Jiong Wang, 1 Dongge Li, 1 Robert A. Bambara, 2 Hongmei Yang 3 and Carrie Dykes 1 INTRODUCTION

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1 Journal of General Virology (213), 94, DOI 1.199/vir L74V increases the reverse transcriptase content of HIV-1 virions with non-nucleoside reverse transcriptase drug-resistant mutations LI+K13N and K11E+G19S, which results in increased fitness Jiong Wang, 1 Dongge Li, 1 Robert A. Bambara, 2 Hongmei Yang 3 and Carrie Dykes 1 Correspondence Carrie Dykes carrie_dykes@urmc.rochester.edu 1 Department of Medicine, University of Rochester Medical Center, Rochester, NY 14642, USA 2 Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA 3 Department of Biostatistics and Computational Biology, University of Rochester Medical Center, Rochester, NY 14642, USA Received 18 December 212 Accepted 25 March 213 The fitness of non-nucleoside reverse transcriptase inhibitor (NNRTI) drug-resistant reverse transcriptase (RT) mutants of HIV-1 correlates with the amount of RT in the virions and the RNase H activity of the RT. We wanted to understand the mechanism by which secondary NNRTI-resistance mutations, LI and K11E, and the nucleoside resistance mutation, L74V, alter the fitness of K13N and G19S viruses. We measured the amount of RT in virions and the polymerization and RNase H activities of mutant RTs compared to wild-type, K13N and G19S. We found that LI, K11E and L74V did not change the polymerization or RNase H activities of K13N or G19S RTs. However, LI and K11E reduced the amount of RT in the virions and subsequent addition of L74V restored RT levels back to those of G19S or K13N alone. We conclude that fitness changes caused by LI, K11E and L74V derive from their effects on RT content. INTRODUCTION The human immunodeficiency virus type-1 (HIV-1) virion contains two copies of genomic RNA. The reverse transcriptase (RT) of HIV-1 converts the genomic RNAs into a single copy of double-stranded DNA through the process of reverse transcription. HIV-1 RT is a multifunctional enzyme that has RNA- and DNA-dependent DNA polymerase activities, which copy RNA or DNA templates, respectively, into double-stranded DNA, plus RNase H activity that cleaves genomic RNA hybridized to DNA (Whitcomb & Hughes, 1992). RT is a heterodimer comprised of a 66 kda (p66) subunit and a 51 kda (p51) subunit (Goff, 199). The p66 subunit has both the polymerase and RNase H domains, which make contact simultaneously with the RNA genome (Ding et al., 1998). The p51 subunit is an N-terminal cleavage of p66 that lacks the RNase H domain and serves as a structural scaffold for the p66 subunit (Amacker & Hübscher, 1998). In view of its essential role in the virus life cycle, the RT enzyme is an important target of antiretroviral therapy (Levy, 27). Efavirenz (EFV) is a non-nucleoside RT inhibitor (NNRTI) that is commonly used in combination with two nucleoside-analogue RT inhibitors (nrtis) for the treatment of antiretroviral-naïve patients (DHHS, 25, Guidelines for the Use of Antiretroviral Agents). One limitation of the NNRTI class of drugs, including EFV, is a low genetic barrier to resistance. A single mutation can cause a high level of drug resistance (Bacheler et al., 2, 21). Our previous studies have shown that the prevalence of NNRTI-resistance mutations in clinical specimens correlates in a complex manner with replication fitness of the viruses with these mutations. In addition, a reduction of replication capacity was associated with defects in the ratio of RNase H to polymerization activity and reduced amounts of RT content in the virion (Archer et al., 2; Gerondelis et al., 1999; Koval et al., 26; Wang et al., 21a; Wang et al., 26). There was no correlation between fitness and the polymerization activities of NNRTI-resistant mutants (Domaoal et al., 26; Gerondelis et al., 1999; Wang et al., 21a). In our studies, K13N, the most common NNRTIresistance mutation, had a modest fitness defect compared to wild-type () HIV-1 virus (Koval et al., 26). K13N 5914 G 213 SGM Printed in Great Britain 1597

