Regulation of Semliki Forest virus RNA replication: a model for the control of alphavirus pathogenesis in invertebrate hosts.

Alphavirus nonstructural proteins are translated as a polyprotein that is ultimately cleaved into four mature proteins called nsP1, nsP2, nsP3, and nsP4 from their order in the polyprotein. The role of this nonstructural polyprotein, of cleavage intermediates, and of mature proteins in synthesis of Semliki Forest virus (SFV) RNA has been studied using mutants unable to cleave one or more of the sites in the nonstructural polyprotein or that had the arginine sense codon between nsP3 and nsP4 changed to an opal termination codon. The results were compared with those obtained for Sindbis virus (SINV), which has a naturally occurring opal codon between nsP2 and nsP3. We found that (1) an active nonstructural protease in nsP2 is required for RNA synthesis. This protease is responsible for all three cleavages in the nonstructural polyprotein. (2) Cleavage between nsP3 and nsP4 (the viral RNA polymerase) is required for RNA synthesis by SFV. (3) SFV mutants that are able to produce only polyprotein P123 and nsP4 synthesize minus-strand RNA early after infection as efficiently as SF wild type but are defective in the synthesis of plus-strand RNA. The presence of sense or opal following nsP3 did not affect this result. At 30 degrees C, they give rise to low yields of virus after a delay, but at 39 degrees C, they are nonviable. (4) SFV mutants that produce nsP1, P23, nsP4, as well as the precursor P123 are viable but produce an order of magnitude less virus than wild type at 30 degrees C and two orders of magnitude less virus at 39 degrees C. The ratio of subgenomic mRNA to genomic RNA is much reduced in these mutants relative to the parental viruses. (5) At 30 degrees C, the variants containing an opal codon grow as well as or slightly better than the corresponding virus with a sense codon. At 39 degrees C, however, the opal variants produce significantly more virus. These results support the conclusion that SFV and SINV, and by extension all alphaviruses, regulate their RNA synthesis in the same fashion after infection. P123 and nsP4 form a minus-strand replicase that synthesizes plus-strand RNA only inefficiently, especially at the higher temperatures found in mammals and birds. A replicase containing nsP1, P23, and nsP4 can make both plus and minus strands, but prefers the promoter for genomic plus sense RNA to that for subgenomic mRNA. The fully cleaved replicase can make only plus-strand RNA, and prefers the promoter for subgenomic mRNA to that for genomic RNA. Alphaviruses alternate between infection of hematophagous arthropods and higher vertebrates. Although the infection of higher vertebrates is acute and often accompanied by disease, continuing transmission of the virus in nature requires that infection of arthropods be persistent and relatively asymptomatic. We propose that this mechanism for control of RNA synthesis evolved to moderate the pathogenicity of the viruses in their arthropod hosts.

Oligomer synthesis by priming deficient polymerase in hepatitis B virus core particle.

Hepadnavirus DNA polymerase functions in DNA synthesis and encapsidation, and acts as a primer for minus-strand DNA synthesis. Through protein priming reaction, a short DNA oligomer synthesized from the bulge of epsilon as template is covalently attached to the Tyr residue in the terminal protein (TP) domain of DNA polymerase. Using endogenous polymerase assays and native agarose gel analysis, we detected endogenous polymerase activity in priming-deficient mutant core particles, but not in reverse transcriptase (RT) reaction- or P protein-deficient mutant core particles. In addition, priming-deficient mutant core particles incorporated radiolabeled (32)P-dATP, (32)P-TTP, and (32)P-dGTP, but not (32)P-dCTP. Our results suggest that the priming-deficient mutant P protein has the ability to synthesize oligomers (presumably nascent minus-strand DNA) in the absence of covalent linkage between TP and the first deoxynucleotide. We propose that the priming-deficient mutant may be defective in minus-strand DNA translocation to direct repeat (DR) 1 at the 3' end of pregenomic RNA (pgRNA) that leads to the elongation of minus-strand DNA.

A phagemid system enabling easy estimation of the combinatorial antibody library size.

