Pierre Fiten

The Hemopexin and O-Glycosylated Domains Tune Gelatinase B/MMP-9 Bioavailability via Inhibition and Binding to Cargo Receptors

Gelatinase B/matrix metalloproteinase-9 (MMP-9), a key regulator and effector of immunity, contains a C-terminal hemopexin domain preceded by a unique linker sequence of ∼64 amino acid residues. This linker sequence is demonstrated to be an extensively O-glycosylated (OG) domain with a compact three-dimensional structure. The OG and hemopexin domains have no influence on the cleavage efficiency of MMP-9 substrates. In contrast, the hemopexin domain contains a binding site for the cargo receptor low density lipoprotein receptor-related protein-1 (LRP-1). Furthermore, megalin/LRP-2 is identified as a new functional receptor for the hemopexin domain of MMP-9, able to mediate the endocytosis and catabolism of the enzyme. The OG domain is required to correctly orient the hemopexin domain for inhibition by TIMP-1 and internalization by LRP-1 and megalin. Therefore, the OG and hemopexin domains down-regulate the bioavailability of active MMP-9 and the interactions with the cargo receptors are proposed to be the original function of hemopexin domains in MMPs.

Cidofovir Resistance in Vaccinia Virus Is Linked to Diminished Virulence in Mice

ABSTRACT Cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC)] is recognized as a promising drug for the treatment of poxvirus infections, but drug resistance can arise by a mechanism that is poorly understood. We show here that in vitro selection for high levels of resistance to HPMPC produces viruses encoding two substitution mutations in the virus DNA polymerase (E9L) gene. These mutations are located within the regions of the gene encoding the 3′-5′ exonuclease (A314T) and polymerase (A684V) catalytic domains. These mutant viruses exhibited cross-resistance to other nucleoside phosphonate drugs, while they remained sensitive to other unrelated DNA polymerase inhibitors. Marker rescue experiments were used to transfer A314T and/or A684V alleles into a vaccinia virus Western Reserve strain. Either mutation alone could confer a drug resistance phenotype, although the degree of resistance was significantly lower than when virus encoded both mutations. The A684V substitution, but not the A314T change, also conferred a spontaneous mutator phenotype. All of the HPMPC-resistant recombinant viruses exhibited reduced virulence in mice, demonstrating that these E9L mutations are inextricably linked to reduced fitness in vivo. HPMPC, at a dose of 50 mg/kg of body weight/day for 5 days, still protected mice against intranasal challenge with the drug-resistant virus with A314T and A684V mutations. Our studies show that proposed drug therapies offer a reasonable likelihood of controlling orthopoxvirus infections, even if the viruses encode drug resistance markers.

Avian papillomaviruses: the parrot Psittacus erithacus papillomavirus (PePV) genome has a unique organization of the early protein region and is phylogenetically related to the chaffinch papillomavirus.

BackgroundAn avian papillomavirus genome has been cloned from a cutaneous exophytic papilloma from an African grey parrot (Psittacus erithacus). The nucleotide sequence, genome organization, and phylogenetic position of the Psittacus erithacus papillomavirus (PePV) were determined. This PePV sequence represents the first complete avian papillomavirus genome defined.ResultsThe PePV genome (7304 basepairs) differs from other papillomaviruses, in that it has a unique organization of the early protein region lacking classical E6 and E7 open reading frames. Phylogenetic comparison of the PePV sequence with partial E1 and L1 sequences of the chaffinch (Fringilla coelebs) papillomavirus (FPV) reveals that these two avian papillomaviruses form a monophyletic cluster with a common branch that originates near the unresolved center of the papillomavirus evolutionary tree.ConclusionsThe PePV genome has a unique layout of the early protein region which represents a novel prototypic genomic organization for avian papillomaviruses. The close relationship between PePV and FPV, and between their Psittaciformes and Passeriformes hosts, supports the hypothesis that papillomaviruses have co-evolved and speciated together with their host species throughout evolution.

Characterization of herpes simplex virus type 1 thymidine kinase mutants selected under a single round of high-dose brivudin.

ABSTRACT A broad variety of herpes simplex virus type 1 clones was selected under a single round of high-dose selection with brivudin. Mutations in the thymidine kinase (TK) genes consisted of 42% frameshift mutations within homopolymer repeats of Gs and Cs and single nucleotide substitutions (58%) that produced stop codons (Q261 and R281) or a new codon at the site of the substitution (A168T, R51W, G59W, G206R, R220H, Y239S, and T287 M). The A168T change, associated with an altered TK phenotype, proved to be the most commonly selected substitution. For the different mutants, a correlation between phenotype, genotype, and in vivo neurovirulence was observed.

Human papillomavirus type 13 and pygmy chimpanzee papillomavirus type 1: comparison of the genome organizations

Human papillomavirus type 13(HPV-13) is associated with oral focal epithelial hyperplasia (FEH) in humans. A recent epidemic of a FEH-like disease in a pygmy chimpanzee (Pan paniscus) colony allowed us to clone a novel papillomavirus genome. To assess the homology between HPV-13 and the pygmy chimpanzee papillomavirus type 1 (PCPV-1), the complete nucleotide sequences of both FEH-related viruses were determined. In both viruses, all eight major open reading frames were located on one strand and the genomic organization was similar to that of other mucosal papillomaviruses. The genomes of PCPV-1 and HPV-13 showed extensive overall sequence homology (85%). They could be classified, using phylogenetic analysis, together with HPV types 6, 11, 43, and 44 in a group associated with benign orogenital lesions. These data indicate that two phylogenetically related papillomaviruses can elicit similar pathology in different primate host species, reflecting viral genomic similarities.

