The Epstein-Barr virus noncoding RNA EBER2 transactivates the UCHL1 deubiquitinase to accelerate cell growth.

The Epstein-Barr virus (EBV) transforms resting B cells and is involved in the development of B cell lymphomas. We report here that the viral noncoding RNA EBER2 accelerates B cell growth by potentiating expression of the UCHL1 deubiquitinase that itself increased expression of the Aurora kinases and of cyclin B1. Importantly, this effect was also visible in Burkitt's lymphoma cells that express none of the virus's known oncogenes. Mechanistically, EBER2 bound the UCHL1 messenger RNA (mRNA), thereby bringing a protein complex that includes PU.1, a UCHL1 transactivator, to the vicinity of its promoter. Although the EBV oncogene LMP1 has been suggested to induce UCHL1, we show here that EBER2 plays a much more important role to reach significant levels of the deubiquitinase in infected cells. However, some viruses that carried a polymorphic LMP1 had an increased ability to achieve full UCHL1 expression. This work identifies a direct cellular target of a viral noncoding RNA that is likely to be central to EBV's oncogenic properties.

RNA-mediated translation regulation in viral genomes: computational advances in the recognition of sequences and structures.

RNA structures are widely distributed across all life forms. The global conformation of these structures is defined by a variety of constituent structural units such as helices, hairpin loops, kissing-loop motifs and pseudoknots, which often behave in a modular way. Their ubiquitous distribution is associated with a variety of functions in biological processes. The location of these structures in the genomes of RNA viruses is often coordinated with specific processes in the viral life cycle, where the presence of the structure acts as a checkpoint for deciding the eventual fate of the process. These structures have been found to adopt complex conformations and exert their effects by interacting with ribosomes, multiple host translation factors and small RNA molecules like miRNA. A number of such RNA structures have also been shown to regulate translation in viruses at the level of initiation, elongation or termination. The role of various computational studies in the preliminary identification of such sequences and/or structures and subsequent functional analysis has not been fully appreciated. This review aims to summarize the processes in which viral RNA structures have been found to play an active role in translational regulation, their global conformational features and the bioinformatics/computational tools available for the identification and prediction of these structures.

A trans-activator-like structure in RCNMV RNA1 evokes the origin of the trans-activator in RNA2.

The Red clover necrotic mosaic virus (RCNMV) genome consists of two plus-strand RNA genome segments, RNA1 and RNA2. RNA2 contains a multifunctional RNA structure known as the trans-activator (TA) that (i) promotes subgenomic mRNA transcription from RNA1, (ii) facilitates replication of RNA2, and (iii) mediates particle assembly and copackaging of genome segments. The TA has long been considered a unique RNA element in RCNMV. However, by examining results from RCNMV genome analyses in the ViRAD virus (re-)annotation database, a putative functional RNA element in the polymerase-coding region of RNA1 was identified. Structural and functional analyses revealed that the novel RNA element adopts a TA-like structure (TALS) and, similar to the requirement of the TA for RNA2 replication, the TALS is necessary for the replication of RNA1. Both the TA and TALS possess near-identical asymmetrical internal loops that are critical for efficient replication of their corresponding genome segments, and these structural motifs were found to be functionally interchangeable. Moreover, replacement of the TA in RNA2 with a stabilized form of the TALS directed both RNA2 replication and packaging of both genome segments. Based on their comparable properties and considering evolutionary factors, we propose that the TALS appeared de novo in RNA1 first and, subsequently, the TA arose de novo in RNA2 as a functional mimic of the TALS. This and other related information were used to formulate a plausible evolutionary pathway to describe the genesis of the bi-segmented RCNMV genome. The resulting scenario provides an evolutionary framework to further explore and test possible origins of this segmented RNA plant virus.

Epstein-Barr virus EBER1 and murine gammaherpesvirus TMER4 share conserved in vivo function to promote B cell egress and dissemination.

The oncogenic gammaherpesviruses, including human Epstein-Barr virus (EBV), human Kaposi's sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, γHV68, MuHV-4) establish life-long latency in circulating B cells. The precise determinants that mediate in vivo gammaherpesvirus latency and tumorigenesis remain unclear. The EBV-encoded RNAs (EBERs) are among the first noncoding RNAs ever identified and have been the subject of decades of studies; however, their biological roles during in vivo infection remain unknown. Herein, we use a series of refined virus mutants to define the active isoform of MHV68 noncoding RNA TMER4 and demonstrate that EBV EBER1 functionally conserves this activity in vivo to promote egress of infected B cells from lymph nodes into peripheral circulation.

