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Journal of Virology, May 2001, p. 4854-4870, Vol. 75, No. 10
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.10.4854-4870.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Analysis and Characterization of the Complete Genome of Tupaia (Tree Shrew) Herpesvirus

Udo Bahr and Gholamreza Darai*

Institut für Medizinische Virologie, Universität Heidelberg, D-69120 Heidelberg, Germany

Received 13 November 2000/Accepted 26 February 2001


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The family Herpesviridae comprises more than 100 different virus species with a worldwide occurrence in all taxonomic groups of vertebrates. The supposed roots of this virus family are in very early evolutionary times, and a long period of development has resulted in the appearance of extremely well host-adapted virus species (47, 61, 62, 63), often more than one in a single host, for example, the eight different human herpesviruses HSV-1 (herpes simplex virus type 1), HSV-2, varizella-zoster virus, HCMV (human cytomegalovirus), EBV (Epstein-Barr virus), HHV-6 (human herpesvirus 6), HHV-7, and HHV-8, that are adapted to different cellular and molecular niches in the same host species (88).

A member of the large herpesvirus family is the Tupaia herpesvirus (THV) that infects tree shrews (Tupaia spp., family Tupaiidae), a group of primitive higher mammals (Proteutheria, Scandentia) that is supposed to have diverged at the base of the primate evolutionary tree (52, 69). Tree shrews were originally distributed in Southeast Asia and are used worldwide as laboratory animals in neurological and physiological research. THV was isolated by Mirkovic et al. in 1970 (68) from a spontaneously degenerating lung tissue culture of a tree shrew and subsequently classified as a herpesvirus by electron microscopic examination (57). From 1977 to 1985, six additional isolates were isolated from malignant lymphoma tissue cultures and degenerating spleen cell cultures of tree shrews (19, 20, 21, 22, 24, 49). The seven THV isolates were grouped into five strains (THV strains 1 to 5) according to their restriction endonuclease cleavage patterns. Molecular cloning and physical mapping of the genome of THV strain 2 was performed, and a complete genome library was established (49). THV strain 2 was isolated in 1979 from a spontaneously degenerating malignant lymphoma cell culture by Darai et al. (19).

THV particles show the classical morphology of herpesviruses (19, 61, 73) and contain a linear double-stranded DNA genome of about 200 kbp (21, 49). The detection of concatemeric viral DNA molecules in infected cells (50) corresponds to the rolling-circle model of herpesvirus genome replication. The herpesviruses have been classified into six genome groups (A to F) (72) according to the presence and arrangement of large repetitive DNA sequences. THV is the prototype species of genome group F, which is characterized by a unique DNA sequence without any extended repetitive DNA elements. The family Herpesviridae is subdivided into the subfamilies Alpha-, Beta-, and Gammaherpesvirinae according to the length of the replication cycle, speed of spreading in cell culture, host range, and location of latency, which is an important biological characteristic of herpesviruses (73). According to its biological properties (19, 20, 22, 24) and in agreement with recent data (5, 82), THV is supposed to be a member of the subfamily Betaherpesvirinae.

THV infections cause a remarkable variety of different clinical pictures in tree shrews, ranging from inapparent to deadly infections and the development of malignant lymphomas (19, 23, 41). Based on the evolutionary stage of tree shrews and the biological and genomic properties of THV, the elucidation of the viral coding strategy is of particular interest. The characterization of the primary structure of the whole genome of THV strain 2 isolated from a malignant lymphoma (19) is the subject of this report. This study allowed the determination of the final phylogenetic placement of THV within the family Herpesviridae.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Viral DNA and genomic library. Propagation of THV on tupaia baby fibroblasts and isolation of viral DNA were carried out as described previously (22). Recombinant plasmids harboring specific DNA sequences of the THV genome were obtained from a defined genome library as described elsewhere (49).

Strategy of determination of the THV genome nucleotide sequence. Determination of the complete nucleotide sequence of the THV genome was accomplished by analysis of the DNA nucleotide sequences of recombinant plasmids harboring specific EcoRI, HindIII, and EcoRI/HindIII fragments of the THV genome (Fig. 1) that form the entirety of the THV genome. The nucleotide sequences of the individual recombinant plasmids were determined by primer walking (86). The correctness of the physical map shown in Fig. 1 was confirmed by amplification and sequencing of the genome regions of original THV DNA around the endonuclease restriction sites, which allowed the assembly of the nucleotide sequences of the individual THV fragments resulting in the nucleotide sequence of the whole THV genome.


