Visualization of Transiently Expressed mRNA in Plants Using MS2.

RNA transport and localization are evolutionarily conserved processes that allow protein translation to occur at specific subcellular sites and thereby having fundamental roles in the determination of cell fates, embryonic patterning, asymmetric cell division, and cell polarity. In addition to localizing RNA molecules to specific subcellular sites, plants have the ability to exchange RNA molecules between cells through plasmodesmata (PD). Plant RNA viruses hijack the mechanisms of intracellular and intercellular RNA transport to establish localized replication centers within infected cells and then to disseminate their infectious genomes between cells and throughout the plant organism with the help of their movement proteins (MP). In this chapter, we describe the transient expression of the tobacco mosaic virus movement protein (TMV-MP) and the application of the MS2 system for the in vivo labeling of the MP-encoding mRNA. The MS2 method is based on the binding of the bacteriophage coat protein (CP) to its origin of assembly (OAS) in the phage RNA. Thus, to label a specific mRNA in vivo, a tandem repetition of a 19-nucleotide-long stem-loop (SL) sequence derived from the MS2 OAS sequence (MSL) is transcriptionally fused to the RNA under investigation. The RNA is detected by the co-expression of fluorescent protein-tagged MS2 CP (MCP), which binds to each of the MSL elements. In providing a detailed protocol for the in vivo visualization of TMV-MP mRNA tagged with the MS2 system in Nicotiana benthamiana epidermal cells, we describe (1) the specific DNA constructs, (2) Agrobacterium tumefaciens-mediated transfection for their transient expression in plants, and (3) imaging conditions required to obtain high-quality mRNA imaging data.

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics.

BACKGROUND: Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES: The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS: The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS: We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

Comparison of internal process control viruses for detection of food and waterborne viruses.

Enteric viruses are pathogens associated with food- and waterborne outbreaks. The recovery of viruses from food or water samples is affected by the procedures applied to detect and concentrate them. The incorporation of an internal process control virus to the analyses allows monitoring the performance of the methodology. The aim of this study was to produce a recombinant adenovirus (rAdV) and apply it together with bacteriophage PP7 as process controls. The rAdV carries a DNA construction in its genome to differentiate it from wild-type adenovirus by qPCR. The stability of both control viruses was evaluated at different pH conditions. The rAdV was stable at pH 3, 7, and 10 for 18 h. PP7 infectious particles were stable at pH 7 and showed a 2.14 log reduction at pH 10 and total decay at pH 3 after 18 h. Three virus concentration methods were evaluated: hollow-fiber tap water ultrafiltration, wastewater ultracentrifugation, and elution-PEG precipitation from lettuce. Total and infectious viruses were quantified and their recoveries were calculated. Virus recovery for rAdV and PP7 by ultrafiltration showed a wide range (2.10-84.42 and 13.54-84.62%, respectively), whereas that by ultracentrifugation was 5.05-13.71 and 6.98-13.27%, respectively. The performance of ultracentrifugation to concentrate norovirus and enteroviruses present in sewage was not significantly different to the recovery of control viruses. For detection of viruses from lettuce, genomic copies of PP7 were significantly more highly recovered than adenovirus (14.74-18.82 and 0.00-3.44%, respectively). The recovery of infectious virus particles was significantly affected during sewage ultracentrifugation and concentration from lettuce. The simultaneous use of virus controls with dissimilar characteristics and behaviors might resemble different enteric viruses.

In Vivo RNA Visualization in Plants Using MS2 Tagging.

