Demonstrating the importance of porcine reproductive and respiratory syndrome virus papain-like protease 2 deubiquitinating activity in viral replication by structure-guided mutagenesis.

Deubiquitination of cellular substrates by viral proteases is a mechanism used to interfere with host cellular signaling processes, shared between members of the coronavirus- and arterivirus families. In the case of Arteriviruses, deubiquitinating and polyprotein processing activities are accomplished by the virus-encoded papain-like protease 2 (PLP2). Several studies have implicated the deubiquitinating activity of the porcine reproductive and respiratory syndrome virus (PRRSV) PLP2 in the downregulation of cellular interferon production, however to date, the only arterivirus PLP2 structure described is that of equine arteritis virus (EAV), a distantly related virus. Here we describe the first crystal structure of the PRRSV PLP2 domain both in the presence and absence of its ubiquitin substrate, which reveals unique structural differences in this viral domain compared to PLP2 from EAV. To probe the role of PRRSV PLP2 deubiquitinating activity in host immune evasion, we selectively removed this activity from the domain by mutagenesis and found that the viral domain could no longer downregulate cellular interferon production. Interestingly, unlike EAV, and also unlike the situation for MERS-CoV, we found that recombinant PRRSV carrying PLP2 DUB-specific mutations faces significant selective pressure to revert to wild-type virus in MARC-145 cells, suggesting that the PLP2 DUB activity, which in PRRSV is present as three different versions of viral protein nsp2 expressed during infection, is critically important for PRRSV replication.

A broadly reactive ultralong bovine antibody that can determine the integrity of foot-and-mouth disease virus capsids.

Foot-and-mouth disease vaccination using inactivated virus is suboptimal, as the icosahedral viral capsids often disassemble into antigenically distinct pentameric units during long-term storage, or exposure to elevated temperature or lowered pH, and thus raise a response that is no longer protective. Furthermore, as foot-and-mouth disease virus (FMDV)'s seven serotypes are antigenically diverse, cross-protection from a single serotype vaccine is limited, and most existing mouse and bovine antibodies and camelid single-domain heavy chain-only antibodies are serotype-specific. For quality control purposes, there is a real need for pan-serotype antibodies that clearly distinguish between pentamer (12S) and protective intact FMDV capsid. To date, few cross-serotype bovine-derived antibodies have been reported in the literature. We identify a bovine antibody with an ultralong CDR-H3, Ab117, whose structural analysis reveals that it binds to a deep, hydrophobic pocket on the interior surface of the capsid via the CDR-H3. Main-chain and hydrophobic interactions provide broad serotype specificity. ELISA analysis confirms that Ab117 is a novel pan-serotype and conformational epitope-specific 12S reagent, suitable for assessing capsid integrity.

Intradermal vaccination with Porcilis® Begonia can clinically protect against fatal PRV challenge with the highly virulent ZJ01 field strain.

Since pseudorabies (PR) re-emerged and rapidly spread in China at the end of 2011, researchers have focused on effective vaccine strategies to prevent and control pseudorabies virus (PRV) infection in pig herds. Due to the extensive application of an attenuated vaccine based on the Bartha-K61 strain isolated in Hungary in 1961 and the variation of the PRV strain, it has been suggested that traditional vaccines based on the Bartha-K61 strain offer only partial protection against variant strains. It was therefore evaluated whether the Porcilis® Begonia vaccine, which is based on the NIA-3 strain with deletions in the gE and TK genes, is efficacious against experimental infection with the virulent, contemporary Chinese PRV strain ZJ01. In this study, piglets were vaccinated with Porcilis® Begonia through either the intradermal (ID) route or the intramuscular (IM) route and subsequently challenged intranasally with strain ZJ01 at 4 weeks post-vaccination. An unvaccinated challenge group and an unvaccinated/nonchallenged group were also included in the study. All animals were monitored for 14 days after challenge. Vaccinated and negative control pigs stayed healthy during the study, while the unvaccinated control animals developed lesions associated with PRV ZJ01 challenge, and 44% of these pigs died before the end of the experiment. This study demonstrated that ID or IM vaccination of pigs with a vaccine based on the NIA-3 strain Porcilis® Begonia clinically protects against fatal PRV challenge with the ZJ01 strain.

