Proteolytic processing of foot-and-mouth disease virus polyproteins expressed in a cell-free system from clone-derived transcripts

All picornaviral genes are expressed as a single, large polyprotein, which is proteolytically processed into the system produces functional proteins, including viral protease 3C, which plays a major role in processing the precursor proteins. To study the function of the two putative proteases 3C and leader (L) in processing, we constructed several cDNA plasmids encoding various regions of the FMDV type A12 genome. These plasmids, containing FMDV cDNA segments under the control of the T7 promoter, were transcribed in vitro by using T7 RNA polymerase and then translated in rabbit reticulocyte lysates. The expressed FMDV gene products were identified by immunoprecipitation with specific antisera and analyzed by gel electrophoresis. The results demonstrate the following: (i) the leader protein, L, is processed from the structural protein precursor, P1, in the absence of any P2 or P3 region proteins; (ii) protein 2A remains associated with the structural protein precursor, P1, rather than the precursor, P2; (iii) the processing of the P1-2A/P2 junction is not catalyzed by 3C or L; (iv) the proteolytic processing of polyproteins from the structural P1 region (except VP4/VP2) and the nonstructural P2 and P3 region is catalyzed by 3C.     

Nucleotide sequence and genome organization of foot-and-mouth disease virus.

A continuous 7802 nucleotide sequence spanning the 94% of foot and mouth disease virus RNA between the 5'-proximal poly(C) tract and the 3'-terminal poly(A) was obtained from cloned cDNA, and the total size of the RNA genome was corrected to 8450 nucleotides. A long open reading frame was identified within this sequence starting about 1300 bases from the 5' end of the RNA genome and extending to a termination codon 92 bases from its polyadenylated 3' end. The protein sequence of 2332 amino acids deduced from this coding sequence was correlated with the 260 K FMDV polyprotein. Its processing sites and twelve mature viral proteins were inferred from protein data, available for some proteins, a predicted cleavage specificity of an FMDV encoded protease for Glu/Gly(Thr, Ser) linkages, and homologies to related proteins from poliovirus. In addition, a short unlinked reading frame of 92 codons has been identified by sequence homology to the polyprotein initiation signal and by in vitro translation studies.     

Impairment of thymus-dependent responses by murine dendritic cells infected with foot-and-mouth disease virus

Foot-and-mouth disease virus (FMDV) is a cytopathic virus that experimentally infects mice, inducing a thymus-independent neutralizing Ab response that rapidly clears the virus. In contrast, vaccination with UV-inactivated virus induces a typical thymus-dependent (TD) response. In this study we show that dendritic cells (DCs) are susceptible to infection with FMDV in vitro, although viral replication is abortive. Infected DCs down-regulate the expression of MHC class II and CD40 molecules and up-regulate the expression of CD11b. In addition, infected DCs exhibit morphological and functional changes toward a macrophage-like phenotype. FMDV-infected DCs fail to stimulate T cell proliferation in vitro and to boost an Ab response in vivo. Moreover, infection of DCs in vitro induces the secretion of IFN-gamma and the suppressive cytokine IL-10 in cocultures of DCs and splenocytes. High quantities of these cytokines are also detected in the spleens of FMDV-infected mice, but not in the spleens of vaccinated mice. The peak secretion of IFN-gamma and IL-10 is concurrent with the suppression of Con A-mediated proliferation of T cells obtained from the spleens of infected mice. Furthermore, the secretion of these cytokines correlates with the suppression of the response to OVA, a typical TD Ag. Thus, infection of DCs with FMDV induces suppression of TD responses without affecting the induction of a protective thymus-independent response. Later, T cell responses are restored, setting the stage for the development of a long-lasting protective immunity.     