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  		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%.

Enzymes and DNA isolation. The restriction endonucleases were purchased from Roche Diagnostics GmbH (Mannheim, Germany). Incubations were carried out according to standard procedures for each enzyme. The recombinant plasmids harboring the DNA sequences of the EcoRI, HindIII, and EcoRI/HindIII fragments that were used to determine the complete nucleotide sequence of the THV genome were purified using the Qiagen Plasmid Midi Prep (Qiagen GmbH, Hilden, Germany) procedure. The PCR products were purified using Micro-Spin S-300 HR columns (Pharmacia Biotech).

DNA sequencing. The recombinant plasmids and the purified PCR products were sequenced using the DyeDeoxy Terminator Taq cycle sequencing technique (Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit; Applied Biosystems GmbH, Weiterstadt, Germany) and a 373A "Extended" DNA sequencer (Applied Biosystems) as described previously (86). The nucleotide sequence of the THV genome was determined by primer walking. The Sequence Navigator software (Version 1.01; Applied Biosystems) was used to assemble the nucleotide sequences obtained from individual sequencing reactions.

Computer-assisted analysis. The strategies used to identify THV genes likely to encode were based on those used in the sequence analysis of other herpesviruses (71). The major criterion for identifying a coding sequence was the presence of an open reading frame (ORF) with a minimum length of 300 bp and less than 60% overlap with adjacent ORFs. The presence of ORFs with more than 60% overlap in Fig. 2 has its explanation in similar lengths and the absence of convincing identification criteria which distinguish between the corresponding ORFs. In addition, analysis of codon usage, the presence of consensus promoter sequences, and homology to known genes were used to support initial ORF selection. Gaps between ORFs were inspected for smaller ORFs and were included in the final ORF map (Fig. 2) if they satisfied the other criteria used for ORF selection. The identification of ORFs was performed with the program TRANSLATE of the the PC/GENE program, release 6.85 (Intelligenetics Inc., Mountain View, Calif.); the decisive criterion for the selection of an ORF was the presence of an ATG start codon and a stop codon (TAA, TGA, TAG) at the end. Searches of potential THV proteins for homology to known proteins were performed by applying BLAST (basic local alignment search tool) (3) and FSTPSCAN of PC/GENE to SWISSPROT database release 39. Protein alignments were carried out with the CLUSTAL program (39). Examination of the DNA sequence for transcription signals was performed by using the search features of the program EUKPROM of PC/GENE 6.85. Protein motif searches were performed with the program PROSITE of PC/GENE 6.85 and PROSITE release 16.25 on the ExPASy Molecular Biology Server (10, 40). The sequence was analyzed for tandem and inverted repeats by using the program REPEATS of PC/GENE 6.85. The phylogenetic trees were calculated using the personal computer programs ClustalW (version 1.64b) (85), Kitsch (version 10.0), Protdist (version 10.0), and Treeview (version 1.5.2).


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  		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.

Nomenclature. All of the ORFs that are likely to be coding sequences are listed in Table 1. They are numbered in the order of their appearance when the genome sequence is analyzed from base pair 1 to base pair 195,859. ORFs that are oriented to the right are marked with the letter R, and ORFs that are oriented to the left are marked with the letter L. In addition, ORFs are marked with the letter T for THV and numbered according to homologous proteins of HCMV strain AD169 (HCMVA) and murine cytomegalovirus strain Smith (MCMVS). THV ORFs that are assumed to be coding sequences but have no homology to known proteins are marked with the letter t and labeled so as to fill the gaps between the homologous ORFs.

