Members of the Closteroviridae and Potyviridae families of the plant positive-strand RNA viruses encode one or two papain-like leader proteinases. In addition to a C-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic N-terminal domain. We compared functions of the several leader proteinases using a gene swapping approach. The leader proteinase (L-Pro) of Beet yellows virus (BYV; a closterovirus) was replaced with L1 or L2 proteinases of Citrus tristeza virus (CTV; another closterovirus), P-Pro proteinase of Lettuce infectious yellows virus (LIYV; a crinivirus), and HC-Pro proteinase of Tobacco etch virus (a potyvirus). Each foreign proteinase efficiently processed the chimeric BYV polyprotein in vitro. However, only L1 and P-Pro, not L2 and HC-Pro, were able to rescue the amplification of the chimeric BYV variants. The combined expression of L1 and L2 resulted in an increased RNA accumulation compared to that of the parental BYV. Remarkably, this L1-L2 chimera exhibited reduced invasiveness and inability to move from cell to cell. Similar analyses of the BYV hybrids, in which only the papain-like domain of L-Pro was replaced with those derived from L1, L2, P-Pro, and HC-Pro, also revealed functional specialization of these domains. In subcellularlocalization experiments, distinct patterns were observed for the leader proteinases of BYV, CTV, and LIYV. Taken together, these results demonstrated that, in addition to a common proteolytic activity, the leader proteinases of closteroviruses possess specialized functions in virus RNA amplification, virus invasion, and cell-to-cell movement. The phylogenetic analysis suggested that functionally distinct L1 and L2 of CTV originated by a gene duplication event.

The Epstein-Barr virus nuclear leader protein LP (EBNALP) and EBNA2 are expressed first in lymphocyte infection, coordinately regulate cell and viral gene transcription, and are critical for lymphocyte outgrowth into lymphoblastoid cell lines (LCLs). We have now found that EBNALP readily associated with EBNA2 or with the EBNA2 C-terminal acidic activation domain (E2AD) when both components were expressed by bacteria. In lymphoblasts, EBNALP and EBNA2 did not stably associate. However, EBNALP deleted for only 10 C-terminal amino acids stably associated with EBNA2 in lymphoblasts or with EBNA2 acidic activating domain from bacteria. The E2AD was essential for EBNALP coactivation of the latent membrane protein 1 promoter in lymphoblasts; EBNALP could coactivate with a deficient mutant EBNA2, EBNA2W 454T, but not with EBNA2 deleted for E2AD. Moreover, EBNALP 31 amino acids (dW2Y1) with 24 C-or N-terminal amino acids was a specific and efficient affinity matrix for EBNA2 or EBNALP. Even an EBNALP 22-aa peptide, dW2, specifically bound EBNALP or EBNA2. These biochemical interactions between EBNALP and EBNA2 enable coordinated transcriptional regulation of cell and viral gene expression in lymphoblasts only when the interaction is unstable; deletion of the EBNALP C-terminal 10 aa stabilized association with EBNA2 and prevented coactivation. Because EBNALPd10 dominantly inhibited EBNALP coactivation with EBNA2, EBNALPd10 expression in LCLs may be useful in assessing the role of EBNALP coactivation in LCL growth or survival.EBV nuclear protein ͉ transcription ͉ association ͉ transformation E pstein-Barr virus (EBV) infection of lymphocytes usually results in a latency III infection, characterized by limited EBV gene expression, genome persistence as an episome, and EBV-driven continuous lymphocyte proliferation (for reviews, see refs. 1 and 2). EBV nuclear leader protein LP (EBNALP) and EBNA2 are the first viral proteins expressed in lymphocytes. Their RNAs are transcribed by the same promoter and differential splicing determines mRNA and protein levels (3-5). EBNALP and EBNA2 up-regulate EBV and cell gene transcription, including the viral oncogene, latent membrane protein 1 (LMP1), and are required for lymphocyte outgrowth into lymphoblastoid cell lines (LCLs) (6 -9). EBNA2 interactions with cellular sequence-specific DNA-binding proteins, such as RBPJkappa, PU.1, and AUF1, are critical for EBNA2 regulation of specific virus and cell promoters (10 -17). Another critical component of EBNA2 is an acidic activating domain (E2AD), which can recruit TFIIB, TAF40, TFIIH, p100, and CBP͞p300, thereby up-regulating cell and virus gene transcription (8, 18 -23).Less is known about the mechanism by which EBNALP strongly and specifically potentiates EBNA2-mediated transcription (24-28). EBNALP mRNA is comprised of four nearly complete W1 and W2 exons, which encode 22-and 44-aa repeats, and Y1 and Y2 exons, which encode 11 and 34 unique amino acids (refs. 29 and 30 and Fig. 1A). Although the four EBNALP W1W2 repeats have substant...

