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Journal of Virology, June 2003, p. 6620-6636, Vol. 77, No. 12
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.12.6620-6636.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Complete Sequence and Genomic Analysis of Rhesus Cytomegalovirus

Scott G. Hansen,1 Lisa I. Strelow,1,2 David C. Franchi,3 David G. Anders,3* and Scott W. Wong1,2,4*

Vaccine and Gene Therapy Institute, Oregon Health & Science University,1 Division of Pathobiology and Immunology, Oregon National Primate Research Center, Beaverton, Oregon 97006,2 The David Axelrod Institute, Wadsworth Center, Albany, New York 12201,3 Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon 972394

Received 21 November 2002/ Accepted 19 March 2003


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The complete DNA sequence of rhesus cytomegalovirus (RhCMV) strain 68-1 was determined with the whole-genome shotgun approach on virion DNA. The RhCMV genome is 221,459 bp in length and possesses a 49% G+C base composition. The genome contains 230 potential open reading frames (ORFs) of 100 or more codons that are arranged colinearly with counterparts of previously sequenced betaherpesviruses such as human cytomegalovirus (HCMV). Of the 230 RhCMV ORFs, 138 (60%) are homologous to known HCMV proteins. The conserved ORFs include the structural, replicative, and transcriptional regulatory proteins, immune evasion elements, G protein-coupled receptors, and immunoglobulin homologues. Interestingly, the RhCMV genome also contains sequences with homology to cyclooxygenase-2, an enzyme associated with inflammatory processes. Closer examination identified a series of candidate exons with the capacity to encode a full-length cyclooxygenase-2 protein. Counterparts of cyclooxygenase-2 have not been found in other sequenced herpesviruses. The availability of the complete RhCMV sequence along with the ability to grow RhCMV in vitro will facilitate the construction of recombinant viral strains for identifying viral determinants of CMV pathogenicity in the experimentally infected rhesus macaque and to the development of CMV as a vaccine vector.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cytomegaloviruses are the prototypic members of the betaherpesvirus subgroup. Like all herpesviruses, they share several characteristics with other viruses of the family, including virion structure and the ability to establish persistent and latent infections. What makes the betaherpesvirus subgroup unique among the herpesviruses is their tropism for the salivary gland, their species specificity, and their slow growth in culture systems (25). Weller et al. coined the term cytomegalovirus to reflect the cytopathic effect caused by virus infection and the virus's role in genitally acquired cytomegalic inclusion disease (75).

Human cytomegalovirus (HCMV) infection is generally asymptomatic in immunocompetent hosts. Typically, HCMV is acquired subclinically during childhood and persists throughout the individual's life. Persistence is characterized by the presence of latent viral genomes that periodically reactivate to produce infectious virus that can be shed intermittently in saliva, urine, semen, cervical secretions, and breast milk (25). Data suggest that persistence is established not only by the virus's ability to infect various cell types within the host, but also by the virus's ability to differentially regulate gene expression and by virally encoded immunomodulators (23, 30, 32, 43, 44, 57, 60, 64). When primary HCMV infection is acquired during adulthood, the virus is capable of doing considerably more damage. In particular, primary infections during pregnancy can lead to congenital abnormalities in the fetus and morbidity and mortality in immunocompromised individuals, including transplant patients and persons with AIDS.

Thus, with advances in medical procedures such as organ allografting and immunosuppressive posttransplant therapies as well as with increasing numbers of people infected with the human immunodeficiency virus (HIV), HCMV is proving to be a significant pathogen. Currently, the only Food and Drug Administration-approved antiviral therapies include the drugs ganciclovir, foscarnet, and cidofovir, each of which targets the viral DNA polymerase, and Vitravene, a novel antisense compound used to treat CMV retinitis (38). Although helpful, these drugs have numerous drawbacks, which include limited efficacy and toxic side effects and frequent emergence of resistant viral isolates during long-term therapy (17, 19, 72).

