Mutagenesis of Gln294 of the reverse transcriptase of human immunodeficiency virus type-2 and its effects on the ribonuclease H activity.

Despite the high homology between human immunodeficiency virus type-1 (HIV-1) and human immunodeficiency virus type-2 (HIV-2) reverse transcriptases (RTs), the ribonuclease H (RNase H) level of HIV-2 RT is lower than that of HIV-1 RT, while the DNA polymerase of both RTs is similar. We conducted mutagenesis of HIV-2 RT Gln294 (shown to control the RNase H activity level when modified to a Pro in the smaller p54 subunit and not in the larger p68 subunit) to various residues, and assayed the activities of all mutants. All exhibited an RNase H that is higher than the wild-type (WT) HIV-2 RT level, although the DNA polymerase of all mutants equals WT HIV-2 RT level. These results represent a unique case, where every mutation induces an increase rather than a decrease in an enzyme's activity.

The ribonuclease H activity of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2 is modulated by residue 294 of the small subunit.

Reverse transcriptases (RTs) exhibit DNA polymerase and ribonuclease H (RNase H) activities. The RTs of human immunodeficiency viruses type 1 and type 2 (HIV-1 and HIV-2) are composed of two subunits, both sharing the same N-terminus (which encompasses the DNA polymerase domain). The smaller subunit lacks the C-terminal segment of the larger one, which contains the RNase H domain. The DNA polymerase domain of RTs resembles a right hand linked to the RNase H domain by a connection subdomain. Despite the high homology between HIV-1 and HIV-2 RTs, the RNase H activity of the latter is substantially lower than that of HIV-1 RT. The thumb subdomain of the small subunit controls the level of RNase H activity. We show here that Gln294, located in this thumb, is responsible for this difference in activity. A HIV-2 RT mutant, where Gln294 in the small subunit was replaced by a proline (present in HIV-1 RT), has an activity almost 10-fold higher than that of the wild-type RT. A comparative in vitro study of the kinetic parameters of the RNase H activity suggests that residue 294 affects the K(m) rather than the kcat value, influencing the affinity for the RNA.DNA substrate.

Clathsterol, a novel anti-HIV-1 RT sulfated sterol from the sponge Clathria species.

As part of a search for novel inhibitors of humandeficiency virus type 1 (HIV-1) reverse transcriptase (RT), the MeOH-EtOAc extract of a Red Sea sponge, Clathria sp., was shown to be active. Bioassay-guided fractionation of the extract yielded a novel sterol sulfate, clathsterol (1), which is responsible for the activity and is active at a concentration of 10 microM. The structure of 1 was established mainly by interpretation of spectral data and a chemical transformation.

The ribonuclease H activity of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2 is affected by the thumb subdomain of the small protein subunits.

Retroviral reverse transcriptases (RTs) have both DNA polymerase and ribonuclease H (RNase H) activities. The RTs of HIV-1 and HIV-2 are heterodimers of p66/p51 and p68/p54 subunits, respectively. The smaller subunit lacks the C-terminal segment of the larger subunit (which is the RNase H domain). The structure of the DNA polymerase domain of HIV-1 RT resembles a right hand (with fingers, palm and thumb subdomains), linked to the RNase H domain via the connection subdomain. The RNase H activity of the Rod strain of HIV-2 RT is about tenfold lower than that of HIV-1 RT, while the DNA polymerase activity of these RTs is similar. A chimeric RT in which residues 227-427 (which constitute a small part of the palm and the entire thumb and connection subdomains) of the Rod strain of HIV-2 RT were replaced by the corresponding segment from HIV-1 RT, has an RNase H activity as high as HIV-1 RT (despite the fact that the RNase H domain is derived from HIV-2 RT). We analyzed the RNase H activity of wild-type HIV-2 RT from the D-194 strain and compared it with this activity of the RT from the Rod strain of HIV-2 and HIV-1 RT. The level of this activity of both HIV-2 RT strains was low; suggesting that low RNase H activity is a general property of HIV-2 isolates. The in vitro RNase H digestion pattern of the three wild-type RTs was indistinguishable, despite the difference in the level of RNase H activity. We constructed new chimeric HIV-1/HIV-2 RTs, in which protein segments and/or subunits were exchanged. The DNA polymerase activity of the parental HIV-1 and HIV-2 RTs was similar; as expected, the specific activity of the polymerases of all the hybrid RTs were also similar. However, the RNase H specific activity of the chimeric RTs was either high (like HIV-1 RT) or low (like HIV-2 RT). The origin of the thumb subdomain in the small subunit of the chimeric RTs (residues 244-322) determines the level of the RNase H activity. The strand-transfer activity of the chimeric RTs is also affected by the thumb subdomain of the small subunit; transfer was much more efficient if this subdomain was derived from HIV-1 RT. The data can be explained from the three-dimensional structure of HIV-1 RT. The thumb of the smaller subunit contacts the RNase H domain; it is through these contacts that the thumb affects the level of the RNase H activity of RT.

