Robust multi-qubit quantum network node with integrated error detection.

Long-distance quantum communication and networking require quantum memory nodes with efficient optical interfaces and long memory times. We report the realization of an integrated two-qubit network node based on silicon-vacancy centers (SiVs) in diamond nanophotonic cavities. Our qubit register consists of the SiV electron spin acting as a communication qubit and the strongly coupled silicon-29 nuclear spin acting as a memory qubit with a quantum memory time exceeding 2 seconds. By using a highly strained SiV, we realize electron-photon entangling gates at temperatures up to 1.5 kelvin and nucleus-photon entangling gates up to 4.3 kelvin. We also demonstrate efficient error detection in nuclear spin-photon gates by using the electron spin as a flag qubit, making this platform a promising candidate for scalable quantum repeaters.

Antiviral RNA interference in mammalian cells.

In antiviral RNA interference (RNAi), the DICER enzyme processes virus-derived double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that guide ARGONAUTE proteins to silence complementary viral RNA. As a counterdefense, viruses deploy viral suppressors of RNAi (VSRs). Well-established in plants and invertebrates, the existence of antiviral RNAi remains unknown in mammals. Here, we show that undifferentiated mouse cells infected with encephalomyocarditis virus (EMCV) or Nodamura virus (NoV) accumulate ~22-nucleotide RNAs with all the signature features of siRNAs. These derive from viral dsRNA replication intermediates, incorporate into AGO2, are eliminated in Dicer knockout cells, and decrease in abundance upon cell differentiation. Furthermore, genetically ablating a NoV-encoded VSR that antagonizes DICER during authentic infections reduces NoV accumulation, which is rescued in RNAi-deficient mouse cells. We conclude that antiviral RNAi operates in mammalian cells.

Animal virus replication and RNAi-mediated antiviral silencing in Caenorhabditis elegans.

The worm Caenorhabditis elegans is a model system for studying many aspects of biology, including host responses to bacterial pathogens, but it is not known to support replication of any virus. Plants and insects encode multiple Dicer enzymes that recognize distinct precursors of small RNAs and may act cooperatively. However, it is not known whether the single Dicer of worms and mammals is able to initiate the small RNA-guided RNA interference (RNAi) antiviral immunity as occurs in plants and insects. Here we show complete replication of the Flock house virus (FHV) bipartite, plus-strand RNA genome in C. elegans. We show that FHV replication in C. elegans triggers potent antiviral silencing that requires RDE-1, an Argonaute protein essential for RNAi mediated by small interfering RNAs (siRNAs) but not by microRNAs. This immunity system is capable of rapid virus clearance in the absence of FHV B2 protein, which acts as a broad-spectrum RNAi inhibitor upstream of rde-1 by targeting the siRNA precursor. This work establishes a C. elegans model for genetic studies of animal virus-host interactions and indicates that mammals might use a siRNA pathway as an antiviral response.

RNA silencing as related to viroid induced symptom expression.

Evidence of post-transcriptional gene silencing (PTGS) in avocado infected by Avocado sunblotch viroid (ASBVd), the type species of family Avsunviroidae, was suggested by detection of ASBVd-specific 22-nucleotide RNAs. PTGS was observed in infected bleached and variegated symptomatic tissues as well as symptomless carrier foliar sources and fruit with typical sunblotch disease lesions. Tissues with the different symptom expressions, characterized by the presence of different predominant ASBVd variants, were found to induce PTGS at differential levels. Detection of the PTGS-associated small interfering RNAs (siRNAs) as well as relative concentration was also related to viroid titer. PTGS induced in Gynura aurantiaca infected with two closely-related variants of Citrus exocortis viroid, a member of family Pospiviroidae, was not directly related to viroid titer with initiation of symptoms.

The suppressor of transgene RNA silencing encoded by Cucumber mosaic virus interferes with salicylic acid-mediated virus resistance.

