Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow Print PDF (158K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (8)
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Quan, Y.
Right arrow Articles by Wainberg, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Quan, Y.
Right arrow Articles by Wainberg, M. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research Pages 5692-5698  

Enhanced impairment of chain elongation by inhibitors of HIV reverse transcriptase in cell-free reactions yielding longer DNA products
Introduction
Materials And Methods
   Enzymes and inhibitors
   Templates and primers
   Primer extension by HIV-1 or M-MuLV RT
   Primer extension by Taq DNA polymerase
Results
   Inhibition of HIV wt RT by AZTTP as a function of product length
   Relationship between inhibition of HIV RT by nevirapine and product length
   Inhibition of M184V RT by 3TCTP
   Inhibition of M-MuLV RT by AZTTP as a function of product length
   Inhibition of Taq DNA polymerase by ddTTP in relation to product length
Discussion
References

Enhanced impairment of chain elongation by inhibitors of HIV reverse transcriptase in cell-free reactions yielding longer DNA products

Enhanced impairment of chain elongation by inhibitors of HIV reverse transcriptase in cell-free reactions yielding longer DNA products

Yudong Quan1, Chen Liang1, Phil Inouye1 and Mark A. Wainberg1,2,*

1McGill University AIDS Centre, Lady Davis Institute-Jewish General Hospital, Montreal, Quebec H3T 1E2, Canada and 2Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada

Received July 16, 1998; Revised and Accepted October 21, 1998

ABSTRACT

We have studied the relationship between the length of HIV-1 reverse transcriptase (RT)-mediated nucleotide polymerization and inhibitors of these reactions in cell-free RT assays performed in the presence of either of two dideoxynucleoside triphosphates (ddNTPs), i.e. AZTTP or 3TCTP, or nevirapine, a non-nucleoside RT inhibitor. These reactions employed a heterologous RNA template and three DNA oligonucleotide primers, i.e. pAR, dPR and PA, that yielded distinct full-length products of 65, 192 and 376 nt, respectively, in the absence of inhibitor. We now show that the extent of inhibition of RT activity was greatest with use of the PA primer, which normally yielded the longest reaction product, and that lesser degrees of inhibition were noted in the reactions that generated shorter products. For example, at a concentration of 5 µM AZTTP, the extent of inhibition was 75% with the PA primer but only 40% and <10% when reactions were primed by the dPR and pAR primers, respectively. Similar results were obtained when either a mutated form of HIV RT (i.e. M184V), associated with resistance to 3TC, was tested in the presence of 3TCTP or when RT derived from Moloney murine leukemia virus (M-MuLV) was tested in the presence of AZTTP.

INTRODUCTION

Reverse transcription of viral genomic RNA into linear DNA is an essential step in retroviral replication and is catalyzed by the viral enzyme reverse transcriptase (RT). The RT of human immunodeficiency virus type 1 (HIV-1) shares with other DNA polymerases the property of being a processive enzyme that elongates newly made DNA (1-5) and has been a primary target of antiviral therapy (6-10). This has involved the use of dideoxynucleoside RT inhibitors (NRTI), e.g. AZT, ddI, ddC and 3TC, that act as chain terminators of nascent DNA synthesis, as well as non-nucleoside RT inhibitors (NNRTI), e.g. nevirapine, that act non-competitively to block DNA synthesis.

Enzyme kinetics assays have revealed that the ability of nucleoside inhibitors to block RT is dependent on the length of the homopolymer template employed in the reaction. For instance, lower rate constants for incorporation by HIV-1 RT of chain terminators such as AZTTP, i.e. Ki values, were observed in reactions performed with long templates than short ones (11). These assays have generally monitored total RT products, including both completed full-length molecules as well as those not completely elongated. Functional RT products are, of course, considered to be those that are elongated to full-length, while chain-terminated DNA fragments are not known to play any physiological role. For this reason, it is sometimes difficult to compare the results of cell-free biochemical analyses with in vivo results. As an example, drug-insensitive viruses commonly display higher levels of resistance in tissue culture than are displayed by equivalent recombinant RTs in vitro (12-15). In spite of the importance of this subject, the influence of the anticipated length of DNA reaction products on NRTI- and NNRTI-mediated impairment of RT-mediated DNA extension has not been well characterized. Nor have experiments been performed to directly assess whether RT-mediated DNA polymerization might be a second order dynamic process, in spite of conjecture on this subject.