New co-metabolites of the streptazolin pathway.

Variation of the culture conditions of Streptomyces sp. strain A1, which produces streptazolin (1), resulted in the isolation of four new co-metabolites: 5-O-(beta-D-xylopyranosyl)streptazolin (3), 9-hydroxystreptazolin (4), 13-hydroxystreptazolin (5), and streptenol E (6). Their structures were established by spectroscopic and chemical methods. The possible biosynthetic relationship between the streptazolins and the streptenols is discussed.

Polycitone A, a novel and potent general inhibitor of retroviral reverse transcriptases and cellular DNA polymerases.

Polycitone A, an aromatic alkaloid isolated from the ascidian Polycitor sp. exhibits potent inhibitory capacity of both RNA- and DNA-directed DNA polymerases. The drug inhibits retroviral reverse transcriptase (RT) [i.e. of human immunodeficiency virus type 1 (HIV), murine leukaemia virus (MLV) and mouse mammary tumour virus (MMTV)] as efficiently as cellular DNA polymerases (i.e. of both DNA polymerases alpha and beta and Escherichia coli DNA polymerase I). The mode and mechanism of inhibition of the DNA-polymerase activity associated with HIV-1 RT by polycitone A have been studied. The results suggest that the inhibitory capacity of the DNA polymerase activity is independent of the template-primer used. The RNase H function, on the other hand, is hardly affected by this inhibitor. Polycitone A has been shown to interfere with DNA primer extension as well as with the formation of the RT-DNA complex. Steady-state kinetic studies demonstrate that this inhibitor can be considered as an allosteric inhibitor of HIV-1 RT. The target site on the enzyme may be also spatially related to the substrate binding site, since this inhibitor behaves competitively with respect to dTTP with poly(rA).oligo(dT) as template primer. Chemical transformations of the five phenol groups of polycitone A by methoxy groups have a determinant effect on the inhibitory potency. Thus, the pentamethoxy derivative which is devoid of all hydroxy moieties, loses significantly, by 40-fold, the ability to inhibit the DNA polymerase function. Furthermore, this analogue lacks the ability to inhibit DNA primer extension as well as the formation of the RT-DNA complex. Indeed, inhibition of the first step in DNA polymerization, the formation of the RT-DNA complex, and hence, of the overall process, could serve as a model for a universal inhibitor of the superfamily of DNA polymerases.

Catalytic features of the recombinant reverse transcriptase of bovine leukemia virus expressed in bacteria.

We have expressed the recombinant reverse transcriptase (RT) of bovine leukemia virus (BLV) in bacteria. The gene encoding the RT was designed to start at its 5' end next to the last codon of the mature viral protease, namely the amino terminus of the RT matches the last 26 codons of the pro gene and is coded for by the pro reading frame. The RT sequence extends into the pol gene, utilizing the pol reading frame after overcoming the stop codon by adding an extra nucleotide (thus imitating the naturally occurring frameshift event). Hence we have generated a transframe polypeptide that is a 584-residues-long protein (see Rice, Stephens, Burny, and Gilden (1985) Virology 142, 357-377). This protein was partially purified after adding a six-histidine tag and studied biochemically testing a variety of parameters. The enzyme exhibits all activities typical of RTs, i.e., both RNA- and DNA-dependent DNA polymerase as well as a ribonuclease H (RNase H) activity. Unlike most RTs, the BLV RT is enzymatically active as a monomer even after binding a DNA substrate. The enzyme shows a preference for Mg2+ over Mn2+ in both its DNA polymerase and RNase H activities. BLV RT is relatively resistant to nucleoside triphosphate analogues, which are known to be potent inhibitors of other RTs such as that of HIV.

The inhibition of the reverse transcriptase of HIV-1 by the natural sulfoglycolipids from cyanobacteria: contribution of different moieties to their high potency.

