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. 2000 Jan;74(1):379-89.
doi: 10.1128/jvi.74.1.379-389.2000.

Sequence and functional analysis of EBNA-LP and EBNA2 proteins from nonhuman primate lymphocryptoviruses

R Peng  1 , A V Gordadze, E M Fuentes Pananá, F Wang, J Zong, G S Hayward, J Tan, P D Ling
Affiliations

Sequence and functional analysis of EBNA-LP and EBNA2 proteins from nonhuman primate lymphocryptoviruses

R Peng et al. J Virol. 2000 Jan.

Abstract

The Epstein-Barr virus (EBV) EBNA-LP and EBNA2 proteins are the first to be synthesized during establishment of latent infection in B lymphocytes. EBNA2 is a key transcriptional regulator of both viral and cellular gene expression and is essential for EBV-induced immortalization of B lymphocytes. EBNA-LP is also important for EBV-induced immortalization of B lymphocytes, but far less is known about the functional domains and cellular cofactors that mediate EBNA-LP function. While recent studies suggest that serine phosphorylation of EBNA-LP and coactivation of EBNA2-mediated transactivation are important, more detailed mutational and genetic studies are complicated by the repeat regions that comprise the majority of the EBNA-LP sequence. Therefore, we have used a comparative approach by studying the EBNA-LP homologues from baboon and rhesus macaque lymphocryptoviruses (LCVs) (baboon LCV and rhesus LCV). The predicted baboon and rhesus LCV EBNA-LP amino acid sequences are 61 and 64% identical to the EBV EBNA-LP W1 and W2 exons and 51% identical to the EBV EBNA-LP Y1 and Y2 exons. Five evolutionarily conserved regions can be defined, and four of eight potential serine residues are conserved among all three EBNA-LPs. The major internal repeat sequence also revealed a highly conserved Wp EBNA promoter with strong conservation of upstream activating sequences important for Wp transcriptional regulation. To test whether transcriptional coactivating properties were common to the rhesus LCV EBNA-LP, a rhesus LCV EBNA2 homologue was cloned and expressed. The rhesus LCV EBNA2 transcriptionally transactivates EBNA2-responsive promoters through a CBF1-dependent mechanism. The rhesus LCV EBNA-LP was able to further enhance rhesus LCV or EBV EBNA2 transactivation 5- to 12-fold. Thus, there is strong structural and functional conservation among the simian EBNA-LP homologues. Identification of evolutionarily conserved serine residues and regions in EBNA-LP homologues provides important clues for identifying the cellular cofactors and molecular mechanisms mediating these conserved viral functions.