Preparation of a cell-free translation system from PC12 cells

The postmitochondrial fraction (S10) contains the cellular components essential for translation, and a high-salt wash (HSW) of the ribosomes is enriched in eukaryotic initiation factors. This report describes the preparation of a cell-free translation system utilizing an S10 extract from PC12 cells. The products synthesized from either firefly luciferase mRNA or PC12 cell poly(A) RNAs in the PC12-S10 extract were increased by the addition of the HSW from PC12 cells. Increases in the translation of luciferase mRNA by the addition of PC12-HSW were dose-dependent and also dependent on the time of incubation. The translation of human epidermal growth factor receptor (hEGFR) mRNA could also be detected in the PC12-S10 extract translation system by immunoprecipitation.N-linked glycosylation of the translation products also was observed. The efficiency of translation was altered by the addition of Mg²⁺ or K⁺, and optimization of the concentrations of these ions was necessary for each mRNA. The translation system made from PC12 cells, then, is capable of the synthesis of proteins of relatively high molecular weight and should be useful for analyzing mechanisms of translational control during proliferation and differentiation of cells from a neuronal lineage. Special issue dedicated to Dr. Hans Thoenen.     

Functional analysis of the internal translation initiation site of foot-and-mouth disease virus.

Mutagenesis of the large untranslated sequence at the 5' end of the genome of foot-and-mouth disease virus revealed that a region of approximately 450 nucleotides preceding the open reading frame of the viral polyprotein is involved in the regulation of translation initiation at two internal start sites. Variations in two domains of this region reduced the translation efficiency up to 10-fold, whereas an intermediate segment seemed to be less essential. A pyrimidine-rich sequence preceding the start codon was most sensitive in that conversion of single pyrimidine residues to purines decreased the translation efficiency strongly. The data are in agreement with a recently proposed general structural model for the internal ribosome entry site of the cardiovirusaphthovirus subgroup of picornaviruses (E. V. Pilipenko, V. M. Blinov, B. K. Chernov, T. M. Dmitrieva, and V. I. Agol, Nucleic Acids Res. 17:5701-5711, 1989). They suggest, however, that this model represents only a core structure for the internal entry of ribosomes and that foot-and-mouth disease virus and other members of the picornaviruses need additional regulatory RNA elements for efficient translation initiation.     

Multi-hour translation of mRNA in a cell-free system

In this study, we demonstrate that mRNA molecules can serve as an efficient template for cell-free translation through a combination of methods to protect them from nucleolytic digestion. Removal of major endonucleases activity from cell extract, the addition of a stemloop structure at the 3??-end of the mRNA and continuous reloading of ribosomes onto mRNA were found to be crucial for maintaining the functional integrity of mRNA during cell-free synthesis. When these three approaches were combined, mRNA-directed protein synthesis continued over 15 h, leading to the production of 2.6 mg/mL of encoded protein. The methods for direct translation of mRNA presented herein will provide a useful option for deciphering genetic information, including the fields of mRNA display and materialization of metagenomic information.     

Synthesis of Newcastle disease virus polypeptides in a wheat germ cell-free system.

We have isolated 18S RNA from cytoplasmic extracts of Newcastle disease virus-infected Chinese hamster ovary cells and tested its ability to direct protein synthesis in extracts derived from wheat germ. The products of the cell-free reaction directed by this RNA contain polypeptides that comigrate with NP, M,F, and 47K roteins from virions. In addition, the products contain a polypeptide (67K) that migrates on polyacrylamide gels slightly faster than the HN protein from virions. Tryptic peptide analysis of the cell-free products and proteins from virions confirms their identity.     

Plasmid recombination intermediates generated in a Saccharomyces cerevisiae cell-free recombination system.

We have developed an assay utilizing Saccharomyces cerevisiae cell extracts to catalyze recombination in vitro between homologous plasmids containing different mutant alleles of the tet gene. Electrophoretic analysis of product DNA indicated that a number of novel DNA species were formed during the reaction. These species migrated through agarose gels as distinct bands with decreased electrophoretic mobility compared with the substrate DNA. The DNA from each individual band was purified and shown to be enriched 5- to 100-fold for tetracycline-resistant recombinants by using a transformation assay. The structure of the DNA molecules present in these bands was determined by electron microscopy. Recombination between circular substrates appeared to involve the formation and processing of figure-eight molecules, while recombination between circular and linear substrates involved the formation of molecules in which a circular monomer had a monomer-length linear tail attached at a region of homology.     

