HIV-1 reverse transcriptase (RT) degrades the plus strand viral RNA genome while synthesizing the minus strand of DNA. Many RNA fragments, including the polypurine tracts, remain annealed to the new DNA. Several RTs are believed to bind after synthesis to degrade all RNA fragments except the polypurine tracts by a polymerization-independent mode of RNase H activity. For this latter process, we found that RT positions the RNase H active site approximately 18 nt from the 5 end of the RNA, making the primary cut. The enzyme rebinds or slides toward the 5 end of the RNA to make a secondary cut creating two products 8 -9 nt long. RT then binds the new 5 end of the RNA created by the first primary or the secondary cuts to make the next primary cut. In addition, we observed another type of RNase H cleavage specificity. RT aligns the RNase H active site to the 3 end of the RNA, cutting 5 residues in. We determined the relative rates of these cuts, defining their temporal order. Results show that the first primary cut is fastest, and the secondary and 5-nt cuts occur next at similar rates. The second primary cuts appear last. Based on these results, we present a model by which RT progressively cleaves RNA fragments.H IV is the causative agent of AIDS (1). To establish an infection, HIV must integrate a double-stranded viral DNA into the host chromosome (2). The virus-encoded reverse transcriptase (RT) converts the single-stranded viral RNA genome into that double-stranded DNA. RT uses several different catalytic activities in this process. The polymerase function catalyzes DNA synthesis on RNA and DNA templates (3). RT also has an RNase H active site that cleaves RNA when annealed to DNA. RNase H activity is required for a number of steps in viral replication, including formation of primers and primer strand transfers (4). An important role for the RNase H is the complete removal of the original genomic RNA during the synthesis of the double-stranded DNA (5, 6).The genomic viral RNA first is converted to an RNA͞DNA hybrid and then to double-stranded DNA. Synthesis of the second DNA strand necessitates complete removal of the original RNA (7,8). The RNA genome is cut into small segments during and after synthesis of the RNA͞DNA hybrid (9-11). Studies show that two different modes of RNase H activity are important for the removal of the RNA (5, 12, 13). The first mode is carried out by the same RT performing DNA synthesis. This is the polymerization-dependent mode of cleavage (9)(10)(11)(14)(15)(16)(17). We showed that the HIV-1 RT synthesizing DNA leaves RNA oligomers in its wake, many still bound to the extended DNA primer (17, 18). These residual RNAs occur because cleavages of the RNA template happen less frequently than nucleotide addition. Kati et al. (19) examined the rates of both catalytic activities of RT and found that the polymerization rate is 7-10 times faster than the RNase H rate. This result demonstrates that the two activities are uncoupled, such that several nucleotides are added in the time required fo...

Human immunodeficiency virus (HIV) DNA synthesis is accompanied by degradation of genomic RNA by the RNase H of reverse transcriptase (RT). Two different modes of RNase H activity appear necessary for complete RNA removal. In one, occurring during minus strand synthesis, positioning of the RNase H is determined by binding of the polymerase active site to the DNA 3-end. In the other, used for removal of remaining RNA fragments, positioning of RT for RNase H-directed cleavage is determined by the RNA 5-ends. We attempted to identify RT amino acids responsible for these modes of positioning. Twelve RT mutants, each with one alanine replacement in residues 224 to 235, known as the primer grip region, were examined for catalytic abilities. Six of the examined primer grip mutants, although distant from the RNase H active site were altered in their ability to cleave RNA. The mutants P226A, F227A, G231A, Y232A, E233A, and H235A failed to perform RNA 5-end-directed RNase H cleavage in heparin-challenged reactions. The last four mutants also lacked DNA synthesis and DNA 3-end-directed RNase H cleavage activities in challenged reactions. Since mutants P226A and F227A carried out these latter reactions normally, these two residues specifically influence 5-RNA-directed RNase H catalysis. Human immunodeficiency virus, type 1 (HIV-1)1 is the causative agent of AIDS. During replication, the virally encoded reverse transcriptase catalyzes the conversion of the singlestranded RNA genome to a double-stranded DNA genome. HIV-RT catalyzes RNA-directed DNA synthesis, DNA-directed DNA synthesis, RNase H, strand displacement, and strand transfer activities (1). The RNA-directed DNA polymerase activity is essential for the formation of minus strand DNA from plus strand genomic RNA. RNase H activity is required for the removal of the RNA portion of the RNA/DNA hybrid formed during minus strand DNA synthesis. Biochemical and structural measurements show that the DNA polymerase and RNase H active sites are separated by a distance of about 18 nt along the template (2-8). Estimates vary from 14 to 20 nt in biochemical studies depending on the sequence of the nucleic acid employed. When the polymerase active site was bound at the 3Ј-OH of a DNA primer on an RNA template, this positioning determined the first site of cleavage of the template at the distance separated by the active sites (2, 3). Hence, this was termed the polymerase-dependent mode of RNase H-directed cleavage. Cleavage at other positions was termed polymeraseindependent. This latter class includes secondary cleavages that occur near the polymerase-dependent cleavage site but closer to the DNA 3Ј-end. Following the initial cleavage, the RT displays a 3Ј 3 5Ј directional processive RNase H activity (9 -12).As suggested by our biochemical studies, cleavage of the plus strand RNA is not completed by the RT that synthesizes the minus DNA strand (13). Fragments of RNA 13-45 nt in length are left behind that stay annealed to the newly synthesized DNA. Two of these are the polypurine t...

