Defective RNA Research Articles

Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells

Infection of mammals by avian influenza viruses requires adaptive mutations to achieve high-level replication in the new host. However, the basic mechanism underlying this adaptation process is still unknown. Here we show that avian polymerases, lacking the human signature PB2-E627K, are incapable of generating usable complementary RNA templates in cultured human cells and therefore require adaptation. Characterization of the highly pathogenic human H5N1 isolate A/Thailand/1(KAN-1)/2004 that retained the avian PB2-E627 reveals that the defect in RNA replication is only partially compensated by mutations in the polymerase. Instead, mutations in the nuclear export protein are required for efficient polymerase activity. We demonstrate that adaptive mutations in nuclear export proteins of several human isolates enhance the polymerase activity of avian polymerases in human cultured cells. In conclusion, when crossing the species barrier, avian influenza viruses acquire adaptive mutations in nuclear export protein to escape restricted viral genome replication in mammalian cells.

Hepatitis D infection in Nigeria

Hepatitis D virus (HDV) is a defective RNA virus that requires the hepatitis B envelope for its replication. Infection with HDVoccurs as co-infection or superinfection in persons infected with hepatitis B virus (HBV). Chronic HDVinfection usually follows superinfection of chronic HBV infection with HDV. Unlike some other infections, persistent HDV infection is associated with presence of HDV RNA, viral antigen (HDAg), and IgM and IgG antibodies to HDV [1]. HDV has a worldwide distribution with nearly 15 million people infected, especially in the Mediterranean Basin, the Middle East, Central Asia, West Africa, the Amazon Basin of South America and certain South Pacific islands [2]. In Nigeria, HBV infection is a major cause of liver disease [3]. In a previous report, HDVantigen was detected in 6.5% of patients with chronic HBV liver disease in Ife, South West Nigeria [4]. With measures for preventing HBV infection (including vaccination and safe blood transfusion), HDV infection may be on the decline [5]. However recent reports, including one from our locality, have tended to indicate otherwise [6, 7]. We set out to investigate the current status of HDV infection. After ethical approval, consecutive consenting patients attending the Gastroenterology Unit of Lagos State University Hospital, Lagos between September 2009 to November 2010 were recruited if they were positive for HBsAg for more than 6 months and had not received any antiviral therapy. A questionnaire was used to collect information on demographics, clinical features, liver biochemical tests and imaging results. Blood (10 mL) was collected in EDTA, and plasma was separated and stored at −20 C. IgG anti-HDV antibodies were assayed using an Elisa method (HDVAb kit; DIA PRO Diagnostic, Milan, Italy) according to the manufacturer’s protocol. The sensitivity and specificity of this assay kit are over 98%. A total of 245 patients (191 male) with age range 13–76 (mean [SD]034.6 [10.6]) years were studied. Of these, 178 were asymptomatic HBV carriers, whereas 67 had features of chronic liver disease (chronic hepatitis 22, cirrhosis 37 and liver cancer 8). Five (2.0%; 2 male) of these 245 patients tested positive to anti-HDV. These included 3 of 178 asymptomatic carriers and 2 of 67 patients with liver disease. Alanine aminotransferase (ALT) levels were mildly elevated in two anti-HDV positive persons and were normal in three. Our study patients with chronic HBV infection had a lower prevalence of anti-HDV antibody as compared to previous reports [4, 7] from our locality with prevalence rates of 6.5% for HDAg and 12.5% for HDV antibody, respectively. Possible explanations for this difference may relate to the characteristics of the study subjects, the assay or an actual reduction in disease burden. Among the previous studies, one by Ojo et al. [4] had smaller sample size, consisted of patients symptomatic for liver disease and the assay was for HDV antigen. The other study [7] included both symptomatic and asymptomatic persons, but had a larger sample size. Our study, as well as both the previous studies, were limited by the fact that testing for HDV RNA, a better marker of HDV infection, was not done. A true reduction in HDV prevalence rates over time is also possible; this could have resulted from measures to prevent HBV infection embarked during the last two decades. A recent study showing HBsAg prevalence of about 8% Part of this work was presented as a poster during the EASL Monothematic Conference in Istanbul, Turkey in September 2010. C. A. Onyekwere (*) : F. Duro-Emmanuel Department of Medicine, Lagos State University Teaching Hospital, Lagos, Nigeria e-mail: ifymobi@yahoo.com

