The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) has two zinc fingers, each containing the invariant metal ion binding residues CCHC. Recent reports indicate that mutations in the CCHC motifs are deleterious for reverse transcription in vivo. To identify reverse transcriptase (RT) reactions affected by such changes, we have probed zinc finger functions in NC-dependent RT-catalyzed HIV-1 minusand plus-strand transfer model systems. Our approach was to examine the activities of wild-type NC and a mutant in which all six cysteine residues were replaced by serine (SSHS NC); this mutation severely disrupts zinc coordination. We find that the zinc fingers contribute to the role of NC in complete tRNA primer removal from minus-strand DNA during plus-strand transfer. Annealing of the primer binding site sequences in plus-strand strong-stop DNA [(؉) SSDNA] to its complement in minus-strand acceptor DNA is not dependent on NC zinc fingers. In contrast, the rate of annealing of the complementary R regions in (؊) SSDNA and 3 viral RNA during minus-strand transfer is approximately eightfold lower when SSHS NC is used in place of wild-type NC. Moreover, unlike wild-type NC, SSHS NC has only a small stimulatory effect on minus-strand transfer and is essentially unable to block TAR-induced self-priming from (؊) SSDNA. Our results strongly suggest that NC zinc finger structures are needed to unfold highly structured RNA and DNA strand transfer intermediates. Thus, it appears that in these cases, zinc finger interactions are important components of NC nucleic acid chaperone activity.Reverse transcription, a critical event in the retrovirus life cycle, consists of a complex series of reactions that culminate in synthesis of a linear, double-stranded DNA copy of the viral RNA genome (27; reviewed in references 4 and 14). This process is catalyzed by the virus-encoded reverse transcriptase (RT) enzyme. However, it is known that in addition to RT, host and other viral factors play important roles in viral DNA synthesis.One of these accessory factors is the viral nucleocapsid protein (NC), a small basic, single-stranded nucleic acid binding protein, which is tightly associated with genomic RNA in the interior of the mature virus particle (for reviews, see references 14, 16, and 59). Studies on the solution structure of free human immunodeficiency virus type 1 (HIV-1) NC indicated that this protein consists of a flexible polypeptide chain and two rigid zinc-binding domains connected by a short basic peptide linker (55-57, 67, 69, 70). Recently, De Guzman et al. (19) solved the three-dimensional nuclear magnetic resonance structure of NC bound to the SL3 RNA stem-loop in the HIV-1 packaging signal. They showed that the N-terminal basic residues of NC in the complex form a helix that binds to the major groove of the RNA stem largely by nonspecific electrostatic interactions, whereas the zinc fingers are involved in specific interactions with the G residues in the GGAG tetraloop (19).The zinc fingers are in cl...

The Gag polyprotein of HIV-1 is essential for retroviral replication and packaging. The nucleocapsid (NC) protein is the primary region for the interaction of Gag with nucleic acids. In this study, we examine the interactions of Gag and its NC cleavage products (NCp15, NCp9 and NCp7) with nucleic acids using solution and single molecule experiments. The NC cleavage products bound DNA with comparable affinity and strongly destabilized the DNA duplex. In contrast, the binding constant of Gag to DNA was found to be ∼10-fold higher than that of the NC proteins, and its destabilizing effect on dsDNA was negligible. These findings are consistent with the primary function of Gag as a nucleic acid binding and packaging protein and the primary function of the NC proteins as nucleic acid chaperones. Also, our results suggest that NCp7's capability for fast sequence-nonspecific nucleic acid duplex destabilization, as well as its ability to facilitate nucleic acid strand annealing by inducing electrostatic attraction between strands, likely optimize the fully processed NC protein to facilitate complex nucleic acid secondary structure rearrangements. In contrast, Gag's stronger DNA binding and aggregation capabilities likely make it an effective chaperone for processes that do not require significant duplex destabilization.

Host proteins are incorporated both on and inside human immunodeficiency virus type 1 (HIV-1) virions. To identify cellular proteins inside HIV-1, virion preparations were treated by a protease-digestion technique that removes external host proteins, allowing for the study of the proteins inside the virus. Treated HIV-1 preparations were analyzed by immunoblot, high-pressure liquid chromatography, and protein sequence analyses. These analyses identified several cellular proteins inside HIV-1: elongation factor 1alpha, glyceraldehyde-3-phosphate dehydrogenase, HS-1, phosphatidylethanolamine-binding protein, Pin1, Lck, Nm23-H1, and the C-terminal tail of CD43. Several of these proteins were found as fragments of their full-sized proteins that appear to be generated by our protease treatment of the virions, the HIV-1 protease, or a cellular protease. Recent advances in cell biology and biochemistry have identified some of these proteins as actin-binding proteins. These results support the hypothesis that actin filaments are incorporated into the virion and may provide additional clues for the understanding of the interaction between viral and cellular proteins during assembly and budding.

The nucleocapsid protein (NC) of human immunodeficiency virus type 1 has two zinc fingers, each containing the invariant CCHC zinc-binding motif; however, the surrounding amino acid context is not identical in the two fingers. Recently, we demonstrated that zinc coordination is required when NC unfolds complex secondary structures in RNA and DNA minus-and plus-strand transfer intermediates; this property of NC reflects its nucleic acid chaperone activity. Here we have analyzed the chaperone activities of mutants having substitutions of alternative zinc-coordinating residues, i.e., CCHH or CCCC, for the wild-type CCHC motif. We also investigated the activities of mutants that retain the CCHC motifs but have mutations that exchange or duplicate the zinc fingers (mutants 1-1, 2-1, and 2-2); these changes affect amino acid context. Our results indicate that in general, for optimal activity in an assay that measures stimulation of minus-strand transfer and inhibition of nonspecific self-priming, the CCHC motif in the zinc fingers cannot be replaced by CCHH or CCCC and the amino acid context of the fingers must be conserved. Context changes also reduce the ability of NC to facilitate primer removal in plus-strand transfer. In addition, we found that the first finger is a more crucial determinant of nucleic acid chaperone activity than the second finger. Interestingly, comparison of the in vitro results with earlier in vivo replication data raises the possibility that NC may adopt multiple conformations that are responsible for different NC functions during virus replication.The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is a small, basic, nucleic acid-binding protein which associates with genomic RNA in the mature virion core (14,15,54); the mature protein is generated by proteolytic cleavage of the Gag precursor (36,47,63). Structural studies have revealed that free HIV-1 NC in solution has two rigid zinc-binding domains or zinc fingers, each containing the invariant CCHC metal ion-binding motif (30,37,59,61). The two fingers are covalently linked to each other by a short flexible basic amino acid region and are flanked by flexible Nor C-terminal "tails" (49-51, 59, 60, 62). The Summers group has recently solved the three-dimensional structures of HIV-1 NC bound to the SL2 (3, 4) and SL3 (18) RNA stem-loops that form part of the larger HIV-1 packaging signal, by nuclear magnetic resonance (NMR) analysis.The two NC zinc fingers are located in close proximity (45, 46, 49, 50) but exhibit only weak interactions with one another (13,43,46,49,66). Interestingly, their structures are similar (58), despite differences in the amino acid sequences surrounding the CCHC motifs (37, 54). Moreover, the biochemical properties (8, 45) and biological activities of the two fingers are not equivalent, and the presence of both fingers is critical for production of replication-competent virus (9,21,26,28,29,48,72); in addition, the positions of the zinc fingers cannot be exchanged (21,26).NC function in vir...