The cytosolic nucleoprotein of the plant-infecting bunyavirus tomato spotted wilt recruits endoplasmic reticulum-resident proteins to endoplasmic reticulum export sites.

In contrast with animal-infecting viruses, few known plant viruses contain a lipid envelope, and the processes leading to their membrane envelopment remain largely unknown. Plant viruses with lipid envelopes include viruses of the Bunyaviridae, which obtain their envelope from the Golgi complex. The envelopment process is predominantly dictated by two viral glycoproteins (Gn and Gc) and the viral nucleoprotein (N).

Noncoding flavivirus RNA displays RNA interference suppressor activity in insect and Mammalian cells.

West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants.

Analysis of the Tomato spotted wilt virus ambisense S RNA-encoded hairpin structure in translation.

Background: The intergenic region (IR) of ambisense RNA segments from animal- and plant-infecting (-)RNA viruses functions as a bidirectional transcription terminator. The IR sequence of the Tomato spotted wilt virus (TSWV) ambisense S RNA contains stretches that are highly rich in A-residues and U-residues and is predicted to fold into a stable hairpin structure. The presence of this hairpin structure sequence in the 3' untranslated region (UTR) of TSWV mRNAs implies a possible role in translation.

Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells.

Background: Chikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively.

Base-pairing promotes leader selection to prime in vitro influenza genome transcription.

The requirements for alignment of capped leader sequences along the viral genome during influenza transcription initiation (cap-snatching) have long been an enigma. In this study, competition experiments using an in vitro transcription assay revealed that influenza virus transcriptase prefers leader sequences with base complementarity to the 3'-ultimate residues of the viral template, 10 or 11 nt from the 5' cap. Internal priming at the 3'-penultimate residue, as well as prime-and-realign was observed.

Preferential use of RNA leader sequences during influenza A transcription initiation in vivo.

In vitro transcription initiation studies revealed a preference of influenza A virus for capped RNA leader sequences with base complementarity to the viral RNA template. Here, these results were verified during an influenza infection in MDCK cells. Alfalfa mosaic virus RNA3 leader sequences mutated in their base complementarity to the viral template, or the nucleotides 5' of potential base-pairing residues, were tested for their use either singly or in competition.

Diverging affinity of tospovirus RNA silencing suppressor proteins, NSs, for various RNA duplex molecules.

The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.

A distinct tospovirus causing necrotic streak on Alstroemeria sp. in Colombia.

A tospovirus causing necrotic streaks on leaves was isolated from Alstroemeria sp. in Colombia. Infected samples reacted positively with tomato spotted wilt virus (TSWV) antiserum during preliminary serological tests.

BNYVV-derived dsRNA confers resistance to rhizomania disease of sugar beet as evidenced by a novel transgenic hairy root approach.

Agrobacterium rhizogenes-transformed sugar beet hairy roots, expressing dsRNA from the Beet necrotic yellow vein virus replicase gene, were used as a novel approach to assess the efficacy of three intron-hairpin constructs at conferring resistance to rhizomania disease. Genetically engineered roots were similar in morphology to wild type roots but were characterized by a profound abundancy, rapid growth rate and, in some cases, plagiotropic development. Upon challenge inoculation, seedlings showed a considerable delay in symptom development compared to untransformed or vector-transformed seedlings, expressing dsRNA from an unrelated source.

Potatovirus X and Tobacco mosaic virus-based vectors compatible with the Gateway cloning system.

Virus-based expression vectors are important tools for high-level production of foreign proteins and for gene function analysis through virus induced gene silencing. To exploit further their advantages as fast, high yield replicons, a set of vectors was produced by converting and adapting Potato virus X (PVX) and Tobacco mosaic virus (TMV)-based vectors to allow easy cloning of foreign sequences by the Gateway cloning system. Target genes were cloned efficiently by recombination and successfully expressed in Nicotiana benthamiana following inoculation by Agrobacterium (agroinfection).

