Dimitrios Topalis

The large tumor antigen: a "Swiss Army knife" protein possessing the functions required for the polyomavirus life cycle.

The SV40 large tumor antigen (L-Tag) is involved in the replication and cell transformation processes that take place during the polyomavirus life cycle. The ability of the L-Tag to interact with and to inactivate the tumor suppressor proteins p53 and pRb, makes this polyfunctional protein an interesting target in the search for compounds with antiviral and/or antiproliferative activities designed for the management of polyomavirus-associated diseases. The severe diseases caused by polyomaviruses, mainly in immunocompromised hosts, and the absence of licensed treatments, make the discovery of new antipolyomavirus drugs urgent. Parallels can be made between the SV40 L-Tag and the human papillomavirus (HPV) oncoproteins (E6 and E7) as they are also able to deregulate the cell cycle in order to promote cell transformation and its maintenance. In this review, a presentation of the SV40 L-Tag characteristics, regarding viral replication and cellular transformation, will show how similar these two processes are between the polyoma- and papillomavirus families. Insights at the molecular level will highlight similarities in the binding of polyoma- and papillomavirus replicative helicases to the viral DNA and in their disruptions of the p53 and pRb tumor suppressor proteins.

In Vitro-Selected Drug-Resistant Varicella-Zoster Virus Mutants in the Thymidine Kinase and DNA Polymerase Genes Yield Novel Phenotype-Genotype Associations and Highlight Differences between Antiherpesvirus Drugs

ABSTRACT Varicella zoster virus (VZV) is usually associated with mild to moderate illness in immunocompetent patients. However, older age and immune deficiency are the most important risk factors linked with virus reactivation and severe complications. Treatment of VZV infections is based on nucleoside analogues, such as acyclovir (ACV) and its valyl prodrug valacyclovir, penciclovir (PCV) as its prodrug famciclovir, and bromovinyldeoxyuridine (BVDU; brivudin) in some areas. The use of the pyrophosphate analogue foscarnet (PFA) is restricted to ACV-resistant (ACVr) VZV infections. Since antiviral drug resistance is an emerging problem, we attempt to describe the contributions of specific mutations in the viral thymidine kinase (TK) gene identified following selection with ACV, BVDU and its derivative BVaraU (sorivudine), and the bicyclic pyrimidine nucleoside analogues (BCNAs), a new class of potent and specific anti-VZV agents. The string of 6 Cs at nucleotides 493 to 498 of the VZV TK gene appeared to function as a hot spot for nucleotide insertions or deletions. Novel amino acid substitutions (G24R and T86A) in VZV TK were also linked to drug resistance. Six mutations were identified in the “palm domain” of VZV DNA polymerase in viruses selected for resistance to PFA, PCV, and the 2-phophonylmethoxyethyl (PME) purine derivatives. The investigation of the contributions of specific mutations in VZV TK or DNA polymerase to antiviral drug resistance and their impacts on the structures of the viral proteins indicated specific patterns of cross-resistance and highlighted important differences, not only between distinct classes of antivirals, but also between ACV and PCV.

Heterogeneity and Evolution of Thymidine Kinase and DNA Polymerase Mutants of Herpes Simplex Virus Type 1: Implications for Antiviral Therapy

BACKGROUND Infections caused by acyclovir-resistant isolates of herpes simplex virus (HSV) after hematopoietic stem cell transplantation (HSCT) are an emerging concern. An understanding of the evolutionary aspects of HSV infection is crucial to the design of effective therapeutic and control strategies. METHODS Eight sequential HSV-1 isolates were recovered from an HSCT patient who suffered from recurrent herpetic gingivostomatitis and was treated alternatively with acyclovir, ganciclovir, and foscavir. The diverse spectra and temporal changes of HSV drug resistance were determined phenotypically (drug-resistance profiling) and genotypically (sequencing of the viral thymidine kinase and DNA polymerase genes). RESULTS Analysis of 60 clones recovered from the different isolates demonstrated that most of these isolates were heterogeneous mixtures of variants, indicating the simultaneous infection with different drug-resistant viruses. The phenotype/genotype of several clones associated with resistance to acyclovir and/or foscavir were identified. Two novel mutations (E798K and I922T) in the viral DNA polymerase could be linked to drug resistance. CONCLUSIONS The heterogeneity within the viral populations and the temporal changes of drug-resistant viruses found in this HSCT recipient were remarkable, showing a rapid evolution of HSV-1. Drug-resistance surveillance is highly recommended among immunocompromised patients to manage the clinical syndrome and to avoid the emergence of multidrug-resistant isolates.