The Cucumber mosaic virus (CMV)-encoded 2b protein (Cmv2b) is a nuclear protein that suppresses transgene RNA silencing in Nicotiana benthamiana. Cmv2b is an important virulence determinant but nonessential for systemic spread in N. glutinosa, in contrast to its indispensable role for systemic infections in cucumber. Here, we report that Cmv2b became essential for systemic infections in older N. glutinosa plants or in young seedlings pretreated with salicylic acid (SA). Expression of Cmv2b from the genome of either CMV or Tobacco mosaic virus significantly reduced the inhibitory effect of SA on virus accumulation in inoculated leaves and systemic leaves. A close correlation is demonstrated between Cmv2b expression and a reduced SA-dependent induction of the alternative oxidase gene, a component of the recently proposed SA-regulated antiviral defense. These results collectively reveal a novel activity of Cmv2b in the inhibition of SA-mediated virus resistance. We used a N. tabacum line expressing a bacterial nahG transgene that degrades SA to provide evidence for a Cmv2b-sensitive antiviral defense mechanism in tobacco in which SA acts as a positive modifier but not as an essential component. We propose that SA induces virus resistance by potentiating a RNA-silencing antiviral defense that is targeted by Cmv2b.

Viral suppressors of RNA silencing.

The suppression of RNA silencing by plant viruses represents a viral adaptation to a novel host antiviral defense. Three types of viral suppressors have been identified through the use of a variety of silencing suppression assays. The first two types of suppressor are capable of a complete or partial reversal of pre-existing RNA silencing; the third type does not reverse RNA silencing but can instead prevent its systemic signaling.

[Effect of xinhe granule on vascular endothelial damage and endothelial expressed endothelin and intercellular adhesion molecule-1 in rats fed with high lipid diet].

OBJECTIVE: To study the protective effect of Xinhe granule (XHG) on vascular endothelial damage in rats fed with high lipid diet. METHODS: Model of vascular endothelial damage was formed by feeding high lipid diet in rats. The model animal were divided into high dosage XHG group, low dosage XHG group, composite Salvia dripping pellet group and model control group, and a blank (normal) control group was also set up. The degree of endothelial damage and positive cell count of synthesizing and secreting endothelin (ET-1) and intercellular adhesion molecule-1 (ICAM-1) were determined by endothelial cell spreading technique and immunohistochemical technique quantitatively. RESULTS: Comparison of the degree on vascular endothelial damage and endothelial secreting ICAM-1 and ET-1 showed model control group > composite Salvia dripping pellet group > low dosage XHG group > high dosage XHG group > blank control group. XHG did not show obvious lowering action on blood lipid. CONCLUSION: XHG could inhibit the endothelial expressed ICAM-1 and ET-1 in hyperlipidemia rats, thus displaying the protective effect on vascular endothelium.

Suppression of post-transcriptional gene silencing by a plant viral protein localized in the nucleus.

Post-transcriptional gene silencing (PTGS) is a homology-dependent RNA degradation process that may target RNA exclusively in the cytoplasm. In plants, PTGS functions as a natural defense mechanism against viruses. We reported previously that the 2b protein encoded by cucumber mosaic cucumovirus (CMV) is a virulence determinant and a suppressor of PTGS initiation in transgenic Nicotiana benthamiana. By fusion with the green fluorescent protein, we now show that the CMV 2b protein localizes to the nuclei of tobacco suspension cells and whole plants via an arginine-rich nuclear localization signal, (22)KRRRRR(27). We further demonstrate that the nuclear targeting of the 2b protein is required for the efficient suppression of PTGS, indicating that PTGS may be blocked in the nucleus. In addition, our data indicate that the PTGS suppressor activity is important, but not sufficient, for virulence determination by the 2b protein.

RNA silencing.

Gene silencing through the increased degradation of mRNA appears to represent a novel cellular pathway that is functional in a broad range of organisms. Recent work has established a role for RNA silencing in host antiviral defense and transposon silencing, suggesting a potential application in plant functional genomics.

Strong host resistance targeted against a viral suppressor of the plant gene silencing defence mechanism.

The 2b protein encoded by cucumber mosaic cucumovirus (Cmv2b) acts as an important virulence determinant by suppressing post-transcriptional gene silencing (PTGS), a natural plant defence mechanism against viruses. We report here that the tomato aspermy cucumovirus 2b protein (Tav2b), when expressed from the unrelated tobacco mosaic tobamovirus (TMV) RNA genome, activates strong host resistance responses to TMV in tobacco which are typical of the gene-for-gene disease resistance mechanism. Domain swapping between Cmv2b, which does not elicit these responses, and Tav2b, revealed functional domains in Tav2b critical for triggering virus resistance and hypersensitive cell death. Furthermore, substitution of two amino acids from Tav2b by those found at the same positions in Cmv2b, Lys21-->Val and Arg28-->Ser, abolished the ability to induce hypersensitive cell death and virus resistance. However, in Nicotiana benthamiana, a species related to tobacco, Tav2b functions as a virulence determinant and suppresses PTGS. Thus, a viral suppressor of the host gene silencing defence mechanism is the target of another independent host resistance mechanism. Our results provide new insights into the complex molecular strategies employed by viruses and their hosts for defence, counter-defence and counter counter-defence.

Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana.

Post-transcriptional gene silencing (PTGS) of a green fluorescent protein (GFP) transgene is suppressed in Nicotiana benthamiana plants infected with potato virus Y (PVY) or with cucumber mosaic virus (CMV), but not in plants infected with potato virus X (PVX). By expressing PVY and CMV-encoded proteins in a PVX vector we have shown that the viral suppressors of gene silencing are the HCPro of PVY and the 2b protein of CMV. The HCPro acts by blocking the maintenance of PTGS in tissues where silencing had already been set, whereas the 2b protein prevents initiation of gene silencing at the growing points of the plants. Combined with previous findings that viruses are both activators and targets of PTGS, these data provide compelling evidence that PTGS represents a natural mechanism for plant protection against viruses.

Ilarviruses encode a Cucumovirus-like 2b gene that is absent in other genera within the Bromoviridae.

We found that RNA 2 of the four ilarviruses sequenced to date encodes an additional conserved open reading frame (ORF), 2b, that overlaps the 3' end of the previously known ORF, 2a. A novel RNA species of 851 nucleotides was found to accumulate to high levels in plants infected with spinach latent virus (SpLV). Further analysis showed that RNA 4A is a subgenomic RNA of RNA 2 and encodes all of ORF 2b. Moreover, a protein species of the size expected for SpLV ORF 2b was translated in vitro from the RNA 4A-containing virion RNAs. The data support the suggestion that the SpLV 2b protein is translated in vivo. The 2b gene of ilarviruses, which is not encoded by alfamoviruses and bromoviruses, shares several features with the previously reported cucumovirus 2b gene; however, their encoded proteins share no detectable sequence similarities. The evolutionary origin of the 2b gene is discussed.

Plasmid vector for cloning infectious cDNAs from plant RNA viruses: high infectivity of cDNA clones of tomato aspermy cucumovirus.

An improved version of the previously obtained cloning vector pCass was constructed by partially duplicating the 35S promoter used to drive the transient transcription of cloned viral cDNAs. Full-length cDNAs of the three genomic RNAs of tomato aspermy cucumovirus (TAV) cloned in this improved pCass (designated pCass2) gave a 3-fold higher infectivity in two plant species tested than the same cDNAs cloned in pCass1 with only a single 35S promoter. Host range, symptoms, morphology of viral particles and viral progeny RNAs induced by these sets of infectious cDNA clones analysed were identical to those induced by the wild-type virus. A mutant of genomic TAV RNA 3 containing a 163 nt deletion in the 3' untranslated region was stably maintained in the progeny RNAs, indicating that these cDNA clones may facilitate a study of virus function. This is the first report of infectious cDNA clones of TAV as well as of infectious cDNA clones with a duplicated 35S promoter of CaMV.

In vivo expression of an overlapping gene encoded by the cucumoviruses.

We recently reported the molecular characterization and functional analysis of an overlapping gene 2b encoded by RNA 2 of the Q strain of cucumber mosaic cucumovirus (Q-CMV). We show here that the homologous gene encoded by the V strain of tomato aspermy cucumovirus (V-TAV) and the WAII strain of CMV (WAII-CMV), which is in a different subgroup to Q-CMV, is also expressed in vivo by demonstrating the accumulation of the mRNA (RNA 4A) and its protein in infected plants. Interestingly, RNA 4A of V-TAV is encapsidated in virions as found previously for Q-CMV whereas WAII-CMV contains very little RNA 4A in virions. As the 2b gene is conserved in all 10 cucumoviral species or strains sequenced to date and the 2b gene is expressed for three of these viruses, we conclude that the 2b gene is a common feature of the Cucumovirus genus.

An interspecies hybrid RNA virus is significantly more virulent than either parental virus.

