594 Nucleic Acids Research, 2006, Vol. 34, No. 2 Figure 1. (A) Schematic representation of the cleavage steps that release the three forms of NC protein from Gag during viral maturation. Primary, second- ary and tertiary cleavage sites are indicated by the numbered arrows, respec- tively. (B) Amino acid sequence of NCp7 (1–55) including its two-CCHC-zinc finger structures. NCp7 has 15 positive residues [5 arginines (R) and 10 lysines (K)] and two aromatic residues (Phe16 and Trp37). In addition, NCp9 (1–71) and NCp15 (1–123) have additional domains in the C-terminus which contain several aromatic residues, whose fluorescence, however, is not quenched upon binding of these proteins to nucleic acid. molecules (9). This recognition appears to rely critically on the interaction of RNA with the two zinc ﬁngers of the nuc- leocapsid (NC) domain and with its cationic residues (9–13), as shown in Figure 1B. Translation of the Gag polyprotein from the mRNA derived from proviral DNA entails ribosomal frameshifting after the NC coding sequence in 5% of cases (14,15). This frame- shifting produces another polyprotein, Gag-Pol, which in addition to the Gag proteins also contains the viral enzymes protease (PR), reverse transcriptase (RT) and integrase (IN) (15). The self-processing of accumulating Gag-Pol, after virus assembly, results in growing levels of PR that begins the sequential cleavage of Gag (15). Extensive experiments have shown that the primary cleavage site of Gag by PR resides between the CA and NC domains after the p2 domain (site 1 in Figure 1A) (16,17). Two secondary cleavage sites, characterized by 9-fold slower cleavage rates, reside between the MA and CA domains (site 2a) and between the p1 and p6 domains (site 2b) of the C-terminal part of Gag. Even slower cleavage events ( 400-fold slower than the prim- ary cleavage) occur at two additional sites, as the p2 and p1 peptides are cleaved from the C-terminal domains of CA and NC, respectively (sites 3a and 3b). In the fully mature, infec- tious virus, all proteins are present predominantly in their completely processed form (18). The three main products of the Gag processing by PR that contain the NC domain of Gag were termed according to their molecular weight and in the order of their appearance as follows: NCp15 (123 amino acids), NCp9 (71 amino acids) and NCp7 (55 amino acids) proteins (17,19). All of the Gag cleavage events appear to be related to the changes in the structure and function of the virus during the course of its life cycle. Thus, the cleavage of NCp15, p2 and MA from Gag results in the formation of the cone- shaped capsid shell composed of CA, which is much smaller than the spherical membrane-bound MA shell (5,20,21). Within this cone-shaped mature core, there is still a dense phase of NC bound to the viral RNA dimer, a complex of the two RNA molecules within the virus (20,22,23). The RNA undergoes a process of further maturation, in which its sec- ondary structure is altered. In a later stage of the retroviral life cycle, this RNA is reverse transcribed into proviral cDNA. The latter process involves several strand transfer steps. These events require major rearrangement of the nucleic acid sec- ondary structure. While this restructuring results in a lower free energy of the nucleic acid molecules, and is therefore spontaneous, it appears to be extremely slow. NCp7 has been shown to signiﬁcantly accelerate these reactions. The capability to facilitate the rearrangements required for these reactions is referred to as nucleic acid chaperone activity. However, it is not known to what extent the complete pro- cessing of Gag to its ﬁnal form, NCp7 (Figure 1B) is required for the enhancement of speciﬁc rearrangements of nucleic acid secondary structure. For example, a recent study showed that while both NCp7 and Gag are effective at annealing tRNALys 3 to the primer-binding site on the HIV-1 genome, Gag is more effective than NC (24). However, only processed NC has been shown to be capable of facilitating minus-strand transfer (25,26). In order to understand the effects of the processing of NC on its nucleic acid chaperone and binding activities, we will examine the interaction of DNA with Gag, NCp7, NCp9 and NCp15 using a single DNA molecule stretching technique with optical tweezers as well as two standard solution tech- niques: tryptophan ﬂuorescence and ﬂuorescence anisotropy (FA) binding assays. In this combined approach, we are able to extract both traditional and novel information on DNA– protein interactions. First, we are able to determine the binding constants to the nucleic acid of the stoichiometrically bound proteins by DNA stretching, obtaining results that agree with both ﬂuorescence studies. In addition, we are able to obtain information on the effect of each protein on force-induced DNA melting, i.e. strand separation due to an external pulling force. In turn, this enables us to determine the effect of each protein on DNA duplex stability and on the kinetics of the strand separation and annealing. While the nucleic acid bind- ing properties of a protein can be compared with previous solution binding data, as is done here, the information on the effect of NC on DNA melting is unique and important for determining the nucleic acid chaperone properties of various forms of NC. Despite our use of long polymeric dsDNA, it is likely that our results are relevant for the binding and duplex destabilizing capabilities of NC to RNA as well. To demonstrate the relevance of our DNA stretching results to RNA binding, we compare binding of both Gag and NCp7 with similar short DNA and RNA oligonucleotides. The major conclusion of this study is that the nucleic acid chaperone activity improves greatly as the Gag precursor is progressively processed to create NCp15, NCp9 and NCp7. MATERIALS AND METHODS Protein preparation NCp7, NCp9 and NCp15 from HIV-1 were expressed and puriﬁed as described (25,27,28). Brieﬂy, HPLC fractions containing the expressed NC sequences (identiﬁed by MALDI-mass spectrometry and N-terminal sequencing) were collected, pooled and lyophilized. NC proteins were resuspended in the presence of 1 equivalent of Zn2+ per ﬁnger,