                              
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  		TABLE 1.   Supposed THV coding ORFsa

RT PCR. Tupaia baby fibroblasts were infected with THV strain 2. Total cellular RNA was isolated 14 h postinfection. RNA isolation was performed using the guanidinium-cesium chloride method as described previously (74). The reverse transcription step was carried out using the RNA LA PCR Kit, version 1.1 (Takara Shuzo Co., Shiga, Japan). PCR was performed using 0.5 fmol of the template DNA in 100-µl volumes containing 1.5 mM MgCl2, 12.5 nmol of each deoxynucleoside triphosphate, 50 pmol of each primer, and 2.5 U of ExTaq DNA polymerase (Takara Shuzo Co.). A total of 35 cycles were run in an automated temperature cycling reactor (Genius; Techne, Cambridge, United Kingdom) under cycling conditions of 96°C for 30 s, 60°C for 1 min, and 72°C for 2 min per cycle. Specific oligonucleotide primers were designed in order to amplify the coding region around the splice sites of THV genes T33 and T89. The following primers were used in the reverse transcriptase PCR (RT-PCR) experiments: 5'-CCATGGACGTCCTGCTGGCTC-3' (primer 1) and 5'-CCCACGGTGCAGCTGGTGTAG-3' (primer 2) for T33 and 5'-AAGCACGTTTCCCAGTTCGTCC-3' (primer 3) and 5'-GAGTTTGGTCAGGAAGCAGGTG-3' (primer 4) for T89. Primers 2 and 4 were used for first-strand synthesis of the cDNA by RT reaction.

Nucleotide sequence accession number. The complete DNA sequence determined in this study has been submitted to the GenBank database and assigned accession number AF281817.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Features of the complete nucleotide sequence of the THV genome. The nucleotide sequences of the accentuated THV fragments in Fig. 1 were determined by automated cycle sequencing and primer walking (86). The assembly of the nucleotide sequences of all of the THV fragments used according to the physical map of the viral genome (Fig. 1) resulted in determination of the complete DNA nucleotide sequence of the THV genome, comprising 195,859 bp. The DNA sequences of the genomic termini were defined by Albrecht et al. in 1985 (2) and were used to determine the left and the right ends of the genome in this study. Altogether, 1,473 sequencing reactions with a total of 684,259 determined bases were performed to obtain the nucleotide sequence of the complete THV genome. Both DNA strands were sequenced independently, and each nucleotide was determined with an average redundancy of 1.75. The average G+C content of the whole THV DNA molecule was found to be 66.5%. As shown in Fig. 1, the G+C distribution is not constant in the viral genome (46.85 to 74.43%). Analysis of the codon usage of THV revealed that the third base of codons of potential viral genes is almost exclusively a G or C.

Repetitive elements of the THV genome. About 2,000 direct and 1,611 inverted repeats were identified. However, all of these repeats were no longer than 25 bp and occurred in short tandem arrangements. Some of the inverted repeats are supposed to form stable hairpin structures and are drawn in the ORF map of THV in Fig. 2. Furthermore, the analysis of the THV nucleotide sequence resulted in the identification of rare direct repeats between 25 and 105 bp in length. They are located between nucleotide positions 64165 and 64334, 108477 and 109442, 140381 and 140452, 186262 and 186305, and 193606 and 194265. The repetitive DNA sequences of the THV genome are restricted and are not comparable to the large repetitive elements of other herpesvirus genomes that are classified into genome groups A to E. This is in agreement with the previous nomination of THV as the prototype species of herpesvirus genome group F (49, 72).

ORFs of the THV genome. THV genome analysis revealed 582 ORFs with a possible coding capacity of more than 40 amino acids. The distribution of the ORFs on the two DNA strands is very regular, with 286 oriented to the left and 296 oriented to the right. Altogether, 158 ORFs were selected to be actual coding sequences according to the criteria used to determine herpesvirus genes (see Materials and Methods) and are listed in Table 1. Potential sites and signatures of the hypothetical viral proteins and the highest homologies to known proteins are also given in Table 1. The 158 ORFs selected to encode viral proteins are depicted in the ORF map of the THV genome in Fig. 2. Those viral gene products that are homologous to known herpesvirus proteins are shown as black or colored arrows. It is evident that the homologous, conserved ORFs are accumulated in the center of the genome. t6-t22.13, t39-t43.4, t58-t63, and t106-t130 are genome regions with ORFs that encode THV-specific proteins with no known homologues. RT-PCR experiments revealed that T33 and T89 consist of two exons. Comparative amino acid analysis to known proteins showed that T36 could be spliced, but it was not possible to prove this by RT-PCR experiments.