Cucumber mosaic cucumovirus (CMV) infects a very wide range of plant species (>1000 species). We recently demonstrated that a previously undescribed gene (2b) encoded by RNA 2 of the tripartite RNA genome of CMV is required for systemic virus spread and disease induction in its hosts. Herein we report that when this CMV gene is replaced by its homologue from tomato aspermy cucumovirus (TAV), the resultant hybrid virus is significantly more virulent, induces earlier onset of systemic symptoms, and accumulates to a higher level in seven host species from three families than either of the parents. Our results indicate that CMV and the TAV 2b protein interact synergistically despite the fact that no synergism occurs in double infections with the two parental viruses. To our knowledge, this is the first example of an interspecific hybrid made from plant or animal RNA viruses that is more efficient in systemic infection of a number of hosts than the naturally occurring parents. As CMV and the hybrid virus accumulated to a similar level in the infected tobacco protoplasts, the observed synergistic responses most likely resulted from an increased efficacy of the hybrid virus in systemic spread in host plants provided by the TAV 2b protein. The relevance of our finding to the application of pathogen-derived resistance is discussed.

A novel naturally occurring hybrid gene encoded by a plant RNA virus facilitates long distance virus movement.

We recently identified a new cucumovirus-specific gene (2b) which is encoded by RNA 2 of the cucumber mosaic cucumovirus (CMV) tripartite RNA genome and whose coding sequence overlaps the C-terminal 69 codons of ORF 2a encoding the RNA polymerase protein. We have now found that although a CMV mutant lacking ORF 2b accumulated in the inoculated cotyledons of cucumber plants, it was unable to spread systemically, demonstrating involvement of 2b in long distance movement. The same mutant infected tobacco systemically with a much reduced virulence and delayed appearance of symptoms, indicating that 2b may contribute to long distance movement in this host. Deletion of the overlapping C-terminal part of ORF 2a did not change infectivity of the mutant in either host species, ruling out 2a mutation as the reason for the change of phenotype. Further infectivity studies with mutants containing partial deletions in ORF 2b further supported the conclusion that 2b encodes a host-specific long distance movement function. Sequence analysis revealed that 2b may represent a novel naturally occurring hybrid gene important to the evolutionary formation of the cucumovirus group and that it could provide a genetic basis for the wide host range of these viruses.

Efficient infection from cDNA clones of cucumber mosaic cucumovirus RNAs in a new plasmid vector.

Full-length cucumber mosaic cucumovirus (CMV) cDNAs were cloned into a new plasmid vector containing a modified plant virus promoter designed to transcribe the inserted sequence from its first nucleotide. cDNA copies of CMV strain Q (Q-CMV) genomic RNAs 1, 2 and 3 cloned into this vector were infectious when inoculated together, producing symptoms indistinguishable from those caused by wildtype Q-CMV infection. The infectivity of the clones could be substantially increased by excision of the viral insert together with the transcriptional promoter and terminator prior to inoculation. A diagnostic but silent mutation was introduced into the RNA 2 cDNA and found to be stably maintained in viral infection, allowing distinction of the recombinant virus from native contaminants. The infectious cDNA clones supported the replication of CMV satellite RNA when co-inoculated with biologically active Q-CMV satellite RNA transcripts. Using the infectious cDNAs described, it was found that a newly-identified overlapping gene (2b) encoded by Q-CMV RNA 2 was not essential for either systemic viral infection of Nicotiana glutinosa or replication of the satellite RNAs.

New overlapping gene encoded by the cucumber mosaic virus genome.

Cucumber mosaic virus (CMV) contains three genomic RNAs (RNAs 1, 2, and 3) and a subgenomic RNA (RNA 4), a shared feature of the Bromoviridae family which includes cucumoviruses, bromoviruses, alfalfa mosaic virus, and ilarviruses. We describe in this paper the molecular characterization of a novel subgenomic RNA of the Q strain of CMV (Q-CMV), RNA 4A, which was first reported in 1973 (K. W. C. Peden and R. H. Symons, Virology 53, 487-492, 1973). RNA 4A is 682 nucleotides and is identical in sequence to the 3'-terminal 682 nucleotides of RNA 2. RNA 4A encodes a small open reading frame (ORF) of 100 codons, which, in RNA 2, overlaps the C-terminal portion of the major 2a gene; thus it is likely that RNA 4A functions as the mRNA for the in vivo expression of the ORF, called ORF 2b. Polyclonal antibodies raised against a 2b fusion protein expressed in Escherichia coli specifically detected the 2b gene product in Q-CMV-infected cucumber plants by Western immunoblotting. Examination of published viral RNA sequences revealed the conservation of ORF 2b in all of the four other cucumoviruses sequenced to date; however, it is absent from the rest of the Bromoviridae. We suggest that the proposed ORF 2b may be expressed in other cucumoviruses, most likely via 4A-like subgenomic RNAs, and that the predicted gene product may have a unique functional role in the infection process of cucumoviruses.