RNAi-mediated transgenic Tospovirus resistance broken by intraspecies silencing suppressor protein complementation.

Extension of an inverted repeat transgene cassette, containing partial nucleoprotein (N) gene sequences from four different tomato-infecting Tospovirus spp. with a partial N gene sequence from the tomato strain of Tomato yellow ring virus (TYRV-t), renders transgenic Nicotiana benthamiana plants additionally resistant to this strain but not to the soybean strain of this Tospovirus sp. (TYRV-s), both strains exhibiting 14.

Requirements for ER-arrest and sequential exit to the golgi of Tomato spotted wilt virus glycoproteins.

The envelope glycoproteins Gn and Gc are major determinants in the assembly of Tomato spotted wilt virus (TSWV) particles at the Golgi complex. In this article, the ER-arrest of singly expressed Gc and the transport of both glycoproteins to the Golgi upon coexpression have been analyzed.While preliminary results suggest that the arrest of Gc at the ER (endoplasmic reticulum) did not appear to result from improper folding, transient expression of chimeric Gc, in which the transmembrane domain (TMD) and/or cytoplasmic tail (CT) were swapped for those from Gn, showed that the TMD of Gn was sufficient to allow ER exit and transport to the Golgi.

Binding of small interfering RNA molecules is crucial for RNA interference suppressor activity of rice hoja blanca virus NS3 in plants.

The NS3 protein of rice hoja blanca virus represents a viral suppressor of RNA interference (RNAi) that sequesters small interfering (si)RNAs in vitro. To determine whether this siRNA binding property is the critical determinant for the suppressor activity of NS3, NS3 was altered by alanine point mutations and the resulting mutant proteins were tested for both siRNA binding ability and RNAi suppressor activity in plants. Alanine substitutions of lysine residues at positions 173-175 resulted in mutant proteins that lost both their affinity for siRNAs and their RNAi suppressor activity in planta.

The NS3 protein of rice hoja blanca virus complements the RNAi suppressor function of HIV-1 Tat.

The question of whether RNA interference (RNAi) acts as an antiviral mechanism in mammalian cells remains controversial. The antiviral interferon (IFN) response cannot easily be distinguished from a possible antiviral RNAi pathway owing to the involvement of double-stranded RNA (dsRNA) as a common inducer molecule. The non-structural protein 3 (NS3) protein of rice hoja blanca virus (RHBV) is an RNA silencing suppressor (RSS) that exclusively binds to small dsRNA molecules.

Tomato spotted wilt virus nucleocapsid protein interacts with both viral glycoproteins Gn and Gc in planta.

Recently, the Tomato Spotted Wilt Virus (TSWV) Gn and Gc glycoproteins were shown to induce the formation of (pseudo-) circular and pleomorphic membrane structures upon transient expression in plant cells. Furthermore, when singly expressed, Gc retains in the ER, while Gn is able to further migrate to the Golgi. Upon co-expression, Gn rescues Gc and co-migrates to the Golgi complex.

A new tomato-infecting tospovirus from iran.

ABSTRACT A new tospovirus species serologically distinct from all other established tospoviruses was found in tomato in Iran. Typical disease symptoms observed include necrotic lesions on the leaves and yellow ring spots on the fruits, hence the name Tomato yellow ring virus (TYRV) was proposed. The S RNA of this virus was cloned and its 3,061 nucleotide long sequence showed features characteristic for tospoviral S RNA segments.

Tomato spotted wilt virus glycoproteins induce the formation of endoplasmic reticulum- and Golgi-derived pleomorphic membrane structures in plant cells.

Tomato spotted wilt virus (TSWV) particles are spherical and enveloped, an uncommon feature among plant infecting viruses. Previous studies have shown that virus particle formation involves the enwrapment of ribonucleoproteins with viral glycoprotein containing Golgi stacks. In this study, the localization and behaviour of the viral glycoproteins Gn and Gc were analysed, upon transient expression in plant protoplasts.

