No abstract

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 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...

During the first strand transfer in reverse transcription, minus-strand strong-stop DNA [(؊) SSDNA] is annealed to the 3 end of the acceptor RNA in a reaction mediated by base-pairing between terminal repeat sequences in the RNA and their complement in the DNA. The large stem-loop structure in the repeat region known as TAR could interfere with this annealing reaction. We have developed an in vitro human immunodeficiency virus type 1 (HIV-1) system to investigate the effect of TAR on strand transfer. Mutational analysis demonstrates that the presence of TAR in the donor and acceptor templates inhibits strand transfer and is correlated with extensive synthesis of heterogeneous DNAs formed by self-priming from (؊) SSDNA. These DNAs are not precursors to the transfer product. Interestingly, products of self-priming are not detected in HIV-1 endogenous reactions; this suggests that virions contain a component which prevents self-priming. Our results show that the viral nucleocapsid protein (NC), which can destabilize secondary structures, drastically reduces self-priming and dramatically increases the efficiency of strand transfer. In addition, the data suggest that the ability to eliminate self-priming is a general property of NC which is manifested during reverse transcriptase pausing at sites of secondary structure in the template. We conclude that this activity of NC is critical for achieving highly efficient and specific viral DNA synthesis. Our findings raise the possibility that inactivation of NC could provide a new approach for targeting reverse transcription in anti-HIV therapy.

No abstract

The HIV-1 nucleocapsid protein (NC) functions as a nucleic acid chaperone during the plus-strand transfer step in reverse transcription by facilitating annealing of the primer binding site (PBS) sequence in the short plus-strand strong-stop DNA fragment [(+) SSDNA] to a complementary site located near the 3' end of the minus-strand DNA [(-) PBS DNA]. To investigate the mechanism by which NC performs this function, we have prepared an 18-nucleotide (-) PBS DNA for nuclear magnetic resonance (NMR) based structural and NC binding studies. The (-) PBS DNA forms a stable hairpin (T(m) approximately 42 +/- 5 degrees C) that contains a five-residue loop and a bulged thymine in a guanosine-cytosine-rich stem. Addition of substoichiometric amounts of NC results in significant broadening and reductions in NMR signal intensities of the Watson-Crick base-paired imino protons and a reduction by 20 degrees C in the upper temperature at which the imino proton signals are detectable, consistent with destabilization of the structure. The results suggest that inefficient annealing in the absence of NC may be due to the intrinsic stability of an internal (-) PBS DNA hairpin and that NC facilitates strand transfer by destabilizing the hairpin and exposing stem nucleotides for base pairing with the PBS sequence in (+) SSDNA.

The RNase H domain of murine leukemia virus (MuLV) reverse transcriptase (RT) was replaced with Escherichia coli RNase H, and the effect on RNase H activity and processive DNA synthesis was studied, using RNA-DNA hybrids containing sequences from the MuLV polypurine tract (PPT). Two chimeric RTs, having the entire polymerase domain or all but the last 19 amino acids, were expressed. In both cases, these RTs made multiple cuts in PPT-containing substrates, whereas wild-type cleavages occurred primarily at sites consistent with the distance between the polymerase and RNase H active sites. Primer extension assays performed with the chimeric RTs, an RNase H-minus RT, and wild-type showed that the presence of a wild-type viral RNase H domain facilitates processive DNA synthesis. When wild-type RT was bound to primer-template, two retarded bands could be detected in band-shift assays. In the absence of primer extension, a high proportion of enzyme-bound primer-template was associated with the faster-migrating band, whereas with DNA synthesis, more of the bound radioactivity was in the super-shifted complex. This suggests that the super-shifted complex contains the active form of RT. The mutant RTs were deficient in formation of this complex, but the chimeric RTs were somewhat less defective than the RNase H-minus mutant. Our results demonstrate that in the wild-type enzyme, the RNase H domain is required to stabilize the interaction between RT and primer-template.

The functional relationship between the polymerase and RNase H domains of reverse transcriptase (RT) was investigated by studying the activities of AKR murine leukemia virus (MuLV) enzymes. In addition to the wild type, an RNase H-minus RT missing the entire RNase H domain and two other mutants having abnormal polymerase:RNase H ratios were expressed. These mutants include (i) a chimeric protein in which the MuLV RNase H domain was replaced by the entire Escherichia coli RNase H sequence and (ii) an RT with a 126 amino acid deletion in a region analogous to the "connection" subdomain in the p66 subunit of human immunodeficiency virus type 1 RT (Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A., & Steitz, T. A. (1992) Science 256, 1783-1790). With the wild-type RT, the major RNase H cleavage reaction was coordinated with DNA synthesis and occurred at a position corresponding to 15 nucleotides from the 3'-terminus of the DNA primer. Additional cleavages closer to the 5'-end of the RNA were explained in terms of a model relating binding of the RNA.DNA hybrid substrate and enzyme structure. The chimeric RT behaved like E. coli RNase H, exhibited 300-fold higher RNase H activity than wild-type RT, and was limited in its ability to synthesize DNA. Qualitative and quantitative changes in the polymerase and RNase H activities of the deletion mutant were also observed. The RNase H domain appeared to function independently of the polymerase domain, supporting the idea that the proper spatial relationship between the two active centers was disrupted by the mutation. Taken together, our results indicate that alteration of the normal polymerase:RNase H ratio can have profound effects on both polymerase and RNase H cleavage activities, as expected for an enzyme with two interdependent domains.