Two types of defective RNAs arising from the tomato black ring virus genome

Short defective RNAs (D-RNAs) associated with tomato black ring virus (TBRV) were isolated, cloned and sequenced. As a result, two types of D-RNAs associated with different TBRV isolates were identified. Both types were derived from RNA1. The first one contained sequences from the 5' and 3' untranslated regions (UTR) and from the 5' region of a single large open reading frame. The second one included a portion of the coding region for the RNA-dependent RNA polymerase flanked by a short fragment of the 5' UTR and the entire 3' UTR. The possible nature and origin of these RNA species is discussed.

Seasonal dynamics and virus translocation of Grapevine leafroll‐associated virus 3 in grapevine cultivars

Grapevine leafroll‐associated virus 3 (GLRaV‐3) is associated with grapevine leafroll disease, one of the most economically important viral diseases of grapevines. This disease impacts on both vine health and grape quality; reduction in yield, brix and wine colour are among its detrimental effects. Many methods, including serological and molecular procedures, have been developed for the detection of GLRaV‐3; however, there is no PCR‐based assay available to quantify virus populations within plant tissues. A real‐time RT‐PCR assay with TaqMan probe was developed for specific and reliable quantitative detection of GLRaV‐3 in infected tissues. The designed primers and probes target the conserved sequence in the RNA‐dependent RNA polymerase (RdRp) domain of the viral genome to prevent amplification of most subgenomic and defective RNAs. This protocol was used to examine the seasonal dynamics and translocation of GLRaV‐3 in field‐grown grapevines. The results showed that the virus spread quickly from trunks to new growing shoots and leaves early in the growing season, and most samples still harboured detectable virus during late summer and autumn. The seasonal progress of one GLRaV‐3 isolate was compared in four grapevine cultivars (Chardonnay, Cabernet Sauvignon, Italia and Thompson Seedless). Within cultivars there was little variability in the distribution and translocation of GLRaV‐3, except for in Thompson Seedless. This quantitative detection assay will be a valuable tool for GLRaV‐3 diagnosis, disease monitoring and population ecology studies.

