Analysis and characterization of the complete genome of tupaia (tree shrew) herpesvirus

Abstract

The tupaia herpesvirus (THV) was isolated from spontaneously degenerating tissue cultures of malignant lymphoma, lung, and spleen cell cultures of tree shrews (Tupaia spp.). The determination of the complete nucleotide sequence of the THV strain 2 genome resulted in a 195,857-bp-long, linear DNA molecule with a G+C content of 66.5%. The terminal regions of the THV genome and the loci of conserved viral genes were found to be G+C richer. Furthermore, no large repetitive DNA sequences could be identified. This is in agreement with the previous classification of THV as the prototype species of herpesvirus genome group F. The search for potential coding regions resulted in the identification of 158 open reading frames (ORFs) regularly distributed on both DNA strands. Seventy-six out of the 158 ORFs code for proteins that are significantly homologous to known herpesvirus proteins. The highest homologies found were to primate and rodent cytomegaloviruses. Biological properties, protein homologies, the arrangement of conserved viral genes, and phylogenetic analysis revealed that THV is a member of the subfamily Betaherpesvirinae. The evolutionary lineages of THV and the cytomegaloviruses seem to have branched off from a common ancestor. In addition, it was found that the arrangements of conserved genes of THV and murine cytomegalovirus strain Smith, both of which are not able to form genomic isomers, are colinear with two different human cytomegalovirus (HCMV) strain AD169 genomic isomers that differ from each other in the orientation of the long unique region. The biological properties and the high degree of relatedness of THV to the mammalian cytomegaloviruses allow the consideration of THV as a model system for investigation of HCMV pathogenicity.

FIG. 1

(A) Physical map of the THV strain 2 genome consisting of EcoRI, HindIII, and EcoRI/HindIII DNA fragments. The THV DNA fragments that are used to determine the nucleotide sequence of the whole THV genome are shaded and bordered in bold. Beneath the physical map, the G+C content over the course of the whole THV genome is shown (B). It varies between 46.85 and 74.43%.

FIG. 2

Coding strategy of the THV genome. ORFs that code for proteins with homologies to known HCMVA and MCMVS proteins are depicted as black arrows, and those with no detected homologies are shown as white arrows. ORFs that show the highest homology only to an HCMVA or an MCMVS protein are in red or green, respectively. The orientation of each arrow corresponds to the direction of transcription. Hairpin loops with a stem length of more than 7 bp and a loop of 3 to 20 bases are marked by little black arrows. ORFs with adjacent numbers are members of corresponding gene families defined by significant homology to one another.

FIG. 3

Graphic representation of the homology value distribution of seven conserved THV protein groups that are classified according to possible functions. Individual groups are marked by distinct colors. The homology values that underlie this graphic are the highest detected homologies to known proteins regardless of the virus species the proteins belong to.

FIG. 4

Arrangement of the seven conserved core gene blocks in herpesvirus genomes. The pattern of these seven clusters is characteristic for the three herpesvirus subfamilies. The assignment of the individual virus species to the subfamily Alpha-, Beta-, or Gammaherpesvirinae is given in parentheses after the particular virus name. U_s (unique short region) and U_L (unique long region) are the descriptions of the two parts of the herpesvirus genomes that are able to form isomers. The inverted spelling of U_L in the genome of HCMVA characterizes a distinct isomer that differs from the prototype L-S isomer in the orientation of the U_L region. The black boxes mark the positions of large repetitive elements. The diagram corresponds to the 1996 publication of Gompels et al. (31) complemented by data on the THV and MCMV genomes.

FIG. 5

Comparative analysis of the arrangement of homologous ORFs among THV, HCMVA, and MCMVS. Each arrow shows the orientation of transcription. The length of each arrow is standardized and does not correspond to the actual length of the ORF. The vertical lines designate the start points of the individual ORFs. The shaded boxes of the HCMVA genome represent repetitive DNA elements that are, in part, responsible for genomic isomerization. These repetitive DNA elements divide the HCMVA genome into U_L (unique long) and U_s (unique short) regions. (A) Comparison of the arrangement of homologous ORFs between THV and HCMVA. The THV ORFs show colinearity with those of a distinct HCMVA genome isomer that is characterized by an inverted U_L region compared to the prototype L-S isomer. (B) Comparison of the arrangement of homologous ORFs between HCMVA and MCMVS. The MCMVS ORFs show colinearity with those of the prototype HCMVA genome isomer.

FIG. 6

Eight phylogenetic trees derived by comparison of the DNA polymerase, DNA polymerase accessory protein, glycoprotein B, probable transport and processing protein, major DNA-binding protein, major capsid protein, viral terminase, and uracil DNA-glycosylase amino acid sequences of different herpesvirus species. The three main branches of the trees represent the evolutionary lineages of the herpesvirus subfamilies Alpha (α)-, Beta (β)-, and Gammaherpesvirinae (γ). The sequences of the individual proteins used to construct the phylogenetic trees were taken from the GenBank and SwissPort release 39 databases.