An octavalent vaccine provides pregnant gilts protection against a highly virulent porcine parvovirus strain.

BACKGROUND: Porcilis® Ery+Parvo+Lepto is an octavalent inactivated ready-to-use vaccine that contains Erysipelothrix rhusiopathiae (Ery), porcine parvovirus (PPV), and six serogroups of Leptospira (Lepto). The efficacy of Porcilis® Ery + Parvo+Lepto against reproductive problems associated with porcine parvovirus (PPV) infection was evaluated in pregnant gilts. For this, a group of ninegilts was vaccinated twice (at 5 and 6 months old) with Porcilis® Ery + Parvo+Lepto (Group 1), while a group of eight gilts was included as unvaccinated controls (Group 2). All pigs were artificially inseminated 4 weeks after the second vaccination. They were challenged during early gestation with PPV-27a, a virulent cluster D strain, and euthanized to collect their offspring by hysterectomy around day 90 in pregnancy. Antibody responses against PPV in gilts were measured, and the presence of PPV in progeny was also determined. RESULTS: No clinical signs were observed after vaccination. After PPV challenge, all foetuses from the vaccinated gilts were alive (132/132), while in the unvaccinated group only 41% were alive (46/112), 19.6% were dead and 39.4% of the offspring (44/112) were mummified. PPV could be detected by qPCR in 14% of the progeny from vaccinated gilts at an average of 4.7 log10/ml, whereas this was significantly higher in the control group, where 90% of the progeny were PPV positive, with titres of 9.8 log10/ml on average. CONCLUSIONS: The present study demonstrates that vaccination of gilts with Porcilis® Ery + Parvo+Lepto was safe and induced an immune response sufficient to protect progeny against PPV by reducing transplacental infection.

The B Cell Response to Foot-and-Mouth Disease Virus in Cattle following Sequential Vaccination with Multiple Serotypes.

Foot-and-mouth disease virus (FMDV) is a highly contagious viral disease. Antibodies are pivotal in providing protection against FMDV infection. Serological protection against one FMDV serotype does not confer interserotype protection. However, some historical data have shown that interserotype protection can be induced following sequential FMDV challenge with multiple FMDV serotypes. In this study, we have investigated the kinetics of the FMDV-specific antibody-secreting cell (ASC) response following homologous and heterologous inactivated FMDV vaccination regimes. We have demonstrated that the kinetics of the B cell response are similar for all four FMDV serotypes tested following a homologous FMDV vaccination regime. When a heterologous vaccination regime was used with the sequential inoculation of three different inactivated FMDV serotypes (O, A, and Asia1 serotypes) a B cell response to FMDV SAT1 and serotype C was induced. The studies also revealed that the local lymphoid tissue had detectable FMDV-specific ASCs in the absence of circulating FMDV-specific ASCs, indicating the presence of short-lived ASCs, a hallmark of a T-independent 2 (TI-2) antigenic response to inactivated FMDV capsid.IMPORTANCE We have demonstrated the development of intraserotype response following a sequential vaccination regime of four different FMDV serotypes. We have found indication of short-lived ASCs in the local lymphoid tissue, further evidence of a TI-2 response to FMDV.

Cell Attachment Domains of the Porcine Epidemic Diarrhea Virus Spike Protein Are Key Targets of Neutralizing Antibodies.