2 J. Wang and others (a) K13N LI+K13N L74V+LI+K13N Substrate 21 Primary 16 Secondary (b) Percentage of substrate remaining K13N LI+K13N L74V+LI+K13N (c) Secondary formation K13N LI+K13N L74V+LI+K13N (d) G19S K11E+G19S L74V+K11E+K13N Substrate 21 Primary 16 Secondary 1598 Journal of General Virology 94

3 RT content in virions alters fitness (e) Percentage of substrate remaining G19S K11E+G19S L74V+K11E+G19S (f). Secondary formation G19S. K11E+G19S L74V+K11E+G19S Fig. 1. Polymerase-dependent RNase H activity of recombinant and mutant RTs. (a, d) Representative polyacrylamide gel of DNA 39 end-directed RNase H activity. The substrate used was made by annealing a 26 nt long DNA oligonucleotide primer to a 59 end-labelled 41 nt long RNA such that the 39 end of the DNA primer was recessed relative to the RNA 59 end. Reactions were allowed to proceed for the indicated lengths of time. Nucleotide size markers are indicated on the left. (b, e) Rate of total substrate degradation (y-axis) versus time (x-axis). (c, f) Rate of secondary formation (y-axis) versus time (x-axis). The ratio of secondary cleavage s to the sum of the total s were quantified by phosphorimaging and plotted on the y- axis versus time on the x-axis. The reactions were performed in triplicate using a single protein prep for each mutant. Results represent the means and standard deviations of three replicates. Bonferroni adjusted P-values were calculated using Students t-test. The amount of substrate remaining or of formed at 15 min for G19S was statistically different to (P,.1). has a fitness value of.94, indicating that the mutant is 6 % less fit than. The emergence in patients of secondary mutations, such as LI, increased drug resistance while reducing the replication fitness of K13N compared to the virus or the K13N variant alone. The fitness of LI+K13N was.84 compared to K13N. Surprisingly, the fitness defect of LI+K13N was compensated completely by the addition of the NRTI resistance mutation L74V. L74V+LI+K13N had a fitness value equal to K13N. L74V had a similar effect on K11E+G19S. The fitness of G19S compared to is.59, indicating that it has a 41 % fitness loss compared to (Wang et al., 26). K11E+G19S is reduced even more, with a fitness value of.57 compared to G19S, indicating an additional loss in fitness of 43 % (Wang et al., 21b). L74V compensated for the fitness defect of the K11E+G19S double mutation without reducing its level of drug resistance. L74V+ K11E+G19S fitness was increased by 32 % compared to K11E+G19S and by 7 % compared to G19S (Wang et al., 21b). In this study we examined the RT content and enzymic properties of the viruses with single and double NNRTI mutations, and in combination with L74V. The intention was to demonstrate how the mutations altered the biochemistry of the mutant viruses to cause the observed changes in fitness. RESULTS Effects of secondary NNRTI mutations LI and K11E on the RNase H activity of RTs with primary mutations K13N and G19S Our previously published data showed that emerging secondary NNRTI drug-resistance mutations, such as LI and K11E, increased the drug resistance of HIV-1 viruses with the primary mutations K13N and G19S, respectively, but decreased their replication efficiency (Koval et al., 26; Wang et al., 21a). In addition, we showed that defects in virus fitness caused by primary NNRTI drug-resistance mutations were associated with reduced RNase H activity, but not polymerase activity, of the purified mutant RTs (Archer et al., 2; Gerondelis et al., 1999; Wang et al., 21a; Wang et al., 26). Therefore, we hypothesized that LI and K11E further decrease the RNase H activity of K13N or G19S, resulting in additional reductions in fitness for these double mutations. In order to test this prediction, we expressed and purified heterodimer RTs having the LI+K13N or K11E+G19S mutations, and then measured their RNase H activities compared with, K13N and G19S RTs. Surprisingly, the polymerasedependent RNase H activity of LI+K13N RT was similar to and K13N alone (Fig. 1a). This result was also true for K11E+G19S RT. Although the polymerasedependent RNase H activities of G19S, K11E+G19S

