Sense RNA Research Articles

Bidirectional nucleolar dysfunction in C9orf72 frontotemporal lobar degeneration

An intronic GGGGCC expansion in C9orf72 is the most common known cause of both frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat expansion leads to the generation of sense and antisense repeat RNA aggregates and dipeptide repeat (DPR) proteins, generated by repeat-associated non-ATG translation. The arginine-rich DPR proteins poly(glycine-arginine or GR) and poly(proline-arginine or PR) are potently neurotoxic and can localise to the nucleolus when expressed in cells, resulting in enlarged nucleoli with disrupted functionality. Furthermore, GGGGCC repeat RNA can bind nucleolar proteins in vitro. However, the relevance of nucleolar stress is unclear, as the arginine-rich DPR proteins do not localise to the nucleolus in C9orf72-associated FTLD/ALS (C9FTLD/ALS) patient brain. We measured nucleolar size in C9FTLD frontal cortex neurons using a three-dimensional, volumetric approach. Intriguingly, we found that C9FTLD brain exhibited bidirectional nucleolar stress. C9FTLD neuronal nucleoli were significantly smaller than control neuronal nucleoli. However, within C9FTLD brains, neurons containing poly(GR) inclusions had significantly larger nucleolar volumes than neurons without poly(GR) inclusions. In addition, expression of poly(GR) in adult Drosophila neurons led to significantly enlarged nucleoli. A small but significant increase in nucleolar volume was also observed in C9FTLD frontal cortex neurons containing GGGGCC repeat-containing RNA foci. These data show that nucleolar abnormalities are a consistent feature of C9FTLD brain, but that diverse pathomechanisms are at play, involving both DPR protein and repeat RNA toxicity.

Using bisulfite conversion of RNA with RT‐PCR to unambiguously quantify expression of overlapping sense and antisense RNA in skeletal muscle

Long non‐coding RNAs (lncRNAs) have been revealed as being regulators of transcription of protein‐coding genes and may play critical roles in the remodeling of skeletal muscle in response to inactivity. We investigated lncRNAs that are transcribed antisense to and overlapping Myh7 (Type I Myosin Heavy Chain). We sought to develop a new RT‐PCR based approach to quantify overlapping sense (S) and antisense (AS) RNA that avoids amplification of non‐specific cDNA (NSC) products. Reverse transcription–polymerase chain reaction (RT‐PCR) is the go‐to method for quantitating RNA transcripts, due in part to its high sensitivity. Quantifying S‐AS gene pairs by RT‐PCR requires gene specific primers to be used in the RT. However, NSC is often transcribed, particularly when overlapping, and thus complementary transcripts are present. NSC is defined as PCR product produced from an RT in which primers are omitted from the RT reaction. On the other hand, gene‐specific cDNA (GSC) is defined as PCR product produced from gene specific primers in the RT reaction. NSC products that amplify during PCR may interfere with accurate quantification of the intended target. We developed an alternative method that utilizes the advantages of RT‐PCR without the specificity issues. We utilized bisulfite conversion of RNA, which converts all cytidine residues to uridine residues, thus rendering overlapping RNA to become non‐complementary and allowing for the unambiguous quantification of S and AS strands when quantified by RT‐PCR. Utilizing this approach with DNase‐treated RNA from Soleus (SOL) muscle of hindlimb suspended (HS) and normal ambulatory (CON) rats (N=6) we examined sense and antisense transcripts in the Myh7 coding region (at +6kb) and upstream from Myh7 at −9kb from the transcription start site. We compared bisulfite converted (BC) and non‐converted (NC) RNA. Data was normalized to beta‐actin mRNA.Using endpoint PCR to detect AS RNA in the coding region of Myh7 we determined that levels of NSC were 66% of the abundance of GSC. Such a high level of cDNA “background” for the AS target may compromise the validity of the quantitation. In contrast, cDNA derived from BC RNA revealed that NSC was undetectable when targeting both the S and the AS transcripts. However, in comparing differential expression of AS Myh7 RNA in HS as compared to CON both BC and NC RNA had the same result; there was not a significant difference. Examination S RNA at −9kb to Myh7 showed that levels of NSC averaged 71% of the abundance of GSC derived from NC RNA. NSC was again undetectable in the BC RNA. Differential expression of S RNA at −9kb in HS as compared to CON was 1.3‐fold (non‐significant) as assessed from NC RNA, and 0.81‐fold (non‐significant) as quantified in BC RNA. Though a significant HS vs CON difference was not apparent with either RNA treatments, the direction of change was not the same in NC as compared to BC RNA. In conclusion, this analysis showed that bisulfite conversion of RNA followed by RT‐PCR yields cDNA free of non‐specific products that may otherwise preclude accurate quantification of lncRNA expression in response to treatments such as altered loading states in muscle.