Strand-Specific Reverse Transcription PCR for Detection of Replicating SARS-CoV-2.

We developed an assay that detects minus-strand RNA as a surrogate for actively replicating severe acute respiratory syndrome coronavirus 2. We detected minus-strand RNA in 41 persons with coronavirus disease up to 30 days after symptom onset. This assay might inform clinical decision-making about patient infectiousness.

Stability of SARS-CoV-2 RNA in Nonsupplemented Saliva.

The expense of saliva collection devices designed to stabilize severe acute respiratory syndrome coronavirus 2 RNA is prohibitive to mass testing. However, virus RNA in nonsupplemented saliva is stable for extended periods and at elevated temperatures. Simple plastic tubes for saliva collection will make large-scale testing and continued surveillance easier.

Polysomally protected viruses.

It is conceivable that an RNA virus could use a polysome, that is, a string of ribosomes covering the RNA strand, to protect the genetic material from degradation inside a host cell. This paper discusses how such a virus might operate, and how its presence might be detected by ribosome profiling. There are two possible forms for such apolysomally protected virus, depending upon whether just the forward strand or both the forward and complementary strands can be encased by ribosomes (these will be termed type 1 and type 2, respectively). It is argued that in the type 2 case the viral RNA would evolve anambigrammaticproperty, whereby the viral genes are free of stop codons in a reverse reading frame (with forward and reverse codons aligned). Recent observations of ribosome profiles of ambigrammatic narnavirus sequences are consistent with our predictions for the type 2 case.

Titer and distribution of little cherry virus 2 in Prunus avium.

Little cherry virus 2 (LChV-2) is a causal agent of little cherry disease, which produces small, misshapen fruit with poor color and taste. As LChV-2 symptoms are only present near harvest, molecular detection is essential for effective control. Therefore, we determined the titer and distribution of this virus in infected trees over time. While initial infections were found to be basipetal, in field trees, early-stage infection was characterized by uneven distribution and low titer, concentrated in woody stems. In contrast, established infections were systemic, and detection was consistent across tissues. These data provide improved sampling recommendations for the detection of LChV-2.

Leader RNA regulates snakehead vesiculovirus replication via interacting with viral nucleoprotein.

Leader RNA, a kind of virus-derived small noncoding RNA, has been proposed to play an important role in regulating virus replication, but the underlying mechanism remains elusive. In this study, snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus causing high mortality to the cultured snakehead fish in China, was used to unveil the molecular function of leader RNA. High-throughput small RNA sequencing of SHVV-infected cells showed that SHVV produced two groups of leader RNAs (named legroup1 and legroup2) during infection. Overexpression and knockout experiments reveal that legroup1, but not legroup2, affects SHVV replication. Mechanistically, legroup1-mediated regulation of SHVV replication was associated with its interaction with the viral nucleoprotein (N). Moreover, the nucleotides 6-10 of legroup1 were identified as the critical region for its interaction with the N protein, and the amino acids 1-45 of N protein were proved to confer its interaction with the legroup1. Taken together, we identified two groups of SHVV leader RNAs and revealed a role in virus replication for one of the two types of leader RNAs. This study will help understand the role of leader RNA in regulating the replication of negative-stranded RNA viruses.

Tricks and threats of RNA viruses - towards understanding the fate of viral RNA.

Human innate cellular defence pathways have evolved to sense and eliminate pathogens, of which, viruses are considered one of the most dangerous. Their relatively simple structure makes the identification of viral invasion a difficult task for cells. In the course of evolution, viral nucleic acids have become one of the strongest and most reliable early identifiers of infection. When considering RNA virus recognition, RNA sensing is the central mechanism in human innate immunity, and effectiveness of this sensing is crucial for triggering an appropriate antiviral response. Although human cells are armed with a variety of highly specialized receptors designed to respond only to pathogenic viral RNA, RNA viruses have developed an array of mechanisms to avoid being recognized by human interferon-mediated cellular defence systems. The repertoire of viral evasion strategies is extremely wide, ranging from masking pathogenic RNA through end modification, to utilizing sophisticated techniques to deceive host cellular RNA degrading enzymes, and hijacking the most basic metabolic pathways in host cells. In this review, we aim to dissect human RNA sensing mechanisms crucial for antiviral immune defences, as well as the strategies adopted by RNA viruses to avoid detection and degradation by host cells. We believe that understanding the fate of viral RNA upon infection, and detailing the molecular mechanisms behind virus-host interactions, may be helpful for developing more effective antiviral strategies; which are urgently needed to prevent the far-reaching consequences of widespread, highly pathogenic viral infections.

