Viral 2A Research Articles

A Novel Methyltransferase MethylatesCucumber Mosaic Virus1a Protein and Promotes Systemic Spread

In mammalian and yeast systems, methyltransferases have been implicated in the regulation of diverse processes, such as protein-protein interactions, protein localization, signal transduction, RNA processing, and transcription. The Cucumber mosaic virus (CMV) 1a protein is essential not only for virus replication but also for movement. Using a yeast two-hybrid system with tobacco plants, we have identified a novel gene encoding a methyltransferase that interacts with the CMV 1a protein and have designated this gene Tcoi1 (tobacco CMV 1a-interacting protein 1). Tcoi1 specifically interacted with the methyltransferase domain of CMV 1a, and the expression of Tcoi1 was increased by CMV inoculation. Biochemical studies revealed that the interaction of Tcoi1 with CMV 1a protein was direct and that Tcoi1 methylated CMV 1a protein both in vitro and in vivo. The CMV 1a binding activity of Tcoi1 is in the C-terminal domain, which shows the methyltransferase activity. The overexpression of Tcoi1 enhanced the CMV infection, while the reduced expression of Tcoi1 decreased virus infectivity. These results suggest that Tcoi1 controls the propagation of CMV through an interaction with the CMV 1a protein.

Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization

Efficient in vitro systems to study the life cycle of hepatitis C virus (HCV) were recently developed for JFH1 (genotype 2a), which has unique replication capacity in Huh7 cells. We developed 4a/JFH1 intergenotypic recombinants containing the structural genes (Core, E1, and E2), p7, and all or part of NS2 of the 4a prototype strain ED43 that, after transfection of Huh7.5 cells with RNA transcripts, produced infectious viruses. Compared with the J6/JFH control virus, production of viruses was delayed. However, efficient spread of infection and high HCV RNA and infectivity titers were obtained in serial passages. Sequence analysis of recovered viruses and subsequent reverse genetic studies revealed a vital dependence on one or two NS2 mutations, depending on the 4a/2a junction. Infectivity of ED43/JFH1 viruses was CD81 dependent. The genotype 4 cell culture systems permit functional analyses as well as drug and vaccine research on an increasingly important genotype in the Middle East, Africa, and Europe. We also developed genotype 1a intergenotypic recombinants from H77C with vital mutations in NS3. Using H77C/JFH1 and ED43/JFH1 viruses, we demonstrated high homologous neutralizing antibody titers in 1a and 4a patient sera, respectively. Furthermore, availability of JFH1 viruses with envelope proteins of the six major HCV genotypes permitted cross-neutralization studies; 1a and 4a serum cross-neutralized 1a, 4a, 5a, and 6a but not 2a and 3a viruses. Thus, the JFH1 intergenotypic recombinants will be of importance for future studies of HCV neutralization and accelerate the development of passive and active immunoprophylaxis.

The Efficacy of Short-Term Interferon-Beta Therapy for Type C Cirrhotic Patients with Genotype 2a and Low Virus Load

The aim of this study was to elucidate the efficacy of short-term interferon (IFN) therapy for type C cirrhotic patients with genotype 2a and low virus load. The present study was retrospective cohort study. Inclusion criteria were liver cirrhosis, hepatitis C virus (HCV) genotype 2a, the serum HCV RNA level of less than 100 KIU/mL, and IFN period of 6 or 8 weeks. Twenty-five consecutive patients who satisfied the above criteria were treated with IFN-beta daily at the dosage of 6 MU for 6 or 8 weeks. Independent factors that might have influenced sustained virologic response (SVR) were studied using multiple logistic regression analysis. Background of clinical profiles were as follows: median (range) age=64 (53-76) years, male/female=13/12, and median (range) HCV-RNA=31 (8-90) KIU/mL. Out of 25, 14 patients (56.0%) had SVR by the intention-to-treat analysis. The SVR was significantly associated with serum HCV RNA level. Logistic analysis showed that SVR occurred when HCV RNA level was <50 KIU/mL (p=0.047). Based on the difference of the serum HCV RNA level, the SVR rate was 68.4% (13/19) in patients with a serum HCV RNA level of <50 KIU/mL and 16.7% (1/6) in patients with a serum HCV RNA level of > or =50 KIU/mL. The 6 or 8-week IFN-beta therapy is a possible selection of therapy for cirrhotic patients with HCV genotype 2a and a serum HCV RNA level of <50 KIU/mL.