P01.13 The role of non-coding RNAs in GBM stem-like cells subtype identity maintenance and transition

AbstractDecades of research on protein coding genes (2% of the genome) led to only incremental improvements of outcomes in glioblastoma multiforme (GBM) patients. Meanwhile, research on non-protein-coding genes, which constitute the vast majority of the human transcriptome, have received comparatively little attention to date. Large scale transcriptomic profiling of GBM into four subtypes has provided remarkable insight about the pathobiology and heterogeneous nature of this disease, including the mechanisms of subtype maintenance and adaptation to tumor anatomic niche and microenvironmental challenges. The co-existence of several molecular subtypes of differentiated tumor cells and GBM stem-like cells (GSCs) within individual tumors is further complicated by their complex speciation, inter-subtype transitions and hybrid stages. The role of non-coding RNAs (such as microRNAs and long, non-coding RNAs) in these processes is poorly understood. Through their unique capability of targeting multiple mRNA targets, microRNAs, whose expression is often de-regulated in cancers, play pivotal role in pathophysiology of tumor. Subtype-specific expression of microRNA-128 (miR-128) affects complex subpopulation dynamics within the heterogeneous GBM tumors. Alterations in miR-128 levels, acting through de-regulation of multiple mRNA targets that control epigenetic landscape, are a potent mechanism of bi-directional transitions between proneural and mesenchymal GSC subpopulations resulting in intermediate hybrid stages and emphasizing highly intricate intra-tumoral networking. Long non-coding RNAs (lncRNAs) have an undefined role in the pathobiology of GBM. Hypoxia Inducible Factor 1 Alpha-Anti Sense RNA 2 (HIF1A-AS2) was identified as a subtype-specific hypoxia-inducible lncRNA, upregulated in mesenchymal GSCs. Knockdown of HIF1A-AS2 affected GSC growth, self-renewal, and hypoxia-dependent molecular reprogramming, leading to delayed growth of mesenchymal GSC tumors, survival benefits, and impaired expression of downstream genes. Our data demonstrate that HIF1A-AS2 contributes to GSCs’ speciation and adaptation to hypoxia within the tumor microenvironment, acting directly through its interactome and targets and indirectly by modulating responses to hypoxic stress depending on the subtype-specific genetic context. These examples highlight immense, yet not fully appreciated, role of non-coding RNAs in pathophysiology of GBM.

Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry

Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers.