The NS3 protein of rice hoja blanca virus suppresses RNA silencing in mammalian cells.

The NS3 protein of the tenuivirus rice hoja blanca virus (RHBV) has previously been shown to represent the viral RNA interference (RNAi) suppressor and is active in both plant and insect cells by binding short interfering RNAs (siRNAs) in vitro. Using a firefly luciferase-based silencing assay it is described here that NS3 is also active in mammalian cells. This activity is independent of the inducer molecule used.

Assessing the expression of chicken anemia virus proteins in plants.

Chicken anemia virus (CAV) is an important pathogen of chicken worldwide, causing severe anemia and immunodeficiency. Its small single-stranded DNA genome (2.3kb) encodes three proteins: VP1, the only structural protein, VP2, a protein phosphatase, and VP3, also known as apoptin, which induces apoptosis.

The nucleoprotein of Tomato spotted wilt virus as protein tag for easy purification and enhanced production of recombinant proteins in plants.

Upon infection, Tomato spotted wilt virus (TSWV) forms ribonucleoprotein particles (RNPs) that consist of nucleoprotein (N) and viral RNA. These aggregates result from the homopolymerization of the N protein, and are highly stable in plant cells. These properties feature the N protein as a potentially useful protein fusion partner.

The NS3 protein of Rice hoja blanca tenuivirus suppresses RNA silencing in plant and insect hosts by efficiently binding both siRNAs and miRNAs.

RNA silencing plays a key role in antiviral defense as well as in developmental processes in plants and insects. Negative strand RNA viruses such as the plant virus Rice hoja blanca tenuivirus (RHBV) replicate in plants and in their insect transmission vector. Like most plant-infecting viruses, RHBV encodes an RNA silencing suppressor, the NS3 protein, and here it is demonstrated that this protein is capable of suppressing RNA silencing in both plants and insect cells.

The cytoplasmic domain of tomato spotted wilt virus Gn glycoprotein is required for Golgi localisation and interaction with Gc.

Envelopment of tomato spotted wilt virus nucleocapsids occurs at the Golgi stacks of infected cells. This is also the place where the two membrane glycoproteins Gn and Gc accumulate upon coexpression. The required Golgi retention signal has previously been demonstrated to reside within Gn.

Tomato chlorotic mottle virus is a target of RNA silencing but the presence of specific short interfering RNAs does not guarantee resistance in transgenic plants.

Tomato chlorotic mottle virus (ToCMoV) is a begomovirus found widespread in tomato fields in Brazil. ToCMoV isolate BA-Se1 (ToCMoV-[BA-Se1]) was shown to trigger the plant RNA silencing surveillance in different host plants and, coinciding with a decrease in viral DNA levels, small interfering RNAs (siRNAs) specific to ToCMoV-[BA-Se1] accumulated in infected plants. Although not homogeneously distributed, the siRNA population in both infected Nicotiana benthamiana and tomato plants represented the entire DNA-A and DNA-B genomes.

Multiple virus resistance at a high frequency using a single transgene construct.

RNA silencing is a natural antiviral defence in plants, which can be exploited in transgenic plants for preprogramming virus recognition and ensuring enhanced resistance. By arranging viral transgenes as inverted repeats it is thus possible to obtain strong repression of incoming viruses. Due to the high sequence specificity of RNA silencing, this technology has hitherto been limited to the targeting of single viruses.

Tomato spotted wilt virus Gc and N proteins interact in vivo.

Tomato spotted wilt virus (TSWV) virions consist of a nucleocapsid core surrounded by a membrane containing glycoproteins Gn and Gc. To unravel the protein interactions involved in the membrane acquisition of RNPs, TSWV nucleocapsid protein (N), Gn and Gc were expressed and analyzed in BHK21 cells. Upon coexpression of Gn, Gc and N, a partial colocalization of N with both glycoproteins was observed in the Golgi region.