Porcine epidemic diarrhea virus (PEDV) causes enteric disease in pigs, resulting in significant economic losses to the swine industry worldwide. Current vaccination approaches against this emerging coronavirus are only partially effective, though natural infection protects pigs against reinfection and provides lactogenic immunity to suckling piglets. The viral spike (S) glycoprotein, responsible for receptor binding and cell entry, is the major target for neutralizing antibodies. However, knowledge of antibody epitopes, their nature and location in the spike structure, and the mechanisms by which the antibodies interfere with infection is scarce. Here we describe the generation and characterization of 10 neutralizing and nonneutralizing mouse monoclonal antibodies raised against the S1 receptor binding subunit of the S protein. By expression of different S1 protein fragments, six antibody epitope classes distributed over the five structural domains of the S1 subunit were identified. Characterization of antibodies for cross-reactivity and cross-neutralization revealed antigenic differences among PEDV strains. The epitopes of potent neutralizing antibodies segregated into two epitope classes and mapped within the N-terminal sialic acid binding domain and in the more C-terminal receptor binding domain. Antibody neutralization escape mutants displayed single amino acid substitutions that impaired antibody binding and neutralization and defined the locations of the epitopes. Our observations picture the antibody epitope landscape of the PEDV S1 subunit and reveal that its cell attachment domains are key targets of neutralizing antibodies.IMPORTANCE Porcine epidemic diarrhea virus (PEDV), an emerging porcine coronavirus, causes an economically important enteric disease in pigs. Effective PEDV vaccines for disease control are currently lacking. The spike (S) glycoprotein on the virion surface is the key player in virus cell entry and, therefore, the main target of neutralizing antibodies. To understand the antigenic landscape of the PEDV spike protein, we developed monoclonal antibodies against the spike protein's S1 receptor binding region and characterized their epitopes, neutralizing activity, and cross-reactivity toward multiple PEDV strains. Epitopes of antibodies segregated into six epitope classes dispersed over the multidomain S1 structure. Monoclonal antibodies revealed antigenic variability in B-cell epitopes between PEDV strains. The epitopes of neutralizing antibodies mapped to two distinct domains in S1 that are involved in binding to carbohydrate and proteinaceous cell surface molecules, respectively, indicating the importance of these cell attachment sites on the PEDV spike protein in eliciting a protective humoral immune response.

Roles of Trm9- and ALKBH8-like proteins in the formation of modified wobble uridines in Arabidopsis tRNA.

Uridine at the wobble position of tRNA is usually modified, and modification is required for accurate and efficient protein translation. In eukaryotes, wobble uridines are modified into 5-methoxycarbonylmethyluridine (mcm(5)U), 5-carbamoylmethyluridine (ncm(5)U) or derivatives thereof. Here, we demonstrate, both by in vitro and in vivo studies, that the Arabidopsis thaliana methyltransferase AT1G31600, denoted by us AtTRM9, is responsible for the final step in mcm(5)U formation, thus representing a functional homologue of the Saccharomyces cerevisiae Trm9 protein. We also show that the enzymatic activity of AtTRM9 depends on either one of two closely related proteins, AtTRM112a and AtTRM112b. Moreover, we demonstrate that AT1G36310, denoted AtALKBH8, is required for hydroxylation of mcm(5)U to (S)-mchm(5)U in tRNA(Gly)(UCC), and has a function similar to the mammalian dioxygenase ALKBH8. Interestingly, atalkbh8 mutant plants displayed strongly increased levels of mcm(5)U, and also of mcm(5)Um, its 2'-O-ribose methylated derivative. This suggests that accumulated mcm(5)U is prone to further ribose methylation by a non-specialized mechanism, and may challenge the notion that the existence of mcm(5)U- and mcm(5)Um-containing forms of the selenocysteine-specific tRNA(Sec) in mammals reflects an important regulatory process. The present study reveals a role in for several hitherto uncharacterized Arabidopsis proteins in the formation of modified wobble uridines.

Discovery of a novel Torque teno sus virus species: genetic characterization, epidemiological assessment and disease association.

