Synergistic reduction of HIV-1 infectivity by 5-azacytidine and inhibitors of ribonucleotide reductase.

Although many compounds have been approved for the treatment of human immunodeficiency type-1 (HIV-1) infection, additional anti-HIV-1 drugs (particularly those belonging to new drug classes) are still needed due to issues such as long-term drug-associated toxicities, transmission of drug-resistant variants, and development of multi-class resistance. Lethal mutagenesis represents an antiviral strategy that has not yet been clinically translated for HIV-1 and is based on the use of small molecules to induce excessive levels of deleterious mutations within the viral genome. Here, we show that 5-azacytidine (5-aza-C), a ribonucleoside analog that induces the lethal mutagenesis of HIV-1, and multiple inhibitors of the enzyme ribonucleotide reductase (RNR) interact in a synergistic fashion to more effectively reduce the infectivity of HIV-1. In these drug combinations, RNR inhibitors failed to significantly inhibit the conversion of 5-aza-C to 5-aza-2'-deoxycytidine, suggesting that 5-aza-C acts primarily as a deoxyribonucleoside even in the presence of RNR inhibitors. The mechanism of antiviral synergy was further investigated for the combination of 5-aza-C and one specific RNR inhibitor, resveratrol, as this combination improved the selectivity index of 5-aza-C to the greatest extent. Antiviral synergy was found to be primarily due to the reduced accumulation of reverse transcription products rather than the enhancement of viral mutagenesis. To our knowledge, these observations represent the first demonstration of antiretroviral synergy between a ribonucleoside analog and RNR inhibitors, and encourage the development of additional ribonucleoside analogs and RNR inhibitors with improved antiretroviral activity.

Dual anti-HIV mechanism of clofarabine.

BACKGROUND: HIV-1 replication kinetics inherently depends on the availability of cellular dNTPs for viral DNA synthesis. In activated CD4(+) T cells and other rapidly dividing cells, the concentrations of dNTPs are high and HIV-1 reverse transcription occurs in an efficient manner. In contrast, nondividing cells such as macrophages have lower dNTP pools, which restricts efficient reverse transcription. Clofarabine is an FDA approved ribonucleotide reductase inhibitor, which has shown potent antiretroviral activity in transformed cell lines. Here, we explore the potency, toxicity and mechanism of action of clofarabine in the human primary HIV-1 target cells: activated CD4(+) T cells and macrophages. RESULTS: Clofarabine is a potent HIV-1 inhibitor in both activated CD4(+) T cells and macrophages. Due to its minimal toxicity in macrophages, clofarabine displays a selectivity index over 300 in this nondividing cell type. The anti-HIV-1 activity of clofarabine correlated with a significant decrease in both cellular dNTP levels and viral DNA synthesis. Additionally, we observed that clofarabine triphosphate was directly incorporated into DNA by HIV-1 reverse transcriptase and blocked processive DNA synthesis, particularly at the low dNTP levels found in macrophages. CONCLUSIONS: Taken together, these data provide strong mechanistic evidence that clofarabine is a dual action inhibitor of HIV-1 replication that both limits dNTP substrates for viral DNA synthesis and directly inhibits the DNA polymerase activity of HIV-1 reverse transcriptase.

5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2'-Deoxycytidine.

5-Azacytidine (5-aza-C) is a ribonucleoside analog that induces the lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1) by causing predominantly G-to-C transversions during reverse transcription. 5-Aza-C could potentially act primarily as a ribonucleotide (5-aza-CTP) or as a deoxyribonucleotide (5-aza-2'-deoxycytidine triphosphate [5-aza-dCTP]) during reverse transcription. In order to determine the primary form of 5-aza-C that is active against HIV-1, Illumina sequencing was performed using proviral DNA from cells treated with 5-aza-C or 5-aza-dC. 5-Aza-C and 5-aza-dC were found to induce highly similar patterns of mutation in HIV-1 in terms of the types of mutations observed, the magnitudes of effects, and the distributions of mutations at individual sequence positions. Further, 5-aza-dCTP was detected by liquid chromatography-tandem mass spectrometry in cells treated with 5-aza-C, demonstrating that 5-aza-C was a substrate for ribonucleotide reductase. Notably, levels of 5-aza-dCTP were similar in cells treated with equivalent effective concentrations of 5-aza-C or 5-aza-dC. Lastly, HIV-1 reverse transcriptase was found to incorporate 5-aza-CTPin vitroat least 10,000-fold less efficiently than 5-aza-dCTP. Taken together, these data support the model that 5-aza-C enhances the mutagenesis of HIV-1 primarily after reduction to 5-aza-dC, which can then be incorporated during reverse transcription and lead to G-to-C hypermutation. These findings may have important implications for the design of new ribonucleoside analogs directed against retroviruses.