The Crossroads between Host Copper Metabolism and Influenza Infection.

Three main approaches are used to combat severe viral respiratory infections. The first is preemptive vaccination that blocks infection. Weakened or dead viral particles, as well as genetic constructs carrying viral proteins or information about them, are used as an antigen. However, the viral genome is very evolutionary labile and changes continuously. Second, chemical agents are used during infection and inhibit the function of a number of viral proteins. However, these drugs lose their effectiveness because the virus can rapidly acquire resistance to them. The third is the search for points in the host metabolism the effect on which would suppress the replication of the virus but would not have a significant effect on the metabolism of the host. Here, we consider the possibility of using the copper metabolic system as a target to reduce the severity of influenza infection. This is facilitated by the fact that, in mammals, copper status can be rapidly reduced by silver nanoparticles and restored after their cancellation.

The critical region for viral RNA encapsidation in leader promoter of Nipah virus.

Encapsidation by nucleocapsid (N) protein is crucial for viral RNA to serve as a functional template for virus replication. However, the potential region that is vital for RNA encapsidation of Nipah virus (NiV) is still unknown. Thus, this study was aimed to identify these regions using a NiV minireplicon system. A series of broad range internal deletion mutations was generated in the 5' non-translated region (NTR) of the N gene mRNA region of NiV leader promoter via site-directed overlapping PCR-mediated mutagenesis. The mutation effects on synthesis and encapsidation of antigenome RNA, transcription, and RNA binding affinity of N protein were evaluated. The deletions of nucleotides 73-108, 79-108, and 85-108 from NiV leader promoter inhibited the encapsidation of antigenome RNA, while the deletion of nucleotides 103-108 suppressed the synthesis and encapsidation of antigenome RNA, implying that these regions are required for genome replication. Surprisingly, none of the mutations had detrimental effect on viral transcription. Using isothermal titration calorimetry, the binding of NiV N protein to genome or antigenome RNA transcript lacking of nucleotides 73-108 was found to be suppressed. Additionally, in silico analysis on secondary structure of genome RNA further supported the plausible cause of inefficient encapsidation of antigenome RNA by the loss of encapsidation signal in genome template. In conclusion, this study suggests that the nucleotides 73-90 within 5' NTR of the N gene mRNA region in NiV leader promoter contain cis-acting RNA element that is important for efficient encapsidation of antigenome RNA.

NXF1 and CRM1 nuclear export pathways orchestrate nuclear export, translation and packaging of murine leukaemia retrovirus unspliced RNA.

Cellular mRNAs are exported from the nucleus as fully spliced RNAs. Proofreading mechanisms eliminate unprocessed and irregular pre-mRNAs to control the quality of gene expression. Retroviruses need to export partially spliced and unspliced full-length RNAs to the cytoplasm where they serve as templates for protein synthesis and/or as encapsidated RNA in progeny viruses. Genetically complex retroviruses such as HIV-1 use Rev-equivalent proteins to export intron-retaining RNA from the nucleus using the cellular CRM1-driven nuclear export machinery. By contrast, genetically simpler retroviruses such as murine leukaemia virus (MLV) recruit the NXF1 RNA export machinery. In this study, we reveal for the first time that MLV hijacks both NXF1 and CRM1-dependent pathways to achieve optimal replication capacity. The CRM1-pathway marks the MLV full-length RNA (FL RNA) for packaging, while NXF1-driven nuclear export is coupled to translation. Thus, the cytoplasmic function of the viral RNA is determined early in the nucleus. Depending on the nature of ribonucleoprotein complex formed on FL RNA cargo in the nucleus, the FL RNA will be addressed to the translation machinery sites or to the virus-assembly sites at the plasma membrane.

Alterations in cellular RNA decapping dynamics affect tomato spotted wilt virus cap snatching and infection in Arabidopsis.