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Figures

FIG. 1
FIG. 1
Restriction enzyme maps of the left end of the EBV, baboon LCV and rhesus LCV genomes. Four IR1 repeats are shown for EBV for purposes of comparison to the other LCVs, and it should be noted that the prototype B95-8 published sequence contains 11 repeats. The restriction enzymes used are shown at the right. Since a complete analysis of the rhesus LCV genome has not been described, the plasmid name containing each XhoI-derived DNA fragment is indicated below the rhesus LCV genome, and the size of each fragment is indicated in kilobases below the plasmid name. Preliminary restriction digests for RcosCC1 indicate approximately four copies of the internal repeats for rhesus LCV (unpublished observations), while similar analysis for the baboon LCV clone JR4 has also indicated four repeats (43). The XhoI restriction enzyme cleaves the homologous IR1 repeats (approximately 3.0 kb) in the rhesus LCV clone twice so that each repeat consists of both pAG5 and pAG6 fragments (1.6 and 1.4 kb, respectively). The approximate boundaries of the rhesus LCV cosmid used for these studies are shown above the rhesus LCV map.
FIG. 2
FIG. 2
Alignment of the predicted amino acid sequences from EBV type 2, baboon LCV, and rhesus LCV EBNA-LP proteins with the EBV type 1 EBNA-LP amino acid sequence. Amino acid residues identical between sequences are indicated by asterisks, and similar nonidentical residues are indicated by a dotted line. Conserved regions are boxed and numbered in consecutive order as CR1 to CR5. Amino acid numbers are indicated at the beginning and end of each line. The boundaries of the W1, W2, Y1, and Y2 exons are shown above the sequences. The triangles indicate conserved serine residues that are potential phosphorylation sites.
FIG. 3
FIG. 3
Alignment of the DNA sequence for the predicted EBV, baboon, and rhesus LCV Wp sequences. (A) UAS1 and UAS2 are indicated by brackets above the sequence. The dark and light shaded sequences in UAS1 indicate important distinct cis-acting elements that bind unknown cellular factors (2). Conserved cis-acting elements that bind cellular factors are boxed. The putative elements are also labeled. The W0 exon is shown by the arrow. The putative W0 splice donor site is indicated by a dashed box. The putative splice acceptor sites are indicated by the underlined bases and black (W1-generated splice acceptor) and white (W1′-generated splice acceptor) boxes. Nucleic acid numbers are indicated at the beginning and end of each line. The first number is arbitrary and begins at the XhoI restriction site for rhesus LCV clone pAG6. The EBV and baboon LCV sequences were then given consistent numbers based on the alignment. For EBV, base 1 corresponds to position 44547 and base 742 corresponds to position 45289 of the last W repeat in the EBV genome. (B) Alignment of EBV splice donor and acceptor sites and the predicted homologous sites for baboon LCV (bLCV) and rhesus LCV (rLCV). Consensus donor and acceptor sites are shown at the top, and the exon junctions are shown at the left.
FIG. 3
FIG. 3
Alignment of the DNA sequence for the predicted EBV, baboon, and rhesus LCV Wp sequences. (A) UAS1 and UAS2 are indicated by brackets above the sequence. The dark and light shaded sequences in UAS1 indicate important distinct cis-acting elements that bind unknown cellular factors (2). Conserved cis-acting elements that bind cellular factors are boxed. The putative elements are also labeled. The W0 exon is shown by the arrow. The putative W0 splice donor site is indicated by a dashed box. The putative splice acceptor sites are indicated by the underlined bases and black (W1-generated splice acceptor) and white (W1′-generated splice acceptor) boxes. Nucleic acid numbers are indicated at the beginning and end of each line. The first number is arbitrary and begins at the XhoI restriction site for rhesus LCV clone pAG6. The EBV and baboon LCV sequences were then given consistent numbers based on the alignment. For EBV, base 1 corresponds to position 44547 and base 742 corresponds to position 45289 of the last W repeat in the EBV genome. (B) Alignment of EBV splice donor and acceptor sites and the predicted homologous sites for baboon LCV (bLCV) and rhesus LCV (rLCV). Consensus donor and acceptor sites are shown at the top, and the exon junctions are shown at the left.
FIG. 4
FIG. 4
Sequence and expression of a rhesus LCV EBNA-LP cDNA clone. (A) Sequence of rLPcDNA-1 5′ untranslated termini. rLPcDNA-1 contains an in-frame splice to generate an initiation codon. The EBV C2 exon sequence is shown below the rLPcDNA-1 sequence for comparison. (B) The entire predicted amino acid sequence of rLPcDNA-1. The W1, W2, Y1, and Y2 boundaries are shown above each line. Amino acid numbers are shown at the beginning and end of each line. Sequence changes in the last W1/W2 exons leading to amino acid changes are indicated in bold. The predicted amino acids at those positions based on genomic sequencing are indicated below in plain type. (C) Western blot analysis of DG75 cells transfected with a rhesus LCV EBNA-LP expression plasmid (lane 1), vector expression plasmid only (lane 2), and an EBV EBNA-LP expression plasmid (lane 3). The blot was probed with a monoclonal antibody reactive with the EBV EBNA-LP protein (JF186). The arrow designates the detected EBNA-LP band. (D) Same as panel C except that the blot was probed with monoclonal antibody M2, which reacts with the Flag epitope that was engineered to be expressed on both EBV and rhesus LCV EBNA-LP proteins. Both EBNA-LP cDNAs used in these assays contain four BamHI W repeats. The two arrows indicate the detected EBV EBNA-LP and rhesus LCV EBNA-LP bands.
FIG. 5
FIG. 5
Alignment of the rhesus LCV EBNA2 amino acid sequence with the EBV type 1 (B95-8), type 2 (AG876), and baboon LCV EBNA2 amino acid sequences. Amino acid residues identical between sequences are indicated by asterisks, and amino acid residues with overall similarity are indicated by a dotted line. Conserved regions have been boxed and shaded and numbered in consecutive order as CR1 to CR9. Amino acid numbers are indicated at the beginning and end of each line.
FIG. 6
FIG. 6
Detection of the rhesus LCV EBNA2 protein in transiently transfected lymphoid cells. Western blot analysis of transfected cell lysates was performed with monoclonal antibody PE2. The cell lysates were prepared from cells transfected with SG5 vector (lane 1), pPDL176A (EBNA2 expression plasmid) (lane 2), and pAG115 (rhesus LCV EBNA2 expression plasmid) (lane 3). The arrows indicate the detected EBNA2 and rhesus LCV EBNA2 (rEBNA2) proteins, which are approximately 87 and 100 kDa, respectively.
FIG. 7
FIG. 7
The rhesus LCV EBNA2 protein stimulates the latency C and LMP2a promoters in transient cotransfections and is dependent on CBF1. Both EBV EBNA2 and rhesus LCV EBNA2 were tested for the ability to activate reporter plasmids containing EBNA2-responsive promoters. The amount of target plasmid in all experiments was 2.0 μg. The results are shown as an average of three experiments; the T-bars indicate standard errors. (A) Transactivation of the EBV Cp (−1024 to +3) (11). EBV EBNA2 is shown as white bars, and rhesus LCV EBNA2 is shown as black bars. (B) Transactivation of the rhesus LCV Cp (−1024 to +3) (12). EBV EBNA2 is shown as white bars, and rhesus LCV EBNA2 is shown as black bars. (C) Transactivation of the EBV LMP2A promoter (60). EBV EBNA2 is shown as white bars, and rhesus LCV EBNA2 is shown as black bars. (D) The rhesus LCV EBNA2 was the only effector plasmid used in these experiments. The white bars indicate rhesus LCV induction of a wild-type rhesus LCV Cp, and the shaded bars indicate the level of rhesus LCV EBNA2 induction of a rhesus LCV Cp containing a mutant CBF1 binding site (12).
FIG. 8
FIG. 8
EBNA-LP from either rhesus LCV or EBV enhances EBNA2-mediated transactivation of the BamCpLUC reporter gene. Plasmids expressing EBNA2 (white bars) or rhesus LCV (rLCV) EBNA2 (black bars) were transfected into DG75 cells (1.0 μg). The reporter plasmid was BamCp8LUC, which contains eight copies of the 100-bp EBNA2 enhancer from the latency C promoter (see Materials and Methods for details). In some samples, EBV EBNA-LP or rhesus LCV EBNA-LP expression plasmids were cotransfected. The presence or absence of the EBNA-LP or EBNA2 plasmids is indicated below the graph.
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