Rapid Determination of HIV-1 Mutant Frequencies and Mutation Spectra Using an mCherry/EGFP Dual-Reporter Viral Vector.

The high mutation rate of human immunodeficiency virus type-1 (HIV-1) has been a pivotal factor in its evolutionary success as a human pathogen, driving the emergence of drug resistance, immune system escape, and invasion of distinct anatomical compartments. Extensive research has focused on understanding how various cellular and viral factors alter the rates and types of mutations produced during viral replication. Here, we describe a single-cycle dual-reporter vector assay that relies upon the detection of mutations that eliminate either expression of mCherry or enhanced green fluorescent protein (EGFP). The reporter-based method can be used to efficiently quantify changes in mutant frequencies and mutation spectra that arise due to a variety of factors, including viral mutagens, drug resistance mutations, cellular physiology, and APOBEC3 proteins.

Characterization of permeability, stability and anti-HIV-1 activity of decitabine and gemcitabine divalerate prodrugs.

BACKGROUND: Over 25 drugs have been approved for the treatment of HIV-1 replication. All but one of these drugs is delivered as an oral medication. Previous studies have demonstrated that two drugs, decitabine and gemcitabine, have potent anti-HIV-1 activities and can work together in synergy to reduce HIV-1 infectivity via lethal mutagenesis. For their current indications, decitabine and gemcitabine are delivered intravenously. METHODS: As an initial step towards the clinical translation of these drugs for the treatment of HIV-1 infection, we synthesized decitabine and gemcitabine prodrugs in order to increase drug permeability, which has generally been shown to correlate with increased bioavailability in vivo. In the present study we investigated the permeability, stability and anti-HIV-1 activity of decitabine and gemcitabine prodrugs and selected the divalerate esters of each as candidates for further investigation. RESULTS: Our results provide the first demonstration of divalerate prodrugs of decitabine and gemcitabine that are readily permeable, stable and possess anti-HIV-1 activity. CONCLUSIONS: These observations predict improved oral availability of decitabine and gemcitabine, and warrant further study of their ability to reduce HIV-1 infectivity in vivo.

Anti-HIV-1 activity of resveratrol derivatives and synergistic inhibition of HIV-1 by the combination of resveratrol and decitabine.

Ribonucleotide reductase inhibitors enhance the anti-HIV-1 activities of a variety of nucleoside analogs, including those that act as chain terminators and those that increase the HIV-1 mutation rate. However the use of these ribonucleotide reductase inhibitors is limited by their associated toxicities. The hydroxylated phytostilbene resveratrol has activity in a host of systems including inhibition of ribonucleotide reductase and has minimal toxicity. Here we synthesized derivatives of resveratrol and examined them for anti-HIV-1 activity and their ability to enhance the antiviral activity of decitabine, a nucleoside analog that decreases viral replication by increasing the HIV-1 mutation rate. The data demonstrates that six of the derivatives have anti-HIV-1 activity greater than resveratrol. However, only resveratrol acted in synergy with decitabine to inhibit HIV-1 infectivity. These results reveal novel resveratrol derivatives with anti-HIV-1 activity that may have mechanisms of action that differ from the drugs currently used to treat HIV-1.

Discovery of drugs that possess activity against feline leukemia virus.

Feline leukemia virus (FeLV) is a gammaretrovirus that is a significant cause of neoplastic-related disorders affecting cats worldwide. Treatment options for FeLV are limited, associated with serious side effects, and can be cost-prohibitive. The development of drugs used to treat a related retrovirus, human immunodeficiency virus type 1 (HIV-1), has been rapid, leading to the approval of five drug classes. Although structural differences affect the susceptibility of gammaretroviruses to anti-HIV drugs, the similarities in mechanism of replication suggest that some anti-HIV-1 drugs may also inhibit FeLV. This study demonstrates the anti-FeLV activity of four drugs approved by the US FDA (Food and Drug Administration) at non-toxic concentrations. Of these, tenofovir and raltegravir are anti-HIV-1 drugs, while decitabine and gemcitabine are approved to treat myelodysplastic syndromes and pancreatic cancer, respectively, but also have anti-HIV-1 activity in cell culture. Our results indicate that these drugs may be useful for FeLV treatment and should be investigated for mechanism of action and suitability for veterinary use.

