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Nucleoside reverse transcriptase inhibitors are an important class of drugs for treatment of human immunodeficiency virus type 1 (HIV-1) infection. Resistance to these drugs is often the result of mutations that increase the transfer of chain-terminating nucleotides from blocked DNA termini to a nucleoside triphosphate acceptor, resulting in the generation of an unblocked DNA chain and synthesis of a dinucleoside polyphosphate containing the chain-terminating deoxynucleoside triphosphate analogue. We have synthesized and purified several dinucleoside tetraphosphates (ddAp 4 ddA, ddCp 4 ddC, ddGp 4 ddG, ddTp 4 ddT, Ap 4 ddG, 2′(3′)- O -( N -methylanthraniloyl)-Ap 4 ddG, and AppNHppddG) and show that these compounds can serve as substrates for DNA chain elongation and termination resulting in inhibition of DNA synthesis. Thymidine analogue-resistant mutants of reverse transcriptase are up to 120-fold more sensitive to inhibition by these compounds than is wild-type enzyme. Drugs based on the dinucleoside tetraphosphate structure could delay or prevent the emergence of mutants with enhanced primer unblocking activity. In addition, such drugs could suppress the resistance phenotype of mutant HIV-1 that is present in individuals infected with resistant virus.

HIV-1 reverse transcriptase can remove chain terminators from blocked DNA ends through a nucleotide-dependent mechanism. We show that the catalytic efficiency of the removal reaction can vary several hundred-fold in different sequence contexts and is most strongly affected by the nature of the base pair at the 3'-primer terminus and the six base pairs upstream of it. Similar effects of the upstream sequence were observed with primer-templates terminated with 2',3'-dideoxy-AMP, 2',3'-dideoxy-CMP, or 2',3'-dideoxy-GMP. However, the removal of 2',3'-dideoxy-TMP or 3'-azido-2',3'-dideoxy-TMP was much less influenced by upstream primer-template sequence, and the rate of excision of these thymidylate analogues was greater than or equal to that of the other chain-terminating residues in each sequence context tested. These results strongly indicate that the primer terminus and adjacent upstream base pairs interact with reverse transcriptase in a sequence-dependent manner that affects the removal reaction. We conclude that primer-template sequence context is a major factor to consider when evaluating the removal of different chain terminators by HIV-1 reverse transcriptase.

Phosphonoformate (foscarnet) is a pyrophosphate (PPi) analogue and a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), acting through the PPibinding site on the enzyme. HIV-1 RT can unblock a chain-terminated DNA primer by phosphorolytic transfer of the terminal residue to an acceptor substrate (PPior a nucleotide such as ATP) which also interacts with the PPibinding site. Primer-unblocking activity is increased in mutants of HIV-1 that are resistant to the chain-terminating nucleoside inhibitor 3′-azido-3′-deoxythymidine (AZT). We have compared the primer-unblocking activity for HIV-1 RT containing various foscarnet resistance mutations (K65R, W88G, W88S, E89K, S117T, Q161L, M164I, and the double mutant Q161L/H208Y) alone or in combination with AZT resistance mutations. The level of primer-unblocking activity varied over a 150-fold range for these enzymes and was inversely correlated with foscarnet resistance and directly correlated with AZT resistance. Based on published crystal structures of HIV-1 RT, many of the foscarnet resistance mutations affect residues that do not make direct contact with the catalytic residues of RT, the incoming deoxynucleoside triphosphate (dNTP), or the primer-template. These mutations may confer foscarnet resistance and reduce primer unblocking by indirectly decreasing the binding and retention of foscarnet, PPi, and ATP. Alternatively, the binding position or orientation of PPi, ATP, or the primer-template may be changed in the mutant enzyme complex so that molecular interactions required for the unblocking reaction are impaired while dNTP binding and incorporation are not.