Conserved THV proteins show the highest homologies to known proteins of HCMVA (13) and MCMVS (71) as representatives of the evolutionary lineages of primate and rodent cytomegaloviruses, respectively. The homology values of the THV proteins to the proteins of these two virus species and the pertinent potential functions are summarized in Table 1. The proteins with the highest homologies are underlined, and it is clear that they are distributed equally between HCMVA and MCMVS.

All of the homologous THV ORFs are classified into functional groups and plotted according to their highest values of identity to known herpesvirus proteins (Fig. 3). The number of proteins gets smaller when the identity values rise, with T89 (viral terminase) being the only protein with 60 to 70% identity to known herpesvirus proteins. None of the functional protein groups show an extraordinary distribution of identities or strikingly low or high conservation. As a rule, they are almost constantly distributed over the range of identity values, with a decreasing number of proteins when the values get higher.


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  		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.

Comparison of the THV ORFs that are supposed to be coding sequences led to the identification of six gene families. The members of each of these six gene families are distinguished by significant identity and similarity to each other, raising the possibility that these gene groups in each case originated from one ancestral gene by duplication events. The gene families are numbered 1 to 6 and drawn in the ORF map in Fig. 2. A striking feature of the members of such gene groups is the fact that they are located in close proximity in the genome, often in a tandem-like arrangement, underlining the hypothesis that they were produced by gene duplication. A concentration of gene family members could be seen in the left part of the THV genome, especially in the nonconserved region between t6 and t22.13. A similar distribution of gene families is present in the genomes of HCMVA (13), MCMVS (71), rat cytomegalovirus (87), or HHV-6 (31), where duplicated genes are also concentrated in nonconserved regions at the left and right ends of the genomes. Significant homology between the members was the main criterion for the formation of the six THV gene families. Another possible way to relate genes to families is by the similarity of the functions of the encoded proteins. In HCMVA and MCMVS, homologues of G protein-coupled receptors (GCRs) form a family. For that reason, T33 and T78, both homologous to G protein-coupled receptors, could also be seen as members of a gene family. This GCR and members of the US22 gene family are conserved among THV, HCMVA, and MCMVS. Both members of the HCMVA UL25-UL35 gene family are conserved in the THV genome. However, homology between the two potential proteins is extremely low.

Gene arrangements of the THV genome. Homologous THV, HCMVA, and MCMVS proteins are summerized in Table 1. All together, 72 proteins are homologous between THV and HCMVA and 73 are homologous between THV and MCMVS. Both cytomegalvirus species are members of the subfamily Betaherpesvirinae. Table 2 shows these homology relationships extended by HHV-6 (Betaherpesvirinae) (31), EBV (Gammaherpesvirinae) (4), and HSV-1 (Alphaherpesvirinae) (58, 59, 60). HHV-6 shares 66, EBV shares 42, and HSV-1 shares 38 homologous proteins with THV. The most homologous proteins are found between THV and the mammalian cytomegaloviruses. The extent of homology between THV and alpha- or gammaherpesviruses is clearly less. Furthermore, Table 2 shows a group of almost 40 homologous proteins that could be found in every herpesvirus species of mammals and birds. These genes are termed core genes and are supposed to be part of the genome of the common ancestor of these herpesviruses. The core genes form seven conserved gene clusters whose arrangements are characteristic of the corresponding herpesvirus subfamily. A comparison of the gene block arrangements among THV, HCMVA, MCMVS, HHV-6, HSV-1, and EBV is shown in Fig. 4. In the genomes of THV, MCMVS, and HCMVA, the arrangement of the seven clusters is almost the same with regard to both the order of appearance and the spaces between the individual blocks. The same order of gene clusters could also be found in HHV-6, but they are more concentrated in the center of the genome. HCMVA, MCMVS, and HHV-6 are members of the subfamily Betaherpesvirinae. The gene cluster arrangement in the genome of HSV-1, and alphaherpesvirus, differs from those of the betaherpesviruses in the position of gene block III, which is localized at the left end of the UL region of the HSV-1 genome. The gammaherpesvirus EBV shows a further different arrangement that is characterized by the localization of gene blocks III and I at the right end of the long unique genome region of EBV. The colinear arrangement of conserved genes within the genomes of THV, HCMVA, and MCMVS is plotted in detail in Fig. 5. HCMVA is able to form four genomic isomers. The arrangement of genes in the prototype HCMVA isomer is colinear with that in the MCMVS genome, which is not able to form isomers. The arrangement and orientation of the homologous THV genes correspond to those of a different HCMVA genome isomer that is characterized by an inverted UL region compared to the prototype HCMVA isomer. The difference in total genome length between THV (195,857 bp) and HCMVA (229,354 bp) is caused mainly by the presence of the repetitive elements in the HCMVA genome. Without these elements, the lengths of the THV and HCMVA genomes are very similar.

