Mutations in the U5 region adjacent to the primer binding site affect tRNA cleavage by human immunodeficiency virus type 1 reverse transcriptase in vivo.

In retroviruses, the first nucleotide added to the tRNA primer defines the end of the U5 region in the right long terminal repeat, and the subsequent removal of this tRNA primer by RNase H exactly defines the U5 end of the linear double-stranded DNA. In most retroviruses, the entire tRNA is removed by RNase H cleavage at the RNA/DNA junction. However, the RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase cleaves the tRNA 1 nucleotide from the RNA/DNA junction at the U5/primer binding site (PBS) junction, which leaves an rA residue at the U5 terminus. We made sequence changes at the end of the U5 region adjacent to the PBS in HIV-1 to determine whether such changes affect the specificity of tRNA primer cleavage by RNase H. In some of the mutants, RNase H usually removed the entire tRNA, showing that the cleavage specificity was shifted by 1 nucleotide. This result suggests that the tRNA cleavage specificity of the HIV-1 RNase domain H depends on sequences in U5.

The nucleoside analogs 4'C-methyl thymidine and 4'C-ethyl thymidine block DNA synthesis by wild-type HIV-1 RT and excision proficient NRTI resistant RT variants.

HIV-1 can become resistant to nucleoside analogs by developing an enhanced ability to excise the analogs after they have been incorporated. Excision requires that the analog be located at the 3' terminus of the primer. We have describe nucleoside analogs that do not block DNA synthesis at the point of incorporation, but only after additional dNTPs have been added to the DNA. These nucleoside analogs are called "delayed chain terminators" and are relatively effective inhibitors of drug-resistant HIV-1 reverse transcriptases (RTs) that are excision proficient. However, the first delayed chain terminator that we characterized was poorly phosphorylated in cultured cells. We have examined other nucleoside analogs to determine whether these compounds also act as delayed chain terminators, but were more efficiently converted to the triphosphate form by cellular kinases. These analogs contain substitutions on the deoxyribose sugar ring at the 4' carbon (4'C-methyl dT and 4'C-ethyl dT). Unlike true delayed chain terminators, which terminate DNA synthesis in a spatial sense (DNA synthesis is halted only after additional dNTPs have been incorporated after the analog), 4'C-methyl dTTP causes a pause in DNA synthesis at the point of incorporation. However, HIV-1 RT can eventually extend the primer blocked by the 4' C-Me dTMP analog. 4'C-methyl dTTP blocks DNA synthesis in a temporal sense, rather than in a spatial sense. A primer blocked by 4'C-ethyl dTMP is not extended by HIV-1 RT, and this compound acts like a conventional chain terminator, despite the presence of a 3'-OH group. These compounds effectively block the replication of an HIV-1-based vector that replicates using wild-type HIV-1 RT, but only in the presence of herpes simplex virus thymidine kinase (HSV TK). These compounds are effective against many NRTI drug-resistant RT variants; however, the M184V mutant is relatively resistant.

Synthesis, biological activity, and crystal structure of potent nonnucleoside inhibitors of HIV-1 reverse transcriptase that retain activity against mutant forms of the enzyme.

In an ongoing effort to develop novel and potent nonnucleoside HIV-1 reverse transcriptase (RT) inhibitors that are effective against the wild type (WT) virus and clinically observed mutants, 1,2-bis-substituted benzimidazoles were synthesized and tested. Optimization of the N1 and C2 positions of benzimidazole led to the development of 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-4-methylbenzimidazole (1) (IC50 = 0.2 microM, EC50 = 0.44 microM, and TC50 >/= 100 against WT). This paper describes how substitution on the benzimidazole ring profoundly affects activity. Substituents at the benzimidazole C4 dramatically enhanced potency, while at C5 or C6 substituents were generally detrimental or neutral to activity, respectively. A 7-methyl analogue did not inhibit HIV-1 RT. Determination of the crystal structure of 1 bound to RT provided the basis for accurate modeling of additional analogues, which were synthesized and tested. Several derivatives were nanomolar inhibitors of wild-type virus and were effective against clinically relevant HIV-1 mutants.

Regulation of nuclear gamma interferon gene expression by interleukin 12 (IL-12) and IL-2 represents a novel form of posttranscriptional control.