The potent in vitro inhibition of the enzymatic activity of the human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT) by the lipophilic extracts of cyanobacteria8 was primarily attributed to the sulfoquinovosylpranosyl lipids, compounds 1-4. These sulfolipids inhibit efficiently and selectively only the DNA polymerase activity of HIV-1 RT (and not the ribonuclease H function) with 50% inhibitory concentration value (IC50) as low as 24 nM exhibited by compound 1. The novel natural compound 4, in which two hydroxy groups on the sugar moiety are substituted by palmitoyl residues, exhibits a significant decrease in the maximal inhibition capacity. It is possible, therefore, that the contribution of acylated groups to the molecule at these positions interferes with inhibition, possibly, by steric hindrance. Both the sulfonic acid moiety and the fatty acid ester side chain have a substantial effect in potentiating the extent of inhibition. For one, the inhibitory effects of all the natural glycolipids tested (5-8) are markedly reduced, and the hydrolysis of the fatty acid side chain, as in derivative 9, has substantially abolished the inhibition of HIV RT.

The processivity of DNA synthesis exhibited by drug-resistant variants of human immunodeficiency virus type-1 reverse transcriptase.

The reverse transcriptase (RT) of human immunodeficiency virus (HIV) undergoes rapid mutagenesis due to selective pressure by RT inhibitors which renders the mutated RT variants resistant to these inhibitors. Resistance to nucleoside analogs during drug therapy results from point mutations that lead to specific variations in the RT sequences. It was recently shown that several well-defined drug-resistant variants of HIV-1 RT (i.e. Leu74Val, Glu89Gly, Tyr183Phe, Met184Lue, Met184Val and Met184Ile) show enhanced accuracy of DNA synthesis relative to wild-type HIV-1 RT (as evident from a reduction in the capacity to introduce mispairs and to elongate them). Since the last two Met184 variants were shown also to possess decreased processivity of DNA synthesis, it was recently suggested that there might be an inverse correlation between the apparent in vitro fidelity and processivity of DNA synthesis in drug-resistant HIV-1 RT mutants. In the present study we have conducted a comparative analysis of the processivity of DNA synthesis on both DNA and RNA templates of the Leu74Val, Glu89Gly, Tyr183Phe and Met184Leu drug-resistant mutants of HIV-1 RT in comparison with wild-type RT. Apart from the Met184 mutant, which shows reduced relative processivity (similar to the other mutants of residue 184 already studied), the other three variants have relative processivity at least as high as that of wild-type RT. This suggests that the inverse correlation between reduced processivity and increased fidelity is restricted only to mutants with modifications of Met184. The results presented may bear on potential mechanistic and structural differences in the involvement of the various mutated residues studied in processivity, fidelity and sensitivity to nucleoside analogs.

Reverse transcriptase of mouse mammary tumour virus: expression in bacteria, purification and biochemical characterization.

We have constructed a plasmid that induces in bacteria the synthesis of an enzymically active reverse transcriptase (RT) of mouse mammary tumour virus (MMTV), a retrovirus with a typical B-type morphology. The highest catalytic activity was detected only when 27 residues from the C-terminus of the protease were included in the N-terminus of the recombinant RT, after an extra deoxyadenosine was added between the pro and pol genes to overcome the -1 frameshift event (which occurs naturally in virus-infected cells). The recombinant protein with a six-histidine tag was purified to homogeneity by a two-column purification procedure, Ni2+ nitriloacetic acid/agarose followed by carboxymethyl-Sepharose chromatography. Unlike most RTs, the purified MMTV RT is enzymically active as a monomer even after binding a DNA substrate. Like all RTs studied, the recombinant MMTV RT possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity, all of which show a preference for Mg2+ over Mn2+ ions. Other features of these enzymic activities, such as extension of DNA primers, processivity of DNA synthesis, pH dependence, steady-state kinetic constants, effects of Na+ or K+ ions and sensitivity to a thiol-specific reagent and to a zinc chelator, have been evaluated. The catalytic properties of MMTV RT were compared with those of the well-studied RT of HIV-1, the causative agent of AIDS. Interestingly, MMTV RT exhibits a high sensitivity to nucleoside triphosphate analogues (which are known to be potent inhibitors of HIV RTs and are being used as the major anti-AIDS drugs), as high as that of HIV-1 and HIV-2 RTs. Furthermore the recombinant MMTV RT shows a processivity of DNA synthesis higher than that of HIV-1 RT.

DNA synthesis exhibited by the reverse transcriptase of mouse mammary tumor virus: processivity and fidelity of misinsertion and mispair extension.