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is essential for EBV episome maintenance, replication, and transcription. These effects are mediated by EBNA1 binding to cognate oriP DNA, which comprise 20 imperfect copies of a 30-bp dyad symmetry enhancer and an origin for DNA replication. To identify cell proteins essential for these EBNA1 functions, EBNA1 associated cell proteins were immune precipitated and analyzed by liquid chromatography-tandem mass spectrometry. Nucleolin (NCL) was identified to be EBNA1 associated. EBNA1's N-terminal 100 aa and NCL's RNA-binding domains were critical for EBNA1/NCL interaction. Lentivirus shRNA-mediated NCL depletion substantially reduced EBNA1 recruitment to oriP DNA, EBNA1-dependent transcription of an EBV oriP luciferase reporter, and EBV genome maintenance in lymphoblastoid cell lines. NCL RNA-binding domain K 429 was critical for ATP and EBNA1 binding. NCL overexpression increased EBNA1 binding to oriP and transcription, whereas NCL K 429 A was deficient. Moreover, NCL silencing impaired lymphoblastoid cell line growth. These experiments reveal a surprisingly critical role for NCL K429 in EBNA1 episome maintenance and transcription, which may be a target for therapeutic intervention.lymphoma | chromatin | oncogenic herpesvirus | nasopharyngeal carcinoma

EBNA2 transcriptional activation and regulated EBNALP coactivation are critical for Epstein-Barr virusinfected primary B-lymphocyte growth transformation. EBNALP coactivation requires the EBNA2 acidic activation domain (E2AD); EBNALP can bind to E2AD. EBNALP has now been found to bind less well to EBNA2 amino acids 1 to 58, which has been identified to be a second transcriptional activation domain, E2AD2. E2AD2 was specifically coactivated by EBNALP. Moreover, E2AD, E2AD2, EBNA2 RG domain, and the intermediate domain between RG and E2AD had significant roles in EBNA2-mediated activation and EBNALP coactivation.Epstein-Barr virus (EBV) infection transforms resting B cells into permanent lymphoblastoid cell lines (for a review, see reference 14). In B lymphocytes, EBV nuclear antigen proteins, EBNA2 and EBNALP are the first two proteins expressed from the EBV genome. EBNA2 up-regulates transcription from specific cell and viral promoters through interactions with cell transcription factors that bind to cognate DNAs. EBNA2 amino acids (aa) 310 to 335 associate at a high level with RBPJk/CBF1, a key factor in Notch receptor gene activation. EBNA2 has a C-terminal acidic transcriptional activation domain (E2AD), which recruits basal and activationrelated cell transcription factors and also binds EBNALP, enabling EBNALP to specifically coactivate transcription with EBNA2 (4, 10-12). In the context of Gal4 DNA binding domain (G4DBD) fusions, E2AD is a strong activator, which is repressed by EBNALP expression (12). Nevertheless, G4DBD-EBNA2 fusion proteins are coactivated by EBNALP, and coactivation by EBNALP is dependent on E2AD (4, 12). The objective of the experiments reported here is to identify the other components of EBNA2 that partially silence E2AD and enable EBNALP to coactivate.EBNA2 aa 1 to 58 fused to G4DBD can activate Gal4-dependent transcription and is coactivated by EBNALP. To identify EBNA2 component(s) other than E2AD that may be coactivated by EBNALP, overlapping parts of the EBNA2 open reading frame were fused to G4DBD, which includes a nuclear localization sequence (2, 8), and were transfected into BJAB B lymphoblasts with a Gal4-dependent luciferase reporter in the presence or absence of an EBNALP expression vector ( Fig. 1) (12). Plasmid CMV-␤gal, a plasmid containing a cytomegalovirus (CMV) promoter and ␤-galactosidase reporter, was used as an internal transfection control. As previously described (12), G4DBD-EBNA2 aa 19 to 483 activated the Gal4-dependent promoter 10-fold, and coactivation with EBNALP resulted in additional 16-fold activation, resulting in 160-fold activation (Fig. 1). G4DBD-EBNA2 aa 1 to 30, 96 to 210, 200 to 334, and 300 to 432 failed to activate the Gal4-dependent promoter and were not coactivated by EBNALP (Fig. 1). Surprisingly, G4DBD-EBNA2 aa 1 to 58 activated transcription 5-to 10-fold, and coactivation with EBNALP resulted in additional 5-to 10-fold activation (Fig. 1). Thus, EBNA2 aa 1 to 58 is an activation domain that can be coactivated by EBNALP. Addition of the C-te...