Despite the growing information on HCMV gene products and pathogenesis, there is a limitation on the ability to develop effective treatments for primary HCMV infection or reactivation of latent infection. To address these limitations, four animal models are being evaluated for antiviral treatments against HCMV and for overall cytomegalovirus pathogenesis. These models are guinea pig cytomegalovirus, murine cytomegalovirus (MCMV), rat cytomegalovirus (RCMV), and more recently, rhesus cytomegalovirus (RhCMV). The guinea pig cytomegalovirus model is well suited for congenital cytomegalovirus infection because the virus can cross the placenta and produce infection in utero (31). Despite this advantage, the guinea pig cytomegalovirus model is not well suited to the analysis of drug therapy, as the virus is resistant to ganciclovir therapy (50). The genomes of MCMV and RCMV have been completely sequenced, and these models are providing valuable information on both cytomegalovirus pathogenicity and antiviral therapies (59, 73). However, MCMV and RCMV are evolutionarily far removed from HCMV, and the results obtained in these systems may not be applicable to treatment and prevention of HCMV infection (51, 61).

Over the past decade, researchers have been working to develop an RhCMV model to study HCMV pathogenesis and antiviral therapies (2, 3, 7, 8, 48, 49, 70, 74). RhCMV was first recognized 70 years ago as an incidental infection of rhesus macaques (15, 16). Several nonhuman primate cytomegaloviruses have been isolated since that time, each of which is different from HCMV in host range and DNA content (3, 18, 63, 69). RhCMV is ubiquitous in captive rhesus macaques, with infection rates of greater than 90% by the first year of life (39, 41, 74). Cytomegalovirus infections in macaques share several features with HCMV infections. The majority of RhCMV infections are subclinical, and healthy adults tend to shed virus in their urine, saliva, semen, cervical secretions, and breast milk for years. Experimental congenital central nervous system disease has been produced in rhesus macaques by inoculating fetuses in utero intracerebrally or intra-amniotically (49).

Like humans infected by HIV, rhesus macaques are susceptible to infection with simian immunodeficiency virus (SIV). Studies utilizing SIV-infected rhesus macaques have demonstrated that disseminated cytomegalovirus disease is quite similar to that observed in HIV-infected humans (8). These symptoms can include orchitis, encephalitis, and respiratory tract disease. In addition, cytomegalovirus infections have been seen in the bone marrow and lungs of rhesus macaques that have been immunosuppressed with antithymocyte globulin, cyclophosphamide, and cortisone and subsequently inoculated with varicella-zoster virus, which is similar to what has been observed in people who acquire HCMV infection after transplant surgery (53). Lastly, studies of the host immune response to RhCMV appear to parallel HCMV infection, as cytomegalovirus-specific cytotoxic T lymphocytes are essential in controlling infection (40).

Studies of a nonhuman primate cytomegalovirus such as RhCMV are relevant to the study of HCMV, as this virus is evolutionarily closer to humans than the rodent cytomegalovirus isolates (51). Until now, only a limited number of reports have demonstrated the similarity of HCMV and RhCMV at the genomic level. In this study. we completely sequenced the RhCMV strain 68-1 genome and confirmed that the virus is significantly homologous to HCMV. Data generated for the sequence analysis revealed that the genome is over 200 kb in size and is structurally similar to previously reported cytomegaloviruses. Like HCMV, RhCMV encodes a complement of immunomodulatory genes as well as structural proteins and enzymes required for replication. Unique to RhCMV is a cyclooxygenase-2 (prostaglandin E2) homologue. Available sequence information on RhCMV will further the development of this virus as a model for HCMV pathogenesis.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of viral DNA. Primary rhesus fibroblasts were grown in two 850-cm2 roller bottles and infected with the 68-1 strain of RhCMV (ATCC VR-677) at a multiplicity of infection of 0.1, and the virus was harvested from the culture supernatant at 12 to 14 days postinfection as previously described (70). Briefly, infected-cell supernatants were clarified to remove cellular debris by centrifugation at 1,000 x g for 10 min. The clarified supernatants were collected and stored, and 10 ml of culture medium was added to the pelleted debris. To remove intracellular virus particles, the cell debris pellet was sonicated until resuspended. Sonication was followed by centrifugation at 1,000 x g for 10 min, after which clarified supernatants were combined.

