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Members of theTranscription factors of the Rel/NF-B family are present in most or all mammalian and avian cells and influence the expression of many genes (for a review, see references 1 and 19). These factors are related to each other over a region of about 300 amino acids called the Rel Homology Domain (RHD), which governs DNA binding and dimerization. Although each protein has a nuclear localization signal (NLS), the various homo-and heterodimers are cytoplasmic by virtue of an inhibitor protein whose binding masks the NLSs. Several different inhibitors (called IBs) are known, and they constitute a family of related proteins. Each IB contains five or six so-called ankyrin repeats, motifs of about 33 amino acids which are abundantly represented in the protein ankyrin. In ankyrin, these repeats apparently function in groups of about six (18) and constitute a protein-protein interaction domain. In addition, the inhibitors contain a C-terminal region which tends to be highly acidic and to resemble PEST sequences associated with rapid protein turnover (22). The IB N-terminal region contains two serine residues which, upon phosphorylation, trigger ubiquitination and proteasomal degradation of the inhibitor, thus freeing the dimer to enter the nucleus (4,5,7,(26)(27)(28)(29).While several crystal structures of Rel family members are known (6, 6a, 10, 20), none is known for the IB family. Nevertheless, studies with various mutants of the Rel family and/or IB proteins have led to some general conclusions about their interaction. First, a Rel family dimer binds a single IB molecule (8,12,14). Second, at least some of the contacts with IB occur in the dimerization domains of the Rel proteins. Based on the crystal structure, each monomer of p50 and RelA consists of two separate domains connected by a short hinge region. The N-terminal domain contains some (but not all) of the residues that contact DNA, while the C-terminal domain contains the remaining DNA contacts and governs dimerization. Partial deletion of the N-terminal domain of RelA did not abolish binding of IB␣ (9). Similar results were obtained with Cactus and N-terminal deletion mutants of Dorsal (Drosophila relatives of IB and Rel proteins, respectively) (11, 25). In addition, mutation of two residues in the dimerization domain of Dorsal prevented interaction with Cactus but did not affect DNA binding or dimerization (16). These studies suggest contact between IB and the C-terminal part of the RHD. The third general conclusion is that the dimer NLSs, which were not resolved in the crystal structures but are immediately downstream of the dimerization domain in the primary structure, are somehow involved in binding to IB. While in at least some cases an intact NLS may not be required for binding to IB (11,23), several studies have shown that altering the NLS can abolish binding (2,9,15,25). Involvement of the NLS would correlate nicely with the fact that the dimer NLSs are masked in a dimer-IB complex. The final generalization is that most of the IB contacts oc...

Although human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) has been extensively studied, there are still significant questions about the effects of mutations on the maturation and stability of RT. We show here that a significant fraction (>80%) of the single point mutations we generated in the thumb subdomain of HIV-1 (RT) affect the stability of RT in virions. Fragments of the unstable mutant RTs can be detected in Western blots of virion proteins; however, the degree of degradation varies. The titers of the mutants whose virions contain degraded RTs are reduced. Some, but not all, of the unstable RT thumb subdomain mutants we analyzed have a temperature-sensitive phenotype. A preliminary survey of mutations in other subdomains of RT shows that some of these mutations also destabilize RT. The stability of the RT mutants is enhanced by the addition of a protease inhibitor, suggesting that the viral protease plays an important role in the degradation of the mutant RTs. These results confirm and extend earlier reports of mutations that affect the stability of RT in virions. The data suggest that the stability of a mutant RT in virions could be a major factor in determining the virus titer and, by extension, viral fitness, which could affect whether a mutation in RT is acceptable to the virus.