DNA Polymerase Delta Exhibits Altered Catalytic Properties on Lysine Acetylation.

DNA polymerase delta is the primary polymerase that is involved in undamaged nuclear lagging strand DNA replication. Our mass-spectroscopic analysis has revealed that the human DNA polymerase δ is acetylated on subunits p125, p68, and p12. Using substrates that simulate Okazaki fragment intermediates, we studied alterations in the catalytic properties of acetylated polymerase and compared it to the unmodified form. The current data show that the acetylated form of human pol δ displays a higher polymerization activity compared to the unmodified form of the enzyme. Additionally, acetylation enhances the ability of the polymerase to resolve complex structures such as G-quadruplexes and other secondary structures that might be present on the template strand. More importantly, the ability of pol δ to displace a downstream DNA fragment is enhanced upon acetylation. Our current results suggest that acetylation has a profound effect on the activity of pol δ and supports the hypothesis that acetylation may promote higher-fidelity DNA replication.

G-quadruplex ligands targeting telomeres do not inhibit HIV promoter activity and cooperate with latency reversing agents in killing latently infected cells.

Altered telomere maintenance mechanism (TMM) is linked to increased DNA damage at telomeres and telomere uncapping. We previously showed that HIV-1 latent cells have altered TMM and are susceptible to ligands that target G-quadruplexes (G4) at telomeres. Susceptibility of latent cells to telomere targeting could potentially be used to support approaches to eradicate HIV reservoirs. However, G4 ligands also target G-quadruplexes in promoters blocking gene transcription. Since HIV promoter sequence can form G-quadruplexes, we investigated whether G4 ligands interfere with HIV-1 promoter activity and virus reactivation from latency, and whether telomere targeting could be combined with latency reversing agents (LRAs) to promote elimination of HIV reservoirs. Our results indicate that Sp1 binding region in HIV-1 promoter can adopt G4 structures in duplex DNA, and that in vitro binding of Sp1 to G-quadruplex is blocked by G4 ligand, suggesting that agents targeting telomeres interfere with virus reactivation. However, our studies show that G4 agents do not affect HIV-1 promoter activity in cell culture, and do not interfere with latency reversal. Importantly, primary memory CD4 + T cells infected with latent HIV-1 are more susceptible to combined treatment with LRAs and G4 ligands, indicating that drugs targeting TMM may enhance killing of HIV reservoirs. Using a cell-based DNA repair assay, we also found that HIV-1 infected cells have reduced efficiency of DNA mismatch repair (MMR), and base excision repair (BER), suggesting that altered TMM in latently infected cells could be associated with accumulation of DNA damage at telomeres and changes in telomeric caps.

Cidofovir Diphosphate Inhibits Adenovirus 5 DNA Polymerase via both Nonobligate Chain Termination and Direct Inhibition, and Polymerase Mutations Confer Cidofovir Resistance on Intact Virus.

Human adenovirus (AdV) can cause fatal disease in immune-suppressed individuals, but treatment options are limited, in part because the antiviral cytidine analog cidofovir (CDV) is nephrotoxic. The investigational agent brincidofovir (BCV) is orally bioavailable, nonnephrotoxic, and generates the same active metabolite, cidofovir diphosphate (CDVpp). However, its mechanism of action against AdV is poorly understood. Therefore, we have examined the effect of CDVpp on DNA synthesis by a purified adenovirus 5 (AdV5) DNA polymerase (Pol). CDVpp was incorporated into nascent DNA strands and promoted a nonobligate form of chain termination (i.e., AdV5 Pol can extend, albeit inefficiently, a DNA chain even after the incorporation of a first CDVpp molecule). Moreover, unlike a conventional mismatched base pair, misincorporated CDVpp was not readily excised by the AdV5 Pol. At elevated concentrations, CDVpp inhibited AdV5 Pol in a manner consistent with both chain termination and direct inhibition of Pol activity. Finally, a recombinant AdV5 was constructed, containing Pol mutations (V303I and T87I) that were selected following an extended passage of wild-type AdV5 in the presence of BCV. This virus had a 2.1-fold elevated 50% effective concentration (EC50) for BCV and a 1.9-fold increased EC50 for CDV; thus, these results confirmed that viral resistance to BCV and CDV can be attributed to mutations in the viral Pol. These findings show that the anti-AdV5 activity of CDV and BCV is mediated through the viral DNA Pol and that their antiviral activity may occur via both (nonobligate) chain termination and (at high concentration) direct inhibition of AdV5 Pol activity.