We have recently expressed in bacteria an enzymatically active reverse transcriptase (RT) of mouse mammary tumor virus (MMTV), a mammalian retrovirus with a typical B-type morphology [Taube, R., Loya, S., Avidan, O., Perach, M. & Hizi, A. (1998) Biochemical J. 329, 579-587]. The purified recombinant protein was shown to possess the catalytic activities characteristic of retroviral reverse transcriptases. In the present study, we have analyzed two basic parameters characteristic of the DNA polymerase activity of the novel MMTV RT, namely the processivity and the fidelity of DNA synthesis. Two features related to fidelity were studied, the capacity to misinsert wrong nucleotides at the 3' end of the nascent DNA strand and the ability to extend 3' mispairs. The studied properties of MMTV RT were compared with those of the RT purified from virions of avian myeloblastosis virus (AMV), since AMV RT shows a relatively high sequence similarity to MMTV RT. MMTV RT shows a relative processivity of DNA synthesis which is as high as the reference AMV RT. Regarding fidelity of DNA synthesis, MMTV RT shows a fidelity of misinsertion lower than that of AMV RT, whereas its capacity to elongate mispaired DNA is lower than that of AMV RT indicating a somewhat higher fidelity. These fidelity properties are discussed also in the context of the RTs of lentiviruses, especially those of HIV, which were reported to exhibit an exceptionally low fidelity of DNA synthesis. It is clear that MMTV RT has a fidelity higher than that of lentiviral RTs.

The fidelity of 3' misinsertion and mispair extension during DNA synthesis exhibited by two drug-resistant mutants of the reverse transcriptase of human immunodeficiency virus type 1 with Leu74-->Val and Glu89-->Gly.

The relatively low fidelity of DNA synthesis characteristic to the reverse transcriptases (RTs) of the AIDS-causing viruses, human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively) was implicated as a dominant factor that contributes to the genetic hypervariability of these viruses. The formation of 3'-mispaired DNA and the subsequent extension of this DNA were shown to be key determinants that lead to the error proneness of these RTs. As part of our goal to study the structure/function relationship in HIV-1 RT, we have conducted mutational studies aimed at identifying amino-acid residues involved in affecting the fidelity of DNA synthesis by the enzyme. We have recently found that two mutants of HIV-1 RT, which show resistance to nucleoside analog inhibitors ([Leu184]RT and [Phe183]RT), exhibit in vitro error proneness of DNA synthesis lower than that of wild-type enzyme [Bakhanshvili, M., Avidan, O. & Hizi, A. (1996) Mutational studies of human immunodeficiency virus type 1 reverse transcriptase: the involvement of residues 183 and 184 in the fidelity of DNA synthesis, FEBS Lett. 391, 257-262]. Using both criteria, the current comparative study suggests that these two mutant RTs display a substantially enhanced fidelity of DNA synthesis relative to the wild-type RT counterpart. In the current study we have analyzed two additional drug-resistant mutants of HIV-1 RT, [Val74]RT and [Gly89]RT, for their in vitro fidelity of DNA synthesis using two parameters of DNA synthesis: 3' mispair formation and elongation of 3'-mismatched DNA. The current comparative study suggests that these two mutant RTs display a substantially enhanced fidelity of DNA synthesis relative to the wild-type RT counterpart, using both criteria. Analysis of the relative frequencies of misinsertion and mispair extension indicates that the overall error proneness of DNA synthesis in HIV-1 RT is wild-type > [Val74]RT > [Gly89]RT mutant. The results further support the possible linkage between the capacity of an enzyme to incorporate a nucleoside analog instead of the correct dNTP (leading to drug sensitivity) and the ability to incorporate and extend a wrong nucleotide (resulting in mutagenesis). Our results may bear on the potential use of selecting and maintaining HIV virions with high fidelity and drug-resistant RTs to suppress the subsequent appearance of virions resistant to other drugs.

Subunit-specific mutagenesis of the cysteine 280 residue of the reverse transcriptase of human immunodeficiency virus type 1: effects on sensitivity to a specific inhibitor of the RNase H activity.

Treatment of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) with N-ethylmaleimide (NEM) selectively inhibits the RNase H activity. The cysteine residue at position 280 (C280) is the target for NEM; HIV-1 RT carrying the mutation C280S is resistant to NEM. Since HIV-1 RT is composed of two related subunits (p66 and p51) that play distinct roles, we asked whether the C280 in p51 or the C280 in p66 is responsible for the sensitivity of the enzyme to NEM. HIV-1 RT versions were prepared that had one mutant and one wild-type subunit. When these chimeric enzymes were tested, both the p51 and p66 subunits were found to contribute to the sensitivity of the enzyme to NEM. The implications of these results are discussed in the context of the structure of the enzyme.