Interferon alpha for chronic hepatitis D

Hepatitis D virus is a small defective RNA virus that requires the presence of hepatitis B virus infection to infect a person. Hepatitis D is a difficult-to-treat infection. Several clinical trials have been published on the efficacy of interferon alpha for hepatitis D virus (HDV) infection. However, there are few randomised trials evaluating the effects of interferon alpha, and it is difficult to judge any benefit of this intervention from the individual trials. To evaluate the beneficial and harmful effects of interferon alpha for patients with chronic hepatitis D. We identified relevant for the review randomised clinical trials by electronic searches in the Cochrane Hepato-Biliary Group Controlled Trials Register, the Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library, MEDLINE, EMBASE, and Science Citation Index Expanded until May 2011. We also checked the bibliographic references of identified randomised trials, textbooks, and review articles in order to find randomised trials not identified by the electronic searches. Randomised clinical trials evaluating interferon alpha versus placebo or no intervention for patients with chronic hepatitis D infection. Two authors assessed the trials and extracted data on mortality, virologic, biochemical, and histological response as well as adverse events at end of treatment and six months or more after completing treatment. The analyses were performed using the intention-to-treat principle including all randomised participants irrespective of follow-up. Drop-outs, withdrawals, and non-compliance were considered as treatment failures. Data were analysed with fixed- and random-effects models. Reported results were based on fixed-effect model except in cases where statistical significance varied between the two models. Six randomised trials fulfilled the inclusion criteria. Two hundred and one randomised participants (male = 174) were included. The risk of bias in all the included trials was high. Five trials compared interferon alpha with no treatment in the control group. One of these trials had two treatment arms with a higher dose and lower dose of interferon alpha and a no-treatment control group. We analysed both treatment regimens as a single group in a primary analysis and as separate groups in the subgroup analysis of different interferon dosages. The sixth trial compared only a higher dose of interferon alpha with a lower dose.Meta-analysis of five trials comparing interferon alpha with no-treatment control group included 169 participants. There were seven drop-outs in the treatment group and nine in the control group. One patient out of 92 (1.1%) died in the interferon alpha group compared with zero out of 77 (0.0%) in the no-intervention control group (risk ratio (RR)) 3.00; 95% confidence interval (CI) 0.14 to 66.5). Interferon alpha led to failure of end of treatment virological response in 62/92 (67.4%) of the patients compared with 71/77 (92.2%) in the untreated controls (RR 0.76, 95% CI 0.66 to 0.87, P = 0.0001 by fixed-effect model and RR 0.71, 95% CI 0.43 to 1.16, P = 0.17 by random-effects model). Failure of normalisation of alanine aminotransferase (ALT) at the end of treatment was seen in 60/92 (65.2%) patients treated with interferon alpha versus 76/77 (98.7%) in the control group (RR 0.69, 95% CI 0.59 to 0.80, P < 0.00001). Sustained virological response was not achieved in 76/92 (82.6%) of patients on interferon compared with 73/77 (94.8%) of controls (RR 0.89, 95% CI 0.80 to 0.98, P = 0.02). Serum alanine aminotransferase was abnormal in 81/92 (88.0%) treated with interferon alpha patients at six months post-treatment follow-up compared with 76/77 (98.7%) in controls (RR 0.92, 95% CI 0.84 to 0.99, P = 0.04). There was no significant histological improvement in 67/92 (72.8%) patients treated with interferon alpha compared with 65/77 (84.4%) in controls (RR 0.86, 95% CI 0.74 to 1.00, P = 0.06).Two trials comparing a higher dose of interferon alpha with the lower dose showed no significant difference in sustained virological response (76.7% compared with 90.0%) (RR 0.85, 95% CI 0.68 to 1.07, P = 0.16). Adverse events such as flu-like symptoms, asthenia, weight loss, alopecia, thrombocytopenia, and leukopenia were reported in all these trials and the adverse events were related to interferon alpha. These were common and sometimes severe. One patient in the treatment group was reported to have died by suicide towards the end of the study period. Interferon alpha does not seem to cure hepatitis D in most patients. The agent seems effective in suppressing viral and liver disease activity in some patients, but this improvement is not sustained in the majority of patients. We cannot exclude overestimation of benefits and underestimation of harms due to high risk of bias (systematic errors) and high risk play of chance (random errors). Therefore, more randomised trials with large sample sizes and less risk of bias are needed before interferon can be recommended or refuted.

Reactions of Nicotiana species to inoculation with monopartite and bipartite begomoviruses

BackgroundSome Nicotiana species are widely used as experimental hosts for plant viruses. Nicotiana species differ in ploidy levels, chromosome numbers and have diverse geographical origins. Thus, these species are useful model systems to investigate virus-host interactions, co-evolution of pathogens and hosts and the effects of ploidy level on virus resistance/susceptibility.ResultsHere we have studied the responses of seven Nicotiana species to inoculation with Cotton leaf curl Multan virus (CLCuMV), a monopartite begomovirus, and Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, both from the Indian subcontinent. All Nicotiana species supported the replication of both begomoviruses in inoculated leaves. However, only three Nicotiana species, namely N. benthamiana, N. tabacum and N. sylvestris showed symptoms when inoculated with ToLCNDV, while N. benthamiana was the only species that developed leaf curl symptoms when inoculated with CLCuMV. CLCuMV accumulated to detectable levels in N. tabacum, but plants remained asymptomatic. A previously identified mutation of RNA dependent RNA polymerase 1 was shown to be present only in N. benthamiana. The finding is in line with earlier results showing that the susceptibility of this species to a diverse range of plant viruses correlates with a defective RNA silencing-mediated host defense.ConclusionsThe results presented show that individual Nicotiana species respond differently to inoculation with begomoviruses. The inability of begomoviruses to systemically infect several Nicotiana species is likely due to inhibition of virus movement, rather than replication, and thus provides a novel model to study virus-host interactions in resistant/susceptible hosts.

