The present review summarizes key progress made in characterizing the small nucleolar RNAs (snoRNAs) of eukaryotic cells. Recent studies have shown snoRNA populations to be substantially more complex than anticipated initially. Many newly discovered snoRNAs are synthesized by an intron-processing pathway, which provides a potential mechanism for coordinating nuclear RNA synthesis. Several snoRNAs and snoRNP proteins are known to be needed for processing of ribosomal RNA, but precise functions remain to be defined. In principle, snoRNAs could have several roles in ribosome synthesis including: folding of pre-rRNA, formation of rRNP substrates, catalyzing RNA cleavages, base modification, assembly of pre-ribosomal subunits, and export of product rRNP particles.

HIV-1 entry into CD4 ؉ cells requires the sequential interactions of the viral envelope glycoproteins with CD4 and a coreceptor such as the chemokine receptors CCR5 and CXCR4. A plausible approach to blocking this process is to use small molecule antagonists of coreceptor function. One such inhibitor has been described for CCR5: the TAK-779 molecule. To facilitate the further development of entry inhibitors as antiviral drugs, we have explored how TAK-779 acts to prevent HIV-1 infection, and we have mapped its site of interaction with CCR5. We find that TAK-779 inhibits HIV-1 replication at the membrane fusion stage by blocking the interaction of the viral surface glycoprotein gp120 with CCR5. We could identify no amino acid substitutions within the extracellular domain of CCR5 that affected the antiviral action of TAK-779. However, alanine scanning mutagenesis of the transmembrane domains revealed that the binding site for TAK-779 on CCR5 is located near the extracellular surface of the receptor, within a cavity formed between transmembrane helices 1, 2, 3, and 7.

To study HIV-1 escape from a coreceptor antagonist, the R5 primary isolate CC1͞85 was passaged in peripheral blood mononuclear cells with increasing concentrations of the CCR5-specific small molecule inhibitor, AD101. By 19 passages, an escape mutant emerged with a >20,000-fold resistance to AD101. This virus was cross-resistant to a related inhibitor, SCH-C, and partially resistant to RANTES but still sensitive to CCR5-specific mAbs. The resistant phenotype was stable; the mutant virus retained AD101 resistance during nine additional passages of culture in the absence of inhibitor. Replication of the escape mutant in peripheral blood mononuclear cells completely depended on CCR5 expression and did not occur in cells from CCR5-⌬32 homozygous individuals. The escape mutant was unable to use CXCR4 or any other tested coreceptor to enter transfected cells. Acquisition of CXCR4 use is not the dominant in vitro escape pathway for a small molecule CCR5 entry inhibitor. Instead, HIV-1 acquires the ability to use CCR5 despite the inhibitor, first by requiring lower levels of CCR5 for entry and then probably by using the drug-bound form of the receptor.

Pseudomonas aeruginosa exoenzyme S is an adenosine diphosphate ribosyltransferase distinct from Pseudomonas toxin A. Exoenzyme S catalyzes the transfer of radioactivity from all portions of radiola led NAD+ except nicotinamide. Digestion of the radiolabeled product(s) formed in the presence of [adenine-14CJNAD+ and exoenzyme S with snake venom phosphodiesterase yields only AMP, suggesting that ADP-ribose is present as monomers and not as poly(ADPribose). Exoenzyme S does not catalyze the transfer of ADPribose from NAD+ to elongation factor 2, as do toxin A and diphtheria toxin, but to one or more other proteins present in crude extracts of wheat germ or rabbit reticulocytes and in partially purified preparations of elongation factor 1. The ADP-ribosyltransferase activity of exoenzyme S is distinct from toxin A by several tests: it is not neutralized by toxin A antibody, it is destroyed rather than potentiated by pretreatment with urea, and it is more heat stable. These latter observations and the substrate specificity suggest that exoenzyme S is different from any previously described prokaryotic ADP-ribosyltransferase.Diphtheria toxin and Pseudomonas toxin A inhibit protein synthesis in eukaryotic cells by catalyzing the transfer of the ADP-ribose (ADP-Rib) moiety of NAD+ to elongation factor 2 (EF-2) (1-3). The only eukaryotic protein known to be modified by these two toxins is EF-2, and all existing information supports the conclusion that the ADP-ribosylation of EF-2 is responsible for the lethality of these two toxins (2, 4). Diphtheria toxin is encoded by a phage gene (2, 5), but the location of the structural gene for Pseudononas toxin A is unknown. Approximately 90% of all isolates of Pseudomonas aeruginosa tested produce toxin A (6, 7).In this report we describe an ADP-ribosyltransferase (exoenzyme S) that is present in the culture supernatant fluid of a strain of P. aeruginosa (Ps 388). Ps 388 was consistently negative in an immune precipitation assay (7) using specific toxin A antibody. In the presence of limiting amounts of EF-2, exoenzyme S catalyzed the transfer of far more ADP-Rib from NAD+ than could be accounted for by the production of ADP-ribosylated EF-2. Data are presented to show that exoenzyme S, unlike diphtheria or Pseudomonas A toxins, does not modify EF-2 but some other eukaryotic protein(s). ADP-Rib appears to be present in the modified proteins(s) as monomeric units rather than as poly(ADP-Rib). We also show that exoenzyme S is distinct from Pseudomonas toxin A by several other tests. It is not neutralized by toxin A antibody, it is destroyed rather than potentiated by pretreatment with urea, and it is more heat stable. (Sigma Chem. Co.). A 25-ml amount of this medium in a 500-ml erlenmeyer flask was inoculated with an overnight culture of Ps 388 to an initial cell density of approximately 5 X 107 cells per ml. The culture was incubated at 320 on a reciprocating shaker (200 linear excursions/min) (Lab.-Line Inst.) for 22 hr. The culture supernatant fluid was obtained by cen...

Recent investigations have identified homologs of eukaryotic box C/D small nucleolar RNAs (snoRNAs) in Archaea termed sRNAs. Archaeal homologs of the box C/D snoRNP core proteins fibrillarin and Nop56/58 have also been identified but a homolog for the eukaryotic 15.5kD snoRNP protein has not been described. Our sequence analysis of archaeal genomes reveals that the highly conserved ribosomal protein L7 exhibits extensive homology with the eukaryotic 15.5kD protein. Protein binding studies demonstrate that recombinant Methanoccocus jannaschii L7 protein binds the box C/D snoRNA core motif with the same specificity and affinity as the eukaryotic 15.5kD protein. Identical to the eukaryotic 15.5kD core protein, archaeal L7 requires a correctly folded box C/D core motif and intact boxes C and D. Mutational analysis demonstrates that critical features of the box C/D core motif essential for 15.5kD binding are also required for L7 interaction. These include stem I which juxtaposes boxes C and D, as well as the sheared G:A pairs and protruded pyrimidine nucleotide of the asymmetric bulge region. The demonstrated presence of L7Ae in the Haloarcula marismortui 50S ribosomal subunit, taken with our demonstration of the ability of L7 to bind to the box C/D snoRNA core motif, indicates that this protein serves a dual role in Archaea. L7 functioning as both an sRNP core protein and a ribosomal protein could potentially regulate and coordinate sRNP assembly with ribosome biogenesis.