Synthesis of the minus strand of viral DNA by human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase is accompanied by RNase H degradation of the viral RNA genome. RNA fragments remain after synthesis and are degraded by the polymerase-independent mode of RNase H cleavage. Recently, we showed that this mode of cleavage occurs by a specific ordered mechanism in which primary cuts are first, secondary and 5-nucleotide cuts are next, and second primary cuts occur last (Wisniewski, M., Balakrishnan, M., Palaniappan, C., Fay, P., J., and Bambara, R., A. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 11978 -11983). Ultimately the RNAs are cleaved into small fragments that can dissociate from the DNA template. Because the cleavage mechanism is an ordered series of events, we determined in this study whether any earlier cut is required for a later cut. By precisely inhibiting cleavage at each site, we examined the dependence of later cuts on cleavage at that site. We found that each cut is independent of the other cuts, demonstrating that the order of this stepwise mechanism is based on the rates of each cut. A mechanism for unlinked ordered cleavage consistent with these results is presented. HIV-11 reverse transcriptase is essential for viral replication. This multi-functional enzyme has RNA-and DNA-dependent DNA polymerase, strand displacement, strand transfer, and RNase H activities, all of which are required for the conversion of the viral RNA genome to double-stranded DNA (1, 2). The RNase H activity, which degrades RNA annealed to DNA, is required for many steps during reverse transcription (2). Several studies have shown this activity to be important in clearing the DNA template of the viral RNA genome for minus strand transfer and plus strand synthesis (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Also this activity is important for the generation of the polypurine tract (PPT) primers that initiate plus strand synthesis and for the removal of the minus and plus strand primers (12,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29).Several reports have examined the spatial arrangement of the polymerase and RNase H active sites of HIV-1 RT (3, 6, 7, 30 -34). Based on crystallographic, biochemical, and cross-linking data, RT positions the polymerase active site to the 3Ј-hydroxyl of the replicating DNA primer and places the RNase H active site18 nt upstream on the RNA (30,(35)(36)(37)(38). This mode of binding allows RT to catalyze DNA synthesis ahead of the RNase H site, which creates the RNA/DNA substrate for degradation. The cleavage of this RNA by the synthesizing RT is referred to as the polymerase-dependent mode of RNase H cleavage. This mode of cleavage by HIV-1 RT degrades the majority of the RNA genome.Several studies have examined the coordination of polymerization and RNase H activities (30,(35)(36)(37)(38)(39)(40). Kati et al. (40) compared the rate of dNTP addition to that of RNase H degradation. This study showed that the nucleotide addition rate was 7-10 times faster than the RNase H ...