Insights into the mechanism of action of cidofovir and other acyclic nucleoside phosphonates against polyoma- and papillomaviruses and non-viral induced neoplasia

Acyclic nucleoside phosphonates (ANPs) are well-known for their antiviral properties, three of them being approved for the treatment of human immunodeficiency virus infection (tenofovir), chronic hepatitis B (tenofovir and adefovir) or human cytomegalovirus retinitis (cidofovir). In addition, cidofovir is mostly used off-label for the treatment of infections caused by several DNA viruses other than cytomegalovirus, including papilloma- and polyomaviruses, which do not encode their own DNA polymerases. There is considerable interest in understanding why cidofovir is effective against these small DNA tumor viruses. Considering that papilloma- and polyomaviruses cause diseases associated either with productive infection (characterized by high production of infectious virus) or transformation (where only a limited number of viral proteins are expressed without synthesis of viral particles), it can be envisaged that cidofovir may act as antiviral and/or antiproliferative agent. The aim of this review is to discuss the advances in recent years in understanding the mode of action of ANPs as antiproliferative agents, given the fact that current data suggest that their use can be extended to the treatment of non-viral related malignancies.

Activities of Different Classes of Acyclic Nucleoside Phosphonates against BK Virus in Primary Human Renal Cells

ABSTRACT BK virus (BKV), a virus belonging to the polyomavirus family, is a circular double-stranded DNA virus that causes nephropathies in immunocompromised patients after kidney or bone marrow transplantation. The occurrence of polyomavirus-associated nephropathy in kidney transplant patients may trigger graft loss, and guidelines for the management of BKV infection have not yet been clearly established. Treatment of BKV nephropathy with cidofovir (CDV) {(S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC)}, an acyclic phosphonate analogue of dCMP with a broad antiviral activity against DNA virus infections, has been proposed. The benefit of this small-molecule-based treatment has been evaluated only with a limited number of cases. In this study, we report the evaluation of three different classes of acyclic nucleoside phosphonates for their activities against BKV replication in two different primary renal cells: renal proximal tubular epithelial cells (RPTECs) and human renal cortical epithelial (HRCE) cells. The data indicate that besides HPMPC and its cyclic form, (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine (HPMP-5-azaC), cyclic HPMP (cHPMP)-5-azaC, hexadecyloxyethyl (HDE)-cHPMP-5-azaC, and 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) are the most selective inhibitors of BKV replication. On the contrary, leflunomide, which has also been proposed for the management of BKV-associated diseases, is not able to inhibit BKV replication at nontoxic concentrations.

Spectrum of Activity and Mechanisms of Resistance of Various Nucleoside Derivatives against Gammaherpesviruses

ABSTRACT The susceptibilities of gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposis sarcoma-associated herpesvirus (KSHV), and animal rhadinoviruses, to various nucleoside analogs was investigated in this work. Besides examining the antiviral activities and modes of action of antivirals currently marketed for the treatment of alpha- and/or betaherpesvirus infections (including acyclovir, ganciclovir, penciclovir, foscarnet, and brivudin), we also investigated the structure-activity relationship of various 5-substituted uridine and cytidine molecules. The antiviral efficacy of nucleoside derivatives bearing substitutions at the 5 position was decreased if the bromovinyl was replaced by chlorovinyl. 1-β-d-Arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU), a nucleoside with an arabinose configuration of the sugar ring, exhibited no inhibitory effect against rhadinoviruses but was active against EBV. On the other hand, the fluoroarabinose cytidine analog 2′-fluoro-5-iodo-aracytosine (FIAC) showed high selectivity indices against gammaherpesviruses that were comparable to those of brivudin. Additionally, we selected brivudin- and acyclovir-resistant rhadinoviruses in vitro and characterized them by phenotypic and genotypic (i.e., sequencing of the viral thymidine kinase, protein kinase, and DNA polymerase) analysis. Here, we reveal key amino acids in these enzymes that play an important role in substrate recognition. Our data on drug susceptibility profiles of the different animal gammaherpesvirus mutants highlighted cross-resistance patterns and indicated that pyrimidine nucleoside derivatives are phosphorylated by the viral thymidine kinase and purine nucleosides are preferentially activated by the gammaherpesvirus protein kinase.