Therefore, we have studied the effects of different DNA primers that yielded products of varying expected lengths in cell-free RT assays performed with either nucleoside triphosphate analog inhibitors of RT, i.e. AZTTP and 3TCTP, or NNRTIs, i.e. nevirapine. We found that the total synthesis of expected products decreased exponentially as DNA elongation proceeded in reactions performed with wild-type (wt) recombinant HIV RT and AZTTP. When either Moloney murine leukemia virus (M-MuLV) recombinant RT or a 3TC-resistant form of HIV RT containing the M184V substitution was used, similar results were obtained in the presence of either AZTTP or 3TCTP, in spite of the fact that the M-MuLV RT and M184V RT were relatively insensitive to AZTTP and 3TCTP, respectively. In the presence of nevirapine, we found that the amount of full-length RT products also decreased with increased product elongation.

MATERIALS AND METHODS

Enzymes and inhibitors

Recombinant wt and M184V mutated RTs were expressed as histidine-tagged heterodimers in Escherichia coli and purified as described (16,17). Briefly, RT expression plasmids were constructed (17,18) and RT expression in bacteria was induced by IPTG. RT molecules were processed into heterodimers by HIV-1 viral protease co-expressed in the bacteria. The cells were lysed, sonicated, centrifuged and supernatants were applied to a nickel nitrilotriacetate-Sepharose column (Qiagen). The column was washed and histidine-tagged RT was then eluted using an imidazole gradient. RT-containing fractions were sequentially passed through DEAE-Sepharose (Pharmacia) and SP-Sepharose (Pharmacia). Fractions containing purified RT were concentrated, dialyzed overnight against storage buffer (50 mM Tris, pH 7.0, 25 mM NaCl, 1 mM EDTA and 50% glycerol) and stored at -20°C. M-MuLV RT and Taq DNA polymerase were purchased from Gibco BRL (Montreal, Canada). AZTTP and 3TCTP were gifts of BioChem Pharma Inc. (Montreal, Quebec, Canada) and nevirapine was a gift of Boehringer-Ingelheim Inc. (Ridgefield, CT).

Templates and primers

HIV RNA template was prepared in vitro from linearized plasmid pHIV-PBS using a MEGAscript[trade] transcription kit (Ambion, Austin, TX) (19). The template consisted of a 497 bp HIV-1 sequence spanning the R region of HIV-1 LTR and a portion of gag. HIV DNA template was prepared using PCR to amplify an HIV DNA fragment in plasmid pHIV-PBS (from the BglII site in the R region to the PstI site in gag) using primer pair PS (sense, nt 15-35) and PST (antisense, nt 964-942). After recovery by phenol/chloroform extraction and ethanol precipitation, the double-stranded DNA was quantified and then served as template for Taq DNA polymerase in primer extension assays.


Figure 1. Schematic representation of templates, primers and products of in vitro reverse transcription reactions. HIV RNA or DNA templates, containing most of the R region, the complete U5 region, the 5[prime]-untranslated leader sequence and part of the gag gene were used in these reactions. The full-length products of DNA polymerization using the RNA template are 65 nt when primed by pAR (45 nt extended), 192 nt when primed by dPR (174 nt extended) and 376 nt when primed by PA (356 nt extended), respectively (details of primer construction are given in Materials and Methods). Numbers in parentheses indicate the lengths of products generated by Taq polymerase from DNA template.

As primers, we employed an 18 nt DNA, termed dPR, complementary to the primer binding site (PBS) and two 20 nt DNA oligomers, pAR and PA, which have complementarity to the R region within the long terminal repeat (LTR) and to the 5[prime]-end of the gag region, respectively (Fig. 1). These primers were [[gamma]-32P]ATP-labeled and filtered through a Sephadex G-25 column. Template-primer complexes were prepared by mixing template and primer at a ratio of 1:2, denaturing at 85°C for 5 min and cooling to 55°C for 8 min and 37°C for 10 min to allow for specific annealing of primer to template.