Reduction of phospholipase D activity during coxsackievirus infection

During enterovirus infection, host cell membranes are rigorously rearranged and modified. One ubiquitously expressed lipid-modifying enzyme that might contribute to these alterations is phospholipase D (PLD). Here, we investigated PLD activity in coxsackievirus-infected cells. We show that PLD activity is not required for efficient coxsackievirus RNA replication. Instead, PLD activity rapidly decreased upon infection and upon ectopic expression of the viral 3A protein, which inhibits the PLD activator ADP-ribosylation factor 1. However, similar decreases were observed during infection with coxsackieviruses carrying defective mutant 3A proteins. Possible causes for the reduction of PLD activity and the biological consequences are discussed.

The use cloned DNA probes for the detection and identification of cocoa swollen shoot virus (CSSV) 1A and CSSV Nsaba

The use cloned DNA probes for the detection and identification of cocoa swollen shoot virus (CSSV) 1A and CSSV Nsaba

Viral 2A peptides allow expression of multiple proteins from a single ORF in transgenic zebrafish embryos

We have adapted a novel multicistronic gene expression system involving viral peptides to the zebrafish. The viral 2A peptide allows production of multiple protein products from a single transgene. Based on highly inefficient peptide bond formation between glycine and proline residues within the 2A peptide, placement of 2A peptide sequence as a linker region between tandem cDNA's allows the stoichiometric translation of multiple unfused protein products. To test this system in zebrafish, we generated two different tandem reporter constructs employing eGFP and mCherry, separated by 2A peptide sequence. Using this system, we produced transgenic zebrafish in which fluorophores were produced as independent proteins from a single transcript. The successful application of this technology in zebrafish will be valuable for visually marking transgenic embryos and transgene-expressing cells, or in any situation where reliable expression of multiple transgenes is desired.

Cardiovirus 2A Protein Associates with 40S but Not 80S Ribosome Subunits during Infection

Host translation shutoff induced in picornavirus-infected cells is a well-known phenomenon. The mechanisms by which separate genera of the picornavirus family achieve this shutoff differ. This study examined alterations in the cellular translational components in HeLa cells infected with encephalomyocarditis virus (EMCV), a cardiovirus. In agreement with previous reports, EMCV induced a marked decrease in host mRNA translation. The inhibition correlated with the appearance of a significantly enhanced 80S peak in cells and a concomitant decrease in polysome abundance. Characterization of the 80S material revealed that these ribosomes were virtually devoid of mRNA. Viral protein 2A was tightly associated with some of the free 40S ribosome subunits, but it was not present in the 80S pool which accumulated after infection. Expression of 2A protein in cells in the absence infection was able to modulate the cellular translational environment to increase the ratio of internal ribosome entry site-dependent translation to cap-dependent translation of a reporter construct. The results provide further evidence for a role of 2A protein in the mechanism of cardiovirus-induced host translational shutoff.

Expression studies of the core+1 protein of the hepatitis C virus 1a in mammalian cells

Recent studies have suggested the existence of a novel protein of hepatitis C virus (HCV) encoded by an ORF overlapping the core gene in the +1 frame (core+1 ORF). Two alternative translation mechanisms have been proposed for expression of the core+1 ORF of HCV-1a in cultured cells; a frameshift mechanism within codons 8-11, yielding a protein known as core+1/F, and/or translation initiation from internal codons in the core+1 ORF, yielding a shorter protein known as core+1/S. To date, the main evidence for the expression of this protein in vivo has been the specific humoral and cellular immune responses against the protein in HCV-infected patients, inasmuch as its detection in biopsies or the HCV infectious system remains elusive. In this study, we characterized the expression properties of the HCV-1a core+1 protein in mammalian cells in order to identify conditions that facilitate its detection. We showed that core+1/S is a very unstable protein, and that expression of the core protein in addition to proteosome activity can downregulate its intracellular levels. Also, we showed that in the Huh-7/T7 cytoplasmic expression system the core+1 ORF from the HCV-1 isolate supports the synthesis of both the core+1/S and core+1/F proteins. Finally, immunofluorescence and subcellular fractionation analyses indicated that core+1/S and core+1/F are cytoplasmic proteins with partial endoplasmic reticulum distribution in interphase cells, whereas in dividing cells they also localize to the microtubules of the mitotic spindle.