Deficiency in DNA damage response, a new characteristic of cells infected with latent HIV-1.

Viruses can interact with host cell molecules responsible for the recognition and repair of DNA lesions, resulting in dysfunctional DNA damage response (DDR). Cells with inefficient DDR are more vulnerable to therapeutic approaches that target DDR, thereby raising DNA damage to a threshold that triggers apoptosis. Here, we demonstrate that 2 Jurkat-derived cell lines with incorporated silent HIV-1 provirus show increases in DDR signaling that responds to formation of double strand DNA breaks (DSBs). We found that phosphorylation of histone H2AX on Ser139 (gamma-H2AX), a biomarker of DSBs, and phosphorylation of ATM at Ser1981, Chk2 at Thr68, and p53 at Ser15, part of signaling pathways associated with DSBs, are elevated in these cells. These results indicate a DDR defect even though the virus is latent. DDR-inducing agents, specifically high doses of nucleoside RT inhibitors (NRTIs), caused greater increases in gamma-H2AX levels in latently infected cells. Additionally, latently infected cells are more susceptible to long-term exposure to G-quadruplex stabilizing agents, and this effect is enhanced when the agent is combined with an inhibitor targeting DNA-PK, which is crucial for DSB repair and telomere maintenance. Moreover, exposing these cells to the cancer drug etoposide resulted in formation of DSBs at a higher rate than in un-infected cells. Similar effects of etoposide were also observed in population of primary memory T cells infected with latent HIV-1. Sensitivity to these agents highlights a unique vulnerability of latently infected cells, a new feature that could potentially be used in developing therapies to eliminate HIV-1 reservoirs.

Acetylation regulates DNA repair mechanisms in human cells.

The p300-mediated acetylation of enzymes involved in DNA repair and replication has been previously shown to stimulate or inhibit their activities in reconstituted systems. To explore the role of acetylation on DNA repair in cells we constructed plasmid substrates carrying inactivating damages in the EGFP reporter gene, which should be repaired in cells through DNA mismatch repair (MMR) or base excision repair (BER) mechanisms. We analyzed efficiency of repair within these plasmid substrates in cells exposed to deacetylase and acetyltransferase inhibitors, and also in cells deficient in p300 acetyltransferase. Our results indicate that protein acetylation improves DNA mismatch repair in MMR-proficient HeLa cells and also in MMR-deficient HCT116 cells. Moreover, results suggest that stimulated repair of mismatches in MMR-deficient HCT116 cells is done though a strand-displacement synthesis mechanism described previously for Okazaki fragments maturation and also for the EXOI-independent pathway of MMR. Loss of p300 reduced repair of mismatches in MMR-deficient cells, but did not have evident effects on BER mechanisms, including the long patch BER pathway. Hypoacetylation of the cells in the presence of acetyltransferase inhibitor, garcinol generally reduced efficiency of BER of 8-oxoG damage, indicating that some steps in the pathway are stimulated by acetylation.

U3 region in the HIV-1 genome adopts a G-quadruplex structure in its RNA and DNA sequence.

Genomic regions rich in G residues are prone to adopt G-quadruplex structure. Multiple Sp1-binding motifs arranged in tandem have been suggested to form this structure in promoters of cancer-related genes. Here, we demonstrate that the G-rich proviral DNA sequence of the HIV-1 U3 region, which serves as a promoter of viral transcription, adopts a G-quadruplex structure. The sequence contains three binding elements for transcription factor Sp1, which is involved in the regulation of HIV-1 latency, reactivation, and high-level virus expression. We show that the three Sp1 binding motifs can adopt different forms of G-quadruplex structure and that the Sp1 protein can recognize and bind to its site folded into a G-quadruplex. In addition, a c-kit2 specific antibody, designated hf2, binds to two different G-quadruplexes formed in Sp1 sites. Since U3 is encoded at both viral genomic ends, the G-rich sequence is also present in the RNA genome. We demonstrate that the RNA sequence of U3 forms dimers with characteristics known for intermolecular G-quadruplexes. Together with previous reports showing G-quadruplex dimers in the gag and cPPT regions, these results suggest that integrity of the two viral genomes is maintained through numerous intermolecular G-quadruplexes formed in different RNA genome locations. Reconstituted reverse transcription shows that the potassium-dependent structure formed in U3 RNA facilitates RT template switching, suggesting that the G-quadruplex contributes to recombination in U3.

GTP is the primary activator of the anti-HIV restriction factor SAMHD1.