Posttranscriptional control of gamma interferon (IFN-gamma) gene expression has not been extensively studied and is poorly understood. Our work describes a posttranscriptional mechanism that modulates IFN-gamma mRNA expression in stimulated natural killer (NK) cells through nuclear retention of the IFN-gamma mRNA. This is evidenced by the elevated and sustained nuclear accumulation of both precursor and processed IFN-gamma mRNAs in NK cells stimulated with interleukin-12 (IL-12). The elevated nuclear mRNA accumulation persists long after transcriptional activity has subsided and the rate of cytoplasmic IFN-gamma mRNA accumulation has dropped. The IL-12-induced nuclear retention of the IFN-gamma mRNA prevails until a secondary cytokine stimulus is received. The secondary stimulus, which is initiated by IL-2, mediates transcription-independent movement of the nuclear IFN-gamma mRNA. Concurrent with the nucleocytoplasmic movement of the IFN-gamma mRNA, we have observed increases in the amount of processed nuclear IFN-gamma mRNA that are greater than that seen for the unprocessed IFN-gamma mRNA. The increase in processed IFN-gamma mRNA appears to be due to increased mRNA stability which then promotes increased nucleocytoplasmic shuttling of the mature IFN-gamma mRNA. These data support a model whereby mobilization of nuclear IFN-gamma mRNA stores allows NK cells to rapidly and robustly respond to secondary cytokine activators in a transcription-independent manner, thus shortening the time for overall cellular response to inflammatory signals.

Fixed conformation nucleoside analogs effectively inhibit excision-proficient HIV-1 reverse transcriptases.

An important mechanism of resistance to nucleoside analogs is the enhanced excision of the analog after it has been incorporated. Excision requires that the analog be located at the 3' terminus of the primer. We have developed nucleoside analogs that do not block DNA synthesis at the point of incorporation, but only after additional normal dNTPs have been added to the DNA. Such "delayed chain terminators" should be relatively resistant to excision and therefore effective against drug-resistant HIV-1 reverse transcriptases (RTs) that are proficient at excision. We tested a class of nucleoside analogs in which a pseudosugar ring is locked in either the North or the South conformation. These analogs have a 3' OH present on the pseudosugar ring, which allows extension of the primer strand after the analog is incorporated. We asked whether these analogs would inhibit polymerization by HIV-1 RT in assays using purified HIV-1 RT and in cell-based assays. HIV-1 RT did not effectively incorporate the analogs in which the pseudosugar is in the South conformation. The North conformation analogs are readily incorporated into the primer; the primer can be extended for two or three additional nucleotides before extension is inhibited. This block to polymerization is not complete; larger extension products are detectable at longer incubation times. Experiments with purified excision-proficient HIV-1 RT mutants suggest that the North conformation analogs are relatively resistant to excision. These analogs can also block the replication of viruses containing excision-proficient RTs. Although the fixed-conformation nucleotides are probably not suitable for development as drugs, other nucleoside analogs that cause delayed chain termination may complement the nucleoside analogs already approved for HIV-1 therapy.

Flexible use of nuclear import pathways by HIV-1.

HIV-1 replication requires transport of nascent viral DNA and associated virion proteins, the retroviral preintegration complex (PIC), into the nucleus. Too large for passive diffusion through nuclear pore complexes (NPCs), PICs use cellular nuclear transport mechanisms and nucleoporins (NUPs), the NPC components that permit selective nuclear-cytoplasmic exchange, but the details remain unclear. Here we identify a fragment of the cleavage and polyadenylation factor 6, CPSF6, as a potent inhibitor of HIV-1 infection. When enriched in the cytoplasm, CPSF6 prevents HIV-1 nuclear entry by targeting the viral capsid (CA). HIV-1 harboring the N74D mutation in CA fails to interact with CPSF6 and evades the nuclear import restriction. Interestingly, whereas wild-type HIV-1 requires NUP153, N74D HIV-1 mimics feline immunodeficiency virus nuclear import requirements and is more sensitive to NUP155 depletion. These findings reveal a remarkable flexibility in HIV-1 nuclear transport and highlight a single residue in CA as essential in regulating interactions with NUPs.

The nucleoside analogue D-carba T blocks HIV-1 reverse transcription.