The study describes a novel Torque teno sus virus (TTSuV) species, provisionally named Torque teno sus virus k2b (TTSuVk2b), originally found in commercial pig sera by applying the rolling-circle amplification technique. Full-length sequences of TTSuVk2b were obtained, annotated and used in the phylogenetic analyses, which revealed that TTSuVk2b is a novel Anellovirus species within the genus Kappatorquevirus of the family Anelloviridae. Quantitative PCR techniques were developed to determine total TTSuV DNA quantities as well as the prevalence and viral DNA quantities of TTSuV1, TTSuVk2a and TTSuVk2b. The mean total TTSuV load in seven commercial sera was determined at 6.3 log(10) DNA copies ml(-1) of serum, with TTSuVk2b loads being the lowest at 4.5 log(10) DNA copies ml(-1) of serum. Subsequently, prevalence and loads of TTSuVs were determined in pig sera from 17 countries. TTSuVk2b prevalence ranged from 0 to 100 % with viral loads from 3.3 to 4.6 log(10) copies ml(-1) of sera. TTSuVk2a, so far the only species in the genus Kappatorquevirus, has been linked to an economically important swine disease, namely post-weaning multisystemic wasting syndrome (PMWS). Considering the grouping of TTSuVk2b in the same genus as TTSuVk2a, TTSuVk2b prevalence and viral DNA load were determined in PMWS-affected animals and healthy counterparts. This revealed that TTSuVk2a and TTSuVk2b are not only genetically related, but also that their viral loads in serum are elevated in PMWS animals compared with those of healthy pen mates. In summary, the present work describes a novel TTSuV species including its genetic characterization, epidemiological assessment and potential disease association.

Bioinformatics and functional analysis define four distinct groups of AlkB DNA-dioxygenases in bacteria.

The iron(II)- and 2-oxoglutarate (2OG)-dependent dioxygenase AlkB from Escherichia coli (EcAlkB) repairs alkylation damage in DNA by direct reversal. EcAlkB substrates include methylated bases, such as 1-methyladenine (m(1)A) and 3-methylcytosine (m(3)C), as well as certain bulkier lesions, for example the exocyclic adduct 1,N(6)-ethenoadenine (epsilonA). EcAlkB is the only bacterial AlkB protein characterized to date, and we here present an extensive bioinformatics and functional analysis of bacterial AlkB proteins. Based on sequence phylogeny, we show that these proteins can be subdivided into four groups: denoted 1A, 1B, 2A and 2B; each characterized by the presence of specific conserved amino acid residues in the putative nucleotide-recognizing domain. A scattered distribution of AlkB proteins from the four different groups across the bacterial kingdom indicates a substantial degree of horizontal transfer of AlkB genes. DNA repair activity was associated with all tested recombinant AlkB proteins. Notably, both a group 2B protein from Xanthomonas campestris and a group 2A protein from Rhizobium etli repaired etheno adducts, but had negligible activity on methylated bases. Our data indicate that the majority, if not all, of the bacterial AlkB proteins are DNA repair enzymes, and that some of these proteins do not primarily target methylated bases.

High Doses of Inactivated African Swine Fever Virus Are Safe, but Do Not Confer Protection against a Virulent Challenge.

African swine fever (ASF) is currently the major concern of the global swine industry, as a consequence of which a reconsideration of the containment and prevention measures taken to date is urgently required. A great interest in developing an effective and safe vaccine against ASF virus (ASFV) infection has, therefore, recently appeared. The objective of the present study is to test an inactivated ASFV preparation under a vaccination strategy that has not previously been tested in order to improve its protective effect. The following have been considered: (i) virus inactivation by using a low binary ethyleneimine (BEI) concentration at a low temperature, (ii) the use of new and strong adjuvants; (iii) the use of very high doses (6 × 109 haemadsorption in 50% of infected cultures (HAD50)), and (iv) simultaneous double inoculation by two different routes of administration: intradermal and intramuscular. Five groups of pigs were, therefore, inoculated with BEI- Pol16/DP/OUT21 in different adjuvant formulations, twice with a 4-week interval. Six weeks later, all groups were intramuscularly challenged with 10 HAD50 of the virulent Pol16/DP/OUT21 ASFV isolate. All the animals had clinical signs and pathological findings consistent with ASF. This lack of effectiveness supports the claim that an inactivated virus strategy may not be a viable vaccine option with which to fight ASF.

Immune efficacy of a candidate porcine reproductive and respiratory syndrome vaccine rHN-NP49 administered by a Needle-free intradermal delivery system in comparison with intramuscular injection.