Infection of human hepatocyte chimeric mouse with genetically engineered hepatitis C virus and its susceptibility to interferon

We developed a reverse genetics system of hepatitis C virus (HCV) genotypes 1a and 2a using infectious clones and human hepatocyte chimeric mice. We inoculated cell culture-produced genotype 2a (JFH-1) HCV intravenously. We also injected genotype 1a CV-H77C clone RNA intrahepatically. Mice inoculated with HCV by both procedures developed measurable and transmissible viremia. Interferon (IFN) alpha treatment resulted in greater reduction of genotype 2a HCV levels than genotype 1a, as seen in clinical practice. Genetically engineered HCV infection system should be useful for analysis of the mechanisms of resistance of HCV to IFN and other drugs.

P21-activated Kinase 1 Is Activated through the Mammalian Target of Rapamycin/p70 S6 Kinase Pathway and Regulates the Replication of Hepatitis C Virus in Human Hepatoma Cells

Cellular mechanisms that regulate the replication of hepatitis C virus (HCV) RNA are poorly understood. p21-activated kinase 1 (PAK1) is a serine/threonine kinase that has been suggested to participate in antiviral signaling. We studied its role in the cellular control of HCV replication. Transfection of PAK1-specific small interfering RNA enhanced viral RNA and protein abundance in established replicon cell lines as well as cells infected with chimeric genotype 1a/2a HCV, despite reducing cellular proliferation, suggesting specific regulation of HCV replication. PAK1 knockdown did not reduce interferon regulatory factor 3-dependent gene expression, indicating that this regulation is independent of the retinoic acid-inducible gene I/interferon regulatory factor 3 pathway. On the other hand, LY294002 and rapamycin abolished PAK1 phosphorylation and enhanced HCV abundance, suggesting that the mammalian target of rapamycin (mTOR) is involved in PAK1 regulation of HCV. Small interfering RNA knockdown of the mTOR substrate p70 S6 kinase abrogated PAK1 phosphorylation and enhanced HCV RNA abundance, whereas overexpression of a constitutively active alternate substrate, eukaryotic translation initiation factor 4E-binding protein 1, increased cap-independent viral translation and viral RNA abundance without influencing PAK1 phosphorylation. Similar data indicated that mTOR is regulated by both phosphatidylinositol 3-kinase/Akt and ERK. Taken together, the data indicate that p70 S6 kinase activates PAK1 and contributes to phosphatidylinositol 3-kinase- and ERK-mediated regulation of HCV RNA replication.

Application of fluorescence resonance energy transfer resolved by fluorescence lifetime imaging microscopy for the detection of enterovirus 71 infection in cells

Timely and effective virus infection detection is critical for the clinical management and prevention of the disease spread in communities during an outbreak. A range of methods have been developed for this purpose, of which classical serological and viral nucleic acids detection are the most popular. We describe an alternative, imaging-based approach that utilizes fluorescence resonance energy transfer (FRET) resolved by fluorescence lifetime imaging microscopy (FLIM) and demonstrate it on the example of enterovirus 71 (EV71) infection detection. A plasmid construct is developed with the sequence for GFP2 and DsRed2 fluorescent proteins, linked by a 12-amino-acid-long cleavage recognition site for the 2A protease (2A(pro)), encoded by the EV71 genome and specific for the members of Picornaviridae family. In the construct expressed in HeLa cells, the linker binds the fluorophores within the Forster distance and creates a condition for FRET to occur, thus resulting in shortening of the GFP2 fluorescence lifetime. On cells infection with EV71, viral 2A(pro) released to the cytoplasm cleaves the recognition site, causing disruption of FRET through separation of the fluorophores. Thus, increased GFP2 lifetime to the native values, manifested by the time-correlated single-photon counting, serves as an efficient and specific indicator of the EV71 virus infection.

Selective Repression of Translation by the Brome Mosaic Virus 1a RNA Replication Protein

Differential expression of viral replication proteins is essential for successful infection. We report here that overexpression of the brome mosaic virus (BMV) 1a protein can repress viral RNA replication in a dosage-dependent manner. Using RNA replication-incompetent reporter constructs, repression of translation from BMV RNA1 and RNA2 was observed, suggesting that the effect on translation of the BMV RNA replication proteins is responsible for the decrease in RNA levels. Furthermore, repression of translation by 1a required the B box in the 5'-untranslated region (5' UTR); BMV RNA3 that lacks a B box in its 5' UTR is not subject to 1a-mediated translational inhibition. Mutations in either the methyltransferase or the helicase-like domains of 1a reduced the repression of replication and translation. These results suggest that in addition to its known functions in BMV RNA synthesis, 1a also regulates viral gene expression.