4 J. Wang and others (a) K13N LI+K13N L74V+LI+K13N Substrate Primary 7 Secondary (b) Percentage of substrate remaining K13N LI+K13N L74V+LI+K13N (c) Secondary formation K13N LI+K13N L74V+LI+K13N (d) G19S K11E+G19S L74V+K11E+G19S Substrate Primary 7 Secondary 16 Journal of General Virology 94

5 RT content in virions alters fitness (e) Percentage of substrate remaining G19S K11E+G19S L74V+K11E+G19S (f) Secondary formation G19S K11E+G19S L74V+K11E+G19S Fig. 2. Polymerase-independent RNase H activity of purified and mutant RTs. (a, d) Representative polyacrylamide gel of RNA 59-end-directed RNase H activity. The substrate used was made by annealing a 59-end-labelled 41 nt RNA to a 77 nt DNA primer such that the 59 end of the RNA was recessed relative to the DNA. The reactions were allowed to proceed for the indicated lengths of time. Nucleotide size markers are indicated on the left. (b, e) Rate of total substrate degradation (y-axis) versus time (x-axis). (c, f) Rate of secondary formation (y-axis) versus time (x-axis). The ratio of secondary cleavage s to the sum of the total s were quantified by phosphorimaging and plotted on the y-axis versus time on the x- axis. The reactions were performed in triplicate using a single protein prep for each mutant. Results represent the means and standard deviations of three replicates. Bonferroni-adjusted significant differences in the amount of substrate remaining or formed at 15 min compared to were calculated using Students t-test. and L74V+K11E+G19S RTs were reduced compared to, there was no change in the polymerase-dependent RNase H activity of K11E+G19S compared to G19S alone (Fig. 1d f). We next tested the polymerase-independent RNase H activities of these enzymes. K13N RT RNase H was slightly slower than, but LI+K13N was similar to K13N alone (Fig. 2a c). For K11E+G19S RT, we saw a modest improvement in RNase H activity compared to G19S alone (Fig. 2d f). These results show that the reduced fitness of LI+K13N and K11E+G19S viruses is not due to decreased RNase H activity. Effects of the NRTI mutation L74V on the RNase H activity of LI+K13N and K11E+G19S RTs As previously published by our group, L74V can improve the fitness of LI+K13N and K11E+G19S viruses. Therefore, we measured the RNase H activity of the triple mutants compared to the double mutants to determine whether the improved viral fitness was the result of improved RT RNase H activity. L74V did not improve the polymerase-dependent (Fig. 1) or the polymerase-independent (Fig. 2) RNase activities of LI+K13N or K11E+G19S (Fig. 1). Effects of L74V, LI and K11E on the RT polymerase activity of K13N and G19S RTs Although previous studies by our group showed that there is no polymerase defect with primary NNRTI drugresistance mutations (Archer et al., 2; Gerondelis et al., 1999; Wang et al., 21a), we measured the polymerization activity of the LI+K13N and K11E+G19S double mutant RTs to determine whether polymerization defects might explain the reduced fitness of viruses with the double mutations compared to the single primary mutations. We measured the RNA-dependent DNA polymerase activity of, L74V, K13N, G19S, LI+K13N and K11E+G19S RTs using the first 2 nt of the HIV-1 genomic RNA as template, and a DNA primer corresponding to the primer binding site (PBS). The results showed that LI and K11E did not decrease the RNAdependent DNA polymerization of K13N and G19S RTs, respectively (Fig. 3). We also determined whether the improved fitness of the triple mutants was due to improved polymerization activity. Fig. 3 shows that L74V did not enhance the polymerase activity of the LI+K13N and K11E+G19S double mutant RTs. These results suggest that the improvement of viral fitness by L74V is not caused by an increase in the polymerase activity of the RT. Effects of 74V, LI and K11E on the amount of IN and RT in the virion of NNRTI-resistant mutants We reported previously that most primary NNRTIresistance mutations have decreased amounts of RT in the virion, and that the level of RT content correlated with replication fitness (Wang et al., 21a). Since the secondary mutations, LI and K11E and the NRTI drug-resistant mutant L74 had no effect on the RNase H and polymerase activities, we hypothesized that NNRTI secondary resistance mutations and L74V affect the RT content in the 161