Porcine reproductive and respiratory syndrome (PRRS) is one of the major drivers of economic loss in the swine industry worldwide. In commercial pig production, vaccination is the first option in an attempt to control infectious diseases. Pigs are therefore often immunized with different vaccines, and almost all of them are delivered via the intramuscular (IM) route. However, the IM injection may result in physical damage, stress reactions, and is labor demanding. An alternative route is urgently needed to reduce the disadvantages of conventional vaccination. In this study, a needle-free intradermal (ID) delivery system was evaluated for delivering a live PRRS vaccine as compared with the traditional needle-syringe method. Fifty-two 4-week-old piglets were divided into six groups: piglets in groups A-C were immunized using ID delivery system with 104, 105 and 106 TCID50 of PRRS candidate vaccine strain rHN-NP49, respectively; piglets in group D were immunized IM with 105 TCID50 of rHN-NP49; and group E and F were used as challenge and control groups, respectively. At 28 days post vaccination, piglets in group A to E were challenged with a lethal dose of highly-pathogenic PRRSV. Similar results were found in viremia and antibody response among the ID and IM groups during the immunization stage. After challenge, similar results were found in average body weight gain, viral shedding, serum viral load, and clinical score among the immunization groups, with a higher protection ratio in the ID group compared with IM group with the same immunization dose. These results demonstrated that the ID delivery system could provide similar or even better protection compared with IM route, and could be an effective route for PRRS vaccination.

Biochemical characterization of arterivirus nonstructural protein 11 reveals the nidovirus-wide conservation of a replicative endoribonuclease.

Nidoviruses (arteriviruses, coronaviruses, and roniviruses) are a phylogenetically compact but diverse group of positive-strand RNA viruses that includes important human and animal pathogens. Nidovirus RNA synthesis is mediated by a cytoplasmic membrane-associated replication/transcription complex that includes up to 16 viral nonstructural proteins (nsps), which carry common enzymatic activities, like the viral RNA polymerase, but also unusual and poorly understood RNA-processing functions. Of these, a conserved endoribonuclease (NendoU) is a major genetic marker that is unique to nidoviruses. NendoU activity was previously verified in vitro for the coronavirus nsp15, but not for any of its distantly related orthologs from other nidovirus lineages, like the arterivirus nsp11. Here, we show that the bacterially expressed nsp11 proteins of two arteriviruses, equine arteritis virus and porcine respiratory and reproductive syndrome virus, possess pyrimidine-specific endoribonuclease activity. RNA cleavage was independent of divalent cations in vitro and was greatly reduced by replacement of residues previously implicated in catalysis. Comparative characterization of the NendoU activity in arteriviruses and severe acute respiratory syndrome coronavirus revealed common and distinct features of their substrate requirements and reaction mechanism. Our data provide the first biochemical evidence of endoribonuclease activity associated with arterivirus nsp11 and support the conclusion that this remarkable RNA-processing enzyme, whose substrate in the infected cell remains to be identified, distinguishes nidoviruses from all other RNA viruses.

Viral AlkB proteins repair RNA damage by oxidative demethylation.

Bacterial and mammalian AlkB proteins are iron(II)- and 2-oxoglutarate-dependent dioxygenases that reverse methylation damage, such as 1-methyladenine and 3-methylcytosine, in RNA and DNA. An AlkB-domain is encoded by the genome of numerous single-stranded, plant-infecting RNA viruses, the majority of which belong to the Flexiviridae family. Our phylogenetic analysis of AlkB sequences suggests that a single plant virus might have acquired AlkB relatively recently, followed by horizontal dissemination among other viruses via recombination. Here, we describe the first functional characterization of AlkB proteins from three plant viruses. The viral AlkB proteins efficiently reactivated methylated bacteriophage genomes when expressed in Escherichia coli, and also displayed robust, iron(II)- and 2-oxoglutarate-dependent demethylase activity in vitro. Viral AlkB proteins preferred RNA over DNA substrates, and thus represent the first AlkBs with such substrate specificity. Our results suggest a role for viral AlkBs in maintaining the integrity of the viral RNA genome through repair of deleterious methylation damage, and support the notion that AlkB-mediated RNA repair is biologically relevant.

Vaccination of pigs reduces Torque teno sus virus viremia during natural infection.