Primer extension by HIV-1 or M-MuLV RT

Reactions contained 50 mM Tris (pH 7.8), 5 mM MgCl2, 60 mM KCl, 10 mM DTT, 100 µM dNTPs, 50 nM HIV RNA template, 100 nM primer, 20 U HIV-1 RT (1 U was able to incorporate 1 nM dTMP into DNA in 10 min) or 30 U M-MuLV RT (unit defined by the manufacturer) in a volume of 20 µl and were incubated for 30 min at 37°C. Figure 1 illustrates the templates and primers used and their full-length products of DNA polymerization. Concentrations of RT inhibitors were varied in different assays as shown in each figure. Reaction products were extracted with phenol/chloroform, boiled for 5 min and electrophoresed in a 5% denaturing polyacrylamide gel. Sequencing reactions were performed with the same primers and homologous DNA to determine the size of RT products. Full-length DNA was analyzed using a molecular imager (Bio-Rad Instruments, Mississauga, Ontario, Canada) and the relative radioactive intensities of product bands were calculated. Curve annealing for relative RT activity versus concentration of inhibitor or expected DNA product length was achieved through use of the GraphPad Prism 2.0 software program (GraphPad Software Inc., San Diego, CA); data were fitted to equations as explained in Discussion.

Primer extension by Taq DNA polymerase

Reactions contained 50 mM Tris (pH 8.0), 2.5 mM MgCl2, 50 mM KCl, 100 µM dNTPs, 50 nM HIV DNA template, 100 nM primer, 0.5 U Taq DNA polymerase (unit defined by the manufacturer) and variable concentrations of ddTTP in a total volume of 20 µl. Figure 1 illustrates the templates and primers used and the full-length products of DNA polymerization. Reaction mixtures were incubated at 94°C for 5 min, followed by 58°C for 1 min and 72°C for 2 min. Products were extracted with phenol/chloroform, boiled for 5 min and electrophoresed in a 5% denaturing polyacrylamide gel. Sequencing and molecular imaging analyses were performed as described above.

RESULTS

Inhibition of HIV wt RT by AZTTP as a function of product length

To test the effect of anticipated DNA product length on reverse transcribed DNA in the presence of RT inhibitors, we required an experimental system that could yield products of different size in the presence of such inhibitors. We therefore designed an in vitro primer extension system to generate three distinct products. The system included a heterogeneous HIV-1 RNA template as well as three primers, pAR, dPR and PA, that initiated RT reactions from different positions on the RNA template to yield full-length products that were extended by 45, 174 or 356 nt, respectively.bac

   a
   b

   c

Figure 2. Inhibition of recombinant wt RT in reactions performed with different primers, pAR, dPR or PA, in the presence of AZTTP. (a) Radioautographic results of reaction mixtures that contained 50 mM Tris (pH 7.8), 5 mM MgCl2, 60 mM KCl, 20 mM DTT, 50 nM HIV RNA template, 100 nM labeled primer, 10 U RT, 100 µM dNTPs and AZTTP concentrations of 0, 5, 10, 25 and 50 µM from left to right in each panel. After 60 min at 37°C, reaction products were boiled, electrophoresed in a denaturing 5% polyacrylamide gel and visualized by radioautography. Numbers identify the nucleotide positions of full-length products. (b and c) The analysis of these results by molecular imaging. Curve annealing was performed through use of the GraphPad Prism program (Materials and Methods).

The effects of AZTTP in this system are shown in Figure 2a and reveal that the lengths of the RT products generated were clearly related to the degree of inhibition displayed by the inhibitor. When the PA primer was used to yield a longer DNA product, we observed that a given concentration of AZTTP displayed a stronger inhibitory effect than when shorter products were initiated by primers PAR and dPR (Fig. 2b). This was also reflected in the IC50 values for AZTTP in these reactions performed with different primers, i.e. 43.0 ± 2.5, 8.8 ± 0.7 and 3.7 ± 0.4 µM for reactions primed by pAR, dPR and PA, respectively.

Figure 2c shows the relationship between relative RT activity and expected full-length product in a different way. Notably, in the presence of equivalent concentrations of AZTTP, the potential for inhibition increased alongside increases in product length. For example, <10% inhibition of synthesis was seen with 5 µM AZTTP when the pAR primer was used to extend the product by 45 nt. However, when the dPR and PA primers were employed to achieve extension by 174 and 356 nt, respectively, this same concentration of AZTTP resulted in inhibition of 40 and 75%. At 50 µM AZTTP, inhibition was 60% with the pAR primer (extension of 45 nt) but was >95% when reactions were primed by dPR and PA. Similar results were obtained with other concentrations of AZTTP (Fig. 2c).