6 J. Wang and others Fig. 3(a) L74V K13N LI+K13N L74V+LI+K13N Marker Primer virion. In order to test this, we measured the amount of RT and integrase (IN) in purified virus stocks using Western blot analysis. A representative Western blot is shown in Fig. 4(a). The amount of virus loaded per lane was normalized by the amount of capsid protein (p24). The amount of IN was significantly reduced for all mutant viruses compared to (P,.1), indicating that these mutations may have a defect in Gag-pol incorporation (Fig. 4b). This reduction in integrase was much more pronounced for the G19S mutants than for the K13N mutants. There was a significant reduction in the amount of IN for LI+K13N and K11E+G19S compared to K13N and G19S, respectively (P,.1), and the addition of L74V improved the amount of IN incorporated into virions compared to the double mutants. There was a significant decrease in the amount of RT for LI+K13N (P5.14) and K11E+G19S (P5.41) compared to K13N alone and G19S alone, respectively (Fig. 4c). This shows that the tested secondary NNRTIresistance mutations further decrease the amount of RT in the infectious virus particle. In addition, the relative amount of RT compared to IN is lower, indicating that the reductions in IN or Gag-pol incorporation do not completely explain the loss of RT in virions. We note that the RT content of all of the mutant viruses is considerably lower than that of the virus. This is an indication that the RT content must be much lower than normal before it limits the rate of virus growth, and that the virus contains a large excess of RT. Moreover, it suggests that the RT content must be much lower than that of the virus before virus growth rate will respond to further decreases or increases in RT content. We also determined whether the improvement in fitness of the triple mutants, L74V+LI+K13N and L74V+ K11E+G19S was due to improvements in the amount of RT in the virions. We measured the RT content of the triple mutants and found that L74V significantly increased the p51/p66 protein level in the virion compared to LI+K13N (P5.21). Although there was a trend for increased RT content of L74V+K11E+G19S compared to K11E+G19S, the results were not significant (P5.48) (Fig. 4a, c). These results suggested that L74V improves the replication fitness of NNRTI drug-resistant viruses by increasing the amount of RT in the mature virion. We have previously shown that the G19S virus has higher amounts of Gag cleavage intermediates, p55 and p41, than, suggesting that reduced RT content results from reduced protease cleavage of the Gag-pol polypeptide (Wang et al., 21a). Here, we determined whether the proteolytic cleavage of Gag in the virions was reduced due to the presence of the secondary mutations. We observed that the K11E+G19S virus had higher levels of Gag intermediates (p55 and p41) than G19S alone (Fig. 4d), but LI+K13N did not. L74V also improved the amount of Gag processing as shown by reduced amounts of 162 Journal of General Virology 94

7 RT content in virions alters fitness Fig. 3(b) L74V K11E+G19S L74V+K11E+G19S Marker Primer Fig. 3. RNA-dependent DNA polymerization activity of purified and mutant RTs. (a, b) Representative polyacrylamide gel of the RNA-dependent DNA polymerization activity of and mutant RTs using a DNA primer. The substrate used was made by annealing a 59 end-labelled PBS DNA primer (26 nt) to the RNA template D199 (RNA containing +1 to+199 of the NL4-3 HIV-1 genomic sequence). Reactions were allowed to proceed for the indicated lengths of time. Nucleotide markers are indicated on the right. Equal amounts of specific activity units of RT as measured by poly(ra)/oligo(dt) template/primer synthesis were used per reaction. pr55 and p41 in the Gag Western blot for K11E+G19S (Fig. 4d). Therefore, reduced protease activity partially explains the reduced amounts of RT in the virions. DISCUSSION We have documented that HIV-1 replication fitness, as measured in cell culture, is reduced for NNRTI-resistant mutants that are found infrequently in the clinic, such as G19A/S and P236L, even though they confer higher levels of resistance than the more common mutants, K13N and Y181C. We interpret this to mean that different NNRTI mutations exact a different and unpredictable price on viral fitness. Moreover, we previously showed that recombinant RTs having NNRTI drug-resistance mutations have reduced RNase H activity and normal polymerization activity (Archer et al., 2; Domaoal et al., 26; Gerondelis et al., 1999; Wang et al., 21a; Wang et al., 26). When tested in culture the mutant viruses had reduced RNase H activity, not only because of the impaired function measured in vitro, but also because the viruses packaged a reduced amount of RT (Wang et al., 21a; Wang et al., 26). The defects in RNase H may reduce fitness because the ratio of polymerization to RNase H is increased, resulting in fewer RNA 59ends created during synthesis for subsequent, and also deficient polymerase-independent RNase H activity. Since reduced content lowers both RT functions, we could not use modelling in vitro to predict its effects on fitness. Although a reduction of replication rates seems likely. Using a different virus system, others have demonstrated that significant 163

8 J. Wang and others reductions in polymerization activity will reduce virus replication (Julias et al., 21). We have previously published that single NNRTI drugresistance mutations do not affect DNA synthesis using a trna primer (Wang et al., 21a). Therefore, we did not test whether these mutations affect initiation, and therefore we have not ruled out this as a possible explanation for the relative fitness of these mutants. Strand transfer efficiency is dependent on the polymerization and RNase H abilities of the enzyme. Since the mutants have similar polymerization activities, we concluded that their strand transfer activities would mirror their relative RNase H activities, and therefore we did not include analysis of strand transfer activity. In this work, we investigated the complex interplay of effects of multiple mutations on growth fitness. Specifically, we probed the biochemical mechanisms underlying the growth inhibitory effects of secondary RT mutations LI and K11E on HIV-1 with K13N and G19S, and the growth restorative effects of the NRTI resistance mutation L74V on the double NNRTI mutant viruses. Our results showed that the combination of LI+K13N and K11E+G19S did not alter the rate or specificity polymerase-dependent or polymerase-independent-rnase H activity of K13N and G19S RTs. This was true despite the fact that purified G19S RT is significantly slowed in both modes of RNase H activity compared to and K13N RTs. In addition, the combination of L74V with LI+K13N or K11E+ G19S did not alter the rate or specificity of both modes of cleavage. We conclude that the decrease in fitness of LI+K13N and K11E+G19S compared to K13N or G19S alone is not due to intrinsic alterations in RNase H activity. However, we cannot rule out the possibility that cleavage of specialized natural substrates, such as the extended PPT or trna primers, is altered by these mutations. We also measured the RNA-dependent DNA polymerization activity of the mutant RTs and did not find any alterations in the patterns or amounts of extension s when normalizing RT input based on specific activity. Since the compensation of fitness by L74V was not due to improvements in intrinsic RNase H or polymerase activities, we measured the RT content of the virions. We found that the two double mutations, LI+K13N and K11E+G19S, had substantially lower virion-associated RT protein levels than those of K13N and G19S alone. Additionally, the presence of the L74V mutation significantly increased the RT content of the LI+K13N double mutant. Work by our group and others has shown that low RT