Mutations in HIV-1 reverse transcriptase (RT) give rise to 3′-azido-3′-deoxythymidine (AZT) resistance by a mechanism that has not been previously reproduced in vitro. We show that mutant RT has increased ability to remove AZTMP from blocked primers through a nucleotide-dependent reaction, producing dinucleoside polyphosphate and extendible primer. In the presence of physiological concentrations of ATP, mutant RT extended 12% to 15% of primers past multiple AZTMP termination sites versus less than 0.5% for wild type. Although mutant RT also unblocked ddAMP-terminated primers more efficiently than wild-type RT, the removal of ddAMP was effectively inhibited by the next complementary dNTP (IC 50 ≈ 12 μM). In contrast, the removal of AZTMP was not inhibited by dNTPs except at nonphysiological concentrations (IC 50 > 200 μM).

HIV-1 replication is inhibited by the incorporation of chain-terminating nucleotides at the 3′ end of the growing DNA chain. Here we show a nucleotide-dependent reaction catalyzed by HIV-1 reverse transcriptase that can efficiently remove the chain-terminating residue, yielding an extendible primer terminus. Radioactively labeled 3′-terminal residue from the primer can be transferred into a product that is resistant to calf intestinal alkaline phosphatase and sensitive to cleavage by snake venom phosphodiesterase. The products formed from different nucleotide substrates have unique electrophoretic migrations and have been identified as dinucleoside tri- or tetraphosphates. The reaction is inhibited by dNTPs that are complementary to the next position on the template ( K i ≈ 5 μM), suggesting competition between dinucleoside polyphosphate synthesis and DNA polymerization. Dinucleoside polyphosphate synthesis was inhibited by an HIV-1 specific non-nucleoside inhibitor and was absent in mutant HIV-1 reverse transcriptase deficient in polymerase activity, indicating that this activity requires a functional polymerase active site. We suggest that dinucleoside polyphosphate synthesis occurs by transfer of the 3′ nucleotide from the primer to the pyrophosphate moiety in the nucleoside di- or triphosphate substrate through a mechanism analogous to pyrophosphorolysis. Unlike pyrophosphorolysis, however, the reaction is nucleotide-dependent, is resistant to pyrophosphatase, and produces dinucleoside polyphosphates. Because it occurs at physiological concentrations of ribonucleoside triphosphates, this reaction may determine the in vivo activity of many nucleoside antiretroviral drugs.

Nondenaturing gel electrophoresis was used to study the nucleotide substrate-induced conformational change in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). Dead-end complex was formed between HIV-1 RT, dideoxynucleotide chain-terminated primer, and DNA template in the presence of deoxynucleotide triphosphate (dNTP) complementary to the next position on the template. Complexes which form in the absence of the next complementary dNTP were disrupted by adding excess poly(rA)/oligo(dT) or heparin just prior to electrophoresis. Dead-end complex formation by noncomplementary dNTP's or ribonucleotides was at least 2000-fold less efficient than with the complementary nucleotide. When dA was the next nucleotide on the template, analogues of dTTP supported dead-end complex formation with increased apparent Kd (dTTPdideoxy-TTP approximately alpha-thio-dTTPdUTP3'-azidothymidine triphosphate). A similar relationship was observed for dGTP analogues across from dC on the template (dGTPdideoxy-GTPalpha-thio-dGTPdITPdideoxy-ITP). The optimal length of the primer/template duplex region for dead-end complex formation was between 20 and 32 base pairs. Primer-template with a mismatched primer terminus did not support dead-end complex formation, and primer terminated with 3'-azidothymidine formed dead-end complex with 25-fold elevated apparent Kd. By contrast, dead-end complex formation on primer terminated with dideoxy-IMP base paired with dC on the template was more efficient than on primer terminated with dideoxy-GMP. Implications for the mechanisms of discrimination between nucleotide analogues by HIV-1 RT are discussed.