We also analyzed our results by curve annealing and found that they fitted an exponential curve. This was true for reactions primed by any of the primers used in the presence of increasing concentrations of AZTTP (Fig. 2b) as well as when RT products were elongated to different lengths (Fig. 2c). Thus, levels of RT products of different lengths can be predicted through use of equations that describe the second order dynamics of these reactions (Discussion).

   a
   b

   c

Figure 3. Inhibition of recombinant wt RT in reactions performed with different primers in the presence of nevirapine. (a) Legend as for Figure 2, except that nevirapine (nev) concentrations were 0, 0.2, 0.5, 1.0 and 2.0 µM from left to right in each panel. (b and c) The analyses of these results by molecular imaging.

Relationship between inhibition of HIV RT by nevirapine and product length

AZTTP acts as a chain terminator by inhibiting RT activity through competition with the natural substrate dTTP. In this context, the relationship between this inhibitory effect and the length of DNA products has been predicted (20). To extend this concept, we decided to assess whether similar effects would be observed with an NNRTI such as nevirapine. Figure 3a shows results obtained with wt RT and different concentrations of nevirapine in the same types of primer extension assays described above. Similar results were obtained as with AZTTP except that nevirapine exerted even stronger inhibitory effects. The greatest degree of inhibition was observed in reactions primed by primer PA, that extended a product of 356 nt, while the least inhibition was seen with primer pAR, that extended a small product of only 45 nt (Fig. 3a). The IC50 values obtained for primer extension of 45, 174 and 356 nt were 0.75 ± 0.05, 0.37 ± 0.04 and 0.19 ± 0.02 µM, respectively. Curve annealing showed that these results could be fitted to an exponential curve (Fig. 3b).

Although slight differences were observed in reactions performed with nevirapine versus AZTTP, the relationship between RT activity and product length fitted the exponential curve well in both cases (Fig. 3c). Thus, the inhibitory effect of the NNRTI was similar to that of the chain terminator, with the total full-length product decreasing alongside an increase in DNA strand length.

Inhibition of M184V RT by 3TCTP

It has been reported that drug-resistant RT molecules may behave differently than wt RT in reactions performed with RT inhibitors and with different lengths of RNA template (21). The M184V substitution in RT confers resistance to 3TC. Therefore, we assessed this enzyme, under the experimental conditions described above, in the presence of the active form of 3TC, i.e. 3TCTP. We found that 3TCTP displayed similar inhibition patterns with regard to M184V RT as did AZTTP for wt RT, except that higher 3TCTP concentrations were required to block the activity of the mutated enzyme (Fig. 4a). IC50 values for 3TCTP with M184V RT were [ge]1000, 380 ± 35 and 220 ± 30 µM, respectively, in reactions performed with primers pAR, dPR and PA, while far lower values were obtained with wt enzyme (results not shown). Curve annealing showed that these results were best fitted to an exponential curve (Fig. 4a). The data of Figure 4b show that the inhibitory effect of 3TCTP on M184V RT was similar to that displayed by AZTTP for wt RT with regard to product length (Fig. 2c). Thus, M184V RT behaves similarly to wt RT in the presence of 3TCTP throughout a range of concentrations high enough to affect its activity.

   a
   b

Figure 4. Inhibition of recombinant mutated M184V RT in reactions performed with different primers, i.e. pAR, dPR or PA, in the presence of 3TCTP. Results were analyzed by molecular imaging. Reactions were performed as described in the legend to Figure 2 for wt RT, except that 3TCTP concentrations were 0, 100, 250, 500 and 1000 µM.

Inhibition of M-MuLV RT by AZTTP as a function of product length

To determine whether the above results might also apply to RTs of viruses other than HIV, we tested M-MuLV RT that contains only a single chain in its primary structure. The results of Figure 5a show that AZTTP had relatively little effect on M-MuLV RT activity in reactions primed by pAR (i.e. 45 nt product). However, significantly greater inhibition was observed in reactions with primers dPR and PA, that extend 174 and 354 nt products, respectively (Fig. 5a). IC50 values were >400, 80 ± 10 and 38 ± 5 µM, respectively, in reactions performed with primers pAR, dPR and PA. Curve analysis showed that these data were best fitted to an exponential curve (Fig. 5b). Therefore, M-MuLV RT behaves similarly to HIV RT, both in the presence and absence of inhibitor.

   a
   b

Figure 5. Inhibition of recombinant M-MuLV RT in reactions performed with different primers, i.e. pAR, dPR or PA, in the presence of AZTTP concentrations of 0, 25, 50, 100, 200 and 400 µM. Results were analyzed by molecular imaging. Reactions were performed as described above for HIV RT except that 30 U enzyme were used in each reaction.