Intracellular trafficking and asymmetric localization of RNA molecules within cells are a prevalent process across phyla involved in developmental control and signaling and thus in the determination of cell fate. In addition to intracellular localization, plants support the trafficking of RNA molecules also between cells through plasmodesmata (PD), which has important roles in the cell-to-cell and systemic communication during plant growth and development. Viruses have developed strategies to exploit the underlying plant RNA transport mechanisms for the cell-to-cell and systemic dissemination of infection. In vivo RNA visualization methods have revolutionized the study of RNA dynamics in living cells. However, their application in plants is still in its infancy. To gain insights into the RNA transport mechanisms in plants, we study the localization and transport of Tobacco mosaic virus RNA using MS2 tagging. This technique involves the tagging of the RNA of interest with repeats of an RNA stem-loop (SL) that is derived from the origin of assembly of the bacteriophage MS2 and recruits the MS2 coat protein (MCP). Thus, expression of MCP fused to a fluorescent marker allows the specific visualization of the SL-carrying RNA. Here we describe a detailed protocol for Agrobacterium tumefaciens-mediated transient expression and in vivo visualization of MS2-tagged mRNAs in Nicotiana benthamiana leaves.

Promising MS2 mediated virus-like particle vaccine against foot-and-mouth disease.

Foot-and-mouth disease (FMD) has caused severe economic losses to millions of farmers worldwide. In this work, the coding genes of 141-160 epitope peptide (EP141-160) of VP1 were inserted into the coat protein (CP) genes of MS2 in prokaryotic expression vector, and the recombinant protein self-assembled into virus-like particles (VLP). Results showed that the CP-EP141-160 VLP had a strong immunoreaction with the FMD virus (FMDV) antigen in vitro, and also had an effective immune response in mice. Further virus challenge tests were carried out on guinea pigs and swine, high-titer neutralizing antibodies were produced and the CP-EP141-160 VLP vaccine could protect most of the animals against FMDV.

Dissecting the role of protein-protein and protein-nucleic acid interactions in MS2 bacteriophage stability.

To investigate the role of protein-protein and protein-nucleic acid interactions in virus assembly, we compared the stabilities of native bacteriophage MS2, virus-like particles (VLPs) containing nonviral RNAs, and an assembly-defective coat protein mutant (dlFG) and its single-chain variant (sc-dlFG). Physical (high pressure) and chemical (urea and guanidine hydrochloride) agents were used to promote virus disassembly and protein denaturation, and the changes in virus and protein structure were monitored by measuring tryptophan intrinsic fluorescence, bis-ANS probe fluorescence, and light scattering. We found that VLPs dissociate into capsid proteins that remain folded and more stable than the proteins dissociated from authentic particles. The proposed model is that the capsid disassembles but the protein remains bound to the heterologous RNA encased by VLPs. The dlFG dimerizes correctly, but fails to assemble into capsids, because it lacks the 15-amino acid FG loop involved in inter-dimer interactions at the viral fivefold and quasi-sixfold axes. This protein was very unstable and, when compared with the dissociation/denaturation of the VLPs and the wild-type virus, it was much more susceptible to chemical and physical perturbation. Genetic fusion of the two subunits of the dimer in the single-chain dimer sc-dlFG stabilized the protein, as did the presence of 34-bp poly(GC) DNA. These studies reveal mechanisms by which interactions in the capsid lattice can be sufficiently stable and specific to ensure assembly, and they shed light on the processes that lead to the formation of infectious viral particles.

Use of pEX and pGEX bacterial heterologous protein expression systems for recombinant oncoprotein production and antisera generation.

In order to study the role played by known and novel genes in growth control and neoplasia, we here compare the pEX and pGEX bacterial expression systems for recombinant oncoprotein production and for generation of specific antisera. The results of five pEX (MS2-c-Fos, MS2-Fra-1, MS2-JunD, bgal-c-Jun and bgal-JunB) and two pGEX [glutathione S-transferase (GSH)-JE/MCP-1 and GST-JunD] fusion-protein productions are presented. Higher (15-43-fold) yields are obtained with the pEX system, but only the pGEX system allows separation of the protein of interest from the fusion moiety by digestion with specific proteases. The degree of fusion-protein purification, as assessed by SDS/PAGE, is similar for both systems. Proteins produced by both systems were successfully used in the generation of specific antisera. The choice between the pEX and pGEX systems is dependent upon the specific recombinant protein produced.