RhCMV was pelleted from supernatants by centrifugation at 12,500 x g for 1 h at 4°C. The virus pellet was resuspended in 1 ml of 1 mM Tris-HCl (pH 8.0)-1 mM EDTA (TE) and added to the top of a six-step sorbitol gradient ranging from 20 to 70%. The gradients were spun in a Beckman SW41 rotor for 2 h at 18,000 rpm at 4°C. The virus-containing band at the 50 to 60% interface was collected and diluted with 15 ml of cold 1 mM Tris-HCl and then pelleted by centrifugation in the SW41 rotor for 50 min at 18,000 rpm at 4°C. The washed virus pellet was resuspended in 9.2 ml of TE (pH 8.0). Virus particles were digested at 37°C overnight with 0.6 ml of 10% sodium dodecyl sulfate and 0.2 ml of proteinase K (10 mg/ml) to release viral DNA. Viral DNA was isolated by CsCl2 gradient centrifugation in a Beckman Ti75 rotor at 38,400 rpm for 72 h. The viral DNA fraction, which routinely corresponds to refractive indices of 1.400 to 1.403, was collected and dialyzed in TE (pH 8.0) to remove CsCl2.

The isolated viral DNA was tested for infectivity by transfection into primary rhesus fibroblasts with the calcium phosphate method without dimethyl sulfoxide shock, and the cells were observed for cytopathic effects. As a negative control, primary rhesus fibroblasts were exposed to transfection reagents and conditions in the absence of viral DNA.

Cloning and DNA sequencing. To facilitate DNA sequencing of the viral genome, a shotgun subclone library was generated. Briefly, purified genomic DNA was hydrodynamically sheared in a high-pressure liquid chromatograph and then separated on a standard 1% agarose gel. A fraction corresponding to fragments 3,000 to 3,500 bp in length was excised from the gel and purified by the GeneClean procedure (Bio101, Inc., Carlsbad, Calif.). The purified DNA fragments were blunt-ended with T4 DNA polymerase. The treated DNA was then ligated to unique BstXI linker adapters. These linkers are complementary to the pGTC vector (Genome Therapeutics Corporation, Waltham, Mass.), while the overhang is not self-complementary. Therefore, the linkers will not concatamerize, nor will the cut vector religate easily. The linker-adapted inserts were separated from the unincorporated linkers on a 1% agarose gel and again purified with GeneClean. The linker-adapted inserts were ligated to BstXI-cut vector to construct a shotgun subclone library.

The library was transformed into Escherichia coli DH10B competent cells (Gibco/Invitrogen, San Diego, Calif.) and assessed on plates containing ampicillin. Bacterial transformants were used for plating of clones and picked for sequencing. The cultures were grown overnight at 37°C, and the DNA was purified by standard methods.

The purified DNA samples were sequenced with ABI (Applied Biosystems Inc., Foster City, Calif.) dye terminator chemistry. All subsequent steps were based on sequencing by automated DNA sequencing methods. The ABI dye terminator sequence reads were run on MegaBace 1000 (Amersham Biosciences, Piscataway, N.J.) machines, and the data were transferred to Unix machines. Base calls and quality scores were determined with the program Phred (21, 22). Reads were assembled with Phrap (29) with default program parameters and quality scores.

Computer-assisted analysis of the RhCMV ORFS and protein coding content. Open reading frames (ORFs) were identified with MacVector version 6.5 (Accelrys, San Diego, Calif.). The target search criterion for an ORF was 100 codons on either the positive or negative strand. The ORFs that were identified in this manner were translated and saved as individual files for further analysis. Putative RhCMV proteins were then compared to GenBank files with the BlastP search engine to look for homology with other known proteins. Significant hits were compiled and are presented in Table 1. Homology with HCMV ORFs was analyzed further with the global alignment algorithm FastA (Table 2). Multiple sequence alignments were done with MacVector's ClustalW feature, with gap penalties of 10 (fixed) and 10 (floating).


