LaXp180, a mammalian ActA-binding protein, identified with the yeast two-hybrid system, co-localizes with intracellular Listeria monocytogenes.

The Listeria monocytogenes surface protein ActA is an important virulence factor required for listerial intracellular movement by inducing actin polymerization. The only host cell protein known that directly interacts with ActA is the phosphoprotein VASP, which binds to the central proline-rich repeat region of ActA. To identify additional ActA-binding proteins, we applied the yeast two-hybrid system to search for mouse proteins that interact with ActA. A mouse cDNA library was screened for ActA-interacting proteins (AIPs) using ActA from strain L. monocytogenes EGD as bait. Three different AIPs were identified, one of which was identical to the human protein LaXp180 (also called CC1). Binding of LaXp180 to ActA was also demonstrated in vitro using recombinant histidine-tagged LaXp180 and recombinant ActA. Using an anti-LaXp180 antibody and fluorescence microscopy, we showed that LaXp180 co-localizes with a subset of intracellular, ActA-expressing L. monocytogenes but was never detected on intracellularly growing but ActA-deficient mutants. Furthermore, LaXp180 binding to intracellular L. monocytogenes was asymmetrical and mutually exclusive with F-actin polymerization on the bacterial surface. LaXp180 is a putative binding partner of stathmin, a protein involved in signal transduction pathways and in the regulation of microtubule dynamics. Using immunofluorescence, we showed that stathmin co-localizes with intracellular ActA-expressing L. monocytogenes.

The human autoantigen La/SS-B accelerates herpes simplex virus type 1 replication in transfected mouse 3T3 cells.

Permanently transfected mouse cell lines which expressed different levels of the human autoantigen La/SS-B were infected with different strains of herpes simplex virus type 1, including the strains ANG, HSZP, 17syn+ and HFEM. During infection the localization of the human La protein was followed using an anti-La MoAb, which recognized only the human La protein but did not cross-react with either the endogenous mouse La protein or any viral encoded protein. After infection La protein was transported from the nucleus to the cytoplasm. The time course of translocation was dependent on the amount of human La protein expressed in the respective cell line. Moreover, acceleration of viral replication was dependent on the level of expression of human La protein, suggesting that La protein is a cellular factor that facilitates virus replication.

Characterization of murine monoclonal antibodies against the Ro52 autoantigen.

Immunization of BALB/c mice with purified recombinant human Ro52 protein resulted in three anti-Ro52 MoAbs termed 2E7, 4C6 and 4F11. All anti-Ro52 MoAbs specifically reacted with recombinant human Ro52 protein, and also with Ro52 protein in total extracts of all human cell lines analysed, including the epithelial cell line HeLa, the B cell line Raji, the bladder carcinoma cell line RT112, and a fibroblast cell line derived from patients with xeroderma pigmentosum. The anti-Ro52 MoAbs were able to immunoprecipitate the recombinant human Ro52 protein expressed in wheat germ extract, but failed to precipitate hY RNAs from cell extracts. The staining pattern of the MoAbs strongly differed between the RT112 cells and the fibroblast cell line. RT112 cells displayed an intense cytoplasmic staining and in addition distinct fine nuclear speckles. In contrast, in the fibroblast cell line no cytoplasmic staining but only staining of distinct nuclear speckles was observed. Using deletion mutants of Ro52 the epitopes recognized by the anti-Ro52 MoAbs 2E7, 4C6 and 4F11 were partially mapped. All three MoAbs appeared to recognize distinct epitopes, that are located in the regions of Ro52 bordered by amino acids 136-164, 208-363 and 136-190, respectively. These MoAbs can be of great use in studying the cellular processes in which the Ro52 protein is involved.

Transfection analysis of expression of mRNA isoforms encoding the nuclear autoantigen La/SS-B.

Transcription of the gene encoding for the nuclear autoantigen La resulted in La mRNA isoforms. A promoter switching combined with an alternative splicing pathway replaced the exon 1 with the exon 1'. The exon 1' contained GC-rich regions and an oligo(U) tail of 23 uridine residues. Moreover, it encoded for three open reading frames upstream of the La protein reading frame. Despite this unusual structure, when exon 1' La mRNAs were expressed in transfected cells, both exon 1 and 1' La mRNAs were translated to La protein, whereas the upstream open reading frames of the exon 1' were not translated. In addition to full-length exon 1' La mRNAs 5'-shortened exon 1' La mRNAs were detected. The exon 1' 5'-starts varied in dependence on the analyzed tissues. Like the full-length exon 1' La mRNA a 5'-shortened exon 1' construct starting downstream of the oligo(U) tail but upstream of the open reading frames 2 and 3 was also well translated when transfected in mouse cells. Thus all La mRNA forms represent functional La mRNAs.