its derivative, FR-900098. Both drugs suppressed the in vitro growth of multidrug-resistant P. falciparum strains. After therapy with these drugs, mice infected with the rodent malaria parasite P. vinckei were cured.

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The gcpE and lytB gene products control the terminal steps of isoprenoid biosynthesis via the 2-C-methyl-D-erythritol 4-phosphate pathway in Escherichia coli. In lytB-deficient mutants, a highly immunogenic compound accumulates significantly, compared to wild-type E. coli, but is apparently absent in gcpE-deficient mutants. Here, this compound was purified from E. coli v vlytB mutants by preparative anion exchange chromatography, and identified by mass spectrometry, 1 H, 13 C and 31 P NMR spectroscopy, and NOESY analysis as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). HMB-PP is 10 4 times more potent in activating human VQ Q9/VN N2 T cells than isopentenyl pyrophosphate. ß

Cell surface molecules that can act as virus receptors may exert an important selective pressure on RNA viral quasispecies. Large population passages of foot-and-mouth disease virus (FMDV) in cell culture select for mutant viruses that render dispensable a highly conserved Arg-Gly-Asp (RGD) motif responsible for integrin receptor recognition. Here, we provide evidence that viability of recombinant FMDVs including a Asp-1433Gly change at the RGD motif was conditioned by a number of capsid substitutions selected upon FMDV evolution in cell culture. Multiply passaged FMDVs acquired the ability to infect human K-562 cells, which do not express integrin ␣ v ␤ 3 . In contrast to previously described cell culture-adapted FMDVs, the RGD-independent infection did not require binding to the surface glycosaminoglycan heparan sulfate (HS). Viruses which do not bind HS and lack the RGD integrin-binding motif replicate efficiently in BHK-21 cells. Interestingly, FMDV mutants selected from the quasispecies for the inability to bind heparin regained sensitivity to inhibition by a synthetic peptide that represents the G-H loop of VP1. Thus, a single amino acid replacement leading to loss of HS recognition can shift preferential receptor usage of FMDV from HS to integrin. These results indicate at least three different mechanisms for cell recognition by FMDV and suggest a potential for this virus to use multiple, alternative receptors for entry even into the same cell type. RNA viruses mutate at rates of 10Ϫ3 to 10 Ϫ5 misincorporations per nucleotide copied; as a consequence, they evolve as complex mutant distributions termed viral quasispecies (17,19,34,35,51,52,54). Evolution of RNA viral quasispecies does not occur by the steady accumulation of mutations as replication proceeds but rather proceeds as the outcome of population disequilibrium in response to population size variations and environmental modifications. This is reflected in frequent fitness variations of RNA viruses as they replicate in cell culture or in vivo (3,12,18,27,29,33,42,64; reviewed in reference 16). Perturbation of equilibrium may lead to the rapid dominance of subsets of variants which were previously present at low frequency in the mutant spectrum. Expression at the cell surface of particular molecules which can act as receptors or coreceptors for the virus may have a major influence on the mutant distributions in viral quasispecies.Foot-and-mouth disease virus (FMDV) has been used in our laboratory as a model system to study viral quasispecies evolution, including the molecular basis of fitness variations (21, 22) and changes in host cell tropism (3,20). FMDV is an important animal pathogen that belongs to the aphthovirus genus of the Picornaviridae family (5, 55) and infects cattle and other cloven-hooved animals (artiodactyls) (2, 9). Integrin ␣ v ␤ 3 was the first molecule identified as a primary receptor for FMDV (4,6,24,38). Recent evidence suggests that integrin ␣ v ␤ 3 is the functional receptor for FMDV infections of cattle (50). The integrin ...

Recombinant LytB protein from the thermophilic eubacterium Aquifex aeolicus produced in Escherichia coli was puri¢ed to apparent homogeneity. The puri¢ed LytB protein catalyzed the reduction of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in a de¢ned in vitro system. The reaction products were identi¢ed as isopentenyl diphosphate and dimethylallyl diphosphate. A spectrophotometric assay was established to determine the steady-state kinetic parameters of LytB protein. The maximal speci¢c activity of 6.6 þ 0.3 W Wmol min 31 mg 31 protein was determined at pH 7.5 and 60 ‡C. The k cat value of the LytB protein was 3.7 þ 0.2 s 31 and the K m value for HMBPP was 590 þ 60 W WM.