We developed a new phagemid system for the generation of combinatorial antibody libraries. This system allows the recombination efficiency to be estimated easily, which aids the accurate measurement of the antibody library size. Two phagemids were constructed. pRTCB bears the structural tetracycline-resistant gene (tetR) and pRTCA bears its promoter region (Ptet). pRTCA and pRTCB were constructed so that recombinase-mediated chain exchange (RMCE) in Cre-expressing bacteria results in the simultaneous acquisition of Tet resistance and newly recombined single chain variable fragment (scFv) genes. PCR and restriction enzyme analysis of randomly selected tetR phagemids showed that all Tet-resistant phagemids have an active tetR gene and a recombined scFv gene. RMCE efficiency was measured by the ratio of the tetR colonies to the total colonies. The scFv proteins expressed from the recombined phagemid vectors were functional. Thus, our phagemid vector system may be useful for making combinatorial antibody libraries.

Selection of scFvs specific for HBV DNA polymerase using ribosome display.

We applied a ribosome display technique to a mouse scFv library to select single chain variable fragments (scFvs) specific for the terminal protein (TP) of hepatitis B virus (HBV) DNA polymerase. Synthetic TP-peptide was used as an antigen to obtain scFvs that recognize specific epitopes within the TP domain, the priming site of HBV DNA polymerase. The scFv DNA library was transcribed in vitro to mRNA for ribosome display. scFv-ribosome-mRNA complexes were produced using a rabbit reticulocyte lysate system, and were panned against TP-peptide under appropriate conditions. After each panning, putative scFv-encoding genes were recovered by RT-PCR, and the analysis showed that scFv-ribosome-mRNA complexes were specifically selected by the TP-peptide. We used a radioimmunoassay to show that the TP-peptide-specific scFv pools were enriched through the selection process. Selected scFvs showed binding activity for both the TP-peptide and the HBV DNA polymerase protein in an ELISA. Sequence analysis showed that each TP-specific scFv had a different sequence, and that random mutagenesis was mediated by ribosome display.

Characterization of N protein self-association in coronavirus ribonucleoprotein complexes.

Mouse hepatitis virus (MHV) nucleocapsid (N) protein binds to the large, single-stranded, positive-sense viral genomic RNA to form a helical nucleocapsid structure in mature virions. In addition N protein binds the intracellular form of the genomic RNA, all of the MHV subgenomic mRNAs, and expressed non-MHV RNA transcripts to form ribonucleoprotein (RNP) complexes in infected cells. Among the intracellular viral RNP complexes, only the genomic RNP complex is packaged into virus particles. The present study demonstrated that N protein in the MHV virion nucleocapsid and in the intracellular genome-length RNP complex that bound to viral envelope M protein was tightly self-associated such that its association was retained even after extensive RNase A-treatment of the RNP complexes. The RNase A-resistant tight N protein association in the virion nucleocapsid was not mediated by an intermolecular disulfide bridge between N proteins. In contrast, N protein association in the majority of the intracellular RNP complexes was susceptible to RNase A-treatment. Because the RNP complexes that specifically interact with the M protein are selectively packaged into MHV particles, the present data suggested that there was a distinct difference between N protein association in viral genomic RNP complexes that undergo packaging into virus particles and the subgenomic RNP complexes that are not packaged into MHV particles.

A visible phagemid system for the estimation of Cre-mediated recombination efficiency.

Diversity in phage antibody libraries is important for obtaining useful high-affinity antibodies. The Cre-lox system is generally employed to increase the size of phage antibody libraries. However, estimation of library sizes after Cre-mediated recombination is difficult, since time-consuming nucleotide sequence analyses are required. We have developed a visible phagemid vector system that facilitates the estimation of recombination efficiency between VH and VL genes. In this system, intermolecular recombination between VL genes flanked by incompatible lox sites is coincident with the expression of functional beta-galactosidase. Recombination efficiency can be calculated simply by counting the number of blue colonies on X-gal-containing medium. Molecular analyses of plasmids isolated from blue colonies reveal a novel VH/VL combination. Our results suggest that this newly developed visible phagemid system may be reliably used for the measurement of recombination efficiency, which would enable precise evaluation of the diversity of phage antibody libraries.

Immunological characterizations of a cloned 13.1-kilodalton protein from pathogenic Naegleria fowleri.