During and after minus-strand DNA synthesis, human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) degrades the RNA genome. To remove RNA left after polymerization, the RT aligns to cut 18 nucleotides in from the 5 RNA end. The enzyme then repositions, making a secondary cut 8 nucleotides from the RNA 5 end. Transfer of the minus strong stop DNA during viral replication requires cleavage of template RNA. Removal of the terminal RNA segment is a special case because the RNA-DNA hybrid forms a blunt end, shown previously to resist cleavage when tested in vitro. We show here that the structure of the substrate extending beyond the RNA 5 end is an important determinant of cleavage efficiency. A short single-stranded DNA extension greatly stimulated the secondary cleavage. Annealing an RNA segment to the DNA extension was even more stimulatory. Recessing the DNA from a blunt end by even one nucleotide caused the RT to reorient its binding, preventing secondary cleavage. The presence of the cap at the 5 end of the viral RNA did not improve the efficiency of secondary cleavage. However, NC protein greatly facilitated the secondary cut on the bluntended substrate, suggesting that NC compensates for the unfavorable substrate structure. HIV-11 reverse transcriptase is required for the conversion of the viral RNA genome to double-stranded DNA. This multifunctional enzyme has RNA-and DNA-dependent DNA polymerase, strand displacement, strand transfer, and RNase H activities. The RNase H activity degrades the RNA genome during and after synthesis of the first or minus DNA strand (1). The RNase H is used to clear the new minus DNA strand of the genomic RNA fragments, in preparation for minus-strand transfer and synthesis of the plus DNA strand. RNase H activity is also required for the generation of the polypurine tract primers that initiate plus-strand synthesis, and for the removal of the minus-and plus-strand primers (Refs. 1 and 2 and reference therein).HIV-1 RT is an asymmetric heterodimer comprising the p66 and p51 subunits (3-5). The structure of the p66 subunit is analogous to that of a right hand with the palm, thumb, fingers, and connection subdomains and an RNase H subdomain. The polymerase active site is located near the amino terminus of the p66, whereas the RNase H active site is near the carboxyl terminus. Biochemical as well as structural analyses show the spatial distance between the two active sites to be about 18ϳ19 nucleotides in length when RT is bound to a duplex substrate (5-10). The active residues of the polymerase domain, Asp-185, Asp-186, and Asp-110, reside within the palm subdomain (5). The fingers, palm, and thumb subdomains of the p66 participate in substrate binding (8, 10). The p51 subunit, a proteolytic product of the p66, folds in a different conformation and does not contain any catalytic sites (5,8,10,11). This subunit primarily serves a structural role in stabilizing the p66 subunit as well as positioning the RNase H subdomain and the tRNA (12-14). The essential active site res...

Mutations in the primer grip region of human immunodeficiency virus reverse transcriptase (HIV-RT) affect its replication fidelity. The primer grip region (residues 227-235) correctly positions the 3-ends of primers. Point mutations were created by alanine substitution at positions 224 -235. Error frequencies were measured by extension of a dG:dA primer-template mismatch. Mutants E224A, P225A, P226A, L228A, and E233A were approximately equal to the wild type in their ability to extend the mismatch. Mutants F227A, W229A, M230A, G231A, and Y232A extended 40, 66, 54, 72, and 76% less efficiently past a dG:dA mismatch compared with the wild type. We also examined the misinsertion rates of dG, dC, or dA across from a DNA template dA using RT mutants F227A and W229A. Mutant W229A exhibited high fidelity and did not produce a dG:dA or dC:dA mismatch. Interestingly, mutant F227A displayed high fidelity for dG:dA and dC:dA mismatches but low fidelity for dA:dA misinsertions. This indicates that F227A discriminates against particular base substitutions. However, a primer extension assay with three dNTPs showed that F227A generally displays higher fidelity than the wild type RT. Clearly, primer grip mutations can improve or worsen either the overall or base-specific fidelity of HIV-RT. We hypothesize that wild type RT has evolved to a fidelity that allows genetic variation without compromising yield of viable viruses. Human immunodeficiency virus (HIV-1) reverse transcriptase (HIV-RT)1 is the enzyme that converts the RNA genome of the virus to a double-stranded DNA, which is ultimately integrated into the host chromosome (1). This enzyme is multifunctional, possessing RNA-and DNA-dependent DNA polymerase, RNase H, strand transfer, and strand displacement activities (2, 3). Studies in vivo and in vitro have addressed the role of RT in the variability of the genome (4 -14). RT contributes to the generation of sequence diversity, partly because it produces frequent replication errors. One reason for its low fidelity is that RT lacks a 3Ј to 5Ј exonuclease activity (15). DNA polymerases in other organisms generally have this activity, which removes incorrectly added nucleotides by recognizing the mismatched base with the template sequence. RT misincorporates nucleotides at an estimated frequency of 1 per 5000 polymerized and extends mismatched termini at varying efficiencies (4, 6, 11, 14 -20). Additionally, RT alters the sequence of viral progeny by participating in recombination between the two copies of the viral genome (13, 21-28). Polymerization errors and recombination produce a high frequency of frameshift, deletion, and deletion with insertion mutations observed both in vivo and in vitro (6,7,11,13,21,29). This proclivity for mutations by RT helps HIV to evade immune responses and drug treatment.HIV-1 RT is a heterodimer composed of p66 and p51 subunits. The p66 subunit contains both the polymerase and RNase H active sites. The p51 subunit is a proteolytic product of p66 lacking the 15-kDa carboxyl-terminal (RNase H) d...