A major pathway for HIV-1 resistance to nucleoside reverse transcriptase inhibitors (NRTIs) involves reverse transcriptase (RT) mutations that enhance ATP-dependent pyrophosphorolysis, which excises NRTIs from the end of viral DNA. We analyzed novel NRTIs for their ability to inhibit DNA synthesis of excision-proficient HIV-1 RT mutants. D-carba T is a carbocyclic nucleoside that has a 3' hydroxyl on the pseudosugar. The 3' hydroxyl group allows RT to incorporate additional dNTPs, which should protect D-carba TMP from excision. D-carba T can be converted to the triphosphate form by host cell kinases with moderate efficiency. D-carba T-TP is efficiently incorporated by HIV-1 RT; however, the next dNTP is added slowly to a D-carba TMP at the primer terminus. D-carba T effectively inhibits viral vectors that replicate using NRTI-resistant HIV-1 RTs, and there is no obvious toxicity in cultured cells. NRTIs based on the carbocyclic pseudosugar may offer an effective approach for the treatment of HIV-1 infections.

The level of reverse transcriptase (RT) in human immunodeficiency virus type 1 particles affects susceptibility to nonnucleoside RT inhibitors but not to lamivudine.

We investigated the relationship between the level of reverse transcriptase (RT) in human immunodeficiency virus type 1 (HIV-1) particles and susceptibility to nonnucleoside reverse transcriptase inhibitors (NNRTIs). HIV-1 virions containing different active levels of RT were generated. Susceptibility to the NNRTIs efavirenz and nevirapine was inversely proportional to the level of enzymatically active RT. However, the sensitivity of HIV-1 to the nucleoside analog 3TC was not affected by the level of RT per particle. These data indicate that the susceptibility of HIV-1 to NNRTIs is influenced by RT activity.

Combining mutations in HIV-1 reverse transcriptase with mutations in the HIV-1 polypurine tract affects RNase H cleavages involved in PPT utilization.

The RNase H cleavages that generate and remove the polypurine tract (PPT) primer during retroviral reverse transcription must be specific to generate linear viral DNAs that are suitable substrates for the viral integrase. To determine if specific contacts between reverse transcriptase (RT) and the PPT are a critical factor in determining the cleavage specificity of RNase H, we made HIV-1 viruses containing mutations in RT and the PPT at the locations of critical contacts between the protein and the nucleic acid. The effects on titer and RNase H cleavage suggest that combining mutations in RT with mutations in the PPT affect the structure of the protein of the RT/nucleic acid complex in ways that affect the specificity and the rate of PPT cleavage. In contrast, the mutations in the PPT (alone) and RT (alone) affect the specificity of PPT cleavage but have much less effect on the overall rate of cleavage.

HIV-1 reverse transcriptase structure with RNase H inhibitor dihydroxy benzoyl naphthyl hydrazone bound at a novel site.

The rapid emergence of drug-resistant variants of human immunodeficiency virus, type 1 (HIV-1), has limited the efficacy of anti-acquired immune deficiency syndrome (AIDS) treatments, and new lead compounds that target novel binding sites are needed. We have determined the 3.15 A resolution crystal structure of HIV-1 reverse transcriptase (RT) complexed with dihydroxy benzoyl naphthyl hydrazone (DHBNH), an HIV-1 RT RNase H (RNH) inhibitor (RNHI). DHBNH is effective against a variety of drug-resistant HIV-1 RT mutants. While DHBNH has little effect on most aspects of RT-catalyzed DNA synthesis, at relatively high concentrations it does inhibit the initiation of RNA-primed DNA synthesis. Although primarily an RNHI, DHBNH binds >50 A away from the RNH active site, at a novel site near both the polymerase active site and the non-nucleoside RT inhibitor (NNRTI) binding pocket. When DHBNH binds, both Tyr181 and Tyr188 remain in the conformations seen in unliganded HIV-1 RT. DHBNH interacts with conserved residues (Asp186, Trp229) and has substantial interactions with the backbones of several less well-conserved residues. On the basis of this structure, we designed substituted DHBNH derivatives that interact with the NNRTI-binding pocket. These compounds inhibit both the polymerase and RNH activities of RT.

Alternate polypurine tracts (PPTs) affect the rous sarcoma virus RNase H cleavage specificity and reveal a preferential cleavage following a GA dinucleotide sequence at the PPT-U3 junction.