Inhibition of Taq DNA polymerase by ddTTP in relation to product length

Since other DNA polymerases besides RT may possess both polymerase and exonuclease activities, we wished to know whether such enzymes might behave similarly to RT in the presence of inhibitors. Therefore, we tested one such enzyme, i.e. Taq DNA polymerase, to determine whether a similar relationship as seen with RT might also exist between primer extension and the inhibitory effects of NRTIs. Since Taq DNA polymerase is insensitive to AZTTP, we employed ddTTP as an inhibitor and found that inhibition of Taq polymerase was less related to product length than was shown for RT, although higher levels of inhibition were still observed in reactions that yielded relatively long products (Fig. 6a). IC50 values were 560 ± 50, 520 ± 55 and 200 ± 15 µM, respectively, in reactions performed with the pAR, dPR and PA primers. Curve annealing showed that these data did not fit well to exponential curves (Fig. 6a and b).

   a
   b

Figure 6. Inhibition of recombinant Taq DNA polymerase in reactions performed with different primers, i.e. pAR, dPR or PA, in the presence of ddTTP concentrations of 0, 100, 300, 600 and 1200 µM. Results were analyzed by molecular imaging.

DISCUSSION

HIV RT is a processive enzyme that catalyzes the polymerization of DNA strands (1,2,5). Unlike most enzymatic reactions that result in increased numbers of products, DNA polymerization reactions generally elongate DNA strands without any increase in product number, i.e. the product of each dNMP incorporation serves as a substrate for each successive reaction until the end of the template is reached. Therefore, the kinetics of such reactions involve both dNMP additions during strand elongation and the steps of each dNMP incorporation. Some RT assays extend just 1 nt to distinguish between these various steps.

In RT assays that extend DNA strands by >1 nt, reaction products may be either full-length or incomplete; the presence of the latter type of product is increased in the presence of enzyme inhibitors. From a functional standpoint, only fully synthesized strands are active, but conventional kinetics assays generally analyze total RT products that include both full-length functional and incomplete products.

The influence of template length on inhibition of RT activity by ddNTPs has been studied. The inhibitory potential of AZTTP is related to the length of the poly(rA) region on a given template (11). The longer the template, the lower was the Ki value obtained. However, Ki determinations are, in general, less meaningful for DNA polymerases than for other enzymes (11,20).

To complement and extend the above studies, we have used an in vitro system that permitted us to distinguish among products of different anticipated length, synthesized through the use of different primers. We found that the length of the anticipated product influenced the degree of inhibition obtained when either ddNTPs or a NNRTI were present in RT reactions. This effect is similar to that of drug concentration, i.e. the longer the product length, the greater the extent of inhibition.

The distribution of nucleotides may vary from one region to another within a template. This may also affect the inhibitory potential of ddNTP inhibitors for enzymes involved in copying different regions of template during synthesis of DNA. Since, the number of A or G residues in any given template may affect the likelihood of incorporation of the inhibitors used in our studies, i.e. AZTTP or 3TCTP, we analyzed our templates and found that A residues were 23-25% and G residues were 25-28% of total nucleotides present. These percentages could not, therefore, have contributed significantly to our results. Furthermore, the inhibitory effect of nevirapine would not have been affected by the nucleotide composition of the template, since NNRTIs bind to and inhibit RT differently than ddNTPs.

Our results with AZTTP are consistent with values predicted by other workers on the basis of theoretical considerations (20). The accumulation of elongated DNA products in RT reactions should follow the equation

T/Pn + E->E·T/Pn + dNTP->E·T/Pn + 1 + dNTP->E·T/Pn + 2 + dNTP->...->E·T/Pn + i

This process is a standard second order dynamic reaction that helps to explain the relationship between amount of product obtained and product length and can be described by the equation (20):

P = P0 · e-ki 1

where P is the product elongated by i nt, P0 is the input template and primer, i is the elongated nucleotide and k is a constant. In the presence of AZTTP, the equation can be rewritten as

P/Pno = (1 - fin)i 2

where fin is the probability that a RT inhibitor at a given concentration may inhibit the ith nucleotide incorporation and Pno represents products generated when no inhibitor was present.