RNA processing and decay pathways have important impacts on RNA viruses, particularly animal-infecting bunyaviruses, which utilize a cap-snatching mechanism to translate their mRNAs. However, their effects on plant-infecting bunyaviruses have not been investigated. The roles of mRNA degradation and non-sense-mediated decay components, including DECAPPING 2 (DCP2), EXORIBONUCLEASE 4 (XRN4), ASYMMETRIC LEAVES2 (AS2) and UP-FRAMESHIFT 1 (UPF1) were investigated in infection of Arabidopsis thaliana by several RNA viruses, including the bunyavirus, tomato spotted wilt virus (TSWV). TSWV infection on mutants with decreased or increased RNA decapping ability resulted in increased and decreased susceptibility, respectively. By contrast, these mutations had the opposite, or no, effect on RNA viruses that use different mRNA capping strategies. Consistent with this, the RNA capping efficiency of TSWV mRNA was higher in a dcp2 mutant. Furthermore, the TSWV N protein partially colocalized with RNA processing body (PB) components and altering decapping activity by heat shock or coinfection with another virus resulted in corresponding changes in TSWV accumulation. The present results indicate that TSWV infection in plants depends on its ability to snatch caps from mRNAs destined for decapping in PBs and that genetic or environmental alteration of RNA processing dynamics can affect infection outcomes.

The RNA genome of hepatitis E virus robustly triggers an antiviral interferon response.

The outcomes of hepatitis E virus (HEV) infection are diverse, ranging from asymptomatic carrier, self-limiting acute infection, and fulminant hepatitis to persistent infection. This is closely associated with the immunological status of the host. This study aimed to understand the innate cellular immunity as the first-line defense mechanism in response to HEV infection. Phosphorylation of signal transducer and activator of transcription 1, a hallmark of the activation of antiviral interferon (IFN) response, was observed in the liver tissues of the majority of HEV-infected patients but not in the liver of uninfected individuals. In cultured cell lines and primary liver organoids, we found that HEV RNA genome potently induced IFN production and antiviral response. This mechanism is conserved among different HEV strains, including genotypes 1, 3, and 7 as tested. Interestingly, single-stranded HEV RNA is sufficient to trigger the antiviral response, without the requirement of viral RNA synthesis and the generation of an RNA replicative form or replicative intermediate. Surprisingly, the m7 G cap and poly A tail are not required, although both are key features of the HEV genome. Mechanistically, this antiviral response occurs in a retinoic acid-inducible gene-I-independent, melanoma differentiation-associated protein 5-independent, mitochondrial antiviral signaling protein-independent, and ß-catenin-independent but IRF3-dependent and IRF7-dependent manner. Furthermore, the integrity of the Janus kinase-signal transducer and activator of transcription pathway is essentially required. CONCLUSION: HEV infection elicits an active IFN-related antiviral response in vitro and in patients, triggered by the viral RNA and mediated by IFN regulatory factors 3 and 7 and the Janus kinase-signal transducer and activator of transcription cascade; these findings have revealed new insights into HEV-host interactions and provided the basis for understanding the pathogenesis and outcome of HEV infection. (Hepatology 2018;67:2096-2112).

Mechanism of Protein Kinase R Inhibition by Human Cytomegalovirus pTRS1.

Double-stranded RNAs (dsRNA) produced during human cytomegalovirus (HCMV) infection activate the antiviral kinase protein kinase R (PKR), which potently inhibits virus replication. The HCMV pTRS1 and pIRS1 proteins antagonize PKR to promote HCMV protein synthesis and replication; however, the mechanism by which pTRS1 inhibits PKR is unclear. PKR activation occurs in a three-step cascade. First, binding to dsRNA triggers PKR homodimerizaton. PKR dimers then autophosphorylate, leading to a conformational shift that exposes the binding site for the PKR substrate eIF2α. Consistent with previous in vitro studies, we found that pTRS1 bound and inhibited PKR. pTRS1 binding to PKR was not mediated by an RNA intermediate, and mutations in the pTRS1 RNA binding domain did not affect PKR binding or inhibition. Rather, mutations that disrupted the pTRS1 interaction with PKR ablated the ability of pTRS1 to antagonize PKR activation by dsRNA. pTRS1 did not block PKR dimerization and could bind and inhibit a constitutively dimerized PKR kinase domain. In addition, pTRS1 binding to PKR inhibited PKR kinase activity. Single amino acid point mutations in the conserved eIF2α binding domain of PKR disrupted pTRS1 binding and rendered PKR resistant to inhibition by pTRS1. Consistent with a critical role for the conserved eIF2α contact site in PKR binding, pTRS1 bound an additional eIF2α kinase, heme-regulated inhibitor (HRI), and inhibited eIF2α phosphorylation in response to an HRI agonist. Together our data suggest that pTRS1 inhibits PKR by binding to conserved amino acids in the PKR eIF2α binding site and blocking PKR kinase activity.IMPORTANCE The antiviral kinase PKR plays a critical role in controlling HCMV replication. This study furthered our understanding of how HCMV evades inhibition by PKR and identified new strategies for how PKR activity might be restored during infection to limit HCMV disease.