Polymorphism analysis suggests that the gelatinase B gene is not a susceptibility factor for multiple sclerosis

The human gelatinase B (MMP-9) gene promoter region contains a CA microsatellite repeat and a single nucleotide polymorphism which are known to influence transcriptional activity. These two polymorphisms were used to investigate the existence of an association between multiple sclerosis (MS) susceptibility and the MMP-9 gene. In a case-control analysis of 345 Swedish individuals and in a study of 125 Sardinian simplex families no genetic associations between the gelatinase B gene polymorphisms and MS susceptibility were found. These data reinforce the suggestion of epistasis in the regulation of the metalloproteinase-inhibitor balance in MS.

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.

Mechanism of antiviral drug resistance of vaccinia virus: identification of residues in the viral DNA polymerase conferring differential resistance to antipoxvirus drugs.

ABSTRACT The acyclic nucleoside phosphonate (ANP) family of drugs shows promise as therapeutics for treating poxvirus infections. However, it has been questioned whether the utility of these compounds could be compromised through the intentional genetic modification of viral sequences by bioterrorists or the selection of drug resistance viruses during the course of antiviral therapy. To address these concerns, vaccinia virus (strain Lederle) was passaged 40 times in medium containing an escalating dose of (S)-1-[3-hydroxy-2-(phosphonomethoxypropyl)-2,6-diaminopurine [(S)-HPMPDAP], which selected for mutant viruses exhibiting a ∼15-fold-increased resistance to the drug. (S)-HPMPDAP-resistant viruses were generated because this compound was shown to be one of the most highly selective and effective ANPs for the treatment of poxvirus infections. DNA sequence analysis revealed that these viruses encoded mutations in the E9L (DNA polymerase) gene, and marker rescue studies showed that the phenotype was produced by a combination of two (A684V and S851Y) substitution mutations. The effects of these mutations on drug resistance were tested against various ANPs, both separately and collectively, and compared with E9L A314T and A684V mutations previously isolated using selection for resistance to cidofovir, i.e., (S)-1-[3-hydroxy-2-(phosphonomethoxypropyl)cytosine]. These studies demonstrated a complex pattern of resistance, although as a general rule, the double-mutant viruses exhibited greater resistance to the deoxyadenosine than to deoxycytidine nucleotide analogs. The S851Y mutant virus exhibited a low level of resistance to dCMP analogues but high-level resistance to dAMP analogues and to 6-[3-hydroxy-2-(phosphonomethoxy)propoxy]-2,4-diaminopyrimidine, which is considered to mimic the purine ring system. Notably, (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-3-deazaadenine retained marked activity against most of these mutant viruses. In vitro studies showed that the A684V mutation partially suppressed a virus growth defect and mutator phenotype created by the S851Y mutation, but all of the mutant viruses still exhibited a variable degree of reduced virulence in a mouse intranasal challenge model. Infections caused by these drug-resistant viruses in mice were still treatable with higher concentrations of the ANPs. These studies have identified a novel mechanism for the development of mutator DNA polymerases and provide further evidence that antipoxviral therapeutic strategies would not readily be undermined by selection for resistance to ANP drugs.

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.

Organotypic Epithelial Raft Cultures as a Model for Evaluating Compounds against Alphaherpesviruses

ABSTRACT The course of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and varicella-zoster virus (VZV) infections in squamous epithelial cells cultured in a three-dimensional organotypic raft culture was tested. In these raft cultures, normal human keratinocytes isolated from neonatal foreskins grown at the air-liquid interface stratified and differentiated, reproducing a fully differentiated epithelium. Typical cytopathic changes identical to those found in the squamous epithelium in vivo, including ballooning and reticular degeneration with the formation of multinucleate cells, were observed throughout the raft following infection with HSV and VZV at different times after lifting the cultures to the air-liquid interface. For VZV, the aspects of the lesions depended on the stage of differentiation of the organotypic cultures. The activity of reference antiviral agents, acyclovir (ACV), penciclovir (PCV), brivudin (BVDU), foscarnet (PFA), and cidofovir (CDV), was evaluated against wild-type and thymidine kinase (TK) mutants of HSV and VZV in the raft cultures. ACV, PCV, and BVDU protected the epithelium against cytopathic effect induced by wild-type viruses in a concentration-dependent manner, while treatment with CDV and PFA proved protective against the cytodestructive effects induced by both TK+ and TK− strains. The quantification of the antiviral effects in the rafts were accomplished by measuring viral titers by plaque assay for HSV and by measuring viral DNA load by real-time PCR for VZV. A correlation between the degree of protection as determined by histological examination and viral quantification could be demonstrated The three-dimensional epithelial raft culture represents a novel model for the study of antiviral agents active against HSV and VZV. Since no animal model is available for the evaluation of antiviral agents against VZV, the organotypic cultures may be considered a model to evaluate the efficacy of new anti-VZV antivirals before clinical trials.