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  		TABLE 1. Summary of the location and features of the 230 ORFs of the RhCMV 68-1 genomea

 

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  		TABLE 2. Similarity between RhCMV genes with amino acid sequence homology to HCMVa

 
Nomenclature and phylogenetic analysis. The nomenclature used for the RhCMV genes is based on the same strategy used in designating both the MCMV and RCMV genomes. This system numbers the ORFs from left to right starting at the first open reading frame on the coding or complementary strand. RhCMV genes homologous to HCMV genes are indicated by uppercase prefixes (e.g., Rh88), whereas ORFs without sequence similarity to HCMV genes are indicated by lowercase prefixes (e.g., rh06).

Sequence data from ClustalW alignments were analyzed with ClustalX version 1.8 and the draw tree feature with bootstrapping. Tree data generated with this method were viewed with the phylogenetic tree-generating software TreeView version 1.6.1 (Logiciels & Duhem, Paris, France). The measure of divergence is presented as a scale at the bottom of each tree. Viral sequences for phylogenetic analysis were acquired from GenBank. The viruses and their accession numbers are as follows: herpes simplex virus type 1 (HSV-1), NC001806; Kaposi's sarcoma-associated herpesvirus (KSHV), NC003409; MCMV, U68299; RCMV, AF232689; chimpanzee cytomegalovirus (ChCMV), NC003521; and HCMV, NC001347. The proteins used for phylogenetic analysis were DNA polymerase, glycoprotein B (gB), helicase, major capsid protein, single-stranded-DNA-binding protein, and uracil N-glycosylase.

Nucleotide sequence accession number. RhCMV 68-1 nucleotide sequence data have been deposited in the GenBank database and assigned accession number AY186194.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Features of nucleotide sequence of RhCMV. The genome of RhCMV 68-1 is 221,459 bp in length, which is less than HCMV at 229,354 bp (52). The overall G+C content is 49% and is evenly distributed throughout the length of the genome. The only exception is a short stretch of sequence from nucleotides 15000 to 20000 (Fig. 1). . This region has a lower G+C content (20 to 40%) and represents ORFs Rh20 through Rh27 inclusive. These proteins have significant homology to the HCMV RL11 family members.



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  		FIG. 1. G+C content of RhCMV 68-1 genome. The distribution of G+C versus A+T content was analyzed over the entire genome. Results are presented as histograms.

 
Like all herpesviruses, the RhCMV genome is a long, double-stranded unique sequence. There are two PacI homologies (virion packaging sites) as defined by HCMV, one found at the start of the genome and the other between what would be the UL and US regions of HCMV. Although our sequence data did not reveal large internal or terminal repeats like those present in the HCMV genome, Southern analysis with probes specific for the termini detected variable copies of a roughly 500-bp terminal direct repeat sequence that may be analogous to the a sequence of HCMV and other herpesviruses (13).

Restriction analysis of RhCMV genomic DNA. To verify the genomic structure of RhCMV DNA, purified viral DNA was digested with the restriction enzymes BamHI and HindIII (Fig. 2). . Both digests produced a large number of distinguishable fragments. The resultant bands correspond to predicted restriction digest patterns (Fig. 2A and B and Table 3). The combination of sequence and restriction digest data, along with data from Chang and Barry (13), supports the hypothesis that the organization presented in this paper is the correct structure for RhCMV and that the virus does not isomerize, unlike HCMV.



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  		FIG. 2. Restriction analysis of RhCMV genome. (A) Restriction digests of the RhCMV 68-1 genome. Purified genomic DNA (10 µg) was digested overnight with 25 U of either BamHI (lane B) or HindIII (lane H) at 37°C. Digests were supplemented with an additional 25 U of enzyme the following day for 4 h prior to separation on a 0.7% agarose gel. Bands were resolved at 20 V overnight. Lanes MW, size markers. (B) Deduced BamHI and HindIII restriction maps. Digested products were numbered based on the sizes shown in Table 3.