Anelloviruses are a group of single-stranded circular DNA viruses infecting several vertebrate species. Four species have been found to infect swine, namely Torque teno sus virus (TTSuV) 1a and 1b (TTSuV1a, TTSuV1b; genus Iotatorquevirus), TTSuVk2a and TTSuVk2b (genus Kappatorquevirus). TTSuV infection in pigs is distributed worldwide, and is characterized by a persistent viremia. However, the real impact, if any, on the pig health is still under debate. In the present study, the impact of pig immunization on TTSuVk2a loads was evaluated. For this, three-week old conventional pigs were primed with DNA vaccines encoding the ORF2 gene and the ORF1-A, ORF1-B, and ORF1-C splicing variants and boosted with purified ORF1-A and ORF2 Escherichia coli proteins, while another group served as unvaccinated control animals, and the viral load dynamics during natural infection was observed. Immunization led to delayed onset of TTSuVk2a infection and at the end of the study when the animals were 15 weeks of age, a number of animals in the immunized group had cleared the TTSuVk2a viremia, which was not the case in the control group. This study demonstrated for the first time that TTSuV viremia can be controlled by a combined DNA and protein immunization, especially apparent two weeks after the first DNA immunization before seroconversion was observed. Further studies are needed to understand the mechanisms behind this and its impact for pig producers.

In vivo assessment of equine arteritis virus vaccine improvement by disabling the deubiquitinase activity of papain-like protease 2.

Arteriviruses are a family of positive-stranded RNA viruses that includes the prototypic equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV). Although several vaccines against these viruses are commercially available there is room for improvement, especially in the case of PRRSV. The ability of arteriviruses to counteract the immune response is thought to decrease the efficacy of the current modified live virus vaccines. We have recently shown that the deubiquitinase (DUB) activity of EAV papain-like protease 2 (PLP2) is important for the inhibition of innate immune activation during infection. A vaccine virus lacking PLP2 DUB activity may therefore be more immunogenic and provide improved protection against subsequent challenge than its DUB-competent counterpart. To test this hypothesis, twenty Shetland mares were randomly assigned to one of three groups. Two groups were vaccinated, either with DUB-positive (n=9) or DUB-negative (n=9) recombinant EAV. The third group (n=2) was not vaccinated. All horses were subsequently challenged with the virulent KY84 strain of EAV. Both vaccine viruses proved to be replication competent in vivo. In addition, the DUB-negative virus provided a similar degree of protection against clinical disease as its DUB-positive parental counterpart. Owing to the already high level of protection provided by the parental virus, a possible improvement due to inactivation of PLP2 DUB activity could not be detected under these experimental conditions. Taken together, the data obtained in this study warrant further in vivo investigations into the potential of using DUB-mutant viruses for the improvement of arterivirus vaccines.

Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins.

AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N(6)-ethenoadenine (ɛA), 3,N(4)-ethenocytosine (ɛC) and 1,N(2)-ethenoguanine (1,N(2)-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N(2)-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N(2)-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins.

Protozoan ALKBH8 oxygenases display both DNA repair and tRNA modification activities.

The ALKBH family of Fe(II) and 2-oxoglutarate dependent oxygenases comprises enzymes that display sequence homology to AlkB from E. coli, a DNA repair enzyme that uses an oxidative mechanism to dealkylate methyl and etheno adducts on the nucleobases. Humans have nine different ALKBH proteins, ALKBH1-8 and FTO. Mammalian and plant ALKBH8 are tRNA hydroxylases targeting 5-methoxycarbonylmethyl-modified uridine (mcm5U) at the wobble position of tRNAGly(UCC). In contrast, the genomes of some bacteria encode a protein with strong sequence homology to ALKBH8, and robust DNA repair activity was previously demonstrated for one such protein. To further explore this apparent functional duality of the ALKBH8 proteins, we have here enzymatically characterized a panel of such proteins, originating from bacteria, protozoa and mimivirus. All the enzymes showed DNA repair activity in vitro, but, interestingly, two protozoan ALKBH8s also catalyzed wobble uridine modification of tRNA, thus displaying a dual in vitro activity. Also, we found the modification status of tRNAGly(UCC) to be unaltered in an ALKBH8 deficient mutant of Agrobacterium tumefaciens, indicating that bacterial ALKBH8s have a function different from that of their eukaryotic counterparts. The present study provides new insights on the function and evolution of the ALKBH8 family of proteins.