Replication of Lettuce chlorosis virus (LCV), a crinivirus in the family Closteroviridae, is accompanied by the production of LCV RNA 1-derived novel RNAs

Cloned infectious complementary DNAs of the bipartite genomic RNAs of Lettuce chlorosis virus (LCV) were constructed. Inoculation of tobacco protoplasts with the in vitro produced RNAs 1 and 2 transcripts, or with RNA 1 transcript alone, resulted in viral replication accompanied by the production of novel LCV RNA 1-derived RNAs. They included the abundantly accumulating LM-LCVR1-1 (~ 0.38 kb) and LM-LCVR1-2 (~ 0.3 kb), and the lowly accumulating HM-LCVR1-1 (~ 8.0 kb) and HM-LCVR1-2 (~ 6.6 kb), all of which reacted with riboprobes specific to the 5′ end of RNA 1 in Northern blot analysis. LM-LCVR1-1 and HM-LCVR1-2 accumulated as positive-stranded RNAs that lacked complementary negative strands, while HM-LCVR1-1 and LM-LCVR1-2 accumulated in both polarities. Additional Northern blot, reverse transcription-polymerase chain reaction, cloning, and sequence analyses revealed LM-LCVR1-2 to be an authentic RNA 1-derived defective (D)RNA, suggesting that its synthesis and maintenance are supported in trans by an RNA 1 encoded replication machinery.

Plastid signalling under multiple conditions is accompanied by a common defect in RNA editing in plastids.

Retrograde signalling from the plastid to the nucleus, also known as plastid signalling, plays a key role in coordinating nuclear gene expression with the functional state of plastids. Inhibitors that cause plastid dysfunction have been suggested to generate specific plastid signals related to their modes of action. However, the molecules involved in plastid signalling remain to be identified. Genetic studies indicate that the plastid-localized pentatricopeptide repeat protein GUN1 mediates signalling under several plastid signalling-related conditions. To elucidate further the nature of plastid signals, investigations were carried out to determine whether different plastid signal-inducing treatments had similar effects on plastids and on nuclear gene expression. It is demonstrated that norflurazon and lincomycin treatments and the plastid protein import2-2 (ppi2-2) mutation, which causes a defect in plastid protein import, all resulted in similar changes at the gene expression level. Furthermore, it was observed that these three treatments resulted in defective RNA editing in plastids. This defect in RNA editing was not a secondary effect of down-regulation of pentatricopeptide repeat protein gene expression in the nucleus. The results indicate that these three treatments, which are known to induce plastid signals, affect RNA editing in plastids, suggesting an unprecedented link between plastid signalling and RNA editing.

Molecular characterization of Drosophila cells persistently infected with Flock House virus

Little is known about the molecular determinants causing and sustaining viral persistent infections at the cellular level. We found that Drosophila cells persistently infected (PI) with Flock House virus (FHV) invariably harbor defective viral RNAs, which are replicated by the FHV RNA-dependent RNA polymerase. Some defective RNAs encoded a functional B2 protein, the FHV suppressor of RNA interference, which might contribute to maintenance of virus persistence. Viral small interfering RNAs (vsiRNAs) of both polarities were detected in PI cells and primarily mapped to regions of the viral genome that were preserved in the isolated defective RNAs. This indicated that defective RNAs could represent major sources of vsiRNAs. Immunofluorescence analysis revealed that mitochondria and viral proteins are differentially distributed in PI cells and lytically infected cells, which may partly explain the reduction in infectious viral progeny. Our results provide a basis for further investigations of the molecular mechanisms underlying persistent infections.