Retroviral replicating vectors (RRVs) have been shown to achieve efficient tumor transduction and enhanced therapeutic benefit in a wide variety of cancer models. An amphotropic murine leukemia virus (MLV)-based RRV encoding the yeast cytosine deaminase (CD) prodrug activator gene, designated Toca 511 (vocimagene amiretrorepvec), is now being investigated in combination with Toca FC (extended-release 5-FC) in multi-center Phase IIB / III clinical trials for patients with recurrent high-grade glioma (clinicaltrials.gov: NCT02414165). RRV-mediated prodrug activator gene therapy represents the ultimate form of “intracellular” chemotherapy, generated selectively and directly from within the infected cancer cells themselves, without incurring systemic toxicity. Moreover, preclinical data support subsequent activation of the immune system selectively against the cancer. We further propose that combination with additional therapies may be desirable to optimize treatment outcomes. Here we first evaluated two different RRVs derived from MLV and gibbon ape leukemia virus (GALV), and expressing either the same fluorescent reporter gene (MLV-GFP and GALV-GFP, respectively), or different reporter genes (MLV-GFP vs. GALV-RFP; MLV-RFP vs. GALV-GFP), in different types of human cancer cells. Individually, RRVs expressing the same reporter gene efficiently infected human glioma, prostate cancer, and ovarian cancer cells, and showed efficient replication and spread in culture. When marked with different fluorescent reporter genes, it was found that MLV-GFP can spread in cells that had been pre-transduced with GALV-RFP, but not in MLV-RFP pre-transduced cells. Similarly, GALV-GFP can spread in MLV-RFP pre-transduced cells, but not in GALV-RFP pre-transduced cells. This mutually exclusive infection pattern is likely due to the phenomenon of receptor interference, which occurs when vectors derived from the same viral strain compete for binding to cell surface receptors, resulting in superinfection resistance. Notably, however, replication and spread of either RRV in culture was not affected by pre-transduction with RRV derived from a different strain, indicating that this approach could overcome receptor interference. In order to investigate the effect of combined prodrug-dependent cell killing in vitro, cells were then transduced with MLV- or GALV-based RRV expressing the yeast cytosine deaminase suicide gene (MLV-CD, GALV-CD), which converts the prodrug 5-fluorocytosine (5-FC) into the active drug 5-fluorouracil (5-FU), or with RRV expressing the Herpes thymidine kinase gene (MLV-TK, GALV-TK) which converts Ganciclovir (GCV) to GCV-monophosphate, either individually or in combination. In vitro cytocidal effects obtained by combining different prodrug activator genes were significantly greater when these genes were delivered with RRV derived from two different strains. These data indicate the potential utility of using RRV pseudotyped with envelopes derived from different viral strains to overcome receptor interference leading to superinfection resistance, and achieve effective combined prodrug activator gene therapy. Citation Format: James Grosso, Sara Collins, Akihito Inagaki, Suzanne Matsuura, Brian Slomovitz, Tan Ince, Noriyuki Kasahara. Alternate pseudotypes overcome receptor interference and enable combination suicide gene therapy with retroviral replicating vectors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A42.

Our studies to date have demonstrated that retroviral replicating vectors (RRV) are capable of highly efficient replication in cancer cells in vitro and in vivo, associated with high levels of tumor-selective gene transfer and significantly enhanced survival benefit when employed for prodrug activator (“suicide”) gene therapy in a variety of cancer models. Clinical testing of RRV-mediated gene therapy using the yeast cytosine deaminase (CD) prodrug activator gene, which mediates conversion of the prodrug 5-fluorocytosine (5-FC) to the anti-cancer drug, 5-fluorouracil (5-FU), is currently underway in multi-center clinical trials being conducted in the United States for patients with recurrent high-grade glioma. In the present study, our goal has been to establish the feasibility of also applying this novel gene therapy strategy to secondary brain tumors arising from metastasis of systemic cancers to the CNS, which occur 5-10 times more frequently than primary brain tumors. Moreover, brain metastases are often accompanied by neurological deficits, which deprive patients of their quality of life. In particular, brain-metastatic breast cancer frequently arises from highly aggressive, treatment-refractory, ‘triple-negative' (ER(-), PR(-), HER2(-)) cells, is associated with a dismal prognosis of 4-6 months, and as treatment options are largely palliative, is the main cause of death in half of these patients. Hence this condition represents an unmet medical need. We first examined the replication kinetics of RRV expressing the GFP reporter gene (AC3-emd) in human and murine breast cancer cell lines by flow cytometry. The replication kinetics of AC3-emd in the triple-negative human breast cancer cell line MDA-MB-231-BR, and in the murine breast cancer cell line JC, showed robust replication activity over time at both MOI = 0.01 and 0.1, resulting in high levels of transduction within 1-2 weeks. Next, we tested in vitro cytotoxicity by MTS assay after 5-FC treatment of 231BR and JC cells that had been fully transduced with RRV expressing the CD prodrug activator gene (clinical-grade vector AC3-yCD2, also designated “Toca 511”). In both of the AC3-yCD2-transduced cell lines, cell viability was reduced approximately 70-85% even after exposure to 0.1 mM 5-FC treatment, and complete cell killing was observed after 6 days (231-BR) or 4 days (JC) exposure to 1 mM 5-FC treatment. We then examined in vivo replication kinetics of AC3-emd in intracranial 231-BR xenograft models. Intratumoral injection of 10e6 AC3-emd on Day 7 after tumor implantation was confirmed to achieve >80% transduction efficiency by flow cytometry on Day 10 post-injection. In survival studies, animals treated with AC3-yCD2+5-FC prodrug activator gene therapy showed statistically significant (p Citation Format: Akihito Inagaki, Sara Collins, Suzanne Matsuura, Noriyuki Kasahara. Therapeutic efficacy of retroviral replicating vectors in orthotopic models of breast cancer metastasis to the brain. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A43.