Abstract IA02: Functional role of endogenous retroviruses in cancer immunity

Abstract Endogenous retroviruses (ERVs) are derived from past exogenous retroviral infections of germ cells, which integrated into the genome of mammals and are inherited in a Mendelian manner. ERVs belong to the class of retro-elements possessing three main viral genes: group-specific antigen (gag), polymerase (pol) and envelope (env) flanked by two long terminal repeats (LTR). More than 8% of the human genome is considered of retroviral origin. Most ERV genes are protein non-functional due to recombination, mutations and deletions; however some ERV gag, pol and env genes produce functional proteins. Although largely gene defective, the LTRs of ERVs retained their regulatory activities resulting in high transcriptional levels; and in addition represent most of the ~10,000 annotated long non-coding (lnc) RNAs. In somatic cells ERVs are generally silent; however we and others demonstrated ERVs become activated during tumorigenesis. Recently we showed that induction of immune responses in human cancer cells by DNA methyltransferase inhibitors (DNMTi), like 5-azacytidine (Aza) and 2'-deoxy-5-azacytidine (Dac) is primarily due to the activation of (hyper)-methylated ERVs (Chiappinelli et al. 2015, Cell 162: 974-86). Furthermore, the unique genomic architecture of ERVs with 5 and 3 LTRs induced sense and antisense RNAs leading to formation of cytosolic dsRNA, which activated the cellular viral RNA sensing and defense pathway (like TLR3 and MAVS) triggering the type I interferon (IFN) response. This kind of response following ERV activation is called viral mimicry or endogenous viremia. Using ovarian carcinoma (OVCA) cell lines as a model, we showed that Aza significantly induced multiple ERV families and IFN stimulated genes (ISG). The induction was due to the formation of dsRNA, which was abolished with RNAse III (hydrolyzing dsRNA) and TLR3 or MAVS specific inhibition. ISGs were also directly induced by transfected ERV genes. Subsequently, a cohort of primary human serous OVCA tissues was analyzed and a significant positive correlation between ERV and ISG expression could be demonstrated. We and others previously showed that treatment with the DNMTi Aza positively contributed to immune checkpoint blockade therapy in a mouse model and in human cancers. Like exogenous retroviruses, which upregulate immune checkpoint genes (ICG), we also evaluated for a possible connection between ERV induction and ICG expression in primary tumors. Presently, we demonstrate that human serous OVCA tissues express significantly higher ICGs than control ovarian tissues, with VTCN-1 (B7-H4) as the highest expressed ICG. Using serous OVCA cell lines transfected with different ERV genes we could show an early induction of ICGs, primarily LGALS9 and PD-L1. Further analysis of tumor infiltrating immune cells using the same serous OVCA cohort and multiplex immunohistochemistry (IHC), we detect in some tumors high levels of tumor-associated CD79a, CD3+/CD8- and CD3+/CD8+ cells. Moreover, we find a direct link between ISG, ICG and infiltrating immune cells in OVCA. Analyzing other human tumors, e.g. brain tumors, like glioblastoma multiforme (GBM) and giant cell GBM (gcGBM) also demonstrate a significant correlation between high ERV induction and ISG as well as ICG expression. In contrast to serous OVCA, PD1 and VTCN-1 were the highest expressed ICG in GBM and gcGBM, which could also be shown with ERV transfected GBM cell lines. Using IHC gcGBM shows a high level of immune cell infiltration, which associated with giant cells and ICG expression, like PD-L1. Interestingly, the highest ERV expression was found in gcGBM, which correlated with endomitosis and cell fusion leading to giant nuclei and multinucleated cells as also shown with a stable ERV transfected GBM cell line. In summary, DNMTi mediated loss of methylation induced ERVs forming dsRNA in tumor cells and stimulated the innate immune system via ISGs. ERV RNA per se induced ISGs, but also ICGs similar to exogenous retroviruses in order to escape the immune system. Different upregulated ERVs, ISGs and ICGs were found in different cancers, like serous OVCA, GBM and gcGBM indicating diverse tumor and tumor microenvironment signatures. Thus ERVs have an ambivalent role in tumorigenesis, where tumor microenvironmental factors tip the balance being detrimental by generating immune evasion or beneficial for cancer therapy by stimulating the innate immune system. Citation Format: Reiner Strick. Functional role of endogenous retroviruses in cancer immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr IA02.

Multiple Transcriptional and Post-transcriptional Pathways Collaborate to Control Sense and Antisense RNAs of Tf2 Retroelements in Fission Yeast.