Detection and characterization of spontaneous internal deletion mutants of Beet Necrotic yellow vein virus RNA3 from systemic host Nicotiana benthamiana

BackgroundBeet Necrotic Yellow Vein virus (BNYVV) is a member of the genus Benyvirus causing a worldwide sugar beet disease rhizomania. BNYVV contains four or five plus-sense single stranded RNAs. In altered selective conditions, multipartite RNA viruses of plant are prone to undergoing internal deletions, thus turning into Defective RNAs (D RNAs). Although several D RNAs have been reported in BNYVV infection, the spontaneous internal deletion mutants responsible for severe symptom in systemic host Nicotiana benthamiana (N. benthamiana) are not described so far.ResultsSystemic host N. benthamiana was inoculated by Chinese BNYVV isolates. RT-PCR and Northern blot showed that the D RNAs forms of BNYVV RNA3 were present in the systemic infection of the N. benthamiana. Three distinct D-RNA3s, named as D-RNA 3α, D-RNA 3β and D-RNA 3γ, were made into infectious clones. When inoculated on the N. benthamiana, the in vitro transcripts of D forms exhibited more stable than that of wild-type RNA3 in systemic movement. Among the detected mutant, the p25 protein frame-shift mutant (D-RNA3α) induced obvious necrotic lesions on Tetragonia.expansa (T. expansa) and pronounced systemic symptom on the N. benthamiana. The D-RNA3α was further mutated artificially to pre-terminate the downstream N protein, leading to the abolishment of the pathogenicity, indicating the N protein was responsible for the necrotic symptom.ConclusionOur studies demonstrated the internal deletion mutants of BNYVV-RNA3 were spontaneously generated in the systemic infection on N. benthamiana. The internal deletions didn't affect the efficient replication of D-RNA3s, instead by improving the stability and pathogenicity of RNA3 in the systemic host N. benthamiana. Besides, our results also suggested the downstream N protein of RNA3, but not the upstream p25 protein, may play an important role in the systemic infection on N. benthamiana.

Role of Defective RNA Processing in the Pathogenesis of Pediatric Diseases

Role of Defective RNA Processing in the Pathogenesis of Pediatric Diseases

Dissection of Dom34–Hbs1 reveals independent functions in two RNA quality control pathways

Eukaryotic cells have several quality control pathways that rely on translation to detect and degrade defective RNAs. Dom34 and Hbs1 are two proteins that are related to translation termination factors and are involved in no-go decay (NGD) and nonfunctional 18S ribosomal RNA (rRNA) decay (18S NRD) pathways that eliminate RNAs that cause strong ribosomal stalls. Here we present the structure of Hbs1 with and without GDP and a low-resolution model of the Dom34-Hbs1 complex. This complex mimics complexes of the elongation factor and transfer RNA or of the translation termination factors eRF1 and eRF3, supporting the idea that it binds to the ribosomal A-site. We show that nucleotide binding by Hbs1 is essential for NGD and 18S NRD. Mutations in Hbs1 that disrupted the interaction between Dom34 and Hbs1 strongly impaired NGD but had almost no effect on 18S NRD. Hence, NGD and 18S NRD could be genetically uncoupled, suggesting that mRNA and rRNA in a stalled translation complex may not always be degraded simultaneously.

Knockout of the plastid RNase E leads to defective RNA processing and chloroplast ribosome deficiency

Ribonuclease E (RNase E) represents a key enzyme in bacterial RNA metabolism. It plays multifarious roles in RNA processing and also initiates degradation of mRNA by endonucleolytic cleavage. Plastids (chloroplasts) are derived from formerly free-living bacteria and have largely retained eubacterial gene expression mechanisms. Here we report the functional characterization of a chloroplast RNase E that is encoded by a single-copy nuclear gene in the model plant Arabidopsis thaliana. Analysis of knockout plants revealed that, unlike in bacteria, RNase E is not essential for survival. Absence of RNase E results in multiple defects in chloroplast RNA metabolism. Most importantly, polycistronic precursor transcripts overaccumulate in the knockout plants, while several mature monocistronic mRNAs are strongly reduced, suggesting an important function of RNase E in intercistronic processing of primary transcripts from chloroplast operons. We further show that disturbed maturation of a transcript encoding essential ribosomal proteins results in plastid ribosome deficiency and, therefore, provides a molecular explanation for the observed mutant phenotype.

