Proper functioning of RNAs requires the formation of complex three-dimensional structures combined with the ability to rapidly interconvert between multiple functional states. This review covers recent advances in isotope-labeling strategies and NMR experimental approaches that have promise for facilitating solution structure determinations and dynamics studies of biologically active RNAs. Improved methods for the production of isotopically labeled RNAs combined with new multidimensional heteronuclear NMR experiments make it possible to dramatically reduce spectral crowding and simplify resonance assignments for RNAs. Several novel applications of experiments that directly detect hydrogen-bonding interactions are discussed. These studies demonstrate how NMR spectroscopy can be used to distinguish between possible secondary structures and identify mechanisms of ligand binding in RNAs. A variety of recently developed methods for measuring base and sugar residual dipolar couplings are described. NMR residual dipolar coupling techniques provide valuable data for determining the long-range structure and orientation of helical regions in RNAs. A number of studies are also presented where residual dipolar coupling constraints are used to determine the global structure and dynamics of RNAs. NMR relaxation data can be used to probe the dynamics of macromolecules in solution. The power dependence of transverse rotating-frame relaxation rates was used here to study dynamics in the minimal hammerhead ribozyme. Improved methods for isotopically labeling RNAs combined with new types of structural data obtained from a growing repertoire of NMR experiments are facilitating structural and dynamic studies of larger RNAs.

T-cell responses were evaluated prospectively in 41 patients with acute human immunodeficiency virus type 1 (HIV-1) infection (30 untreated and 11 receiving zidovudine, lamivudine, and indinavir) and in 38 uninfected adults. By 6-12 months, treated patients had significantly greater median Candida and tetanus lymphoproliferative responses (stimulation index [SI], 76 and 55, respectively) than did untreated patients (SI, 7 and 6, P=.02 and.001, respectively), and the responses of treated patients surpassed those of uninfected adults (SI, 19 and 32, P= .002 and .101, respectively). Unlike the patients in the untreated group, the patients in the treated group mounted a 6-fold increased HIV-1 p24 response (SI increase, 1.0 to 5.7, P= .01) within 3 months. HIV-1-specific cytotoxicity remained detectable in most treated patients. Thus, combination therapy administered within 3-4 months of infection was associated with improved T-cell memory responses that were distinct from those of untreated patients. The amplified HIV-1-specific T-cell responses may help maintain cytotoxic activities.

Although multiple viruses utilize host cell cyclophilins, including severe acute respiratory syndrome (SARS) and human immunodeficiency virus type-1(HIV-1), their role in infection is poorly understood. To help elucidate these roles, we have characterized the first virally encoded cyclophilin (mimicyp) derived from the largest virus discovered to date (the Mimivirus) that is also a causative agent of pneumonia in humans. Mimicyp adopts a typical cyclophilin-fold, yet it also forms trimers unlike any previously characterized homologue. Strikingly, immunofluorescence assays reveal that mimicyp localizes to the surface of the mature virion, as recently proposed for several viruses that recruit host cell cyclophilins such as SARS and HIV-1. Additionally mimicyp lacks peptidyl-prolyl isomerase activity in contrast to human cyclophilins. Thus, this study suggests that cyclophilins, whether recruited from host cells (i.e. HIV-1 and SARS) or virally encoded (i.e. Mimivirus), are localized on viral surfaces for at least a subset of viruses.

SummaryResidual dipolar couplings (RDCs) complement standard NOE distance and J-coupling torsion angle data to improve the local and global structure of biomolecules in solution. One powerful application of RDCs is for domain orientation studies, which are especially valuable for structural studies of nucleic acids, where the local structure of a double helix is readily modeled and the orientations of the helical domains can then be determined from RDC data. However, RDCs obtained from only one alignment media generally result in degenerate solutions for the orientation of multiple domains. In protein systems, different alignment media are typically used to eliminate this orientational degeneracy, where the combination of RDCs from two (or more) independent alignment tensors can be used to overcome this degeneracy. It is demonstrated here for native E. coli tRNA Val that many of the commonly used liquid crystalline alignment media result in very similar alignment tensors, which does not eliminate the 4-fold degeneracy for orienting the two helical domains in tRNA. The intrinsic magnetic susceptibility anisotropy (MSA) of the nucleobases in tRNA Val was also used to obtain RDCs for magnetic alignment at 800 and 900 MHz. While these RDCs yield a different alignment tensor, the specific orientation of this tensor combined with the high rhombicity for the tensors in the liquid crystalline media only eliminates two of the four degenerate orientations for tRNA Val . Simulations are used to show that, in optimal cases, the combination of RDCs obtained from liquid crystalline medium and MSA-induced alignment can be used to obtain a unique orientation for two helical domains in tRNA Val .