Retrotransposons are mobile genetic elements that colonize eukaryotic genomes by replicating through an RNA intermediate. As retrotransposons can move within the host genome, defense mechanisms have evolved to repress their potential mutagenic activities. In the fission yeast Schizosaccharomyces pombe, the mRNA of Tf2 long terminal repeat retrotransposons is targeted for degradation by the 3'-5' exonucleolytic activity of the exosome-associated protein Rrp6. Here, we show that the nuclear poly(A)-binding protein Pab2 functions with Rrp6 to negatively control Tf2 mRNA accumulation. Furthermore, we found that Pab2/Rrp6-dependent RNA elimination functions redundantly to the transcriptional silencing mediated by the CENP-B homolog, Abp1, in the suppression of antisense Tf2 RNA accumulation. Interestingly, the absence of Pab2 attenuated the derepression of Tf2 transcription and the increased frequency of Tf2 mobilization caused by the deletion of abp1 Our data also reveal that the expression of antisense Tf2 transcripts is developmentally regulated and correlates with decreased levels of Tf2 mRNA. Our findings suggest that transcriptional and post-transcriptional pathways cooperate to control sense and antisense RNAs expressed from Tf2 retroelements.

The IMPORTance of the Nucleus during Flavivirus Replication.

Flaviviruses are a large group of arboviruses of significant medical concern worldwide. With outbreaks a common occurrence, the need for efficient viral control is required more than ever. It is well understood that flaviviruses modulate the composition and structure of membranes in the cytoplasm that are crucial for efficient replication and evading immune detection. As the flavivirus genome consists of positive sense RNA, replication can occur wholly within the cytoplasm. What is becoming more evident is that some viral proteins also have the ability to translocate to the nucleus, with potential roles in replication and immune system perturbation. In this review, we discuss the current understanding of flavivirus nuclear localisation, and the function it has during flavivirus infection. We also describe—while closely related—the functional differences between similar viral proteins in their nuclear translocation.

Detection of Viral RNA in Tissues following Plasma Clearance from an Ebola Virus Infected Patient.

An unprecedented Ebola virus (EBOV) epidemic occurred in 2013–2016 in West Africa. Over this time the epidemic exponentially grew and moved to Europe and North America, with several imported cases and many Health Care Workers (HCW) infected. Better understanding of EBOV infection patterns in different body compartments is mandatory to develop new countermeasures, as well as to fully comprehend the pathways of human-to-human transmission. We have longitudinally explored the persistence of EBOV-specific negative sense genomic RNA (neg-RNA) and the presence of positive sense RNA (pos-RNA), including both replication intermediate (antigenomic-RNA) and messenger RNA (mRNA) molecules, in the upper and lower respiratory tract, as compared to plasma, in a HCW infected with EBOV in Sierra Leone, who was hospitalized in the high isolation facility of the National Institute for Infectious Diseases “Lazzaro Spallanzani” (INMI), Rome, Italy. We observed persistence of pos-RNA and neg-RNAs in longitudinally collected specimens of the lower respiratory tract, even after viral clearance from plasma, suggesting possible local replication. The purpose of the present study is to enhance the knowledge on the biological features of EBOV that can contribute to the human-to-human transmissibility and to develop effective intervention strategies. However, further investigation is needed in order to better understand the clinical meaning of viral replication and shedding in the respiratory tract.

Genetic variation and population structure of Cucumber green mottle mosaic virus

Cucumber green mottle mosaic virus (CGMMV) is a single-stranded, positive sense RNA virus infecting cucurbitaceous plants. In recent years, CGMMV has become an important pathogen of cucurbitaceous crops including watermelon, pumpkin, cucumber and bottle gourd in China, causing serious losses to their production. In this study, we surveyed CGMMV infection in various cucurbitaceous crops grown in Zhejiang Province and in several seed lots purchased from local stores with the dot enzyme-linked immunosorbent assay (dot-ELISA), using a CGMMV specific monoclonal antibody. Seven CGMMV isolates obtained from watermelon, grafted watermelon or oriental melon samples were cloned and sequenced. Identity analysis showed that the nucleotide identities of the seven complete genome sequences ranged from 99.2 to 100%. Phylogenetic analysis of seven CGMMV isolates as well as 24 other CGMMV isolates from the GenBank database showed that all CGMMV isolates could be grouped into two distinct monophyletic clades according to geographic distribution, i.e. Asian isolates for subtype I and European isolates for subtype II, indicating that population diversification of CGMMV isolates may be affected by geographical distribution. Site variation rate analysis of CGMMV found that the overall variation rate was below 8% and mainly ranged from 2 to 5%, indicating that the CGMMV genomic sequence was conservative. Base substitution type analysis of CGMMV showed a mutational bias, with more transitions (A↔G and C↔T) than transversions (A↔C, A↔T, G↔C and G↔T). Most of the variation occurring in the CGMMV genome resulted in non-synonymous substitutions, and the variation rate of some sites was higher than 30% because of this mutational bias. Selection constraint analysis of CGMMV ORFs showed strong negative selection acting on the replication-associated protein, similar to what occurs for other plant RNA viruses. Finally, potential recombination analysis identified isolate Ec as a recombinant with a low degree of confidence.