Human immunodeficiency virus type I (HIV-1) exploits various host cellular pathways for efficient infection. Here we report that the absence of mitochondrial DNA (mtDNA) in ρ(0) cells markedly attenuates HIV-1 infection. Importantly, reduced infection efficiency in ρ(0) cells is not simply the result of impaired oxidative phosphorylation (OXPHOS) because pharmacological OXPHOS inhibition did not inhibit HIV-1 infection. Analysis of the early steps of virus infection by real-time PCR quantification of stage-specific HIV-1 DNA products in the infected ρ(0) and parental cell line have allowed us to conclude that HIV-1 infection in ρ(0) cells is blocked at the steps that occur after reverse transcription and prior to nuclear import. Additionally, confocal fluorescence microscope analysis showed that the majority of viral complexes containing HIV-1 p24 co-localize with mitochondria in target cells, suggesting an interaction between the two. Collectively, our data strongly indicate that mitochondria play an important role during early stages of HIV-1 infection, probably through direct association with HIV-1 intracellular complexes.

Resistance to nucleoside reverse transcriptase (RT) inhibitors is conferred on human immunodeficiency virus type 1 through thymidine analogue resistance mutations (TAMs) that increase the ability of RT to excise chain-terminating nucleotides after they have been incorporated. The RT mutation M184V is a potent suppressor of TAMs. In RT containing TAMs, the addition of M184V suppressed the excision of 3′-deoxy-3′-azidothymidine monophosphate (AZTMP) to a greater extent on an RNA template than on a DNA template with the same sequence. The catalytically inactive RNase H mutation E478Q abolished this difference. The reduction in excision activity was similar with either ATP or pyrophosphate as the acceptor substrate. Decreased excision of AZTMP was associated with increased cleavage of the RNA template at position −7 relative to the primer terminus, which led to increased primer-template dissociation. Whether M184V was present or not, RT did not initially bind at the −7 cleavage site. Cleavage at the initial site was followed by RT dissociation and rebinding at the −7 cleavage site, and the dissociation and rebinding were enhanced when the M184V mutation was present. In contrast to the effect of M184V, the K65R mutation suppressed the excision activity of RT to the same extent on either an RNA or a DNA template and did not alter the RNase H cleavage pattern. Based on these results, we propose that enhanced RNase H cleavage near the primer terminus plays a role in M184V suppression of AZT resistance, while K65R suppression occurs through a different mechanism.