SAMHD1 (SAM domain- and HD domain-containing protein 1) is a dGTP-dependent dNTP triphosphohydrolase that converts dNTPs into deoxyribonucleosides and triphosphates. Therefore, SAMHD1 expression, particularly in non-dividing cells, can restrict retroviral infections such as HIV and simian immunodeficiency virus by limiting cellular dNTPs, which are essential for reverse transcription. It has previously been established that dGTP acts as both an activator and a substrate of this enzyme, suggesting that phosphohydrolase activity of SAMHD1 is regulated by dGTP availability in the cell. However, we now demonstrate biochemically that the NTP GTP is equally capable of activating SAMHD1, but GTP is not hydrolyzed by the enzyme. Activation of SAMHD1 phosphohydrolase activity was tested under physiological concentrations of dGTP or GTP found in either dividing or non-dividing cells. Because GTP is 1000-fold more abundant than dGTP in cells, GTP was able to activate the enzyme to a greater extent than dGTP, suggesting that GTP is the primary activator of SAMHD1. Finally, we show that SAMHD1 has the ability to hydrolyze base-modified nucleotides, indicating that the active site of SAMHD1 is not restrictive to such modifications, and is capable of regulating the levels of non-canonical dNTPs such as dUTP. This study provides further insights into the regulation of SAMHD1 with regard to allosteric activation and active site specificity.

Mechanism of HIV-1 RNA dimerization in the central region of the genome and significance for viral evolution.

The genome of HIV-1 consists of two identical or nearly identical RNA molecules. The RNA genomes are held in the same, parallel orientation by interactions at the dimer initiation site (DIS). Previous studies showed that in addition to interactions at DIS, sequences located 100 nucleotides downstream from the 5' splice site can dimerize in vitro through an intermolecular G-quartet structure. Here we report that the highly conserved G-rich sequence in the middle portion of the HIV-1 genome near the central polypurine tract (cPPT) dimerizes spontaneously under high ionic strength in the absence of protein. The antisense RNA does not dimerize, strongly indicating that RNA dimerization does not exclusively involve A:U and G:C base pairing. The cation-dependent reverse transcriptase pausing profile, CD spectra profile, and cation-dependent association and thermal dissociation characteristics indicate G-quartet structures. Different forms of G-quartets are formed including monomers and, significantly, intermolecular dimers. Our results indicate that RNA genome dimerization and parallel alignment initiated through interactions at DIS may be greatly expanded and stabilized by formation of an intermolecular G-quartet at a distant site near the cPPT. It is likely that formation of G-quartet structure near the cPPT in vivo keeps the RNA genomes in proximity over a long range, promoting genetic recombination in numerous hot spots.

Reverse transcriptase backbone can alter the polymerization and RNase activities of non-nucleoside reverse transcriptase mutants K101E+G190S.

Previous work by our group showed that human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) containing non-nucleoside RT inhibitor (NNRTI) drug resistance mutations has defects in RNase H activity as well as reduced amounts of RT protein in virions. These deficits correlate with replication fitness in the absence of NNRTIs. Viruses with the mutant combination K101E+G190S replicated better in the presence of NNRTIs than in the absence of drug. Stimulation of virus growth by NNRTIs occurred during the early steps of the virus life cycle and was modulated by the RT backbone sequence in which the resistance mutations arose. We wanted to determine what effects RT backbone sequence would have on RT content and polymerization and RNase H activities in the absence of NNRTIs. We compared a NL4-3 RT with K101E+G190S to a patient-isolate RT sequence D10 with K101E+G190S. We show here that, unlike the NL4-3 backbone, the D10 backbone sequence decreased the RNA-dependent DNA polymerization activity of purified recombinant RT compared to WT. In contrast, RTs with the D10 backbone had increased RNase H activity compared to WT and K101E+G190S in the NL4-3 backbone. D10 virions also had increased amounts of RT compared to K101E+G190S in the NL4-3 backbone. We conclude that the backbone sequence of RT can alter the activities of the NNRTI drug-resistant mutant K101E+G190S, and that identification of the amino acids responsible will aid in understanding the mechanism by which NNRTI drug-resistant mutants alter fitness and NNRTIs stimulate HIV-1 virus replication.

Efavirenz stimulates HIV-1 reverse transcriptase RNase H activity by a mechanism involving increased substrate binding and secondary cleavage activity.