Hepatitis D virus: an update

Hepatitis D virus (HDV) infection involves a distinct subgroup of individuals simultaneously infected with the hepatitis B virus (HBV) and characterized by an often severe chronic liver disease. HDV is a defective RNA agent needing the presence of HBV for its life cycle. HDV is present worldwide, but the distribution pattern is not uniform. Different strains are classified into eight genotypes represented in specific regions and associated with peculiar disease outcome. Two major specific patterns of infection can occur, i.e. co-infection with HDV and HBV or HDV superinfection of a chronic HBV carrier. Co-infection often leads to eradication of both agents, whereas superinfection mostly evolves to HDV chronicity. HDV-associated chronic liver disease (chronic hepatitis D) is characterized by necro-inflammation and relentless deposition of fibrosis, which may, over decades, result in the development of cirrhosis. HDV has a single-stranded, circular RNA genome. The virion is composed of an envelope, provided by the helper HBV and surrounding the RNA genome and the HDV antigen (HDAg). Replication occurs in the hepatocyte nucleus using cellular polymerases and via a rolling circle process, during which the RNA genome is copied into a full-length, complementary RNA. HDV infection can be diagnosed by the presence of antibodies directed against HDAg (anti-HD) and HDV RNA in serum. Treatment involves the administration of pegylated interferon-α and is effective in only about 20% of patients. Liver transplantation is indicated in case of liver failure.

Tackling the pathogenesis of RNA nuclear retention in myotonic dystrophy

DM1 (myotonic dystrophy type I) is a common form of muscular dystrophy that affects mainly adults. It is a disease that belongs to the group of defective RNA export diseases, since a major part of the pathogenic mechanism of the disease is the retention of the mutant transcripts in the cell nucleus. The presence of an expanded CUG trinucleotide repeat in the 3'-UTR (3'-untranslated region) of the DMPK (myotonic dystrophy protein kinase) gene causes the attraction of RNA-binding proteins by the nuclear-located mutant transcripts. As a result of the occupation of the RNA-binding proteins, there is defective mis-splicing of several cellular transcripts. This is believed to be a major pathogenic mechanism of the disease and any attempt to repair the activities of the RNA-binding proteins or target the mutant transcripts should be beneficial for the patients. Certain approaches have been described in the literature and they demonstrate progress in various directions. The purpose of the present review is to summarize the successful attempts to tackle the pathogenesis caused by nuclear retention of mutant transcripts in myotonic dystrophy and to discuss the possible gains from such approaches.

A link between nuclear RNA surveillance, the human exosome and RNA polymerase II transcriptional termination

In eukaryotes, the production of mature messenger RNA that exits the nucleus to be translated into protein in the cytoplasm requires precise and extensive modification of the nascent transcript. Any failure that compromises the integrity of an mRNA may cause its retention in the nucleus and trigger its degradation. Multiple studies indicate that mRNAs with processing defects accumulate in nuclear foci or ‘dots’ located near the site of transcription, but how exactly are defective RNAs recognized and tethered is still unknown. Here, we present evidence suggesting that unprocessed β-globin transcripts render RNA polymerase II (Pol II) incompetent for termination and that this quality control process requires the integrity of the nuclear exosome. Our results show that unprocessed pre-mRNAs remain tethered to the DNA template in association with Pol II, in an Rrp6-dependent manner. This reveals an unprecedented link between nuclear RNA surveillance, the exosome and Pol II transcriptional termination.

Role of a novel zebrafish nup98 during embryonic development

The nucleoporin NUP98 is a component of the nuclear pore complex that regulates nucleocytoplasmic trafficking. It has been characterized in acute myeloid leukemia as a fusion partner during chromosomal translocation. In this study, we identified a zebrafish nup98 gene and examined its role in embryonic development. Two expressed sequence tags with translated sequences homologous to human NUP98 were identified. The gene was cloned by polymerase chain reaction from complementary DNA of zebrafish embryos. Cellular functions of zebrafish NUP98 were investigated in HeLa cells. nup98 expression and developmental functions in zebrafish embryos were investigated by whole-mount in situ hybridization and morpholino knockdown. Protein sequence of zebrafish nup98 shared 65% identity with its human homolog. Ectopic expression of zebrafish nup98 rescued the defective messenger RNA export due to human NUP98 knockdown in HeLa cells. In zebrafish embryos, nup98 was expressed diffusely in eyes and the developing brain since 18 hours postfertilization. Knockdown of nup98 with morpholino upregulated pu.1 expression by 39% ± 15% (p = 0.0153) and scl expression by 36% ± 7.6% (p = 0.0017). Expression of genes associated with erythropoiesis was unchanged. The morphants also developed intracranial hemorrhage at 48 hours postfertilization due to defective blood vessel development. A novel zebrafish nup98 was identified and it serves a role in nucleocytoplasmic trafficking similar to human NUP98. During development, it modulates hematopoietic stem cell and early myeloid development and maintains the integrity of cranial vasculature in the developing central nervous system.