Multi Epitope Peptide Vaccine Prediction against Sudan Ebola Virus Using Immuno-Informatics Approaches

Sudan Ebola virus is single stranded negative sense RNA genome belonging to Filovirus Filoviridae family that causes hemorrhagic fever. There is no treatment or vaccine for it, thus the aim of this study is to design a peptide vaccine using immuoinformatics approaches to analyse the glycoprotein of the all strain of SUDV, to determine the conserved region which is further studied to predict all possible epitopes that can be used as a peptide vaccine. A total of 21 Sudan Ebola virus glycoprotein retrieved from NCBI database were aligned to determine the conservancy and to predict the epitopes using IEDB analysis resource. Three epitopes predicted as a peptide vaccine for B cell (PPPPDGVR, ETFLQSPP, LQSPPIRE). For T cell four epitopes showed high affinity to MHC class I (FLYDRLAST, IIIAIIALL, MHNQNALVC and RTYTILNRK) and high coverage against Sudan and the whole world population. Also in MHC class II, Four epitopes that interact with most frequent MHC class II alleles (FAEGVIAFL, FLRATTELR, FLYDRLAST and FVWVIILFQ) with high coverage against Sudan and the whole world population. We recommend in vivo and in vitro study to prove the effectiveness of these predicted epitopes as a peptide vaccine.

A REVIEW PAPER ON POTATO MOP-TOP VIRUS (PMTV): OCCURRENCE, PROPERTIES AND MANAGEMENT

Potato mop-top virus (PMTV) is a plant pathogenic virus that affects potatoes. The virus was initially reported from Germany but now it has spread throughout Europe, Asia, South America and North America. It is responsible for spraing symptoms (brown arcs/lines, blemishes, and rings) on potato tubers and yellow chevrons or mopping (Shortened internodes) in the leaves and stems of plants grown from infected potato tubers. PMTV causes huge economic losses due to poor tuber quality. It is an important disease in the potato growing areas of the world. PMTV is tubular rod shape and has a single stranded positive sense RNA (+ssRNA) tripartite genome. RNA 1 encodes RdRp (viral RNA-dependent RNA polymerase). Coat protein (20kDa) and a larger protein (91kDa) is encoded by RNA2. RNA2 encodes larger protein (91 kDa) by read through (RT) of the amber termination codon of the coat protein. There are three conserved moldular sets of genes known as triple gene block (TGB) which are coded by RNA3. These TGBs are involved in cell to cell or long distance movement of PMTV. In nature, PMTV is vectored and transmitted by a soil born pathogen (Plasmodiophorid (Spongospora subterranean f.sp. subterranean abbreviated as ‘Sss’) that itself causes the powdery scab disease on tubers. The disease caused by PMTV and Sss are favored by cool and damp conditions. PMTV remain in spore balls of Sss for several years even if the potato is not grown in the field. There are no efficient means to manage the virus nor its vector in an infested field, therefore, preventive measures are essential. Since PMTV along with its vector is causing important disease of potato, so understanding its molecular, biological, physical properties and management strategies is very important.