This equation does not, however, consider the effects of template on RT reactions since we have used a controlled parameter, i.e. P/Pno, rather than P/P0. The equation also presupposes that inhibitors have no significant effects on interactions between RT and the template/primer. Indeed, this is probably the case for AZTTP, since significant changes in RT pausing sites along the template were not observed in a drug concentration-dependent manner in our studies (Fig. 2a).

Equation 2 fits the results of observed interactions between HIV-1 wt RT and AZTTP and also results obtained with the M184V mutated HIV-1 RT and 3TCTP (Fig. 4) and M-MuLV RT and AZTTP (Fig. 5). These results are expected, since these inhibitors are chain terminators and share a common mechanism of action (20,22) and also because the enzymes studied are similar (1-2).

In contrast, inhibition by ddTTP of Taq DNA polymerase did not fit equation 2, although the inhibitory effect of this compound was increased in reactions that yielded longer DNA strands. This may reflect the possibility that Taq polymerase is functionally distinct from RT, notably in regard to higher processivity and its weak 3[prime] exonuclease activity. This proof-reading activity can remove dideoxynucleosides after ddNTPs have blocked DNA polymerization, thus enabling a proportion of chain-terminated DNA strands to be further extended. Theoretically, shorter DNA strands have the best chance of completing polymerization, since there are fewer potential sites at which a given inhibitor might be incorporated.


Figure 7. Theoretical prediction of the relationship between the observed length of DNA strands and lengths of anticipated products. The curves are deduced from equation 1 with various [alpha] values in the form of k = 1 - [alpha].

The effects of nevirapine on RT and product length can also be fitted to equation 2, which can therefore predict both the effects of chain terminators and NNRTIs in RT reactions. The inhibitory effect of NNRTIs may be exerted through slowing the speed of RT catalysis, hence, longer DNA strands should result in fewer full-length products. Equation 1 predicts that factors that affect polymerase activity should also alter the k value and thereby influence DNA strand elongation. However, residual RT activity obtained in the presence of AZTTP was lower than that observed in reactions performed with nevirapine (compare Figs 2c and 3c). Thus, nevirapine might have less of an inhibitory effect than AZTTP with regard to synthesis of long DNA strands, although both possess similar inhibitory potential with regard to short strands. This may reflect the fact that NNRTIs are not chain terminators but non-competitive inhibitors that decrease catalytic efficiency (23-25). Some nevirapine molecules might randomly have dissociated from the RT·template-primer complex, allowing further elongation of once inhibited DNA strands to take place. This may account for the higher inhibition of pAR-primed products but lower inhibition of both dPR- and PA-primed products in Figure 3c than in Figure 2c. In addition, nevirapine may affect the interaction between RT and template-primer because it increases RT pausing at some places on the template (26).

Our results distinguish between RT inhibition experiments in which short versus long DNA products are synthesized. This may help to explain why some RT inhibitors, e.g. AZT and 3TC, are much more effective in vivo than are their derivative ddNTPs in vitro (12-15,27). Figure 7 displays the profound effect of DNA strand sequential elongation on efficiency of overall product synthesis based on equation 1. Using [alpha] = 0.90 as an example, 90% of total DNA strands can be successfully elongated to 200 nt, corresponding approximately to (-) strong-stop DNA, but only ~0.5% of DNA strands will be elongated to 10 kb, the length of the HIV genome. This is consistent, as well, with reports that AZT blocked viral replication by >90% without significantly affecting formation of (-) strong-stop DNA within cells (28-31). Of course, the effects of any given drug in vivo are also dependent on other factors, including intracellular distribution and metabolism (32-34).