                              
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  		TABLE 2.   HCMVA, MCMVS, HHV-6, EBV, and HSV-1 proteins homologous to potential THV proteinsa



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  		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. Us (unique short region) and UL (unique long region) are the descriptions of the two parts of the herpesvirus genomes that are able to form isomers. The inverted spelling of UL in the genome of HCMVA characterizes a distinct isomer that differs from the prototype L-S isomer in the orientation of the UL 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.


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  		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 UL (unique long) and Us (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 UL 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.

Phylogenetic classification of THV. The phylogenetic trees derived from the 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 herpesviruses are shown in Fig. 6A to H. The selected proteins are those with the highest levels of homology between different members of the family Herpesviridae. The phylogenetic trees show a distinct subdivision into three main branches corresponding to the herpesvirus subfamilies Alpha-, Beta-, and Gammaherpesvirinae, which are groups of herpesvirus species with similar biological properties and phylogenetic relatedness. In all eight trees, THV is a member of the subfamily Betaherpesvirinae. In addition, the evolutionary lineage of this subfamily is divided into two branches that correspond to the genus Roseolovirus with HHV-6 and HHV-7, the so-called Beta2 herpesviruses, and the mammalian cytomegaloviruses, the so-called Beta1 herpesviruses. Within the subfamily Betaherpesvirinae, THV is most closely related to the mammalian cytomegaloviruses, with similar evolutionary distances to the phylogenetic lineages of primate and rodent cytomegaloviruses.


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  		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 (alpha )-, Beta (beta )-, and Gammaherpesvirinae (gamma ). The sequences of the individual proteins used to construct the phylogenetic trees were taken from the GenBank and SwissPort release 39 databases.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The complete nucleotide sequence (195,857 bp) and the coding capacity of the THV genome were determined. The position of THV as the prototype species of herpesvirus genome group F (49, 72) was confirmed. The G+C content (66.5%) of the THV genome varies over the course of the genome. The highest values were found at the termini of the viral genome and within the DNA sequences of the conserved genes. The mechanism for G+C accumulation is not known. It occurs in the genomes of different herpesvirus species (e.g., EBV [4] or HSV-1 [58, 59, 60]) regardless of phylogenetic relatedness and is supposed to be an adaptation to an unknown evolutionary pressure.

Seventy-six out of 158 potential gene products of THV were identified as significantly homologous to known herpesvirus proteins of primate and rodent cytomegaloviruses, mainly those of HCMVA and MCMVS. The thorough examination of many of these homologous proteins allows the assignment of functions to the potential THV proteins. T1 to T5, T28, T29, and T36 are homologues of the US22 gene family of HCMVA. The members of the US22 gene family are known to regulate gene expression (13). m139 to m143 are the corresponding MCMVS homologues to THV T1 to T5. They are supposed to play an essential role in pathogenicity in the natural host and seem to be important for genome replication (35). HCMVA UL122 and UL123 are the main components of the so-called major immediate-early region (32, 45, 83, 84). These genes correspond to M122, M123, and T122 of MCMVS (12, 65) and THV, respectively. The mRNAs of UL122, UL123, M122, and M123 consist of several exons composed by alternative splicing events. It is very probable that the corresponding THV genes have similar exon structures. However, the transcription of these viral genes will be the subject of future studies. HCMVA immediate-early genes play an essential role in the regulation of the expression of early and late viral genes and are indispensable for the correct course of the lytic replication cycle. THV T36 to T38 and T115 are homologous to HCMVA immediate-early proteins UL36 to UL38 and UL115 (14, 15). UL37 is an integral membrane protein (1) that is supposed to be located in mitochondria and to inhibit Fas-mediated apoptosis of the host cell (30).

