PMC

ORF73-encoded proteins of rhadinoviruses: homology to LANA and EBNA1. Shown is a graphical representation of the results presented in Table . Hatched boxes, repeat regions within LANA and ORF73-encoded proteins of other rhadinoviruses. Primary amino acid homology of the ORF73-encoded proteins to LANA was evaluated by BLASTP pairwise sequence alignment. Black bars, regions showing significant homology to LANA; grey boxes, regions which show predicted structural homology to EBNA1, as judged by 3D-PSSM analysis (see Table for details). The ORF73-encoded proteins of alcelaphine herpesvirus 2 and ovine herpesvirus 2 are not depicted since they do not show homology to either LANA or EBNA1.

3D-PSSM alignment of EBNA1 and LANA. The carboxy-terminal domain of LANA (aa 932 to 1162; GenBank accession no. AAC57158) was used to search the 3D-PSSM database (program version 2.6.0; Imperial College of Science, Technology and Medicine [http://www.sbg.bio.ic.ac.uk/∼3dpssm/]). Shown is a modified version of the alignment of the relevant regions in LANA (aa 1010 to 1144) and EBNA1 (aa 461 to 607) generated by 3D-PSSM. The primary sequences of LANA and EBNA1 (accession no. NP_039875) are given. Dots, gaps introduced into the aligned sequences. Identities and positive (+) or negative (−) scores for aligned residues as assigned by 3D-PSSM are shown between the primary sequence data for LANA and EBNA1. The secondary structure of LANA (LANA-PSS) as predicted by the 3D-PSSM algorithm and the known secondary structure of EBNA1 (EBNA1-SS) are shown above or below the primary sequences of LANA and EBNA1, respectively. The CORE values shown below the aligned sequences provide an index for the contribution of residues to hydrophobic interactions (on a scale from 0 to 9) within the resolved EBNA1 structure. High values indicate buried residues important for the core structure, whereas low values indicate residues on the surface of the protein.

The carboxy-terminal region of LANA is required for replication activity. (A) Cartoon showing the proteins encoded by the expression vectors pCMN:73 (full-length LANA) and pCMN:L1-1036 (lacking the carboxy-terminal 126 aa of LANA). Mutant L1-1036 is truncated within the region showing structural homology to EBNA1. All amino acid positions conform to the LANA sequence with GenBank accession no. AAC57158. (B) DpnI resistance assays. Expression construct pCMN:73 or pCMN:L1-1036 or the empty expression vector pCMN was transfected into BJAB cells together with the reporter construct pGTR4. Episomes were recovered 72 h later by Hirt extraction and analyzed as described for Fig. . (C) Western blot analysis of LANA expression. Aliquots of the transfected BJAB cells (4 × 10 ⁶ cells) described above were harvested 48 h posttransfection, and extracts containing 40 μg of total protein were analyzed by Western blotting using a polyclonal antiserum against LANA (). The positions of size markers (in kilodaltons) are shown. Note that LANA shows multiple bands and runs higher than expected from its calculated molecular mass (135 kDa). The protein shows similar behavior in KSHV-positive BCBL1 cells (data not shown). (D) Immunofluorescence analysis of endothelial SLK cells transfected with the expression construct pCMN:73 (left) or pCMN:L1-1036 (right). LANA was detected with polyclonal antibodies, and DNA was stained with DAPI (4′,6′-diamidino-2-phenylindole).

Replication of TR-containing plasmids in B cells. (A) Functional elements of reporter and expression constructs. The reporter constructs used in this study are based on the vector backbone pGFP. The vector contains a GFP expression cassette driven by the CMV promoter and a multiple-cloning site (MCS) upstream of the CMV promoter. The linker inserted into the BsaI site of pGFP and the cleavage sites of BsmBI and BbsI are shown enlarged. Cleavage with either enzyme generates identical NotI-compatible overhangs. These sites together with the indicated ClaI site were used to assemble the four head-to-tail-oriented TR units of pGTR4 (see text for details). The reporter construct pGTR4:73 contains the CMV-driven ORF73 expression cassette of pCDNA3:73 inserted into the MCS of pGTR4. The LANA expression constructs pCDNA3:73 and pCMN:73 were used to provide LANA in trans. EGFP, enhanced GFP. (B to D) DpnI resistance assays. The indicated constructs were introduced into either BJAB cells (B and C) or the stable LANA-expressing cell line BJAB:73 (D) as described in the text. Episomal plasmids were recovered 72 h later by Hirt extraction and subjected to digestion with either XhoI (which cuts once in the reporters) (−) or XhoI and DpnI (+). Standards were analyzed by Southern blotting, and reporter constructs were detected with a radioactively marked GFP probe. The positions of size markers (in kilobase pairs) are shown. Quantitation of linearized versus DpnI-digested plasmids was carried out with a phosphorimager (Molecular Dynamics Storm860 and ImageQuant software). A relatively large proportion of cells undergo cell death following the electroporation procedure; the smears below the linearized bands result from degraded DNA released from dead cells as well as degraded, adherent extracellular plasmid DNA and thus were not included in the quantitation.