Folding of firefly luciferase during translation in a cell-free system.

In vitro synthesis of firefly luciferase and its folding into an enzymatically active conformation were studied in a wheat germ cell-free translation system. A novel method is described by which the enzymatic activity of newly synthesized luciferase can be monitored continuously in the cell-free system while this protein is being translated from its mRNA. It is shown that ribosome-bound polypeptide chains have no detectable enzymatic activity, but that this activity appears within a few seconds after luciferase has been released from the ribosome. In contrast, the renaturation of denatured luciferase under identical conditions occurs with a half-time of 14 min. These results support the cotranslational folding hypothesis which states that the nascent peptides start to attain their native tertiary structure during protein synthesis on the ribosome.     

Functional analysis of the two alternative translation initiation sites of foot-and-mouth disease virus.

The effect of deletion of each of the two authentic polyprotein translation initiation sites of foot-and-mouth disease virus on viral protein synthesis and replication was analyzed. Deletion of either the first or the second initiation site led to the expression of only one form of the leader protein, L or L', respectively, but in vitro processing of the viral polyprotein and cleavage of eIF-4 gamma were not affected by either deletion. Whereas RNA in which the first translation initiation site had been deleted led to the production of viruses in transfected BHK cells, deletion of the second translation initiation site abolished virus replication.     

Ribavirin cures cells of a persistent infection with foot-and-mouth disease virus in vitro.

Ribavirin (1-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide) eliminates foot-and-mouth disease virus from persistently infected cell cultures. The latter are 10-fold more sensitive to ribavirin than lytically infected cells. In treated cells no viral RNA or proteins could be detected by dot-blot hybridization to cDNA probes, virus and RNA infectivity assays, or immunofluorescence. A potential application of the drug for the treatment of animals carrying the virus is suggested.     

Borna disease virus: immunoelectron microscopic characterization of cell-free virus and further information about the genome.

The etiological agent of Borna disease, a persistent virus infection of the central nervous system with differently expressed symptomatology, was morphologically unknown. Here we provide the first convincing data on its phenotypic architecture. Salt-released virus comprising the biological parameters of Koch's postulates has an unsegmented single-stranded RNA. A dense band (1.22 g/cm3) in CsCl contains 90-nm particles which appear to be enveloped and a fraction of 50- to 60-nm particles. Labeling of the virions with neutralizing antisera and colloidal gold conjugates indicates that the 90-nm particles most likely represent the causative agent.     

Human immunodeficiency virus integration in a cell-free system.

Integration of the viral genome into the nuclear DNA of a host cell plays a pivotal role in the replication of retroviruses. We have developed an in vitro method for studying the biochemistry of human immunodeficiency virus (HIV) integration by using extracts from HIV-infected cells. Analysis of the reaction products showed that HIV integration in vitro accurately reproduces the in vivo process. Integration occurred without apparent specificity for the target sequence, and the integrated provirus was directly flanked by a 5-base-pair duplication of DNA from the target site. HIV integration did not require a high-energy cofactor, and the enzymatic activities required for integration were recovered with the viral DNA when cell extracts were fractionated by gel exclusion chromatography.     

Interaction of foot-and-mouth disease virus with dendritic cells

Despite several decades of investigation, the manner in which foot-and-mouth disease virus (FMDV) interacts with the innate and adaptive immune compartments is not completely understood. The importance of elucidating this relationship is emphasized by the inability of current FMDV vaccines to provide long-term protection and the recent outbreaks of FMDV in formerly disease-free countries. Dendritic cells (DCs) are professional antigen-presenting cells that have evolved to monitor the environment and provide a link between the innate and adaptive immune systems. Comprehending the cross-talk between DC and FMDV will provide valuable information towards understanding the host response to the virus and will aid in the design of effective tools and vaccines to block virus spread.