Binding of the next complementary dNTP by the binary complex containing HIV-1 reverse transcriptase (RT) and primer-template induces conformational changes that have been implicated in catalytic function of RT. We have used DNase I footprinting, gel electrophoretic mobility shift, and exonuclease protection assays to characterize the interactions between HIV-1 RT and chain-terminated primer-template in the absence and presence of various ligands. Distinguishable stable complexes were formed in the presence of foscarnet (an analogue of pyrophosphate), the dNTP complementary to the first (+1) templating nucleotide or the dNTP complementary to the second (+2) templating nucleotide. The position of HIV-1 RT on the primer-template in each of these complexes is different. RT is located upstream in the foscarnet complex, relative to the +1 complex, and downstream in the +2 complex. These results suggest that HIV-1 RT can translocate along the primer-template in the absence of phosphodiester bond formation. The ability to form a specific foscarnet complex might explain the inhibitory properties of this compound. The ability to recognize the second templating nucleotide has implications for nucleotide misincorporation.

Finger insertion mutations of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (T69S mutations followed by various dipeptide insertions) have a multinucleoside resistance phenotype that can be explained by decreased sensitivity to deoxynucleoside triphosphate (dNTP) inhibition of the nucleotide-dependent unblocking activity of RT. We show that RTs with SG or AG (but not SS) insertions have three- to fourfold-increased unblocking activity and that all three finger insertion mutations have threefold-decreased sensitivity to dNTP inhibition. The additional presence of M41L and T215Y mutations increased unblocking activity for all three insertions, greatly reduced the sensitivity to dNTP inhibition, and resulted in defects in in vitro DNA chain elongation. The DNA chain elongation defects were partially repaired by additional mutations at positions 210, 211, and 214. These results suggest that structural communication between the regions of RT defined by these mutations plays a role in the multinucleoside resistance phenotype.

Nucleotide-dependent unblocking of chain-terminated DNA by human immunodeficiency virus type 1 reverse transcriptase (RT) is enhanced by the presence of mutations associated with 3′-azido-3′-deoxythymidine (AZT) resistance. The increase in unblocking activity was greater for mutant combinations associated with higher levels of in vivo AZT resistance. The difference between mutant and wild-type activity was further enhanced by introduction of a methyl group into the nucleotide substrate and was decreased for a nonaromatic substrate, suggesting that π-π interactions between RT and an aromatic structure may be facilitated by these mutations.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutations were detected in DNA from peripheral blood mononuclear cells from 11 of 12 HIV-1-infected children after 11-20 months of zidovudine monotherapy. The number of children with mutations detected at each codon were as follows: codon 41, 4; codon 67, 2; codon 70, 7; codon 215, 7; codon 219, 0. Codon 41 mutations were found only in the presence of a codon 215 mutation and in the absence of a codon 70 mutation. The codon 41/215 mutant combination was associated with decline in weight-for-age z score during therapy, weight10th percentile, CD4+ cell counts3rd percentile, and immune-complex dissociated HIV-1 p24 antigen (ICD p24 Ag) levels100 pg/ml. Patients developing the codon 70 mutation tended to have body weight30th percentile, CD4+ cell counts25th percentile, and ICD p24 Ag100 pg/ml. The codon 41 mutation was associated with clinical deterioration during a 6-month followup period.

Thirteen monoclonal antibodies which react with the major capsid protein (VP1) of simian virus 40 (SV40) have been isolated. Of these, five neutralized viral infectivity when added in sufficient concentration. Seven of the antibodies reacted with denatured VP1 and also recognized fragments generated by protease or cyanogen bromide cleavage. The region of VP1 recognized by all seven antibodies was mapped within a nine-amino-acid segment located in the carboxyl portion of the protein (from amino acid positions 312 to 321). This region is likely to protrude from the surface of the protein as judged by high hydrophilicity and low hydropathy predicted from the amino acid sequence and lack of secondary structure by contrast with the rest of the protein for which predominantly β-sheet structure is predicted. Competition between these antibodies and synthetic peptides for binding to virus particles confirmed that the continuous epitope is contained within the nine-amino-acid sequence. Competition between the different monoclonal antibodies suggested that the continuous epitope was also part of more complex discontinuous epitopes recognized by some of the other antibodies. These results support a model in which a segment of the carboxyl-terminal portion of VP1 protrudes from the surface of the virus to form an antigenic structure.