KAY-2-41, a novel nucleoside analogue inhibitor of orthopoxviruses in vitro and in vivo

ABSTRACT The availability of adequate treatments for poxvirus infections would be valuable not only for human use but also for veterinary use. In the search for novel antiviral agents, a 1′-methyl-substituted 4′-thiothymidine nucleoside, designated KAY-2-41, emerged as an efficient inhibitor of poxviruses. In vitro, KAY-2-41 was active in the micromolar range against orthopoxviruses (OPVs) and against the parapoxvirus orf. The compound preserved its antiviral potency against OPVs resistant to the reference molecule cidofovir. KAY-2-41 had no noticeable toxicity on confluent monolayers, but a cytostatic effect was seen on growing cells. Genotyping of vaccinia virus (VACV), cowpox virus, and camelpox virus selected for resistance to KAY-2-41 revealed a nucleotide deletion(s) close to the ATP binding site or a nucleotide substitution close to the substrate binding site in the viral thymidine kinase (TK; J2R) gene. These mutations resulted in low levels of resistance to KAY-2-41 ranging from 2.7- to 6.0-fold and cross-resistance to 5-bromo-2′-deoxyuridine (5-BrdU) but not to cidofovir. The antiviral effect of KAY-2-41 relied, at least in part, on activation (phosphorylation) by the viral TK, as shown through enzymatic assays. The compound protected animals from disease and mortality after a lethal challenge with VACV, reduced viral loads in the serum, and abolished virus replication in tissues. In conclusion, KAY-2-41 is a promising nucleoside analogue for the treatment of poxvirus-induced diseases. Our findings warrant the evaluation of additional 1′-carbon-substituted 4′-thiothymidine derivatives as broad-spectrum antiviral agents, since this molecule also showed antiviral potency against herpes simplex virus 1 in earlier studies.

Tenofovir Activating Kinases May Impact the Outcome of HIV Treatment and Prevention.

Tenofovir (TFV), an acyclic nucleoside phosphonate analogue of 2′-deoxyadenosine, remains a first-line choice for the treatment of HIV infections under its prodrug form (i.e. tenofovir disoproxil fumarate, TDF). During the last decade, the use of TFV in pre-exposure prophylaxis (PrP) was first investigated as a vaginal microbicide gel (Mayer et al., 2006). More recently, rectal microbicide preparations and oral TDF alone or in combination with emtricitabine have been evaluated in PrP (Marrazzo et al., 2015), opening a new era in the fight against HIV. Clinical studies using TFV-based regimens in PrP provided inconsistent results, which have been primarily explained by poor adherence. The study by Lade et al. (2015) explored whether differences in the mechanism of TFV activation in peripheral blood mononuclear cells (PBMCs), vaginal and colorectal tissues exist. Also, they examined whether genetic variation in the nucleotide kinases that activate the drug could be responsible for the discrepant results observed in PrP studies using TFV-based regimens. To be active, TFV requires two successive phosphorylation catalyzed by cellular kinases. Then, its active diphosphate form (TFV-DP) targets the HIV reverse transcriptase leading to inhibition of viral replication. The first step of TFV activation is substrate-specific and requires phosphorylation by a cellular adenylate kinase (AK). The isoform AK2 is known to be localized in the mitochondrial intermembrane space and to catalyze TFV phosphorylation in a more efficient manner than its cytoplasmic counterpart AK1. Lade et al. (2015) found that AK2 activates TFV to TFV-MP in PBMCs and vaginal and colon tissues from healthy, HIV-uninfected donors that were not administered with the drug. Two questions rise regarding an AK2-dependent activation of TFV. The first issue is whether AK2 exists as a cytosolic form in PBMCs and in vaginal and colorectal tissues. Several studies reported the existence of small amounts of AK2 in the cytosol of porcine and rat tissues (Nobumoto et al., 1998; Watanabe et al., 1979). In contrast, Jurkat cells, a human leukemic T cell line, showed the absence of AK2 in the cytosol (Kohler et al., 1999). If small amounts of AK2 are present in the cytosol, will these low AK2 levels produce sufficient TFV-MP to afford anti-HIV activity? If AK2 is only expressed in the mitochondrial intermembrane space, then the activation of TFV will be performed exclusively in the mitochondria. Hence, the second issue is that TFV activation in the mitochondria will necessitate export of the phosphorylated form (TFV-MP or TFV-DP) into the cytosol via an active transport in order to exert its anti-HIV activity and to avoid mitochondrial toxicity. Several reports described TFV mitochondrial toxicity in renal proximal tubular cells (Lebrecht et al., 2009), while others showed no toxicity in HepG2 cells and skeletal muscle cells (Birkus et al., 2002). Furthermore, it is known that the use of TFV can be associated, in some cases, with renal dysfunction and Fanconi syndrome. Therefore, it will be interesting to investigate in PBMCs, vaginal and colorectal tissues the localization of AK2 and the mitochondrial transporter(s) that might contribute to the export of TFV-MP and/or TFV-DP. The second step of TFV activation involves kinases that recognize a wide range of substrates. Several nucleoside diphosphate kinases (NDPKs) can be responsible for the production of TFV-DP, including NDPK1, NDPK2, pyruvate kinase (PK), phosphoglycerate kinase (PGK), and creatine kinase (CK). Interestingly, the analysis by Lade and colleagues points to a role of PK in the conversion of TFV-MP to TFV-DP in PBMCs and vaginal tissue, while CK appeared to be responsible for the formation of TFV-DP in colon tissue. These findings are in agreement with enzymatic results reported by Varga et al. showing a CK-mediated phosphorylation of TFV-MP, without excluding a possible role for PK (Varga et al., 2013). In their study, Lade et al. (2015) also highlight the notion that genetic variants within the nucleotide kinases that exhibit enzymatic activity towards TFV exist, which may influence drug pharmacokinetics and drug efficacy. By means of next-generation targeted sequencing of the Microbicide Trials Network MTN-001 clinical samples, they identified genetic variants in the genes encoding for the kinases involved in TFV activation. The functional impact of the detected genetic variants was predicted using in silico tools, therefore, their attempt to correlate the detected genetic variants to enzyme function is purely predictive. Because the relative frequency of the genetic variants detected among the participants of the MTN-001 study was low, the authors could not make strong correlations with published TFV pharmacokinetic data (Hendrix et al., 2013). Further research should focus on the expression of recombinant enzymes produced by site-directed mutagenesis in order to test the impact of the identified genetic variants on TFV activation. Also, future studies that would correlate genotype with TFV pharmacology are also important in order to predict clinical outcomes. Taken together, the study reported in this issue of E-BioMedicine (Lade et al., 2015) is an important step in two new concepts. First, the activation of TFV is tissue specific due to differential nucleotide kinase expression and activity which may be linked to potential differential pharmacology of TDF. Second, genetic variants of the kinases that activate TFV could impact TFV activation, influencing the efficacy of TFV in the prophylaxis and treatment of HIV.