Retroviral polypurine tracts (PPTs) serve as primers for plus-strand DNA synthesis during reverse transcription. The generation and removal of the PPT primer requires specific cleavages by the RNase H activity of reverse transcriptases; removal of the PPT primer defines the left end of the linear viral DNA. We replaced the endogenous PPT from RSVP(A)Z, a replication-competent shuttle vector based on Rous sarcoma virus (RSV), with alternate retroviral PPTs and the duck hepatitis B virus "PPT." Viruses in which the endogenous RSV PPT was replaced with alternate PPTs had lower relative titers than the wild-type virus. 2-LTR circle junction analysis showed that the alternate PPTs caused significant decreases in the fraction of viral DNAs with complete (consensus) ends and significant increases in the insertion of part or all of the PPT at the 2-LTR circle junctions. The last two nucleotides in the 3' end of the RSV PPT are GA. Examination of the (mis)cleavages of the alternate PPTs revealed preferential cleavages after GA dinucleotide sequences. Replacement of the terminal 3' A of the RSV PPT with G caused a preferential miscleavage at a GA sequence spanning the PPT-U3 boundary, resulting in the deletion of the terminal adenine normally present at the 5' end of the U3. A reciprocal G-to-A substitution at the 3' end of the murine leukemia virus PPT increased the relative titer of the chimeric RSV-based virus and the fraction of consensus 2-LTR circle junctions.

Roles of viral and cellular proteins in the expression of alternatively spliced HTLV-1 pX mRNAs.

The human T cell leukemia virus type 1 (HTLV-1) genome contains a cluster of at least five open reading frames (ORFs) near the 3' terminus within the pX region. The pX ORFs are encoded by mono- or bicistronic mRNAs that are generated by alternative splicing. The various pX mRNAs result from skipping of the internal exon (2-exon versus 3-exon isofoms) or from the utilization of alternative splice acceptor sites in the terminal exon. The Rex and Tax proteins, encoded by ORFs X-III and X-IV, have been studied intensively and are encoded by the most abundant of the alternative 3-exon mRNAs. The protein products of the other pX ORFs have not been detected in HTLV-1-infected cell lines and the levels of the corresponding mRNAs have not been accurately established. We have used real-time RT-PCR with splice-site specific primers to accurately measure the levels of individual pX mRNA species in chronically infected T cell lines. We have asked whether virus regulatory proteins or ectopic expression of cellular factors influence pX mRNA splicing in cells that were transfected with HTLV-1 provirus clones. In chronically infected cell lines, the pX-tax/rex mRNA was present at 500- to 2500-fold higher levels than the pX-tax-orfII mRNA and at approximately 1000-fold higher levels than pX-rex-orfI mRNA. Chronically infected cell lines that contain numerous defective proviruses expressed 2-exon forms of pX mRNAs at significantly higher levels compared to cell lines that contain a single full-length provirus. Cells transfected with provirus expression plasmids expressed similar relative amounts of 3-exon pX mRNAs but lower levels of 2-exon mRNA forms compared to cells containing a single, full-length provirus. The pX mRNA expression patterns were nearly identical in cells transfected with wild-type, Tax-minus, or Rex-minus proviruses. Cotransfection of cells with HTLV-1 provirus in combination with SF2/ASF expression plasmid resulted in a relative increase in pX-tax/rex mRNA compared to pX-tax-orfII and pX-rex-orfI mRNAs, but did not affect exon skipping. Ectopic expression of hnRNP A1 did not affect pX splice site utilization, but increased exon skipping, as the level of pX-p21rex mRNA was increased by almost 10-fold.

Construction and characterization of a replication-competent retroviral shuttle vector plasmid.

We constructed two versions of an RCASBP-based retroviral shuttle vector, RSVP (RCASBP shuttle vector plasmid), containing either the zeocin or blasticidin resistance gene. In this vector, the drug resistance gene is expressed in avian cells from the long terminal repeat (LTR) promoter, whereas in bacteria the resistance gene is expressed from a bacterial promoter. The vector contains a bacterial origin of replication (ColE1) to allow circular viral DNA to replicate as a plasmid in bacteria. The vector also contains the lac operator sequence, which binds to the lac repressor protein, providing a simple and rapid way to purify the vector DNA. The RSVP plasmid contains the following sequence starting with the 5" end: LTR, gag, pol, env, drug resistance gene, lac operator, ColE1, LTR. After this plasmid was transfected into DF-1 cells, we were able to rescue the circularized unintegrated viral DNA from RSVP simply by transforming the Hirt DNA into Escherichia coli. Furthermore, we were able to rescue the integrated provirus. DNA from infected cells was digested with an appropriate restriction enzyme (ClaI) and the vector-containing segments were enriched using lac repressor protein and then self-ligated. These enriched fractions were used to transform E. coli. The transformation was successful and we did recover integration sites, but higher-efficiency rescue was obtained with electroporation. The vector is relatively stable upon passage in avian cells. Southern blot analyses of genomic DNAs derived from successive viral passages under nonselective conditions showed that the cassette (drug resistance gene-lac operator-ColE1) insert was present in the vector up to the third viral passage for both resistance genes, which suggests that the RSVP vectors are stable for approximately three viral passages. Together, these results showed that RSVP vectors are useful tools for cloning unintegrated or integrated viral DNAs.