 

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  		TABLE 3. Predicted fragment sizes

 
Protein coding content. The arrangement of the RhCMV genes is shown in Fig. 3. The genome contains 230 ORFs that are arranged colinearly with those of previously sequenced betaherpesviruses, including HCMV, MCMV, and RCMV. All of the conserved herpesvirus gene blocks are retained in RhCMV in both position and orientation. Of the 230 ORFs, 138 (60%) encode products that are homologous to HCMV proteins (Table 2). These homologues are dispersed throughout the genome. Less than half of the ORFs are predicted to encode genes unique to RhCMV; these ORFs are possibly host range specific. Unlike MCMV, RhCMV encodes almost all classified HCMV gene families, including RL11, UL25, UL82, seven transmembrane, US1, US2/6, US12, and US22 families.



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  		FIG. 3. Map of RhCMV genome. Map of the RhCMV 68-1 genome showing the ORFs on the coding strand (dark arrows) and the noncoding strand (light arrows). Putative RhCMV genes are numbered as shown in Table 1 from Rh01 to Rh236. A selected number of HCMV homologues are listed: pp150, large structural phosphoprotein (UL32); DNA pol, DNA-dependent DNA polymerase (UL54); gB, glycoprotein B (UL55); MDBP, major DNA-binding protein (UL57); pp71, phosphoprotein 71 (UL82); pp65, phosphoprotein 65 (UL83); MCP, major capsid protein (UL86).

 
Genome and individual ORFs. (i) Proteins essential for origin-dependent replication. RhCMV encodes six of the seven proteins necessary for HSV-1 replication. These proteins are the DNA polymerase (Rh87), the polymerase accessory protein (Rh70), the single-stranded-DNA-binding protein (Rh92), and the helicase-primase complex (Rh100, Rh139, and Rh142). The DNA helicase (Rh142) contains the six motifs required for function in congruent positions described for HSV and HCMV (77). RhCMV lacks the origin-binding protein (UL9) of HSV-1 that is required for HSV replication. The only betaherpesvirus known to encode an HSV-1 UL9-like protein is human herpesvirus 6 (28). RhCMV does encode all of the remaining trans-activating factors required for transient complementation of HCMV origin of lytic replication-dependent DNA replication (54). These include UL36 to 38 (Rh61, Rh62, Rh64, and Rh66), TRS1 (Rh230), and IE1/IE2 (Rh156 exons 4 and 5, respectively), which are known regulatory proteins, and ORFs UL84 (Rh114) and UL112/UL113 (Rh144/Rh145), which encode early temporal class nucleus-associated proteins. HCMV UL84 and IE2 have been shown to interact and are thought to be responsible for transcriptional control (65). The DNase gene (Rh134) shown to be required for HSV-1 replication in vitro is 65% conserved with HCMV UL98 (Table 2) (51).

An RhCMV origin of replication (ori) is situated between homologues of HCMV UL57 and UL69 (between nucleotides 88161 and 90525). The ori shares several structural features with HCMV oriLyt, most notably the Y-R element (78), and mediates RhCMV-dependent replication in transient assays (David Anders, unpublished results).

(ii) Other genes involved in DNA and nucleotide metabolism. RhCMV encodes all of the enzymes found in HCMV that are required for nucleotide metabolism, replication, and repair. These are uracil-DNA glycosylase (Rh146), ribonucleotide reductase (Rh72), and dUTPase (Rh101). The enzymes all have very high homologies to their HCMV counterparts, UL114 (69%), UL45 (61%), and UL72 (57%), respectively (Table 2). The ribonucleotide reductase homologue is structurally identical in size (97 kDa) to both the HCMV and MCMV proteins. This is in contrast to HSV-1, which encodes a functional protein of 124 kDa. The RhCMV protein lacks a homologue of the small subunit of ribonucleotide reductase and thus may not be functional. Also, like HCMV and MCMV, the RhCMV genome does not have an ORF that encodes a thymidylate synthetase or a thymidine kinase.