Structural basis for the specificity of thymidylate kinases from human pathogens: implications for nucleotide analogues activation.

Several human pathogens possess nucleoside or nucleotide kinases with large substrate specificity compared to their human counterparts. This phenomenon has been successfully exploited for the specific targeting of prodrugs such as Acyclovir against herpes virus. Combined structural and biochemical studies of these enzymes can thus provide essential information for the rational design of specific antimicrobial agents. Here we studied the structural basis for the specificity of a thymidylate kinase from the poxvirus family. Poxvirus thymidylate kinase has unusual substrate specificity and can accept bulky analogues such as 5-bromo-vinyl-dUMP (BVdUMP). The 2 A crystal structure of the thymidylate kinase bound to this compound now gives the structural basis for its specific molecular recognition.

The Anti–Human Immunodeficiency Virus Drug Tenofovir, a Reverse Transcriptase Inhibitor, Also Targets the Herpes Simplex Virus DNA Polymerase

Background Genital herpes is an important cofactor for acquisition of human immunodeficiency virus (HIV) infection, and effective prophylaxis is a helpful strategy to halt both HIV and herpes simplex virus (HSV) transmission. The antiretroviral agent tenofovir, formulated as a vaginal microbicide gel, was shown to reduce the risk of HIV and HSV type 2 (HSV-2) acquisition. Methods HSV type 1 (HSV-1) and HSV-2 mutants were selected for resistance to tenofovir and PMEO-DAPy (6-phosphonylmethoxyethoxy-2,4-diaminopyrimidine, an acyclic nucleoside phosphonate with dual anti-HSV and anti-HIV activity) by stepwise dose escalation. Several plaque-purified viruses were characterized phenotypically (drug resistance profiling) and genotypically (sequencing of the viral DNA polymerase gene). Results Tenofovir resistant and PMEO-DAPy-resistant viruses harbored specific amino acid substitutions associated with resistance not only to tenofovir and PMEO-DAPy but also to acyclovir and foscarnet. These amino acid changes (A719V, S724N, and L802F [HSV-1] and M789T and A724V [HSV-2]) were also found in clinical isolates recovered from patients refractory to acyclovir and/or foscarnet therapy or in laboratory-derived strains. A total of 10 (HSV-1) and 18 (HSV-2) well-characterized DNA polymerase mutants had decreased susceptibility to tenofovir and PMEO-DAPy. Conclusions Tenofovir and PMEO-DAPy target the HSV DNA polymerase, and clinical isolates with DNA polymerase mutations emerging under acyclovir and/or foscarnet therapy showed cross-resistance to tenofovir and PMEO-DAPy.