Mutations in the RNase H domain of HIV-1 reverse transcriptase affect the initiation of DNA synthesis and the specificity of RNase H cleavage in vivo.

Retroviral reverse transcriptases contain a DNA polymerase activity that can copy an RNA or DNA template and an RNase H activity that degrades the viral RNA genome during reverse transcription. RNase H makes both specific and nonspecific cleavages; specific cleavages are used to generate and remove the polypurine tract primer used for plus-strand DNA synthesis and to remove the tRNA primer used for minus-strand DNA synthesis. We generated mutations in an HIV-1-based vector to change amino acids in the RNase H domain that contact either the RNA and DNA strands. Some of these mutations affected the initiation of DNA synthesis, demonstrating an interdependence of the polymerase and RNase H activities of HIV-1 reverse transcription during viral DNA synthesis. The ends of the linear DNA form of the HIV-1 genome are defined by the specific RNase H cleavages that remove the plus- and minus-strand primers; these ends can be joined to form two-long-terminal repeat circles. Analysis of two-long-terminal repeat circle junctions showed that mutations in the RNase H domain affect the specificity of RNase H cleavage.

Effects of mutations in the G tract of the human immunodeficiency virus type 1 polypurine tract on virus replication and RNase H cleavage.

The RNase H cleavages that generate and remove the polypurine tract (PPT) primer during retroviral reverse transcription must be specific in order to create a linear viral DNA that is suitable for integration. Lentiviruses contain a highly conserved sequence consisting of six guanine residues at the 3' end of the PPT (hereafter referred to as the G tract). We introduced mutations into the G tract of a human immunodeficiency virus type 1-based vector and determined the effects on the virus titer and RNase H cleavage specificity. Most mutations in the G tract had little or no effect on the virus titer. Mutations at the second and fifth positions of the G tract increased the proportion of two-long-terminal-repeat (2-LTR) circle junctions with one or two nucleotide insertions. The second and fifth positions of the G tract make specific contacts with amino acids in the RNase H domain that are important for RNase H cleavage specificity. These complementary data define protein-nucleic acid interactions that help control the specificity of RNase H cleavage. When the G-tract mutants were analyzed in a viral background that was deficient in integrase, in most cases the proportion of consensus 2-LTR circle junctions increased. However, in the case of a mutant with Ts at the second and fifth positions of the G tract, the proportion of 2-LTR circle junctions containing the one-nucleotide insertion increased, suggesting that linear viral DNAs containing an extra base are substrates for integration. This result is consistent with the idea that the 3' end-processing reactions of retroviral integrases may help to generate defined ends from a heterogenous population of linear viral DNAs.

Mutations at position 184 of human immunodeficiency virus type-1 reverse transcriptase affect virus titer and viral DNA synthesis.

Methionine at position 184 of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) was changed to valine, isoleucine, threonine, or alanine in an HIV-1-based vector. The vectors were analyzed for replication capacity and for resistance to the nucleoside analog 2',3'-dideoxy-3'thiacytidine (3TC) using a single-cycle assay. Viruses containing the valine or isoleucine mutations were highly resistant to 3TC and replicated almost as well as the wild-type virus. The virus containing the threonine mutation was resistant to 3TC, but replicated about 30% as well as the wild-type. The alanine mutation conferred partial resistance to 3TC, but replicated poorly. The amounts of viral DNA synthesized decreased in 3TC-treated cells when the cells were infected with wild-type virus and the M184A mutant. The effect of these mutations on the generation of the ends of the linear viral DNA was determined using the sequence of the 2-LTR circle junctions. The M184T mutation increased the proportion of 2-LTR circle junctions containing a tRNA insertion, suggesting that the mutation affected the RNase H activity of RT.