content in virions correlates with the reduced replication efficiency of NNRTI drug-resistant variants (Huang et al., 23; Wang et al., 21a). Huang et al. tested the effect of L74V on several variants of G19, including G19S and determined that L74V increased the fitness of G19 mutations by increasing the amount of RT in virions (Huang et al., 23). They did not test the effect of L74V on the RNase H activity of G19 mutations; therefore, it is possible that L74V may increase the fitness of G19 mutations or K11E+G19S through a direct effect on G19S. The reason is evidently based on reduced RT catalytic activity, since virions with lower amounts of RT have reduced viral genome synthesis rates (Julias et al., 21; Wang et al., 21a). We conclude that secondary mutations and L74V alter the replication fitness of NNRTI drug-resistant variants by altering the amount of virionassociated RT content. We also concluded that if the mutations affect Gag processing, then Gag-pol processing would also be affected. However, we did not see a correlation between reduced RT content and reduced Gag-pol processing in our Western blot data. This may be due to the inability of the anti-rt antibodies we used to detect RT when part of the Gag-pol polyprotein. L74V is an uncommon NRTI-resistant mutation, seen only in approximately 11 % of isolates (McColl et al., 28). However, analysis of the Stanford HIV Drug Resistance Database shows that L74V is present in 57 % of samples containing LI+K13N (78/138), but only in 12 % of samples containing K13N alone (14/1143; a comparison P,.1) (Rhee et al., 23; Shafer, 26). In addition, L74V is present in 25.5 % of samples containing K11E+G19S (14/55). These results suggest that K13N+LI or K11E+G19S double mutants promote the selection of the L74V mutation. These results are consistent with those of a genotypic study of antiretroviral drug-resistance-associated mutations in clinical isolates from vertically infected children and adolescents, which show that LI was significantly associated with K13N and that L74V was significantly associated with LI+K13N (Aulicino et al., 21). L74V in an NL4-3 or HXBc2 background results in impaired replication capacity compared with and a more common NRTI mutation, M184V (Deval et al., 24; Sharma & Crumpacker, 1999; Sharma et al., 29). One defect reported for L74V RT is a reduced processivity of primer extension, as discovered from analysis of the purified RT. However, a purified RT having M184V showed an even greater decrease in processivity than the L74V mutant, despite the ability of M184V virus to replicate better than an L74V virus in cell culture assays (Sharma & Crumpacker, 1999; Sharma et al., 29). Additionally, conflicting reports did not find a significant difference in processivity between and L74V RTs, suggesting that there may be other biochemical mechanisms that contribute to the L74V fitness defect (Boyer et al., 1998; Caliendo et al., 1996). Logically, processivity should not be a significant factor in viral DNA synthesis if the RT is in sufficient excess that a new RT binds the primer terminus nearly immediately after one dissociates. We show here that L74V can increase the fitness of NNRTIresistant genotypes by increasing the amount of RT in virions and that this is a more consistent explanation for the fitness of L74V alone or in combination with other drug-resistant mutants. 164 Journal of General Virology 94

9 RT content in virions alters fitness (a) p66-- p51-- (c) G19S K11E+G19S L74V+11E+G19S K13N LI+K13N L74V+LI+K13N Marker kda -75 a RT mabs a IN Ab -25 a p24 mab (b) Percentage of IN in virions relative to 12 (d) % 25% 5% % K13N LI+K13N L74V+LI+K13N G19S K11E+G19S L74V+K11E+G19S Percentage of RT in virions relative to % 25% 5% % K13N LI+K13N L74V+LI+K13N G19S K11E+G19S L74V+K11E+G19S p55- p41- p24- G19S K11E+G19S L74V+11E+G19S K13N LI+K13N L74V+LI+K13N Marker kda Fig. 4. Western blot showing the relative amounts of RT in and mutant virions. The G19S proteins were run on separate gels than the K13N proteins. Each gel was stripped and reprobed for RT, IN and