Bidirectional Transcriptional Inhibition as Therapy for ALS/FTD Caused by Repeat Expansion in C9orf72

Hexanucleotide repeat expansion in the bi-directionally transcribed C9orf72 gene is the most frequent cause of familial ALS and frontotemporal dementia (FTD). Kramer etal. (2016) report in Science that targeted reduction in the transcription elongation factor SUPT4H1/SUPT5H reduces both sense and antisense repeat-containing RNAs and their associated neurodegeneration.

What is the role of TDP-43 in C9orf72-related amyotrophic lateral sclerosis and frontemporal dementia?

This scientific commentary refers to ‘Timing and significance of pathological features in C9orf72 expansion-associated frontotemporal dementia’, by Vatsavayai et al. (doi:10.1093/brain/aww250) . The C9orf72 hexanucleotide repeat expansion mutation is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), accounting for up to 12% of all patients in an average ALS clinic population. At post-mortem, patients with the C9orf72 hexanucleotide repeat expansion have the typical pathology seen in sporadic ALS and FTD, in which transactive response DNA-binding protein of 43 kDa (TDP-43; encoded by TARDBP ) is translocated from its normal nuclear location to form cytoplasmic aggregates. However, they also show additional pathological features in the form of RNA foci, containing the transcribed hexanucleotide repeats, and aggregates, which stain with antibodies against dipeptide repeat proteins, the product of non-ATG translation. The order in which these distinct molecular abnormalities develop, and whether each of these pathological features is a necessary or sufficient condition for the development of clinical disease, are currently key questions in ALS/FTD research. In this issue of Brain , Vatsavayai et al. describe two strikingly different pathological cases of C9orf72 -positive FTD with excellent ante-mortem characterization that provide novel and provocative contributions to this debate (Vatsavayai et al. , 2016). Transcription of the C9orf72 hexanucleotide repeat expansion can be detected as sense and antisense RNA foci in the nucleus and cytoplasm. Despite being located in the non-coding region of the C9orf72 gene, the repeat sequence is translated through a non-canonical process called ‘repeat associated non-ATG’ (RAN) translation, into five different species of dipeptide …

Detection of viral RNA in tissues following plasma clearance from an Ebola virus infected patient

Purpose: An unprecedented Ebola outbreak occurred in 2014-2015 in West Africa. A better understanding of the EBOV life cycle is fundamental to develop new countermeasures, as well as to fully comprehend the pathways of inter-human transmission. We have explored the possibility of viral persistence in different body fluids’ samples obtained from a Health Care Worker (HCW) infected in Sierra Leone and treated at INMI L. Spallanzani, Italy. To evaluate whether the virus was in a replicative status or simply derived from blood spill over, we compared the trends of EBOV-specific negative sense genomic RNA (neg-RNA), positive sense RNA (pos-RNA) and total viral RNA in different clinical samples. Methods & Materials: Clinical samples were inactivated in BSL4 facility and RNA was extracted with QIAamp Viral-RNA Mini-Kit (Qiagen). Quantification of total viral RNA was performed with the reference test Altona-FilovirusScreen Kit1.0 (Altona Diagnostics). To measure EBOV-specific negative sense genomic RNA (neg-RNA) and positive sense RNA (pos-RNA) [including both replication intermediate (cRNA) and messenger RNA (mRNA)], L-gene specific reverse or forward primers were used in the reverse transcription step Results: The clinical samples analyzed (sputum, nasopharyngeal swab, ocular swab, urine and plasma) show different trends in neg-RNA, pos-RNA and total viral RNA levels. In Nasopharyngeal swab, Ocular Swab and Urine, total viral RNA is the only one detectable, until Day 12, 5 and 15 after hospitalization, respectively. On the contrary, in plasma, total viral RNA, neg-RNA and pos-RNA levels simultaneously decreased, starting from day 3 and becoming undetectable at Day 6. In Sputum, pos-RNA levels decreased since Day 8, persisting at detectable levels up to Day 10, coherently with total viral RNA and neg-RNA levels, which start decreasing at Day 10 and become undetectable at Day 11. Conclusion: The presence of viral RNA and replication markers (pos-RNA and neg-RNA) in sputum might be of relevance in future analysis regarding the persistence of the virus in the upper respiratory tract, even after viral clearance from plasma. These results should be taken under further investigation in order to better understand the role of the respiratory tract for possible involvement in viral shedding, viral replication site or as a viral reservoir.