Genome characterization of a debilitation-associated mitovirus infecting the phytopathogenic fungus Botrytis cinerea

The full-length sequences of B otrytis c inerea mito virus 1 (BcMV1) and an associated RNA (BcMV1-S) in strain CanBc-1c-78 of Botrytis cinerea were determined. Sequence analysis showed that BcMV1 is 2804 nt long and AU-rich (66.8%). BcMV1 shares 95% nucleotide sequence identity with O phiostoma n ovo- u lmi mito virus 3b (OnuMV3b). However, it is 472 nt longer than OnuMV3b. Mitochondrial codon usage revealed that BcMV1 contains one open reading frame encoding RdRp, which is 96% identical to the RdRp of OnuMV3b. These findings confirm that BcMV1 belongs to the genus Mitovirus and is a strain of OnuMV3b. BcMV1-S is 2171 nt long and derived from BcMV1 through a single internal in-frame deletion of 633 nt, suggesting that it is a defective RNA of BcMV1. BcMV1-S was found to suppress the replication of BcMV1 and to be co-transmissible with BcMV1 through hyphal anastomosis. Its presence, however, did not alleviate the BcMV1-associated debilitation phenotypes of B. cinerea.

Hereditary diffuse gastric cancer due to a previously undescribed CDH1 splice site mutation

Our patient was a 52-year-old man who was diagnosed with signet ring cell gastric adenocarcinoma. An extensive family history of gastric cancer raised suspicion for hereditary diffuse gastric cancer. Sequencing of the patient's CDH1 gene revealed a novel point mutation in a strictly conserved splice site within intron 6, c.833-2 A > G. This mutation was predicted to result in loss of function due to defective RNA splicing. To characterize the pathogenic mechanism of this mutation, we amplified the patient's CDH1 gene products by reverse transcriptase polymerase chain reaction. Primers flanking the region of the mutation detected 3 distinct transcripts. In addition to the wild-type product, a larger product consistent with activation of a cryptic splice site within intron 6 and a smaller product shown to result from exon 7 skipping were detected. In summary, we have identified a novel CDH1 mutation in a large hereditary diffuse gastric cancer kindred and identified its pathogenic mechanism.

Protein Aggregation and Defective RNA Metabolism as Mechanisms for Motor Neuron Damage

The presence of protein inclusions within the central nervous system is a characteristic of most neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Aggregates may induce cell death trough several mechanisms, such as sequestration of essential cellular components, clogging of the proteasome system, and/or disruption of axonal transport. The neuropathological signature of ALS is represented by the presence of ubiquitinated inclusions immunoreactive for the protein TDP-43 in the cytoplasm of motor neurons. Recent studies demonstrated that a significant percentage of familial ALS cases are caused by pathogenic mutations in the TAR DNA binding protein and fused in sarcoma/translocated in liposarcoma genes encoding, respectively, for TDP-43 and FUS proteins. Both TDP-43 and FUS are DNA/RNA-binding proteins involved in transcriptional regulation and splicing, shuttling, maturation and transport of mRNA molecules. Mutations in the two genes seem to induce a nucleo-cytoplasmic redistribution of FUS and TDP-43, possibly promoting aggregate formation and/or disrupting their physiological nuclear functions or their interactions with specific RNA targets. Those findings collectively suggest that alterations in cellular RNA metabolism may trigger motor neuron degeneration.