p24 quantitations. (a) Representative Western blot probed with antibodies that recognized HIV-1 RT p66 and p51 subunits, IN, or Gag capsid p24 protein. Virus stocks were prepared by transfecting 293 cells with either or mutant plasmid DNA. Mutant virus pellets were prepared with an equal amount (2 ng) of capsid protein. (b) Relative amounts of IN for each mutant compared to as percentages on the y-axis. The bars represent the means and standard deviations of triplicate Western blots. Bonferroni adjusted P-values were calculated using one way ANOVA. vs mutants P,.1; LI+K13N vs K13N P,.1; K11E+G19S vs G19S P,.1 (c) Relative amounts of RT for each mutant compared to as percentages on the y-axis. The bars represent the means and standard deviations of triplicate Western blots. Bonferroni adjusted P-values were calculated using one way ANOVA. LI+K13N vs K13N P5.14; K11E+G19S vs G19S P5.41; L74V+LI+K13N vs LI+K13N P5.21; L74V+K11E+G19S vs K11E+G19S P5.48. (d) Representative Western blot of the relative amounts of Gag processing intermediates. Equal amounts of p24 capsid protein were loaded for each virus (2 ng). The molecular masses in kilodaltons of the markers are shown on the right side

10 J. Wang and others Understanding the properties of viruses with multiple interacting drug-resistant mutations of this type that arise after different classes of therapy will allow clinicians to make more informed choices about which antiviral therapies to combine. More long-term, understanding how drug-resistant mutants alter virus replication will aid in the development of new antiviral therapies. METHODS AND METHODS Cell culture and antibodies. The 293 cell line (ATCC, Rockville, MD) was grown in Dulbecco s modified Eagle s medium (Cellgro, Herndon, VA) with 1 % FBS, penicillin ( U ml 21 ) and streptomycin ( U ml 21 ). The following antibodies were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: the HIV-1 RT monoclonal antibody 8C4 was obtained from Dr D. Helland and Dr A. M. Szilvay and recognizes both subunits of RT (Szilvay et al., 1992); p24 monoclonal antibody 183- H12-5C was obtained from Dr Bruce Chesebro and Kathy Wehrly (Chesebro et al., 1992; Toohey et al., 1995); polyclonal HIV-1 integrase antiserum recognizing epitopes mapping to amino acids 1 16 was obtained from Dr Duane P. Grandgenett (Bukrinsky et al., 1993). Site-directed mutagenesis. Each of the following drug-resistant mutations, K13N, L74V, double mutations LI+K13N, and triple mutations L74V+LI+K13N was introduced into prha1 using the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA). A molecular clone of HIV-1, pnl4-3, was obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, from Malcolm Martin. The drugresistant mutations were subcloned from prha1 into pnl4-3 using ApaI and AgeI restriction sites, as previously described (Koval et al., 26). Expression and purification of recombinant RTs. The full-length 6X His-tagged p51, p66 subunits of and mutant RT sequences were expressed with pet21a(+) vector (Novagen), and were purified with Q-Sepharose column, Talon column, and Sesource S column used the AKTAprime plus system (Amersham/GE, Piscataway, NJ), as previously described (Hou et al., 24; Wang et al., 21a). The fractions with equal relative amounts of p51 and p66 were determined using SDS-PAGE and chosen to measure DNA polymerization and RNase H activities. RNase H assays and RNA-dependent DNA polymerization of recombinant RTs. Specific DNA polymerization activity of each RT preparation was measured by using a poly(ra)/oligo(dt) template/ primer and an [a- 32 P] dttp substrate (Archer et al., 2). A unit was defined as the amount of enzyme required to incorporate 1 nmol of dttp into nucleic acid in 1 min at 37 uc. Specific activity is a measure of the inherent polymerization activity of the mutant as well as the quality of the protein preparation. In order to prevent protein prep quality from influencing the results, an equivalent number of units of specific polymerization activity were added to both the polymerization rate and RNase H assays. Using this