Several lines of evidence are compatible with the hypothesis that Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) or leader protein (EBNA-LP) affects expression of the EBV latent infection membrane protein LMP1. We now demonstrate the following. (i) Acute transfection and expression of EBNA-2 under control of simian virus 40 or Moloney murine leukemia virus promoters resulted in increased LMP1 expression in P3HR-1-infected Burkitt's lymphoma cells and the P3HR-1 or Daudi cell line. (ii) Transfection and expression of EBNA-LP alone had no effect on LMP1 expression and did not act synergistically with EBNA-2 to affect LMP1 expression. (iii) LMP1 expression in Daudi and P3HR-l-infected cells was controlled at the mRNA level, and EBNA-2 expression in Daudi cells increased LMP1 mRNA. (iv) No other EBV genes were required for EBNA-2 transactivation of LMP1 since cotransfection of recombinant EBNA-2 expression vectors and genomic LMP1 DNA fragments enhanced LMP1 expression in the EBV-negative B-lymphoma cell lines BJAB, Louckes, and BL30. (v) An EBNA-2-responsive element was found within the-512 to +40 LMP1 DNA since this DNA linked to a chloramphenicol acetyltransferase reporter gene was transactivated by cotransfection with an EBNA-2 expression vector. (vi) The EBV type 2 EBNA-2 transactivated LMP1 as well as the EBV type 1 EBNA-2. (vii) Two deletions within the EBNA-2 gene which rendered EBV transformation incompetent did not transactivate LMP1, whereas a transformation-competent EBNA-2 deletion mutant did transactivate LMP1. LMP1 is a potent effector of B-lymphocyte activation and can act synergistically with EBNA-2 to induce cellular CD23 gene expression. Thus, EBNA-2 transactivation of LMP1 amplifies the biological impact of EBNA-2 and underscores its central role in EBV-induced growth transformation.

b; Queen Victoria NHS Foundation Trust, East Grinstead, West Sussex, United Kingdom c Proteus mirabilis forms extensive crystalline biofilms on urethral catheters that occlude urine flow and frequently complicate the management of long-term-catheterized patients. Here, using random transposon mutagenesis in conjunction with in vitro models of the catheterized urinary tract, we elucidate the mechanisms underpinning the formation of crystalline biofilms by P. mirabilis. Mutants identified as defective in blockage of urethral catheters had disruptions in genes involved in nitrogen metabolism and efflux systems but were unaffected in general growth, survival in bladder model systems, or the ability to elevate urinary pH. Imaging of biofilms directly on catheter surfaces, along with quantification of levels of encrustation and biomass, confirmed that the mutants were attenuated specifically in the ability to form crystalline biofilms compared with that of the wild type. However, the biofilm-deficient phenotype of these mutants was not due to deficiencies in attachment to catheter biomaterials, and defects in later stages of biofilm development were indicated. For one blocking-deficient mutant, the disrupted gene (encoding a putative multidrug efflux pump) was also found to be associated with susceptibility to fosfomycin, and loss of this system or general inhibition of efflux pumps increased sensitivity to this antibiotic. Furthermore, homologues of this system were found to be widely distributed among other common pathogens of the catheterized urinary tract. Overall, our findings provide fundamental new insight into crystalline biofilm formation by P. mirabilis, including the link between biofilm formation and antibiotic resistance in this organism, and indicate a potential role for efflux pump inhibitors in the treatment or prevention of P. mirabilis crystalline biofilms.

To delineate the cis-acting element through which EBNA-2 transactivates latent membrane protein 1 (LMP1), we assayed the effect of EBNA-2 on the activity of LMP1 promoter upstream deletion mutants in the context of the LMP1 or heterologous promoters controlling chloramphenicol acetyltransferase (CAT) reporter gene expression in Epstein-Barr virus-negative Burkitt lymphoma cells. Assays of progressive 5' deletions of the LMP1 promoter revealed low constitutive and at least eightfold EBNA-2-stimulated activity from-512 to +40 (-512/+40),-334/+40, and-234/+40 LMP1CAT plasmids. More extensive 5'-deleted-205/+40,-155/ +40, and-147/+40 LMP1CAT plasmids also had low constitutive activity but were not EBNA-2 responsive. The most 5'-deleted-551+40 LMP1CAT plasmid had moderate constitutive activity and was not EBNA-2 inducible. Either orientation of the-334/+40 LMP1 sequence conferred EBNA-2 responsiveness when positioned upstream of an enhancerless simian virus 40 or herpes simplex virus thymidine kinase (TK) promoter. EBNA-2 and the cis-acting LMP1 DNA were both required to increase TK promoter-initiated mRNA, indicating that the EBNA-2 effect is at the transcriptional level. Further deletion analysis of the EBNA-2-responsive cis-acting element defined a-234/-92 LMP1 DNA fragment which conveyed EBNA-2 responsiveness to the herpes simplex virus TK promoter. The 5' 30 bp between-234 and-205 were essential for EBNA-2 responsiveness. Thus, these experiments define a 142-bp cis-acting element which is sufficient for conveying EBNA-2 responsiveness and an essential 30-bp component of that element. The role of this element in LMP1 and LMP2B expression and its possible role in LMP2A expression are discussed.