The structure of the icosahedral bacteriophage MS2 has been determined to 3.3 A resolution by X-ray crystallography. The phase determination involved both molecular replacement at low resolution using a known structure and heavy-atom substitution. The coat protein has no structural similarity to that of any other known RNA virus.

Flaviviridae are small enveloped viruses hosting a positive-sense single-stranded RNA genome. Besides yellow fever virus, a landmark case in the history of virology, members of the Flavivirus genus, such as West Nile virus and dengue virus, are increasingly gaining attention due to their re-emergence and incidence in different areas of the world. Additional environmental and demographic considerations suggest that novel or known flaviviruses will continue to emerge in the future. Nevertheless, up to few years ago flaviviruses were considered low interest candidates for drug design. At the start of the European Union VIZIER Project, in 2004, just two crystal structures of protein domains from the flaviviral replication machinery were known. Such pioneering studies, however, indicated the flaviviral replication complex as a promising target for the development of antiviral compounds. Here we review structural and functional aspects emerging from the characterization of two main components (NS3 and NS5 proteins) of the flavivirus replication complex. Most of the reviewed results were achieved within the European Union VIZIER Project, and cover topics that span from viral genomics to structural biology and inhibition mechanisms. The ultimate aim of the reported approaches is to shed light on the design and development of antiviral drug leads.

Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate. This activity is universally required for fatty acid biosynthesis as well as for the production of a number of small molecules, pH homeostasis, and other functions. At least three different carbonic anhydrase families are known to exist, of which the ␣-class found in humans has been studied in most detail. In the present work, we describe the structures of two of the three ␤-class carbonic anhydrases that have been identified in Mycobacterium tuberculosis, i.e. Rv1284 and Rv3588c. Both structures were solved by molecular replacement and then refined to resolutions of 2.0 and 1.75 Å, respectively. The active site of Rv1284 is small and almost completely shielded from solvent, whereas that of Rv3588c is larger and quite open to solution. Differences in coordination of the active site metal are also observed. In Rv3588c, an aspartic acid side chain displaces a water molecule and coordinates directly to the zinc ion, thereby closing the zinc coordination sphere and breaking the salt link to a nearby arginine that is a feature of Rv1284. The two carbonic anhydrases thus exhibit both of the metal coordination geometries that have previously been observed for structures in this family. Activity studies demonstrate that Rv3588c is a completely functional carbonic anhydrase. The apparent lack of activity of Rv1284 in the present assay system is likely exacerbated by the observed depletion of zinc in the preparation.

The double mutant enzyme (D151N/E222Q) has lost its ability to bind the metal and, thereby, also its activity. Our structural information complemented with molecular dynamics simulations and free energy calculations provides the framework for the design of new inhibitors and gives new insights into the reaction mechanism. The conformation of fosmidomycin bound to the metal ion is different from that reported in a previously published structure and indicates that a rearrangement of the intermediate is not required during catalysis.

The K103N substitution is a frequently observed HIV-1 RT mutation in patients who do not respond to combinationtherapy. The drugs Efavirenz, MSC194 and PNU142721 belong to the recent generation of NNRTIs characterized by an improved resistance profile to the most common single point mutations within HIV-1 RT, including the K103N mutation. In the present study we present structural observations from Efavirenz in complex with wild-type protein and the K103N mutant and PNU142721 and MSC194 in complex with the K103N mutant. The structures unanimously indicate that the K103N substitution induces only minor positional adjustments of the three inhibitors and the residues lining the binding pocket. Thus, compared to the corresponding wild-type structures, these inhibitors bind to the mutant in a conservative mode rather than through major rearrangements. The structures implicate that the reduced inhibitory efficacy should be attributed to the changes in the chemical environment in the vicinity of the substituted N103 residue. This is supported by changes in hydrophobic and electrostatic interactions to the inhibitors between wild-type and K103N mutant complexes. These potent inhibitors accommodate to the K103N mutation by forming new interactions to the N103 side chain. Our results are consistent with the proposal by Hsiou et al. [Hsiou, Y., Ding, J., Das, K., Clark, A.D. Jr, Boyer, P.L., Lewi, P., Janssen, P.A., Kleim, J.P., Rosner, M., Hughes, S.H. & Arnold, E. (2001) J. Mol. Biol. 309, 437-445] that inhibitors with good activity against the K103N mutant would be expected to have favorable interactions with the mutant asparagines side chain, thereby compensating for resistance caused by stabilization of the mutant enzyme due to a hydrogen-bond network involving the N103 and Y188 side chains.Keywords: drug-resistance; HIV; NNRTI; reverse transcriptase.The use of highly active antiretroviral therapy (HAART) involving multidrug combinations has significantly reduced the death rates of HIV-1 infected individuals receiving such treatment [1]. Inhibitors of the HIV-1 reverse transcriptase (RT) constitute a cornerstone in this therapy and are commonly used in combination with inhibitors of the HIV-1 protease. The RT inhibitors belong to two classes, the nucleoside inhibitors and the non-nucleoside inhibitors (NNRTI). Whereas the NRTIs are nucleoside analogues with chain-terminating properties and affinity to active site residues, the NNRTIs include a wide range of series of chemical compounds characterized by noncompetitive binding to an allosteric site some 10 Å away from the active site. Structural comparison of RT in complex with template/primer and NNRTIs together with native RT complexes have shown that the NNRTIs inhibit the polymerase activity through long-range and short-range structural distortions in several of the RT subdomains. The distortions involve repositioning of residues in the nonnucleoside binding pocket (NNIBP) that impose steric impediments on the thumb subdomain flexibility forcing it to remain in the ...