Complete genomic sequence of an Epstein-Barr virus-related herpesvirus naturally infecting a new world primate: a defining point in the evolution of oncogenic lymphocryptoviruses

Abstract

Callitrichine herpesvirus 3 (CalHV-3) was isolated from a B-cell lymphoma arising spontaneously in the New World primate Callithrix jacchus, the common marmoset. Partial genomic sequence analysis definitively identified CalHV-3 as a member of the Epstein-Barr virus (EBV)-related lymphocryptovirus (LCV) genus and extended the known host range of LCVs beyond humans and Old World nonhuman primates. We have now completed the first genomic sequence of an LCV infecting a New World primate by describing the unique short region, the major internal repeat, and a portion of the unique long region. This portion of the genome contains the putative latent origin of replication and 13 additional open reading frames (ORFs), 5 of which show no homology to any viral or cell genes. One of the novel genes, C5, is a positional homologue for the transformation-essential EBV gene EBNA-2. The marmoset LCV genome is also notable for the absence of viral interleukin-10 and small nonpolyadenylated RNA homologues. Marmoset LCV transcripts encoding putative latent infection nuclear proteins have a common leader sequence that is spliced from the major internal repeat in a manner similar to that of the EBV EBNA-LP, suggesting strong conservation of a common promoter and splicing of these latent infection mRNAs. An EBV LMP2A-like spliced transcript crossing the terminal repeats encodes a unique ORF, C7, with multiple transmembrane domains and tyrosine kinase phosphorylation sites functionally reminiscent of EBV LMP2A. However, the carboxy-terminal location of the candidate phosphotyrosine residues is more reminiscent of the Kaposi's sarcoma-associated herpesvirus K15 gene and provides potential evidence of an evolutionary transition from rhadinoviruses to lymphocryptoviruses. The unusual gene repertoire of the marmoset LCV differentiates ancestral viral genes likely present in an LCV progenitor from viral genes acquired later as primates and LCV coevolved, providing a defining point in the evolution of oncogenic LCVs.

FIG. 1.

Partial map of EBV and marmoset LCV genomes, showing the U_S, IR1, and a portion of the U_L region. (A) Map of EBV U_S, IR1, and a portion of U_L. The map is drawn to scale, and the nucleotide coordinates as numbered in GenBank are shown at the top. U_S and U_L regions are delimited by arrows, and repeat regions are boxed. The lytic origin of replication (Orilyt) is shown, as well as the latent origin of replication (OriP) with the family of repeats (FR) and dyad symmetry element (DS). Each ORF is represented as a black arrow. The orientation of the ORFs is shown by the direction of the arrow. EBERs are represented by short lines. (B) Map of marmoset LCV U_S, IR1, and a portion of U_L. Each ORF is represented as an open arrow.

FIG. 2.

Nucleotide and amino acid sequences of marmoset LCV C0 and C5 genes. Arrowheads indicate C0 exon boundaries. Each C0 exon is labeled above the nucleotide sequence. Translational start and stop codons are shown in bold. C0 and C5 nuclear localization signals are boxed. C0 serine residues predicted to be phosphorylated are indicated by asterisks. A cluster of acidic residues in the C5 carboxy terminus is shown in bold.

FIG. 3.

Marmoset LCV C7 gene. (A) Nucleotide and amino acid sequences of the longest RT-PCR product. Potential translational start and stop codons are shown in bold. C7 exon boundaries are identified with arrowheads. Predicted transmembrane domains are shaded. Potential SH2 domains are boxed. (B) Comparison of the marmoset LCV C7, EBV LMP2, and Kaposi's sarcoma-associated herpesvirus K15 putative secondary structures. Predicted transmembrane domains are shown with shaded boxes. Tyrosine residues predicted to be phosphorylated in SH2 and immunoreceptor tyrosine-based activation motifs are boxed and identified as SH2 and ITAM, respectively. Other tyrosine residues not predicted to be phosphorylated are identified as Y.

FIG. 4.

Putative marmoset LCV Orip and comparison with EBV, rhesus LCV, and baboon LCV OriPs. (A) Scheme of the EBV, rhesus LCV, baboon LCV, and marmoset LCV Orip elements. Monomers of the family of repeats (FR) and the dyad symmetry element (DS) are represented as open boxes. The number and length of the monomers are indicated. Note that the marmoset LCV FR element contains two types of monomers, repeat A and repeat B. (B) Comparison of the consensus sequence of EBV, rhesus LCV, baboon LCV, and marmoset LCV repeat A and repeat B FR monomers. Consensus sequences for a given virus are shown as follows: a nucleotide conserved in all monomers is in boldface uppercase; a nucleotide conserved in more than 75% of the monomers is shown in lightface uppercase; and a nucleotide conserved in less than 75% of the monomers is shown in lowercase. X designates shorter monomers. The EBNA-1 binding site consensus sequence (TAGCATATGCTA) is boxed. (C) Comparison of the consensus sequence of EBV, rhesus LCV, and baboon LCV DS monomers. Consensus sequences are displayed with the criteria defined in B. No DS element could be identified in the marmoset LCV genome.

FIG. 5.

Absence of EBERs in marmoset LCV-infected cells. Total RNA from EBV-negative BJAB cells, rhesus (Rh) LCV-infected LCL 8664 cells, or marmoset LCV-infected CJ0149 cells were separated by gel electrophoresis and visualized by ethidium bromide staining (top panels). Northern blots were hybridized with ³²P-labeled rhesus LCV cosmid CD1 DNA containing the entire U_S and IR1 and a portion of the U_L region of rhesus LCV (bottom left) or ³²P-labeled marmoset LCV cosmid E4 DNA containing the entire U_S and IR1 and a portion of the U_L region of marmoset LCV (bottom right). The positions of the rhesus LCV EBERs are shown by an arrow.

FIG. 6.

Structure of the marmoset LCV C0, C5, C3, and ORF39 transcripts. Schemes for the marmoset LCV genome (A) and C5, C3, and ORF39 spliced mRNAs (B) are shown. Genome and mRNAs are not drawn to scale. Exons 1a and 1b of the common leader sequence (C0) are derived from the repeats in IR1, and C0 exons 2, 3, and 4 are derived from U_L. Alternative splicing of an additional fifth exon at nucleotides 102177 to 102007 for C3 and ORF39 transcripts is shown. RT-PCR amplification products for the respective transcripts are shown on the right. Locations of the primers used for the RT-PCR analysis are indicated by arrowheads. Control amplifications with all reagents except reverse transcriptase (−RT) and with water substituted for cDNA (W) are shown. The positions of size markers are shown in base pairs.

FIG. 7.

Marmoset LCV genome and ORFs and homology to EBV ORFs. (A) Organization of the marmoset LCV genome. Major repeat regions IR1 (nt 142148 to 120197), IR2 (nt 117265 to 116619), IR3 (nt 69982 to 69841), IR4 (nt 2792 to 2469), and terminal repeats (TR, nt 161345 to 152171) are identified in the marmoset LCV genome. (B) Marmoset LCV ORFs and amino acid homology to EBV ORFs. The percent amino acid similarity is shown on the y axis. Putative latent, immediate-early, early, and late lytic ORFs are represented by black, dark grey, light grey, and white arrows, respectively. The ORFs are numbered from right to left, and the orientation of the ORFs is shown by the direction of the arrow. ORFs common to other herpesviruses are shown with a bold outline. The initiator codon for each ORF is positioned accurately, but the ORF size is not drawn to scale. (C) Marmoset LCV unique genes. ORFs are represented as defined for B.