Efavirenz is a non-nucleoside reverse transcriptase inhibitor used for treating HIV/AIDS. We found that polymerization activity of a reverse transcriptase (RT) with the E478Q mutation that inactivates the RNase H catalytic site is much more sensitive to efavirenz than wild-type RT, indicating that a functional RNase H attenuates the effectiveness of efavirenz. Moreover, efavirenz actually stimulated wild-type RNase H binding and catalytic functions, indicating another link between efavirenz action and RNase H function. During reverse transcription in vivo, the RT that is extending the DNA primer also periodically cleaves the genomic RNA. The RNase H makes primary template cuts ~18 nucleotides from the growing DNA 3'-end, and when the RT pauses synthesis, it shifts to make secondary cuts ~9 nucleotides from the DNA 3'-end. After synthesis, RTs return to bind the remaining template RNA segments at their 5'-ends and make primary and secondary cuts, 18 and 9 nucleotides in, respectively. We found that efavirenz stimulates both 3'- and 5'-directed RNase H activity. Use of specific substrates revealed a particular acceleration of secondary cuts. Efavirenz specifically promoted binding of the RT to RNase H substrates, suggesting that it stabilizes the shifting of RTs to make the secondary cuts. We further showed that efavirenz similarly stimulates the RNase H of an RT from a patient-derived virus that is highly resistant and grows more rapidly in the presence of low concentrations of efavirenz. We suggest that for efavirenz-resistant RTs, stimulated RNase H activity contributes to increased viral fitness.

Anti-HIV host factor SAMHD1 regulates viral sensitivity to nucleoside reverse transcriptase inhibitors via modulation of cellular deoxyribonucleoside triphosphate (dNTP) levels.

Newly identified anti-HIV host factor, SAMHD1, restricts replication of lentiviruses such as HIV-1, HIV-2, and simian immunodeficiency virus in macrophages by enzymatically hydrolyzing and depleting cellular dNTPs, which are the substrates of viral DNA polymerases. HIV-2 and some simian immunodeficiency viruses express viral protein X (VPX), which counteracts SAMHD1 and elevates cellular dNTPs, enhancing viral replication in macrophages. Because nucleoside reverse transcriptase inhibitors (NRTIs), the most commonly used anti-HIV drugs, compete against cellular dNTPs for incorporation into proviral DNA, we tested whether SAMHD1 directly affects the efficacy of NRTIs in inhibiting HIV-1. We found that reduction of SAMHD1 levels with the use of virus-like particles expressing Vpx- and SAMHD1-specific shRNA subsequently elevates cellular dNTPs and significantly decreases HIV-1 sensitivity to various NRTIs in macrophages. However, virus-like particles +Vpx treatment of activated CD4(+) T cells only minimally reduced NRTI efficacy. Furthermore, with the use of HPLC, we could not detect SAMHD1-mediated hydrolysis of NRTI-triphosphates, verifying that the reduced sensitivity of HIV-1 to NRTIs upon SAMHD1 degradation is most likely caused by the elevation in cellular dNTPs.

Flap endonuclease 1.

First discovered as a structure-specific endonuclease that evolved to cut at the base of single-stranded flaps, flap endonuclease (FEN1) is now recognized as a central component of cellular DNA metabolism. Substrate specificity allows FEN1 to process intermediates of Okazaki fragment maturation, long-patch base excision repair, telomere maintenance, and stalled replication fork rescue. For Okazaki fragments, the RNA primer is displaced into a 5' flap and then cleaved off. FEN1 binds to the flap base and then threads the 5' end of the flap through its helical arch and active site to create a configuration for cleavage. The threading requirement prevents this active nuclease from cutting the single-stranded template between Okazaki fragments. FEN1 efficiency and specificity are critical to the maintenance of genome fidelity. Overall, recent advances in our knowledge of FEN1 suggest that it was an ancient protein that has been fine-tuned over eons to coordinate many essential DNA transactions.

L74V increases the reverse transcriptase content of HIV-1 virions with non-nucleoside reverse transcriptase drug-resistant mutations L100I+K103N and K101E+G190S, which results in increased fitness.

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, L100I and K101E, and the nucleoside resistance mutation, L74V, alter the fitness of K103N and G190S viruses. We measured the amount of RT in virions and the polymerization and RNase H activities of mutant RTs compared to wild-type, K103N and G190S. We found that L100I, K101E and L74V did not change the polymerization or RNase H activities of K103N or G190S RTs. However, L100I and K101E reduced the amount of RT in the virions and subsequent addition of L74V restored RT levels back to those of G190S or K103N alone. We conclude that fitness changes caused by L100I, K101E and L74V derive from their effects on RT content.