RhCMV ORF Rh132 is the HCMV UL97 homologue. The UL97 protein of HCMV is a phosphotransferase and has been shown to phosphorylate ganciclovir (47). Unlike the alphaherpesviruses, RhCMV does not encode any additional protein kinases. However, RhCMV does encode a homologue to the pyruvoyl decarboxylase gene (Rh106) found in HCMV. Consistent with HCMV, the putative protein homologue contains a single pyruvoyl decarboxylase enzyme prosthetic group represented by the sequence ALGSSLFN (single-amino-acid code) from amino acids 566 to 574.

(iii) Regulatory genes. Examination of RhCMV ORFs for viral regulatory genes involved in the control of viral gene expression reveals that the virus encodes homologues of the major immediate-early region (Rh156), the UL36 to UL38 region (Rh60 to Rh66), one of the TRS1 transactivator genes (Rh230), the UL69 gene (Rh97), and the US3 transcription unit (Rh184). The major immediate-early region is colinear with that of HCMV and has matching splicing patterns (7). Transcription for the HCMV major immediate-early genes is controlled by a strong enhancer or promoter sequence at positions -733 to +625 (relative to the start site of transcription at +1) (2). In HCMV, TRS1 and IRS transactivate UL44 as well as other early genes (9, 37, 66).

(iv) Structural proteins. Capsid assembly proteins are very similar among the betaherpesviruses. RhCMV is no exception, encoding homologues to the major capsid protein (Rh118), the large tegument protein (Rh78), and a minor capsid protein (Rh75). Also present in RhCMV is a UL80 (Rh109) homologue that is thought to be involved in assembly and packaging of DNA into the virion. RhCMV also encodes other structural proteins common to the betaherpesvirus group, including the upper matrix protein pp71 (Rh110), the lower matrix protein pp65 (Rh111 and Rh112), the large phosphoprotein pp150 (Rh55), and the small phosphoprotein pp28 (Rh136). Rh91 is homologous to the HCMV UL56 gene product. However, the predicted size of Rh91 is 88.2 kDa, compared with 130 kDa for HCMV. Although gaps are found throughout the protein, accounting for the size difference, the protein is still highly conserved at 70% identity. The HCMV UL56 gene product has been found associated with the nucleocapsid tegument fraction and is thought to be involved in capsid maturation (10).

(v) Glycoproteins. A total of 21 glycoproteins were found within the RhCMV genome. These predictions were based on known homologies within GenBank. RhCMV encodes homologues to the gB (Rh89), gH (Rh104), gL (Rh147), gM (Rh138), gN (Rh102), and gO (Rh103) glycoproteins. In addition, homologues to UL37 (Rh62), UL42 (Rh68), UL116 (Rh148), UL121 (Rh155), US02 (Rh182), US03 (Rh184), and US11 (Rh189) are present in RhCMV. No homologues to HCMV glycoproteins gpUL04, gpUL16, or gpUS10 were found. Glycoprotein B (gB) is a prominent component of the viral envelope and is involved in virus attachment, penetration, and cellular spread (12). Many gB homologues carry the motif RXK/RR, which is the specific recognition motif for cleavage by the cellular protease furin. The gB homologues of varizella-zoster virus, bovine herpesvirus type 1, pseudorabies virus, equine herpesvirus, Marek's disease virus, and all known betaherpesviruses, including HCMV and human herpesvirus 6, contain this cleavage site (reviewed in reference 68). The RhCMV gB sequence contains several potential furin sites; however, none of these sites is an exact match to the sequence RXK/RR.

In HCMV, glycoprotein O (gO) is encoded by the UL74 gene (36). gO has homologues only among the betaherpesviruses (36). Similar to HCMV, the RhCMV gO homologue has numerous putative N- and O-linked glycosylation sites. gO has been shown to be dispensable for growth of the virus; however, a gO knockout virus is severely attenuated in growth and spread in culture (33). A tripartite complex of glycoproteins H, L, and O (gH/gL/gO) forms through disulfide bonds and has been implicated in viral entry (via membrane fusion), cell-to-cell spread, and virion maturation (36, 55, 71). Recent studies have found that gO represents a hypervariable region of the genome, with some sequences varying from each other by as much as 45% (55, 58). Currently, we do not know if RhCMV gO is a hypervariable region. Sequencing of primary isolates will indicate if RhCMV is similar to HCMV in this respect.