Mutations in the 5' end of the human immunodeficiency virus type 1 polypurine tract affect RNase H cleavage specificity and virus titer.

The RNase H activity of retroviral reverse transcriptases (RTs) degrades viral genomic RNA after it has been copied into DNA, removes the tRNA used to initiate negative-strand DNA synthesis, and generates and removes the polypurine tract (PPT) primer used to initiate positive-strand DNA synthesis. The cleavages that remove the tRNA and that generate and remove the PPT primer must be specific to generate linear viral DNAs with ends that are appropriate for integration into the host cell genome. The crystal structure of human immunodeficiency virus type 1 (HIV-1) RT in a complex with an RNA/DNA duplex derived from the PPT revealed that the 5' end of the PPT deviates from traditional Watson-Crick base pairing. This unusual structure may play a role in the proper recognition of the PPT by HIV-1 RT. We made substitution mutations in the 5' end of the PPT and determined their effects on virus titer. The results indicated that single and double mutations in the 5' end of the PPT had modest effects on virus replication in a single-cycle assay. More complex mutations had stronger effects on virus titer. Analysis of the two-long-terminal-repeat circle junctions derived from infecting cells with the mutant viruses indicated that the mutations affected RNase H activity, resulting in the retention of PPT sequences on viral DNA. The mutants tested preferentially retained specific segments of the PPT, suggesting an effect on cleavage specificity. These results suggest that structural features of the PPT are important for its recognition and cleavage in vivo.

Mutation of amino acids in the connection domain of human immunodeficiency virus type 1 reverse transcriptase that contact the template-primer affects RNase H activity.

The crystal structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase in a complex with an RNA-DNA template-primer identified amino acids in the connection domain that make specific contacts with the nucleic acid. We analyzed the effects of mutations in these amino acids by using a one-round HIV-1 vector. Mutations in amino acids in the connection domain generally had small effects on virus titers. To determine whether the mutations affected the level of RNase H activity or the specificity of RNase H cleavage, we used the two-long-terminal-repeat circle junction as a surrogate for the ends of linear viral DNA; specific RNase H cleavages determine the ends of the viral DNA. Several of the mutations in the connection domain affected the frequency of the generation of viral DNAs with aberrant ends. The mutation H361A had the largest effect on the titer and on the generation of DNAs with aberrant ends. H361 contacts the phosphate backbone of the nucleic acid in the same location as amino acid Y501 in the RNase H primer grip. Mutations at Y501 have been shown to decrease the virus titer and affect the specificity of RNase H cleavage. H361A affected the frequency of the generation of linear viral DNAs with aberrant ends, but in general the connection domain mutations had subtle effects on the efficiency of RNase H cleavage. The results of this study suggest that, in addition to its primary role in linking the polymerase and RNase H domains, the connection subdomain has a modest role in binding and positioning the nucleic acid.

Altering the RNase H primer grip of human immunodeficiency virus reverse transcriptase modifies cleavage specificity.

Recent crystallographic data suggest that conserved residues in the connection subdomain and C-terminal ribonuclease H (RNase H) domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) contact the nascent DNA primer and modulate the trajectory of the template relative to the RNase H catalytic center. Within the RNase H domain, these residues include Thr473, Glu475, Lys476, Tyr501, and Ile505, while His539 and Asn474 interact with the scissile phosphate of the RNA template. Amino acid substitutions at several of these positions were evaluated in the context of hydrolysis of nonspecific RNA-DNA hybrids and substrates mimicking specific RNase H-mediated events. With the exception of mutant I505G, which exhibited a dimerization defect, substituting alanine at positions 473-476 and 501 had minimal consequences for DNA synthesis on duplex and hybrid DNA and RNA substrates. In contrast, the efficiency with which most mutants catalyzed polymerization-independent RNase H cleavage was sharply reduced. This deficiency was more pronounced when mutant enzymes were challenged to process the (+) strand polypurine tract (PPT) primer from either (+) RNA or a PPT/(+) DNA RNA/DNA chimera. Reduced polymerization-independent RNase H activity also significantly influenced the rate of DNA strand transfer, suggesting the donor template must be reduced in size below 13 nt before this event proceeds.