Activity of a novel combined antiretroviral therapy of gemcitabine and decitabine in a mouse model for HIV-1.

The emergence of drug resistance threatens to limit the use of current anti-HIV-1 drugs and highlights the need to expand the number of treatment options available for HIV-1-infected individuals. Our previous studies demonstrated that two clinically approved drugs, decitabine and gemcitabine, potently inhibited HIV-1 replication in cell culture through a mechanism that is distinct from the mechanisms for the drugs currently used to treat HIV-1 infection. We further demonstrated that gemcitabine inhibited replication of a related retrovirus, murine leukemia virus (MuLV), in vivo using the MuLV-based LP-BM5/murine AIDS (MAIDS) mouse model at doses that were not toxic. Since decitabine and gemcitabine inhibited MuLV and HIV-1 replication with similar potency in cell culture, the current study examined the efficacy and toxicity of the drug combination using the MAIDS model. The data demonstrate that the drug combination inhibited disease progression, as detected by histopathology, viral loads, and spleen weights, at doses lower than those that would be required if the drugs were used individually. The combination of decitabine and gemcitabine exerted antiviral activity at doses that were not toxic. These findings indicate that the combination of decitabine and gemcitabine shows potent antiretroviral activity at nontoxic doses and should be further investigated for clinical relevance.

Broadening the use of antiretroviral therapy: the case for feline leukemia virus.

Antiretroviral drugs have saved and extended the lives of millions of individuals infected with HIV. The major classes of anti-HIV drugs include reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, and entry/fusion inhibitors. While antiretroviral drug regimens are not commonly used to treat other types of retroviral infections, there are instances where there is a perceived need for re-evaluation of the benefits of antiretroviral therapy. One case in point is that of feline leukemia virus (FeLV), an infection of companion felines. While vaccines exist to prevent FeLV infection and spread, they have not eliminated FeLV infection. For FeLV-infected felines and their human companions, antiretroviral therapy would be desirable and of practical importance if good options were available. Here, we discuss FeLV biology and current treatment options, and propose that there is a need for antiretroviral treatment options for FeLV infection. The comparative use and analysis of antiretroviral therapy can provide new insights into the mechanism of antiretroviral drug action.

Analysis of the ex vivo and in vivo antiretroviral activity of gemcitabine.

Replication of retroviral and host genomes requires ribonucleotide reductase to convert rNTPs to dNTPs, which are then used as substrates for DNA synthesis. Inhibition of ribonucleotide reductase by hydroxyurea (HU) has been previously used to treat cancers as well as HIV. However, the use of HU as an antiretroviral is limited by its associated toxicities such as myelosuppression and hepatotoxicity. In this study, we examined the ribonucleotide reductase inhibitor, gemcitabine, both in cell culture and in C57Bl/6 mice infected with LP-BM5 murine leukemia virus (LP-BM5 MuLV, a murine AIDS model). Gemcitabine decreased infectivity of MuLV in cell culture with an EC50 in the low nanomolar range with no detectable cytotoxicity. Similarly, gemcitabine significantly decreased disease progression in mice infected with LP-BM5. Specifically, gemcitabine treatment decreased spleen size, plasma IgM, and provirus levels compared to LP-BM5 MuLV infected, untreated mice. Gemcitabine efficacy was observed at doses as low as 1 mg/kg/day in the absence of toxicity. Higher doses of gemcitabine (3 mg/kg/day and higher) were associated with toxicity as determined by a loss in body mass. In summary, our findings demonstrate that gemcitabine has antiretroviral activity ex vivo and in vivo in the LP-BM5 MuLV model. These observations together with a recent ex vivo study with HIV-1, suggest that gemcitabine has broad antiretroviral activity and could be particularly useful in vivo when used in combination drug therapy.

Human pluripotent stem cells produce natural killer cells that mediate anti-HIV-1 activity by utilizing diverse cellular mechanisms.