method means we are comparing the ratio of polymerization activity on a complex template to a simple template [poly(ra)/oligo(dt)] and the ratio of polymerization to RNase H rather than the inherent polymerization or RNase H activity per mass of protein. DNA 39 end-directed and RNA 59 end-directed RNase H activities were measured as previously described (Wang et al., 26). To determine the rate of RNA-dependent DNA polymerization activity of each RT, the polymerization activities were measured using a 59-radiolabelled PBS oligomer DNA primer annealed to the RNA template D199, +1 to +199 of the pnl4-3 genomic RNA, which included the PBS binding site. The primer/template (15 nm/25 nm) was pre-bound with purified RT normalized by relative specific activities of polymerization in reaction buffer containing 25 mm Tris/ HCl ph 8., 25 mm NaCl, and.5 mm EDTA and DTT at 37 uc for 2 min. The reactions were initiated by adding 6 mm MgCl 2 and 5 mm dntps and quenched at various times by an equal volume of 26 DNA loading buffer (Ambion). Extension s were analysed by SDS PAGE and quantified by phosphorimaging. Generation of NNRTI-resistant virus stock. A human cell line, 293, was transiently transfected with NL4-3 or the drug-resistant mutants of NL4-3 using Superfect (Qiagen, Santa Clarita, CA). After 72 h, clarified supernatants were harvested and the p24 capsid protein of each stock was measured using ELISA (Perkin Elmer, Wellesley, MA). and mutant stocks in the pnl4-3 background were used for Western blot analysis to measure the relative RT content. Quantification of RT content in virions using Western blotting. Western blotting was performed as previously described (Wang et al., 21a). Briefly, supernatant solutions from transfected 293 cells with or drug-resistant mutants were pelleted by centrifugation at 5 g for 1 h at 4 uc. In order to control for differences in transfection efficiency between stocks, the amount of virus used for Western blot was normalized by p24; 2 ng of p24 antigen was loaded per lane for and mutant viruses. The virus pellets were resuspended with 15 ml NuPAGE 26 sample buffer (Invitrogen) and fractionated by electrophoresis according to the NuPAGE manufacturer s instructions. The proteins were separated using a 4 12 % Bistris gel with 16 MOPS running buffer (Invitrogen) and transferred to nitrocellulose membranes. In order to reduce the adverse effect of striping on Western blot quality, membranes were cut into sections according to the sizes of RT, IN and capsid (p24), then blocked by 5 % milk in Tris buffered saline (TBS) with.5 % Tween TBST (.1 % Tween 2) overnight, and then probed with the appropriate antibodies. Viral proteins were visualized using SuperSignal West Femto chemiluminescence substrate (Thermo Scientific) and quantified by 1D image analysis software (Kodak Digital Science). p51 and p66 subunits of RT were recognized by the RT monoclonal antibody pool (8C4 and 5B2B2). The level of p24 and Gag processing was visualized using an antibody specific to p24 (183- H12-5C) and the levels of integrase were determined using a polyclonal antibody that recognized amino acids 1 16 of integrase. ACKNOWLEDGEMENTS This work was supported by the NIH (grant R1 AI-41387) and the University of Rochester Developmental Center for AIDS Research (NIH P3AI78498). Disclosures: the authors do not have any conflicts of interests to disclose. REFERENCES Amacker, M. & Hübscher, U. (1998). Chimeric HIV-1 and feline immunodeficiency virus reverse transcriptases: critical role of the p51 subunit in the structural integrity of heterodimeric lentiviral DNA polymerases. J Mol Biol 278, Archer, R. H., Dykes, C., Gerondelis, P., Lloyd, A., Fay, P., Reichman, R. C., Bambara, R. A. & Demeter, L. M. (2). Mutants of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase resistant to nonnucleoside reverse transcriptase inhibitors demonstrate altered rates of RNase H cleavage that correlate with HIV-1 replication fitness in cell culture. J Virol 74, Journal of General Virology 94

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