Herpesvirus tegument proteins have structural functions in viral morphogenesis and are involved in the regulation of gene expression immediately after penetration of host cells. In addition, some of them are responsible for the activation of the cellular immune response (33). THV gene products T25, T32, T47, T48, T69, T82, and T99 are homologous to tegument proteins of HCMVA and MCMVS (6, 16, 17, 18, 37, 38, 64, 91, 94). HCMVA UL32, UL82, and UL83 are the main components of the viral tegument. UL32 was designated the major tegument phosphoprotein and makes up 15% of the total protein mass of a virion and plays an important role in viral morphogenesis (34, 66). HCMVA UL83 is essential for the viral life cycle in the natural host but not in cell culture (77). A homologue to UL83 is present in MCMVS (71) and rat cytomagalovirus (87) but absent in HHV-6 (31) and THV. HCMVA UL69, which is homologous to THV T69, has been supposed to be a transactivator (90) and plays a role in the G1 phase of the host cell cycle (36, 55).

THV T46 (minor capsid protein), T85, T86 (major capsid protein), and T94 are homologous to the corresponding capsid proteins of HCMVA and MCMVS (13, 29, 71, 89). The transcription unit of the smallest capsid protein of HCMVA is 225 bp in length and located between UL48 and UL49 (28). A homologous protein in THV could not be detected. However, a small ORF (t48.1) of 312 bp located between ORFs T48 and T49 has, moreover, a potential Bowman-Birk serine protease inhibitor family signature. THV T80 is the homologue of the viral protease, one of the most important herpesvirus enzymes, which is a scaffolding protein with essential functions in the morphogenesis of the viral capsid (7, 11, 54, 75, 92).

Eleven gene loci were identified within the HCMVA genome, which are essential for DNA replication. UL54 (DNA polymerase), UL44 (DNA polymerase accessory protein), UL57 (major DNA-binding protein), UL70 (primase), UL102 (primase-helicase complex-associated protein), and UL105 (helicase) (46, 70, 80, 81) are replication fork proteins and are also present in THV. T54, the DNA polymerase of THV, is highly conserved and possesses characteristic sites and signatures of the B family of DNA polymerases (82). T57 shows a similar strong conservation and possesses sequence motifs essential for DNA binding (5). Homologues of THV T36-38, T84, T112, and T122 are also essential for DNA replication. They are supposed to activate the expression of the replication fork genes (27, 76, 79).

In the course of herpesvirus DNA replication, which proceeds by the rolling-circle mechanism, concatemeric genomes are formed (50). HCMVA UL51, UL52, UL56, UL77, UL89, and UL104 were identified as the vital components of the cleavage and packaging processes that are essential for the formation of unit length viral genomes and correct packaging (51). THV possesses individual homologues to these six proteins.

THV homologues of nucleic acid metabolism are T45, T72 (dUTPase), and T114 (uracil DNA-glycosylase). T45 is the large chain of the ribonucleotide reductase. All betaherpesviruses, including THV, have no homologous ORF for the small chain of the ribonucleotide reductase that actually possesses the active site of the enzyme. It is not known how the betaherpesvirus ribonucleotide reductases retain their function. Like other G+C-rich herpesviruses like EBV (4) or HSV-1 (58, 59, 60), THV is missing important nucleic acid metabolism enzymes like thymidylate synthase or dihydrofolate reductase. In addition, THV possesses no thymidine kinase, the gene for which is absent from all betaherpesvirus genomes. An important putative THV gene product is T97, which is homologous to HCMVA UL97 (ganciclovir kinase). This protein is responsible for the ganciclovir effect caused by chemotherapy of HCMV infection (53). THV T98 is homologous to the herpesvirus alkaline exonuclease that plays a role in DNA processing and capsid transport from the nucleus to the cytoplasm (26, 78).