REFERENCES

1. Skalka,A.M. and Goff,S.P. (1993) Reverse Transcriptase. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 163-191.

2. Weiss,R., Teich,N., Varmus,H.E. and Coffin,J. (1985) RNA Tumor Viruses, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

3. Hsieh,J., Zinnen,S. and Modrich,P. (1993) J. Biol. Chem., 268, 24607-24613. MEDLINE Abstract

4. Kati,W.M., Johnson,K.A., Jerva,L.F. and Anderson,K.S. (1992) J. Biol. Chem., 267, 25988-25997. MEDLINE Abstract

5. Patel,P.H., Jacobo-Molina,A., Ding,J., Taillo,C.,Jr, Clark,A.D., Raag,R., Nanni,R.G., Hughes,S.H. and Arnold,E. (1995) Biochemistry, 34, 5351-5356. MEDLINE Abstract

6. Merluzzi,V.J. et al.) (1990) Science, 250, 1411-1413. MEDLINE Abstract

7. Mitsuya,H. and Broder,S. (1986) Proc. Natl Acad. Sci. USA, 83, 1911-1915. MEDLINE Abstract

8. St Clair,M.H., Martin,J.L., Tudor-Williams,G., Bach,M.C., Vavro,C.L., King,D.M., Kellam,P., Kemp,S.D. and Larder,B.A. (1991) Science, 253, 1557-1559. MEDLINE Abstract

9. St. Clair,M.H., Richards,C.A., Spector,T., Weinhold,K.J. and Miller,W.H. (1987) Antimicrob. Agents Chemother., 31, 1972-1977.

10. Larder,B.A., Darby,G. and Richman,D.D. (1989) Science, 243, 1731-1734. MEDLINE Abstract

11. Ma,Q.F., Bathurst,I.C., Barr,P.J. and Kenyon,G.L. (1992) Biochemistry, 31, 1375-1379. MEDLINE Abstract

12. Gao,Q., Gu,Z., Parniak,M.A., Cameron,J., Cammack,N., Boucher,C. and Wainberg,M.A. (1993) Antimicrob. Agents Chemother., 37, 1390-1392. MEDLINE Abstract

13. Gu,Z., Gao,Q., Fang,H., Salomon,H., Parniak,M.A., Goldberg,E., Cameron,J. and Wainberg,M.A. (1994) Antimicrob. Agents Chemother., 38, 275-281. MEDLINE Abstract

14. Quan,Y., Gu,Z., Li,X., Morrow,C.D. and Wainberg,M.A. (1996) J. Virol., 70, 5642-5645. MEDLINE Abstract

15. Faraj,A., Agrofoglio,L.A., Wakefield,J.K., McPherson,S., Morrow,C.D., Gosselin,G., Mathe,C., Imbach,J.-L., Schinazi,R.F. and Sommadossi,J.-P. (1994) Antimicrob. Agents Chemother., 38, 2300-2305. MEDLINE Abstract

16. Le Grice,S.F.J. and Gruninger-Leitch,F. (1990) Eur. J. Biochem., 187, 307-314. MEDLINE Abstract

17. Le Grice,S.F.J., Cameron,C.E. and Benkovic,S.J. (1995) Methods Enzymol., 262, 130-144. MEDLINE Abstract

18. Quan,Y., Inouye,P., Liang,C., Rong,L., Gotte,M. and Wainberg,M.A. (1998) J. Biol. Chem., 273, 21918-21925. MEDLINE Abstract

19. Arts,E.J., Li,X., Gu,Z., Kleiman,L., Parniak,M.A. and Wainberg,M.A. (1994) J. Biol. Chem., 269, 14672-14680. MEDLINE Abstract

20. Goody,R.S., Muller,B. and Restle,T. (1991) FEBS Lett., 291, 1-5. MEDLINE Abstract

21. Boyer,P., Tantillo,C., Jacobo-Molina,A., Nanni,R.G., Ding,J., Arnold,E. and Hughes,S.H. (1994) Proc. Natl Acad. Sci. USA, 91, 4882-4886. MEDLINE Abstract