Mode of inhibition of HIV reverse transcriptase by 2-hexaprenylhydroquinone, a novel general inhibitor of RNA-and DNA-directed DNA polymerases.

A natural compound from the Red Sea sponge Ircinia sp., 2-hexaprenylhydroquinone (HPH), has been shown to be a general inhibitor of retroviral reverse transcriptases (from HIV-1, HIV-2 and murine leukaemia virus) as well as of cellular DNA polymerases (Escherichia coli DNA polymerase I, and DNA polymerases alpha and beta). The pattern of inhibition was found to be similar for all DNA polymerases tested. Thus the mode of inhibition was studied in detail for HIV-1 reverse transcriptase. HPH is a non-competitive inhibitor and binds the enzyme irreversibly with high affinity (Ki=0. 62 microM). The polar hydroxy groups have been shown to be of key importance. A methylated derivative, mHPH, which is devoid of these polar moieties, showed a significantly decreased capacity to inhibit all DNA polymerases tested. Like the natural product, mHPH binds the enzyme independently at an allosteric site, but with reduced affinity (Ki=7.4 microM). We show that HPH does not interfere with the first step of the polymerization process, i.e. the physical formation of the reverse-transcriptase-DNA complex. Consequently, we suggest that the natural inhibitor interferes with the subsequent steps of the overall reaction. Since HPH seems not to affect the affinity of dNTP for the enzyme (the Km is unchanged under conditions where the HPH concentration is increased), we speculate that its inhibitory capacity is derived from its effect on the nucleotidyl-transfer catalytic reaction. We suggest that such a mechanism of inhibition is typical of an inhibitor whose mode of inhibition should be common to all RNA- and DNA-directed polymerases.

Analysis of HIV-2 RT mutants provides evidence that resistance of HIV-1 RT and HIV-2 RT to nucleoside analogs involves a repositioning of the template-primer.

Mutations that confer resistance to nucleoside analogs do not cluster around the deoxynucleotide triphosphate (dNTP) binding site. Instead, these mutations appear to lie along the groove in the enzyme where the template-primer binds. Based on such structural data and on complementary biochemical analyses, it has been suggested that resistance to nucleoside analogs involves repositioning of the template-primer. We have prepared mutations in HIV-2 RT that are the homologs of mutations that confer resistance to nucleoside analogs in HIV-1 RT. Analysis of the behavior of HIV-2 RT mutants (Leu74Val, Glu89Gly, Ser215Tyr, Leu74Val/Ser215Tyr and Glu89Gly/Ser215Tyr) in vitro confirms the results obtained with HIV-1 RT: resistance is a function of the length of the template overhang. These analyses also suggest that the homolog in HIV-2 RT of one of the mutations that confers resistance to AZT in HIV-1 RT (Thr215Tyr) confers resistance by repositioning of the template-primer.

Mutagenesis by human immunodeficiency virus reverse transcriptase: incorporation of O6-methyldeoxyguanosine triphosphate.

The high frequency of incorporation of non-complementary nucleotides by HIV-1 reverse transcriptase is likely to be a major factor in the exceptionally rapid accumulation of viral mutations during the course of AIDS infections. To investigate whether this high level of infidelity is also associated with the incorporation of nucleotide analogs, we analyzed O6-methyldeoxyguanosine triphosphate and compared the incorporation of this analog by HIV-1 reverse transcriptase to that catalyzed by other DNA synthesizing enzymes. Our results indicate that O6-methyldeoxyguanosine triphosphate serves as a substrate for DNA synthesized in vitro by HIV-1 RT on both DNA and RNA templates. The product DNA contains the modified purine; it is sensitive to the repair enzyme, O6-methylguanine methyltransferase, which specifically reacts with DNA containing methylated guanines at the O6 position. Using a forward mutation assay we demonstrated that the nucleotide analog incorporated by HIV-1 RT is mutagenic. The mutations produced are single-base substitutions opposite template thymidines and result in A:T --> G:C transitions. The incorporation of a mutagenic nucleotide by HIV-1 RT highlights the possibility of increasing the rate of mutagenesis of HIV by the use of nucleotides that form non-complementary base pairs at high frequency.

Incorporation of the guanosine triphosphate analogs 8-oxo-dGTP and 8-NH2-dGTP by reverse transcriptases and mammalian DNA polymerases.