Cell-based therapies against HIV/AIDS have been gaining increased interest. Natural killer (NK) cells are a key component of the innate immune system with the ability to kill diverse tumor cells and virus-infected cells. While NK cells have been shown to play an important role in the control of HIV-1 replication, their functional activities are often compromised in HIV-1-infected individuals. We have previously demonstrated the derivation of NK cells from human embryonic stem cells (hESCs) with the ability to potently kill multiple types of tumor cells both in vitro and in vivo. We now demonstrate the derivation of functional NK cells from human induced pluripotent stem cells (iPSCs). More importantly, both hESC- and iPSC-derived NK cells are able to inhibit HIV-1 NL4-3 infection of CEM-GFP cells. Additional studies using HIV-1-infected human primary CD4(+) T cells illustrated that hESC- and iPSC-derived NK cells suppress HIV-1 infection by at least three distinct cellular mechanisms: killing of infected targets through direct lysis, antibody-dependent cellular cytotoxicity, and production of chemokines and cytokines. Our results establish the potential to utilize hESC- and iPSC-derived NK cells to better understand anti-HIV-1 immunity and provide a novel cellular immunotherapeutic approach to treat HIV/AIDS.

The role of Rab5a GTPase in endocytosis and post-endocytic trafficking of the hCG-human luteinizing hormone receptor complex.

This study examined the role of Rab5a GTPase in regulating hCG-induced internalization and trafficking of the hCG-LH receptor complex in transfected 293T cells. Coexpression of wild-type Rab5a (WT) or constitutively active Rab5a (Q79L) with LHR significantly increased hCG-induced LHR internalization. Conversely, coexpression of dominant negative Rab5a (S34N) with LHR reduced internalization. Confocal microscopy showed LHR colocalizing with Rab5a (WT) and Rab5a (Q79L) in punctuate structures. Coexpression of Rab5a (WT) and Rab5a (Q79L) with LHR significantly increased colocalization of LHR in early endosomes. Conversely, dominant negative Rab5a (S34N) decreased this colocalization. While Rab5a stimulated internalization of LHR, it significantly decreased LHR recycling to the cell surface and increased degradation. Dominant negative Rab5a (S34N) increased LHR recycling and decreased degradation. These results suggest that Rab5a plays a role in LHR trafficking by facilitating internalization and fusion to early endosomes, increasing the degradation of internalized receptor resulting in a reduction in LHR recycling.

Exploiting drug repositioning for discovery of a novel HIV combination therapy.

The development of HIV drugs is an expensive and a lengthy process. In this study, we used drug repositioning, a process whereby a drug approved to treat one condition is used to treat a different condition, to identify clinically approved drugs that have anti-HIV activity. The data presented here show that a combination of two clinically approved drugs, decitabine and gemcitabine, reduced HIV infectivity by 73% at concentrations that had minimal antiviral activity when used individually. Decreased infectivity coincided with a significant increase in mutation frequency and a shift in the HIV mutation spectrum. These results indicate that an increased mutational load is the primary antiviral mechanism for inhibiting the generation of infectious progeny virus from provirus. Similar results were seen when decitabine was used in combination with another ribonucleotide reductase inhibitor. Our results suggest that HIV infectivity can be decreased by combining a nucleoside analog that forms noncanonical base pairs with certain ribonucleotide reductase inhibitors. Such drug combinations are relevant since members of these drug classes are used clinically. Our observations support a model in which increased mutation frequency decreases infectivity through lethal mutagenesis.

5-Azacytidine can induce lethal mutagenesis in human immunodeficiency virus type 1.

Ribonucleosides inhibit human immunodeficiency virus type 1 (HIV-1) replication by mechanisms that have not been fully elucidated. Here, we report the antiviral mechanism for the ribonucleoside analog 5-azacytidine (5-AZC). We hypothesized that the anti-HIV-1 activity of 5-AZC was due to an increase in the HIV-1 mutation rate following its incorporation into viral RNA during transcription. However, we demonstrate that 5-AZC's primary antiviral activity can be attributed to its effect on the early phase of HIV-1 replication. Furthermore, the antiviral activity was associated with an increase in the frequency of viral mutants, suggesting that 5-AZC's primary target is reverse transcription. Sequencing analysis showed an enrichment in G-to-C transversion mutations and further supports the idea that reverse transcription is an antiviral target of 5-AZC. These results indicate that 5-AZC is incorporated into viral DNA following reduction to 5-aza-2'-deoxycytidine. Incorporation into the viral DNA leads to an increase in mutant frequency that is consistent with lethal mutagenesis during reverse transcription as the primary antiviral mechanism of 5-AZC. Antiviral activity and increased mutation frequency were also associated with the late phase of HIV-1 replication; however, 5-AZC's effect on the late phase was less robust. These results reveal that the primary antiviral mechanism of 5-AZC can be attributed to its ability to increase the HIV-1 mutation frequency through viral-DNA incorporation during reverse transcription. Our observations indicate that 5-AZC can affect two steps in HIV-1 replication (i.e., transcription and reverse transcription) but that its primary antiviral activity is due to incorporation during reverse transcription.