22. Arts,E.J. and Wainberg,M.A. (1996) Antimicrob. Agents Chemother., 40, 527-540. MEDLINE Abstract

23. Spence,R., Anderson,K.S. and Johnson,K.A. (1996) Biochemistry, 35, 1054-1063. MEDLINE Abstract

24. Spence,R.A., Kati,W.M., Anderson,K.S. and Johnson,K.A. (1995) Science, 267, 988-993. MEDLINE Abstract

25. Rittinger,K., Divta,G. and Goody,R.S. (1995) Proc. Natl Acad. Sci. USA, 92, 8046-8049. MEDLINE Abstract

26. Gu,Z., Quan,Y., Li,Z., Arts,E.J. and Wainberg,M.A. (1995) J. Biol. Chem., 270, 31046-31051. MEDLINE Abstract

27. Quan,Y., Gu,Z., Li,X., Liang,C., Parniak,M.A. and Wainberg,M.A. (1998) J. Mol. Biol., 277, 237-247. MEDLINE Abstract

28. Zack,J.A., Arrigo,S.J., Weitman,S.R., Go,A.S., Haislip,A. and Chen,I.S.Y. (1990) Cell, 61, 213-222. MEDLINE Abstract

29. Zack,J.A., Haosip,A.M., Knogstad,P. and Chen,I.S.Y. (1992) J. Virol., 66, 1717-1725. MEDLINE Abstract

30. Li,X., Liang,C., Quan,Y., Chandok,R., Laughrea,M., Parniak,M.A., Kleiman,L. and Wainberg,M.A. (1997) J. Virol., 71, 6003-6010. MEDLINE Abstract

31. Arts,E.J. and Wainberg,M.A. (1994) Antimicrob. Agents Chemother., 38, 1008-1016. MEDLINE Abstract

32. Back,N.K.T., Nijhuis,M., Keulen,W., Boucher,C.A.B., Oude Essink,B.B., van Kuilenburg,A.B.P., van Gennip,A.H. and Berkhout,B. (1996) EMBO J., 15, 4040-4049.

33. Gao,W.Y., Shirasaka,T., Johns,D.G., Broder,S. and Mitsuya,H. (1993) J. Clin. Invest., 91, 2326-2333. MEDLINE Abstract

34. Allain,B., Lapadat-Tapolsky,M., Berlioz,C. and Darlix,J.L. (1994) EMBO J., 13, 973-981. MEDLINE Abstract

*To whom correspondence should be addressed at: McGill AIDS Centre, Lady Davis Institute-Jewish General Hospital, 3755 Cote Ste-Catherine Road, Montreal, Quebec H3T 1E2, Canada. Tel: +1 514 340 8260; Fax: +1 514 340 7537; Email: mdwa@musica.mcgill.ca

This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 3 Dec 1998
Copyright©Oxford University Press, 1998.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
J. A. Grobler, G. Dornadula, M. R. Rice, A. L. Simcoe, D. J. Hazuda, and M. D. Miller
HIV-1 Reverse Transcriptase Plus-strand Initiation Exhibits Preferential Sensitivity to Non-nucleoside Reverse Transcriptase Inhibitors in Vitro
J. Biol. Chem., March 16, 2007; 282(11): 8005 - 8010.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
P. R. Meyer, A. J. Smith, S. E. Matsuura, and W. A. Scott
Chain-Terminating Dinucleoside Tetraphosphates Are Substrates for DNA Polymerization by Human Immunodeficiency Virus Type 1 Reverse Transcriptase with Increased Activity against Thymidine Analogue-Resistant Mutants
Antimicrob. Agents Chemother., November 1, 2006; 50(11): 3607 - 3614.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
K. Diallo, M. Gotte, and M. A. Wainberg
Molecular Impact of the M184V Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase
Antimicrob. Agents Chemother., November 1, 2003; 47(11): 3377 - 3383.
[Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
Y. Quan, B. G. Brenner, M. Oliveira, and M. A. Wainberg
Lamivudine Can Exert a Modest Antiviral Effect against Human Immunodeficiency Virus Type 1 Containing the M184V Mutation
Antimicrob. Agents Chemother., February 1, 2003; 47(2): 747 - 754.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. Pascolo, C. Wenz, J. Lingner, N. Hauel, H. Priepke, I. Kauffmann, P. Garin-Chesa, W. J. Rettig, K. Damm, and A. Schnapp
Mechanism of Human Telomerase Inhibition by BIBR1532, a Synthetic, Non-nucleosidic Drug Candidate
J. Biol. Chem., May 3, 2002; 277(18): 15566 - 15572.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
Y. Quan, L. Rong, C. Liang, and M. A. Wainberg
Reverse Transcriptase Inhibitors Can Selectively Block the Synthesis of Differently Sized Viral DNA Transcripts in Cells Acutely Infected with Human Immunodeficiency Virus Type 1
J. Virol., August 1, 1999; 73(8): 6700 - 6707.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Print PDF (158K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (8)
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Quan, Y.
Right arrow Articles by Wainberg, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Quan, Y.
Right arrow Articles by Wainberg, M. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?