ALKBH8-mediated formation of a novel diastereomeric pair of wobble nucleosides in mammalian tRNA.

Mammals have nine different homologues (ALKBH1-9) of the Escherichia coli DNA repair demethylase AlkB. ALKBH2 is a genuine DNA repair enzyme, but the in vivo function of the other ALKBH proteins has remained elusive. It was recently shown that ALKBH8 contains an additional transfer RNA (tRNA) methyltransferase domain, which generates the wobble nucleoside 5-methoxycarbonylmethyluridine (mcm(5)U) from its precursor 5-carboxymethyluridine (cm(5)U). In this study, we report that (R)- and 5-methoxycarbonylhydroxymethyluridine (mchm(5)U), hydroxylated forms of mcm(5)U, are present in mammalian tRNA-Arg(UCG), and tRNA-Gly(UCC), respectively, representing the first example of a diastereomeric pair of modified RNA nucleosides. Through in vitro and in vivo studies, we show that both diastereomers of mchm(5)U are generated from mcm(5)U, and that the AlkB domain of ALKBH8 specifically hydroxylates mcm(5)U into (S)-mchm(5)U in tRNA-Gly(UCC). These findings expand the function of the ALKBH oxygenases beyond nucleic acid repair and increase the current knowledge on mammalian wobble uridine modifications and their biogenesis.

Mammalian ALKBH8 possesses tRNA methyltransferase activity required for the biogenesis of multiple wobble uridine modifications implicated in translational decoding.

Uridines in the wobble position of tRNA are almost invariably modified. Modifications can increase the efficiency of codon reading, but they also prevent mistranslation by limiting wobbling. In mammals, several tRNAs have 5-methoxycarbonylmethyluridine (mcm5U) or derivatives thereof in the wobble position. Through analysis of tRNA from Alkbh8-/- mice, we show here that ALKBH8 is a tRNA methyltransferase required for the final step in the biogenesis of mcm5U. We also demonstrate that the interaction of ALKBH8 with a small accessory protein, TRM112, is required to form a functional tRNA methyltransferase. Furthermore, prior ALKBH8-mediated methylation is a prerequisite for the thiolation and 2'-O-ribose methylation that form 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) and 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um), respectively. Despite the complete loss of all of these uridine modifications, Alkbh8-/- mice appear normal. However, the selenocysteine-specific tRNA (tRNASec) is aberrantly modified in the Alkbh8-/- mice, and for the selenoprotein Gpx1, we indeed observed reduced recoding of the UGA stop codon to selenocysteine.

An infectious recombinant equine arteritis virus expressing green fluorescent protein from its replicase gene.

Thus far, systems developed for heterologous gene expression from the genomes of nidoviruses (arteriviruses and coronaviruses) have relied mainly on the translation of foreign genes from subgenomic mRNAs, whose synthesis is a key feature of the nidovirus life cycle. In general, such expression vectors often suffered from relatively low and unpredictable expression levels, as well as genome instability. In an attempt to circumvent these disadvantages, the possibility to express a foreign gene [encoding enhanced green fluorescent protein (eGFP)] from within the nidovirus replicase gene, which encodes two large polyproteins that are processed proteolytically into the non-structural proteins (nsps) required for viral RNA synthesis, has now been explored. A viable recombinant of the arterivirus Equine arteritis virus, EAV-GFP2, was obtained, which contained the eGFP insert at the site specifying the junction between the two most N-proximal replicase-cleavage products, nsp1 and nsp2. EAV-GFP2 replication could be launched by transfection of cells with either in vitro-generated RNA transcripts or a DNA launch plasmid. EAV-GFP2 displayed growth characteristics similar to those of the wild-type virus and was found to maintain the insert stably for at least eight passages. It is proposed that EAV-GFP2 has potential for arterivirus vector development and as a tool in inhibitor screening. It can also be used for fundamental studies into EAV replication, which was illustrated by the fact that the eGFP signal of EAV-GFP2, which largely originated from an eGFP-nsp2 fusion protein, could be used to monitor the formation of the membrane-bound EAV replication complex in real time.