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Retrovirus integrase (IN) integrates the viral linear DNA genome ( approximately 10 kb) into a host chromosome, a step which is essential for viral replication. Integration occurs via a nucleoprotein complex, termed the preintegration complex (PIC). This article focuses on the reconstitution of synaptic complexes from purified components whose molecular properties mirror those of the PIC, including the efficient concerted integration of two ends of linear viral DNA into target DNA. The methods described herein permit the biochemical and biophysical analyses of concerted integration. The methods enable (1) the study of interactions between purified recombinant IN and its viral DNA substrates at the molecular level; (2) the identification and characterization of nucleoprotein complexes involved in the human immunodeficiency virus type-1 (HIV-1) concerted integration pathway; (3) the determination of the multimeric state of IN within these complexes; (4) dissection of the interaction between HIV-1 IN and cellular proteins such as lens epithelium-derived growth factor (LEDGF/p75); (5) the examination of HIV-1 Class II and strand transfer inhibitor resistant IN mutants; (6) the mechanisms associated with strand transfer inhibitors directed against HIV-1 IN that have clinical relevance in the treatment of HIV-1/AIDS.

The small nucleolar RNAs (snoRNAs) are associated with proteins in ribonucleoprotein complexes called snoRNPs ("snorps"). These complexes create modified nucleotides in preribosomal RNA and other RNAs and participate in nucleolytic cleavages of pre-rRNA. The various reactions occur in site-specific fashion, and the mature rRNAs are ultimately incorporated into cytoplasmic ribosomes. Most snoRNAs exist in two structural classes, and most members in each class are involved in nucleotide modification reactions. Guide snoRNAs in the "box C/D" class target methylation of the 2'-hydroxyl moiety, to form 2'-O-methylated nucleotides (Nm), whereas guide snoRNAs in the "box H/ACA" class target specific uridines for conversion to pseudouridine (Psi). The rRNA nucleotides modified in this manner are numerous, totaling approximately 100 in yeast and twice that number in humans. Although the chemistry of the modifications and the factors involved in their formation are largely explained, very little is known about the influence of the copious snoRNA-guided nucleotide modifications on rRNA activity and ribosome function. Among eukaryotic organisms the sites of rRNA modification and the corresponding guide snoRNAs have been best characterized in S. cerevisiae, making this a model organism for analyzing the consequences of modification. This chapter presents approaches to characterizing rRNA modification effects in yeast and includes strategies for evaluating a variety of specific rRNA functions. To aid in planning, a package of bioinformatics tools is described that enables investigators to correlate guide function with targeted ribosomal sites in several contexts. Genetic procedures are presented for depleting modifications at one or more rRNA sites, including ablation of all Nm or Psi modifications made by snoRNPs, and for introducing modifications at novel sites. Methods are also included for characterizing modification effects on cell growth, antibiotic sensitivity, rRNA processing, formation of various rRNP complexes, translation activity, and rRNA structure within the ribosome.