Okazaki fragment metabolism.

Cellular DNA replication requires efficient copying of the double-stranded chromosomal DNA. The leading strand is elongated continuously in the direction of fork opening, whereas the lagging strand is made discontinuously in the opposite direction. The lagging strand needs to be processed to form a functional DNA segment. Genetic analyses and reconstitution experiments identified proteins and multiple pathways responsible for maturation of the lagging strand. In both prokaryotes and eukaryotes the lagging-strand fragments are initiated by RNA primers, which are removed by a joining mechanism involving strand displacement of the primer into a flap, flap removal, and then ligation. Although the prokaryotic fragments are ~1200 nucleotides long, the eukaryotic fragments are much shorter, with lengths determined by nucleosome periodicity. The prokaryotic joining mechanism is simple and efficient. The eukaryotic maturation mechanism involves many enzymes, possibly three pathways, and regulation that can shift from high efficiency to high fidelity.

Msh2-Msh3 interferes with Okazaki fragment processing to promote trinucleotide repeat expansions.

Trinucleotide repeat (TNR) expansions are the underlying cause of more than 40 neurodegenerative and neuromuscular diseases, including myotonic dystrophy and Huntington's disease. Although genetic evidence points to errors in DNA replication and/or repair as the cause of these diseases, clear molecular mechanisms have not been described. Here, we focused on the role of the mismatch repair complex Msh2-Msh3 in promoting TNR expansions. We demonstrate that Msh2-Msh3 promotes CTG and CAG repeat expansions in vivo in Saccharomyces cerevisiae. Furthermore, we provide biochemical evidence that Msh2-Msh3 directly interferes with normal Okazaki fragment processing by flap endonuclease1 (Rad27) and DNA ligase I (Cdc9) in the presence of TNR sequences, thereby producing small, incremental expansion events. We believe that this is the first mechanistic evidence showing the interplay of replication and repair proteins in the expansion of sequences during lagging-strand DNA replication.

Biochemical analyses indicate that binding and cleavage specificities define the ordered processing of human Okazaki fragments by Dna2 and FEN1.

In eukaryotic Okazaki fragment processing, the RNA primer is displaced into a single-stranded flap prior to removal. Evidence suggests that some flaps become long before they are cleaved, and that this cleavage involves the sequential action of two nucleases. Strand displacement characteristics of the polymerase show that a short gap precedes the flap during synthesis. Using biochemical techniques, binding and cleavage assays presented here indicate that when the flap is ∼ 30 nt long the nuclease Dna2 can bind with high affinity to the flap and downstream double strand and begin cleavage. When the polymerase idles or dissociates the Dna2 can reorient for additional contacts with the upstream primer region, allowing the nuclease to remain stably bound as the flap is further shortened. The DNA can then equilibrate to a double flap that can bind Dna2 and flap endonuclease (FEN1) simultaneously. When Dna2 shortens the flap even more, FEN1 can displace the Dna2 and cleave at the flap base to make a nick for ligation.

Frequent incorporation of ribonucleotides during HIV-1 reverse transcription and their attenuated repair in macrophages.

Macrophages are well known long-lived reservoirs of HIV-1. Unlike activated CD4(+) T cells, this nondividing HIV-1 target cell type contains a very low level of the deoxynucleoside triphosphates (dNTPs) required for proviral DNA synthesis whereas the ribonucleoside triphosphate (rNTP) levels remain in the millimolar range, resulting in an extremely low dNTP/rNTP ratio. Biochemical simulations demonstrate that HIV-1 reverse transcriptase (RT) efficiently incorporates ribonucleoside monophosphates (rNMPs) during DNA synthesis at this ratio, predicting frequent rNMP incorporation by the virus specifically in macrophages. Indeed, HIV-1 RT incorporates rNMPs at a remarkable rate of 1/146 nucleotides during macrophage infection. This greatly exceeds known rates for cellular replicative polymerases. In contrast, little or no rNMP incorporation is detected in CD4(+) T cells. Repair of these rNMP lesions is also substantially delayed in macrophages compared with CD4(+) T cells. Single rNMPs embedded in a DNA template are known to induce cellular DNA polymerase pausing, which mechanistically contributes to mutation synthesis. Indeed, we also observed that embedded rNMPs in a dsDNA template also induce HIV-1 RT DNA synthesis pausing. Moreover, unrepaired rNMPs incorporated into the provirus during HIV-1 reverse transcription would be generally mutagenic as was shown in Saccharomyces cerevisiae. Most importantly, the frequent incorporation of rNMPs makes them an ideal candidate for development of a new class of HIV RT inhibitors.