Ancient recombination events and the origins of hepatitis E virus.

BackgroundHepatitis E virus (HEV) is an enteric, single-stranded, positive sense RNA virus and a significant etiological agent of hepatitis, causing sporadic infections and outbreaks globally. Tracing the evolutionary ancestry of HEV has proved difficult since its identification in 1992, it has been reclassified several times, and confusion remains surrounding its origins and ancestry.ResultsTo reveal close protein relatives of the Hepeviridae family, similarity searching of the GenBank database was carried out using a complete Orthohepevirus A, HEV genotype I (GI) ORF1 protein sequence and individual proteins. The closest non-Hepeviridae homologues to the HEV ORF1 encoded polyprotein were found to be those from the lepidopteran-infecting Alphatetraviridae family members. A consistent relationship to this was found using a phylogenetic approach; the Hepeviridae RdRp clustered with those of the Alphatetraviridae and Benyviridae families. This puts the Hepeviridae ORF1 region within the “Alpha-like” super-group of viruses. In marked contrast, the HEV GI capsid was found to be most closely related to the chicken astrovirus capsid, with phylogenetic trees clustering the Hepeviridae capsid together with those from the Astroviridae family, and surprisingly within the “Picorna-like” supergroup. These results indicate an ancient recombination event has occurred at the junction of the non-structural and structure encoding regions, which led to the emergence of the entire Hepeviridae family.The Astroviridae capsid is also closely related to the Tymoviridae family of monopartite, T = 3 icosahedral plant viruses, whilst its non-structural region is related to viruses of the Potyviridae; a large family of plant-infecting viruses with a flexible filamentous rod-shaped virion. Thus, we identified a separate inter-viral family recombination event, again at the non-structural/structural junction, which likely led to the creation of the Astroviridae.ConclusionsIn summary, we have shown that new viral families have been created though recombination at the junction of the genome that encodes non-structural and structural proteins, and such recombination events are implicated in the genesis of important human pathogens; HEV, astrovirus and rubella virus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0785-y) contains supplementary material, which is available to authorized users.

Interaction of Host Nucleolin with Influenza A Virus Nucleoprotein in the Early Phase of Infection Limits the Late Viral Gene Expression.

Influenza A virus nucleoprotein, is a multifunctional RNA-binding protein, encoded by segment-5 of the negative sense RNA genome. It serves as a key connector between the virus and the host during virus replication. It continuously shuttles between the cytoplasm and the nucleus interacting with various host cellular factors. In the current study, host proteins interacting with nucleoprotein of Influenza A virus of H1N1 2009 pandemic strain were identified by co-immunoprecipitation studies followed by MALDI-TOF/MS analysis. Here we report the host nucleolin, a major RNA-binding protein of the nucleolus as a novel interacting partner to influenza A virus nucleoprotein. We thus, explored the implications of this interaction in virus life cycle and our studies have shown that these two proteins interact early during infection in the cytoplasm of infected cells. Depletion of nucleolin in A549 cells by siRNA targeting endogenous nucleolin followed by influenza A virus infection, disrupted its interaction with viral nucleoprotein, resulting in increased expression of gene transcripts encoding late viral proteins; matrix (M1) and hemagglutinin (HA) in infected cells. On the contrary, over expression of nucleolin in cells transiently transfected with pEGFP-NCL construct followed by virus infection significantly reduced the late viral gene transcripts, and consequently the viral titer. Altered expression of late viral genes and titers following manipulation of host cellular nucleolin, proposes the functional importance of its interaction with nucleoprotein during influenza A virus infection.

Organization, Function, and Therapeutic Targeting of the Morbillivirus RNA-Dependent RNA Polymerase Complex.