As far as viral glycoproteins are concerned, THV T37, T50, T55 (glycoprotein B), T73, T74 (glycoprotein O), T75 (glycoprotein H), T100 (glycoprotein M), and T115 (glycoprotein L) are homologous to HCMVA and MCMVS glycoproteins. HCMVA UL74, UL75, and UL115 form the gCIII glycoprotein complex (42, 43, 44, 48). The gCIII glycoprotein complex and glycoprotein B are the most important structural surface proteins of HCMV. HCMVA UL55 and UL75 are supposed to start pathways that lead to the activation of cellular transcription factors Sp1 and NFkappa B by binding to special cellular receptors (93). In the THV genome, 10 potential glycoproteins (t7, t11, t17, t22, t22.1, t22.2, t22.3, t22.5, t22.7, and t22.8) with no homologies to known proteins were identified according to characteristic sites and signatures.

THV T33 and T78 are GCR homologues and are supposed to be homologous to host proteins. It is presumed that the function of viral GCR homologues is to catch extracellular signals and block the pertinent intracellular pathways. HCMVA UL33 is a CC chemokine receptor (9) and is not essential for growth in cell culture (56). UL33 homologues are conserved only in betaherpesviruses (8, 25). The HHV-6 homologue of THV T78 was found to be a CC chemokine receptor in vitro (67). t90 is not homologous to any known protein but was found to hold a GCR signature. However, further work is necessary to determine if t90 is actually a GCR homologue. US27 and US28 of HCMVA are CC chemokine receptor homologues and also members of the GCR family (9, 95). However, no corresponding homologous proteins could be identified in the THV genome.

The THV genome possesses a number of ORFs that code for proteins with no homologies to known proteins. These genes seem to code for virus species-specific functions in the natural host. t16 was found to possess a potential aminoacyl-transfer RNA synthetase class II signature, and t119 holds an ATP-dependent DNA ligase AMP-binding site. However, it has to be verified whether these signatures are actually functional. Interestingly, the locations of the unconserved genes are almost in the same genomic areas within the genomes of the members of the subfamily Betaherpesvirinae. The majority of these ORFs code for glycoproteins and are members of gene families. HCMVA UL24, UL43, UL83, UL116, UL118, and UL121 have corresponding homologues in MCMVS but not in THV. The latter five seem to have functions that are specific to cytomegaloviruses.

The biological and genomic properties of THV correspond to the criteria prepared for the classification of herpesviruses in the subfamily Betaherpesvirinae (73). THV is evolutionarily placed within the group of mammalian cytomegaloviruses. There is good reason to suppose that the separation of the three evolutionary lineages which lead to THV and the primate and the rodent cytomegaloviruses has taken place in a very short evolutionary period. This assumption is in accordance with the phylogenetic tree of the hosts of these virus species and confirms the accepted hypothesis that herpesviruses follow the development of their hosts, a process known as coevolution (47, 61, 62, 63). The identification of the genetic structure of the viral ancestor of these three herpesviruses is very difficult. It is certain that the homologous genes of THV, HCMVA, and MCMVS were also present in the ancestral genome. However, it is not clear why the gene arrangements of THV and MCMVS correspond to two different HCMVA genome isomers. A simple explanation would be that the viral ancestor had repetitive DNA elements similar to those of HCMVA and the ability to form genome isomers. However, one can assume that the repetitive DNA elements disappeared in the MCMVS and THV phylogenetic lineages due to distinct evolutionary pressures. In view of the high degree of relatedness of THV to the mammalian cytomegaloviruses, THV can be considered a model system for the investigation of HCMV infection and pathogenesis.


    ACKNOWLEDGMENTS

We thank the Deutsche Forschungsgemeinschaft, project Da-142/10-1-5, for providing the automatic DNA sequencing equipment (373 "Extended" DNA sequencer; Perkin-Elmer Corporation, Applied Biosystems).

We thank Michaela Handermann and Nurith J. Jakob for critical comments and helpful discussion.


    FOOTNOTES

* Corresponding author. Mailing address: Institut für Medizinische Virologie, Universität Heidelberg, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany. Phone: 49-6221-56-5008. Fax: 49-6221-56-4104. E-mail: g.darai{at}urz.uni-heidelberg.de.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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