We previously cloned an antigenic gene (named nfa1) from a cDNA library of Naegleria fowleri by immunoscreening. The nfa1 gene had a coding nucleotide sequence consisting of 357 bases and produced a recombinant 13.1-kDa protein (Nfa1). In this study, to get more information regarding the recombinant Nfa1 protein (rNfa1), we produced an anti-Nfa1 polyclonal antibody from mice immunized with rNfa1 and used a peroxidase staining method to carry out immunocytochemistry experiments. In addition, we observed the effect of the presence of an anti-Nfa1 antibody on the in vitro cytotoxicity of N. fowleri against Chinese hamster ovary (CHO) cells. Trophozoites of N. fowleri in cultivation reacted strongly with a peroxidase-labeled anti-Nfa1 antibody. In inflammatory and necrotic regions of brain tissue infected with N. fowleri, labeled trophozoites that were stained brown were also observed. When examined using a transmission electron microscope, the Nfa1 protein showed pseudopodium-specific immunolocalization on a trophozoite of N. fowleri. When examined using a light microscope, CHO cells grown in cocultures with N. fowleri trophozoites (group I) for 48 h showed morphologically severe destruction but CHO cells grown in cocultures with N. fowleri trophozoites and an anti-Nfa1 polyclonal antibody (group II) showed less destruction. The results of a lactate dehydrogenase release assay showed that group I CHO cells exhibited 81% cytotoxicity and group II CHO cells exhibited 13.8% cytotoxicity.

The putative transcriptional activator MSN1 promotes chromium accumulation in Saccharomyces cerevisiae.

Yeast is a good system for studying molecular mechanisms of metal tolerance. Using a mini-Tn mutagenized yeast pool, we isolated a chromate-tolerant mutant, CrT9, that displayed metal-specific tolerance since it was only tolerant to Cr(VI), not to Cr(III), Cd, As, or Fe. The Cr-tolerance of CrT9 appeared to be due to reduced Cr accumulation as it accumulated only 56% as much as WT (Y800). Using IPCR (inverse PCR), we found that the mini-Tn had been inserted at nt 741 of the transcriptional activator, MSN1. MSN1 is a multifunctional protein involved in invertase activity, iron uptake, starch degradation, pseudohyphal growth, and osmotic gene expression. We found that there was only one mini-Tn insertion in CrT9 since MSN1 and mini-Tn probes hybridized to the same DNA fragment, and the MSN1 probe detected an enlarged MSN1 mRNA. When we over-expressed MSN1 in CrT9 and WT, both accumulated larger amounts of Cr. We conclude that Cr accumulation in S. cerevisiae is promoted by the transcriptional activator MSN1.

Production and characterization of an anti-idiotypic single chain Fv that recognizes an anti-DNA antibody.

A well-characterized recombinant anti-idiotype to an anti-DNA antibody can be useful for studies of the regulation of anti-DNA-producing B cells. Using a hybridoma technique, a monoclonal anti-idiotypic antibody, designated O2F3, was obtained, and its scFv gene was constructed. O2F3 single chain Fv (scFv) was produced against an idiotope of a monoclonal anti-DNA antibody, 3D8, that was obtained from an autoimmune-prone mouse, MRL-lpr/lpr. Here we describe the production and in vitro characterization of the O2F3 scFv, and compare it with its parent monoclonal antibody, O2F3 IgM. To characterize O2F3 scFv and O2F3 IgM, we generated recombinant 3D8 fragments, including 3D8 scFv, 3D8 VH, and 3D8 VL, that were used as antigens in several assays. ELISA and Western blot analysis showed that both O2F3 scFv and O2F3 IgM recognized a conformational determinant formed by the association of the variable region heavy and light chains of the 3D8 antibody, suggesting that O2F3 scFv retained a similar binding pattern to its parent O2F3 antibody. The idiotope recognized by O2F3 was shown by competitive ELISA to be outside of the DNA binding site of the 3D8 antibody. This characterized O2F3 scFv could be applied for the regulation of anti-DNA antibody production and the manipulation of recombinant antibody-based proteins to which toxins, enzymes, and chemical agents can be connected.