We have measured the efficiencies of utilization of 8-oxo-dGTP and 8-NH2-dGTP by human immunodeficiency virus type 1 and murine leukemia virus reverse transcriptases and compared them to those of DNA polymerases alpha and beta. Initially, we carried out primer extension reactions in the presence of dGTP or a dGTP analog and the remaining three dNTPs using synthetic DNA and RNA templates. These assays revealed that, in general, 8-NH2-dGTP is incorporated and extended more efficiently than 8-oxo-dGTP by all enzymes tested. Second, we determined rate constants for the incorporation of each analog opposite a template cytidine residue using steady state single nucleotide extension kinetics. Our results demonstrated the following. 1) Both reverse transcriptases incorporate the nucleotide analogs; discrimination against their incorporation is a function primarily of Km or Vmax depending on the analog and the enzyme. 2) Discrimination against the analogs is more stringent with the DNA template than with a homologous RNA template. 3) Polymerase alpha exhibits a mixed kinetic phenotype, with a large discrimination against 8-oxo-dGTP but a comparatively higher preference for 8-NH2-dGTP. 4) Polymerase beta incorporates both analogs efficiently; there is no discrimination with respect to Km and a significantly lower discrimination with respect to Vmax when compared with the other polymerases.

Inefficient repair of RNA x DNA hybrids.

RNA x DNA hybrids are commonly observed during normal biological processes. We tested the ability of three DNA-repair enzymes to remove lesions from the DNA strand of RNA x DNA heteroduplexes. Three nucleotide analogs, 5-hydroxy-2'-deoxycytidine triphosphate, 8-oxo-2'-deoxyguanosine triphosphate, and O6-methyl-2'-deoxyguanosine triphosphate, representative of lesions generated by oxygen damage and methylating agents, were incorporated into the DNA strand synthesized using either a DNA or RNA template. The extended DNA x DNA and RNA x DNA hybrids were used as substrates for bacterial formamidopyrimidine-DNA glycosylase, Nth protein (endonuclease III) and O6-methylguanine-DNA methyltransferase. We show that all three lesions are readily cleaved from the DNA strand of a DNA x DNA duplex but are relatively resistant to cleavage when present in the DNA strand of an RNA x DNA hybrid. Our in vitro studies suggest that damaged DNA in RNA x DNA hybrids is less likely to be repaired in vivo.

The fidelity of misinsertion and mispair extension throughout DNA synthesis exhibited by mutants of the reverse transcriptase of human immunodeficiency virus type 2 resistant to nucleoside analogs.

The AIDS-causing retroviruses, human immunodeficiency virus types 1 and type 2 (HIV-1 and HIV-2, respectively) undergo extensive genetic variations, which effect their pathogenesis and resistance to drug therapy. It was postulated that this genetic hypervariability results from high rates of viral replication in conjugation with a relatively low fidelity of DNA synthesis [typical to the reverse transcriptases (RT) of these retroviruses]. As part of studying structure/function relationship in HIV RT, mutational analyses were conducted to identify amino acid residues which are involved in affecting the fidelity of DNA synthesis. The formation of 3'-mispaired DNA due to nucleotide misinsertions, and the subsequent elongation of this mismatched DNA were shown to be major determinants in affecting those substitutions during DNA synthesis (exhibited in vitro by HIV RT). It was interesting to find a correlation between sensitivity to nucleoside analogs (due to the ability to incorporate or reject an incoming analog) and the fidelity of DNA synthesis (which depends on the capacity to incorporate and extend a wrong nucleotide). Such a connection has already been found for several drug-resistant mutants of HIV-1 RT, with an increased fidelity of DNA synthesis relative to the wild-type RT. In the present study we have examined the fidelity of DNA synthesis using the same parameters of misinsertion and mispair extension for five novel drug-resistant mutants of HIV-2 RT; i.e. the single mutants [Val74]RT, [Gly89]RT and [Tyr215]RT and the double mutants [Val74,Tyr215]RT and [Gly89, Tyr215]RT. This comparative study suggests that unlike the Val74 mutant of HIV-1 RT, which was shown earlier to display a substantially enhanced fidelity, the comparable mutant of HIV-2 RT has fidelity similar to that of the wild-type RT. Depending on the assay employed and the DNA sequences extended, most other mutants of HIV-2 RT display moderate effects on the enzyme, leading to mild increases in fidelity of DNA synthesis. This implies a more complex and less distinctive correlation between drug-resistance, misinsertion and mispair extension in HIV-2 RT in contrast to HIV-1 RT, providing evidence for potential biochemical differences between these two related RT.