Suzanne E. Matsuura

Effects of Specific Zidovudine Resistance Mutations and Substrate Structure on Nucleotide-Dependent Primer Unblocking by Human Immunodeficiency Virus Type 1 Reverse Transcriptase

ABSTRACT 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.

Relationship between 3′-Azido-3′-Deoxythymidine Resistance and Primer Unblocking Activity in Foscarnet-Resistant Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

ABSTRACT 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 PPi binding 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 (PPi or a nucleotide such as ATP) which also interacts with the PPi binding 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.

Effects of dipeptide insertions between codons 69 and 70 of human immunodeficiency virus type 1 reverse transcriptase on primer unblocking, deoxynucleoside triphosphate inhibition, and DNA chain elongation

ABSTRACT 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.

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

ABSTRACT 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 (ddAp4ddA, ddCp4ddC, ddGp4ddG, ddTp4ddT, Ap4ddG, 2′(3′)-O-(N-methylanthraniloyl)-Ap4ddG, 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 Infection Is Blocked at an Early Stage in Cells Devoid of Mitochondrial DNA

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 ρ0 cells markedly attenuates HIV-1 infection. Importantly, reduced infection efficiency in ρ0 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 ρ0 and parental cell line have allowed us to conclude that HIV-1 infection in ρ0 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.

A Role of Template Cleavage in Reduced Excision of Chain-Terminating Nucleotides by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Containing the M184V Mutation.

ABSTRACT 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.

Reverse transcriptase mutations in HIV-1 : Infected children treated with zidovudine

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, weight < 10th percentile, CD4+ cell counts < 3rd percentile, and immune-complex dissociated HIV-1 p24 antigen (ICD p24 Ag) levels > 100 pg/ml. Patients developing the codon 70 mutation tended to have body weight > 30th percentile, CD4+ cell counts > 25th percentile, and ICD p24 Ag < 100 pg/ml. The codon 41 mutation was associated with clinical deterioration during a 6-month followup period.

Abstract A42: Alternate pseudotypes overcome receptor interference and enable combination suicide gene therapy with retroviral replicating vectors

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.