The morbillivirus genus comprises major human and animal pathogens, including the highly contagious measles virus. Morbilliviruses feature single stranded negative sense RNA genomes that are wrapped by a plasma membrane-derived lipid envelope. Genomes are encapsidated by the viral nucleocapsid protein forming ribonucleoprotein complexes, and only the encapsidated RNA is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRp). In this review, we discuss recent breakthroughs towards the structural and functional understanding of the morbillivirus polymerase complex. Considering the clinical burden imposed by members of the morbillivirus genus, the development of novel antiviral therapeutics is urgently needed. The viral polymerase complex presents unique structural and enzymatic properties that can serve as attractive candidates for druggable targets. We evaluate distinct strategies for therapeutic intervention and examine how high-resolution insight into the organization of the polymerase complex may pave the path towards the structure-based design and optimization of next-generation RdRp inhibitors.

An efficient and rapid method to detect and verify natural antisense transcripts of animal genes

High-throughput sequencing has identified a large number of sense-antisense transcriptional pairs, which indicates that these genes were transcribed from both directions. Recent reports have demonstrated that many antisense RNAs, especially lncRNA (long non-coding RNA), can interact with the sense RNA by forming an RNA duplex. Many methods, such as RNA-sequencing, Northern blotting, RNase protection assays and strand-specific PCR, can be used to detect the antisense transcript and gene transcriptional orientation. However, the applications of these methods have been constrained, to some extent, because of the high cost, difficult operation or inaccuracy, especially regarding the analysis of substantial amounts of data. Thus, we developed an easy method to detect and validate these complicated RNAs. We primarily took advantage of the strand specificity of RT-PCR and the single-strand specificity of S1 endonuclease to analyze sense and antisense transcripts. Four known genes, including mouse β-actin and Tsix (Xist antisense RNA), chicken LXN (latexin) and GFM1 (G elongation factor, mitochondrial 1), were used to establish the method. These four genes were well studied and transcribed from positive strand, negative strand or both strands of DNA, respectively, which represented all possible cases. The results indicated that the method can easily distinguish sense, antisense and sense-antisense transcriptional pairs. In addition, it can be used to verify the results of high-throughput sequencing, as well as to analyze the regulatory mechanisms between RNAs. This method can improve the accuracy of detection and can be mainly used in analyzing single gene and was low cost.

Interplay between Inflammation and Cellular Stress Triggered by Flaviviridae Viruses.

The Flaviviridae family comprises several human pathogens, including Dengue, Zika, Yellow Fever, West Nile, Japanese Encephalitis viruses, and Hepatitis C Virus. Those are enveloped, single-stranded positive sense RNA viruses, which replicate mostly in intracellular compartments associated to endoplasmic reticulum (ER) and Golgi complex. Virus replication results in abundant viral RNAs and proteins, which are recognized by cellular mechanisms evolved to prevent virus infection, resulting in inflammation and stress responses. Virus RNA molecules are sensed by Toll-like receptors (TLRs), RIG-I-like receptors (RIG-I and MDA5) and RNA-dependent protein kinases (PKR), inducing the production of inflammatory mediators and interferons. Simultaneously, the synthesis of virus RNA and proteins are distinguished in different compartments such as mitochondria, ER and cytoplasmic granules, triggering intracellular stress pathways, including oxidative stress, unfolded protein response pathway, and stress granules assembly. Here, we review the new findings that connect the inflammatory pathways to cellular stress sensors and the strategies of Flaviviridae members to counteract these cellular mechanisms and escape immune response.

Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts.

An expanded hexanucleotide repeat in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). Therapeutics are being developed to target RNAs containing the expanded repeat sequence (GGGGCC); however, this approach is complicated by the presence of antisense strand transcription of expanded GGCCCC repeats. We found that targeting the transcription elongation factor Spt4 selectively decreased production of both sense and antisense expanded transcripts, as well as their translated dipeptide repeat (DPR) products, and also mitigated degeneration in animal models. Knockdown of SUPT4H1, the human Spt4 ortholog, similarly decreased production of sense and antisense RNA foci, as well as DPR proteins, in patient cells. Therapeutic targeting of a single factor to eliminate c9FTD/ALS pathological features offers advantages over approaches that require targeting sense and antisense repeats separately.