Telomere proteins POT1, TRF1 and TRF2 augment long-patch base excision repair in vitro.

Human telomeres consist of multiple tandem hexameric repeats, each containing a guanine triplet. Guanosine-rich clusters are highly susceptible to oxidative base damage, necessitating base excision repair (BER). Previous demonstration of enhanced strand displacement synthesis by the BER component DNA polymerase ß in the presence of telomere protein TRF2 suggests that telomeres employ long-patch (LP) BER. Earlier analyses in vitro showed that efficiency of BER reactions is reduced in the DNA-histone environment of chromatin. Evidence presented here indicates that BER is promoted at telomeres. We found that the three proteins that contact telomere DNA, POT1, TRF1 and TRF2, enhance the rate of individual steps of LP-BER and stimulate the complete reconstituted LP-BER pathway. Thought to protect telomere DNA from degradation, these proteins still apparently evolved to allow selective access of repair proteins.

Altered strand transfer activity of a multiple-drug-resistant human immunodeficiency virus type 1 reverse transcriptase mutant with a dipeptide fingers domain insertion.

Prolonged highly active anti-retroviral therapy with multiple nucleoside reverse transcriptase inhibitors for the treatment of patients infected with human immunodeficiency virus type 1 (HIV-1) can induce the development of an HIV-1 reverse transcriptase (RT) harboring a dipeptide insertion at the RT fingers domain with a background thymidine analog mutation. This mutation renders viral resistance to multiple nucleoside reverse transcriptase inhibitors. We investigated the effect of the dipeptide fingers domain insertion mutation on strand transfer activity using two clinical RT variants isolated during the pre-treatment and post-treatment of an infected patient, termed pre-drug RT without dipeptide insertion and post-drug RT with Ser-Gly insertion, respectively. First, the post-drug RT displayed elevated strand transfer activity compared to the pre-drug RT, with two different RNA templates. Second, the post-drug RT exhibited less RNA template degradation than the pre-drug RT but higher polymerization-dependent RNase H activity. Third, the post-drug RT had a faster association rate (k(on)) for template binding and a lower equilibrium binding constant K(d) for the template, leading to a template binding affinity tighter than that of the pre-drug RT. The k(off) values for the pre-drug RT and the post-drug RT were similar. Finally, the removal of the dipeptide insertion from the post-drug RT abolished the elevated strand transfer activity and RNase H activity, in addition to the loss of azidothymidine resistance. These biochemical data suggest that the dipeptide insertion elevates strand transfer activity by increasing the interaction of the RT with the RNA donor template, promoting cleavage that generates more invasion sites for the acceptor template during DNA synthesis.

Nonnucleoside reverse transcriptase inhibitor-resistant HIV is stimulated by efavirenz during early stages of infection.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are potent and commonly prescribed antiviral agents used in combination therapy (CART) of human immunodeficiency virus type 1 (HIV-1) infection. The development of drug resistance is a major limitation of CART. Reverse transcriptase (RT) genotypes with the NNRTI resistance mutations K101E+G190S are highly resistant to efavirenz (EFV) and can develop during failure of EFV-containing regimens in patients. We have previously shown that virus with K101E+G190S mutations can replicate more efficiently in the presence of EFV than in its absence. In this study, we evaluated the underlying mechanism for drug-dependent stimulation, using a single-cycle cell culture assay in which EFV was added either during the infection or the virus production step. We determined that EFV stimulates K101E+G190S virus during early infection and does not affect late steps of virus replication, such as increasing the amount of active RT incorporated into virions. Additionally, we showed that another NNRTI, nevirapine (NVP), stimulated K101E+G190S virus replication during the early steps of infection similar to EFV, but that the newest NNRTI, etravirine (ETR), did not. We also showed that EFV stimulates K101E+Y188L and K101E+V106I virus, but not K101E+L100I, K101E+K103N, K101E+Y181C, or K101E+G190A virus, suggesting that the stimulation is mutation specific. Real-time PCR of reverse transcription intermediates showed that although the drug did not stimulate minus-strand transfer, it did stimulate minus-strand strong-stop DNA synthesis. Our results indicate that stimulation most likely occurs through a mechanism whereby NNRTIs stimulate priming or elongation of the tRNA.