Hepatitis C Virus Induces the Localization of Lipid Rafts to Autophagosomes for Its RNA Replication.

Autophagy plays important roles in maintaining cellular homeostasis. It uses double- or multiple-membrane vesicles termed autophagosomes to remove protein aggregates and damaged organelles from the cytoplasm for recycling. Hepatitis C virus (HCV) has been shown to induce autophagy to enhance its own replication. Here we describe a procedure that combines membrane flotation and affinity chromatography for the purification of autophagosomes from cells that harbor an HCV subgenomic RNA replicon. The purified autophagosomes had double- or multiple-membrane structures with a diameter ranging from 200 nm to 600 nm. The analysis of proteins associated with HCV-induced autophagosomes by proteomics led to the identification of HCV nonstructural proteins as well as proteins involved in membrane trafficking. Notably, caveolin-1, caveolin-2, and annexin A2, which are proteins associated with lipid rafts, were also identified. The association of lipid rafts with HCV-induced autophagosomes was confirmed by Western blotting, immunofluorescence microscopy, and immunoelectron microscopy. Their association with autophagosomes was also confirmed in HCV-infected cells. The association of lipid rafts with autophagosomes was specific to HCV, as it was not detected in autophagosomes induced by nutrient starvation. Further analysis indicated that the autophagosomes purified from HCV replicon cells could mediate HCV RNA replication in a lipid raft-dependent manner, as the depletion of cholesterol, a major component of lipid rafts, from autophagosomes abolished HCV RNA replication. Our studies thus demonstrated that HCV could specifically induce the association of lipid rafts with autophagosomes for its RNA replication.IMPORTANCE HCV can cause severe liver diseases, including cirrhosis and hepatocellular carcinoma, and is one of the most important human pathogens. Infection with HCV can lead to the reorganization of membrane structures in its host cells, including the induction of autophagosomes. In this study, we developed a procedure to purify HCV-induced autophagosomes and demonstrated that HCV could induce the localization of lipid rafts to autophagosomes to mediate its RNA replication. This finding provided important information for further understanding the life cycle of HCV and its interaction with the host cells.

HSV-1-encoded microRNA miR-H1 targets Ubr1 to promote accumulation of neurodegeneration-associated protein.

Herpes simplex virus 1 (HSV-1) encodes various microRNAs (miRNAs), whose targets are largely unknown. miR-H1 is the first discovered HSV-1 miRNA and is expressed predominantly in productive infection. Here we show that ubiquitin protein ligase E3 component n-recognin 1 (Ubr1) is a cellular target of miR-H1. Ubr1 is a RING-type E3 ubiquitin ligase of the Arg/N-end rule pathway, which causes the degradation of proteins bearing "destabilizing" N-terminal residues, such as neurodegeneration-associated protein fragment ß-amyloid. Using model substrates, we found that miR-H1 significantly repressed the expression and activity of Ubr1. Consequently, miR-H1-mediated Ubr1 silencing resulted in the accumulation of ß-amyloid, which might contribute to the neurodegenerative pathogenesis enhanced by HSV-1. Our results provide novel insights into the mechanism by which HSV-1-encoded miR-H1 functions in neurodegenerative pathogenesis through targeting Ubr1-mediated Arg/N-end rule degradation pathway.

Sofosbuvir Inhibits Hepatitis E Virus Replication In Vitro and Results in an Additive Effect When Combined With Ribavirin.

Infection with hepatitis E virus genotype 3 may result in chronic hepatitis in immunocompromised patients. Reduction of immunosuppression or treatment with ribavirin or pegylated interferon-α can result in viral clearance. However, safer and more effective treatment options are needed. Here, we show that sofosbuvir inhibits the replication of hepatitis E virus genotype 3 both in subgenomic replicon systems as well as a full-length infectious clone. Moreover, the combination of sofosbuvir and ribavirin results in an additive antiviral effect. Sofosbuvir may be considered as an add-on therapy to ribavirin for the treatment of chronic hepatitis E in immunocompromised patients.