Arabidopsis tonoplast proteins TIP1 and TIP2 interact with the cucumber mosaic virus 1a replication protein

The cucumber mosaic virus (CMV) replication complex has previously been shown to associate with cellular membranes. However, it remains unknown whether any host factors participate in this process. In this study, five groups of Arabidopsis tonoplast intrinsic protein (TIP) genes were isolated and the proteins they encoded were evaluated with regard to their interactions with CMV proteins. TIP1 and TIP2 were found to interact with the CMV 1a protein in the Sos recruitment system, whereas no interactions with the other three TIP subgroups were observed in this assay. The interaction of CMV 1a with the TIP1 and TIP2 proteins was confirmed via co-immunoprecipitation assays. Additionally, CMV 1a co-localized with TIP1 and TIP2 in transfected Arabidopsis protoplasts. The findings of this study suggest that members of two TIP subfamilies might affect CMV replication via interaction with CMV 1a in the tonoplasts.

Compensatory Mutations in E1, p7, NS2, and NS3 Enhance Yields of Cell Culture-Infectious Intergenotypic Chimeric Hepatitis C Virus

There is little understanding of mechanisms underlying the assembly and release of infectious hepatitis C virus (HCV) from cultured cells. Cells transfected with synthetic genomic RNA from a unique genotype 2a virus (JFH1) produce high titers of virus, while virus yields are much lower with a prototype genotype 1a RNA containing multiple cell culture-adaptive mutations (H77S). To characterize the basis for this difference in infectious particle production, we constructed chimeric genomes encoding the structural proteins of H77S within the background of JFH1. RNAs encoding polyproteins fused at the NS2/NS3 junction ("H-NS2/NS3-J") and at a site of natural, intergenotypic recombination within NS2 ["H-(NS2)-J"] produced infectious virus. In contrast, no virus was produced by a chimera fused at the p7-NS2 junction. Chimera H-NS2/NS3-J virus (vH-NS2/NS3-J) recovered from transfected cultures contained compensatory mutations in E1 and NS3 that were essential for the production of infectious virus, while yields of infectious vH-(NS2)-J were enhanced by mutations within p7 and NS2. These compensatory mutations were chimera specific and did not enhance viral RNA replication or polyprotein processing; thus, they likely compensate for incompatibilities between proteins of different genotypes at sites of interactions essential for virus assembly and/or release. Mutations in p7 and NS2 acted additively and increased the specific infectivity of vH-(NS2)-J particles, while having less impact on the numbers of particles released. We conclude that interactions between NS2 and E1 and p7 as well as between NS2 and NS3 are essential for virus assembly and/or release and that each of these viral proteins plays an important role in this process.

Modeling-based characterization of the elicitor function of amino acid 461 of Cucumber mosaic virus 1a protein in the hypersensitive response

The Ns strain of Cucumber mosaic virus (CMV) induces hypersensitive response (HR) on Nicotiana tabacum cv. Xanthi-nc and on Nicotiana glutinosa. The genetic determinant of the HR induction was localized earlier to amino acid 461 of the 1a protein. The 3D structure of the 1a protein is still unknown and building a homology model is impossible. Nevertheless, on the basis of secondary structure predictions we have created partial protein models for the region surrounding residue 461 which can account structurally for the effect of aa 461 on elicitor function. Seven different amino acid mutations were designed and introduced to the position 461 of the 1a protein in RNA 1. Three of the mutations (proline, glutamic acid, asparagine) inhibited virus replication. Two of the mutants caused systemic symptom development (lysine and arginine). Two mutants (alanine and serine) resulted in localization of the virus, but strong necrosis similar to the original Ns-CMV strain was not observed. Inoculation of purified Ns-CMV virions at extremely high concentration provoked systemic symptoms.

Functional analysis of human T lymphotropic virus type 2 Tax proteins

BackgroundThe Tax proteins encoded by human T lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) are transcriptional activators of both the viral long terminal repeat (LTR) and cellular promoters via the CREB and NFkB pathways. In contrast to HTLV-1, HTLV-2 has been classified into four distinct genetic subtypes A, B, C and D defined by phylogenetic analysis of their nucleotide sequences and the size and amino acid sequence of their Tax proteins. In the present study we have analysed and compared the transactivating activities of three Tax 2A and one Tax 2B proteins using LTR and NFkB reporter assays.ResultsWe found that with the exception of the prototype Tax 2A Mo protein, the other two Tax 2A proteins failed to transactivate either the viral LTR or NFkB promoter in Jurkat and 293T cells. Loss of activity was not associated with either expression levels or an alteration in subcellular distribution as all Tax 2 proteins were predominantly located in the cytoplasm of transfected cells. Analysis of the sequence of the two inactive Tax 2A proteins relative to Mo indicated that one had six amino acid changes and the other had one change in the central region of the protein. Mutations present at the amino and the extreme carboxy termini of Mo resulted in the loss of LTR but not NFkB activation whereas those occurring in the central region of the protein appeared to abolish transactivation of both promoters. Analysis of the transactivation phenotypes of Tax 1, Tax 2A Mo and Tax 2B containing mutations identified in the present study or previously characterised Tax mutations showed that domains required for LTR and NFkB activation are very similar but not identical in all three Tax proteins.ConclusionOur results suggest that loss of activity of two Tax 2A proteins derived from different isolates is associated with multiple amino acid changes relative to Mo in domains required for the activation of the CREB or CREB and NFkB pathways and that these domains are very similar but not identical in Tax 2B and Tax 1. The loss of Tax function in 2A viruses may have implications for their biological and pathogenic properties.

Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells.

Infections with hepatitis C virus (HCV) are marked by frequent viral persistence, chronic liver disease, and extraordinary viral genetic diversity. Although much has been learned about HCV since its discovery, progress has been slowed by a lack of permissive cell culture systems supporting its replication. Productive infections have been achieved recently with genotype 2a virus, but cirrhosis and liver cancer are typically associated with genotype 1 HCV, which is more prevalent and relatively resistant to IFN therapy. We describe production of infectious genotype 1a HCV in cells transfected with synthetic RNA derived from a prototype virus (H77-S). Viral proteins accumulated more slowly in H77-S transfected cells than in cells transfected with genotype 2a (JFH-1) RNA, but substantially more H77-S RNA was secreted into supernatant fluids. Most secreted RNA was noninfectious, banding in isopycnic gradients at a density of 1.04-1.07 gm/cm(3), but infectivity was associated with H77-S particles possessing a density of 1.13-1.14 gm/cm(3). The specific infectivity of H77-S particles (5.4 x 10(4) RNA copies per focus-forming unit) was significantly lower than JFH-1 virus (1.4 x 10(2) RNA copies per focus-forming unit). Infection with either virus was blocked by CD81 antibody. Sera from genotype 1a-infected individuals neutralized H77-S virus, but had little activity against genotype 2a virus, suggesting that these genotypes represent different serotypes. The ability of this genotype 1a virus to infect cultured cells will substantially benefit antiviral and vaccine discovery programs.

In vivo interaction between Tobacco mosaic virus RNA-dependent RNA polymerase and host translation elongation factor 1A

Several host translation elongation factors have been suggested to play essential roles in the replication and translation of viral RNAs in plants, animals and bacteria. Here, we show the interaction between eukaryotic translation elongation factor 1A (eEF1A) and Tobacco mosaic virus (TMV) RNA-dependent RNA polymerase (RdRp) in vivo by immunoprecipitation. The tobacco eEF1A interacted not only with 3′-untranslated region (3′-UTR) of TMV RNA but also directly with RdRp without mediation by the 3′-UTR. The methyltransferase domain of TMV RdRp was indicated to be responsible for the interaction with eEF1A in vitro and in yeast. These results suggest that eEF1A is a component of the virus replication complex of TMV.

O.012 Cross-reactive epitopes identified in hepatitis C virus envelope proteins induce antibodies that capture virions from infected patients' sera and neutralize HCV/HIV pseudotypes

We separated E1 and E2 of hepatitis C virus (HCV) genotypes 1a, 1b, and 2a into two individual expression plasmids and replaced the transmembrane domains of 1b and 2a E1 and E2 with that of genotype 1a. The complementation features of E1 and E2 as well as the contributions of both the ecto- and transmembrane domains to the formation of the E1E2 complex were evaluated using the HCV pseudoparticle(s) (HCVpp(s)) system.We demonstrated that 1aE2 could not only complement its native 1aE1, but could also complement 1bE1 as well; in genotype 1b, glycoprotein complex formation is primarily dependent on the overall biological characteristics of the intact native E1 and E2; in genotype 2a, although the interaction of intact native E1 and E2 is critical for the formation of the glycoprotein complex, the ectodomain made a greater contribution than that of the transmembrane domain.Our study provides valuable findings regarding HCV E1 and E2 biology and will be of use in both anti-HCV strategy and understanding on the mechanisms of coinfection of different HCV strains.

P.172 Functional comparison of the antagonism of hepatitis C virus genotype 1b and 2a nonstructural protein 5a to interferon antiviral activity

P.172 Functional comparison of the antagonism of hepatitis C virus genotype 1b and 2a nonstructural protein 5a to interferon antiviral activity