Gonadotropin receptors: role of post-translational modifications and post-transcriptional regulation.

This review focuses on the post-translational modifications of LH and FSH receptors and recent studies on the regulation of LH receptor expression mediated by an RNA binding protein. Both LH and FSH receptors undergo extensive post-translational modifications. N-linked glycosylation occurs co-translationally and plays a role in the maturation and processing of the receptor, while palmitoylation is involved in receptor endocytosis and post-endocytic trafficking. A third type of post-translational modification is phosphorylation and its function has been reviewed. Finally, the regulation of LH receptor at the mRNA level by an RNA binding protein is discussed in the context of ovarian function.

N-linked glycosylation facilitates processing and cell surface expression of rat luteinizing hormone receptor.

The extracellular domain of the luteinizing hormone (LH) receptor has six potential N-linked glycosylation sites. Although previous studies have shown that mutation of the first three sites results in decreased ligand binding at the cell surface, the role of glycosylation in LH receptor processing is not understood. In the present study, we examined whether mutation of the first three sites has any affect on receptor synthesis, processing, and degradation of the mutant receptors. The data show that mutation of N77, N152, or N173 did not affect receptor synthesis, but did significantly reduce processing of the receptor precursor to the mature, cell surface form. Furthermore, defective processing was due to increased degradation of the precursor rather than increased turnover of cell surface receptors. Thus, lack of glycosylation decreases LH receptor processing and targets the receptors for degradation thereby leading to decreased cell surface expression. These results show that glycosylation of the LH receptor plays an important role in receptor processing and cell surface expression.

Evidence that palmitoylation of carboxyl terminus cysteine residues of the human luteinizing hormone receptor regulates postendocytic processing.

Palmitoylation is a well-conserved posttranslational modification among members of the G protein-coupled receptor superfamily. The present study examined the role of palmitoylation in endocytosis and postendocytic trafficking of the human LH receptor (LHR). Palmitoylation of the LHR was determined by incorporation of [3H]palmitic acid into wild-type (WT) or mutant receptor in which the potential palmitoylation sites, C643 and C644, were mutated to glycine residues. The WT receptor showed incorporation of [3H]palmitic acid into the mature 90-kDa form of the receptor whereas mutation of the two Cys residues abrogated this incorporation, indicating that Cys 643 and C644 are the sites of palmitoylation. The role of palmitoylation on endocytosis and postendocytic processing was examined by testing the ability of the WT and mutant receptor to undergo internalization, recycling, and lysosomal degradation. Compared with the WT receptor, the mutant receptor showed increased internalization and decreased recycling, suggesting that retention of palmitic acid residues at Cys 643 and 644 promotes LHR recycling. The role of palmitoylation on receptor recycling was substantiated by demonstrating that a different mutant, D578H LHR, which is deficient in palmitoylation, also recycled less efficiently. Furthermore, the data show that palmitoylation, not the rate of internalization, determines the efficiency of recycling. The present study shows that palmitoylation of cysteine residues 643 and 644 of the human LHR is a determinant of recycling.

Regulation of luteinizing hormone/human chorionic gonadotropin receptor expression: a perspective.

The LH/hCG receptor, a member of the G protein coupled receptor family mediates the cellular actions of LH in the ovary. A considerable amount of information regarding its structure, mechanism of activation, and regulation of expression has emerged in recent years. Here we provide a brief overview of the current information on the structural organization of the receptor and the mechanism of receptor mediated signaling as well as an in-depth discussion on recent developments pertaining to the regulation of receptor expression. Specifically, we describe studies from our laboratory showing that the posttranscriptional regulation of the receptor involves an LH/hCG receptor mRNA-binding protein. We also propose a model to explain the loss of steady-state LH/hCG receptor mRNA levels during receptor down-regulation.