HCMV gH remains membrane bound and is the anchor for the gH/gL/gO fusion complex. RhCMV gL has two putative N-linked glycosylation sites defined by the motif NXT/S, where X is any amino acid (46). HCMV gL has one glycosylation site, whereas MCMV is predicted to have five. The RhCMV gM homologue (Rh138) is predicted to have eight putative transmembrane domains, based on the hydrophobicity plot. A similar profile is seen with both HCMV and MCMV gM proteins. The HCMV gM protein has five putative glycosylation sites, only one of which is present in RhCMV gM. However, the characteristic HCMV gM acidic C-terminal tail is present in Rh138. Unlike MCMV, RhCMV encodes a gN protein. Therefore, the gM/gN structural complex is preserved in RhCMV. In HCMV, gN has been designated a hypervariable region, with at least four distinct subtypes identified (reviewed in reference 56).

(vi) Immunomodulators. Like most DNA viruses, RhCMV encodes a total of 13 putative immunomodulators. Six of these show significant homology to known HCMV immunomodulatory proteins, including UL111 (Rh143), UL144 (Rh163), US02 (Rh182), US03 (Rh184), and US11 (Rh189). The UL36 gene of HCMV encodes a cell death suppressor, denoted vICA (62). Immediately downstream of the HCMV UL36 gene locus is a mitochondrion-localized inhibitor of apoptosis (vMIA), a product of the UL37 gene (27). RhCMV ORFs Rh62 and Rh66 show homology to UL37. Recently, the HCMV UL118/119 spliced transcripts have been found to encode viral Fc{gamma} receptor homologues (4). RhCMV potentially encodes these receptors through the Rh151 and Rh152 ORFs. RhCMV encodes two genes that show homology to the HCMV UL36 genes, Rh60 and Rh61. Rh60 and Rh61 may be spliced to form a functional RNA transcript. The putative ORFs rh96 and rh98 show homology with the immunoglobulin heavy chain. While rh186 is structurally similar to the US6 family members, the protein does not have an HCMV homologue by Blast analysis. Rh143 is an interleukin-10 (IL-10) homologue that has previously been shown to be expressed and may act as a cytokine synthesis inhibitory factor in vivo (48). Rh163 has homology to a tumor necrosis factor (TNF) receptor, which may play a role in preventing apoptosis. The US2, US3, US8, and US11 homologues have been shown to downregulate major histocompatibility complex (MHC) class I (24).

(vii) Unique to RhCMV. RhCMV encodes a putative cyclooxygenase-2 or prostaglandin E2 homologue. A total of six exons make the putative cyclooxygenase-2 transcript. RhCMV has several gene duplications, one of which is a duplication of the pp65 homologue. In addition, the UL11 gene appears to be present in three copies (Rh21 to Rh23), the US14 homologue has four copies (Rh194 to Rh197), and the US28 gene homologue has six copies (Rh214 to Rh216, Rh218, and Rh220).

RhCMV gene families. RhCMV encodes eight different gene families that are also present in HCMV (Table 4).


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  		TABLE 4. RhCMV gene families homologous with HCMV genes

 
(i) RL11 family. The RL11 family is represented by seven putative ORFs. The RL11 family of membrane glycoproteins is represented by RhCMV ORFs Rh05 (UL153), Rh17 (UL11), Rh20 (UL06), Rh21 (UL11), Rh22 (UL11), Rh23 (UL11), and Rh25 (UL09). The function of these proteins in HCMV has not been completely elucidated. However, the RL11 sequences are fairly conserved within the long repeat sequence of herpesviruses.

(ii) UL25. The UL25 family of proteins is represented by two RhCMV ORFs that are also conserved in MCMV and RCMV. These are RhCMV ORFs Rh43 (UL25) and Rh59 (UL35). The HCMV UL25 family members may be virion associated, although the data are controversial (6, 79).

(iii) Seven-transmembrane family. Proteins within this family have seven transmembrane domains; some family members have been shown to act as G protein-coupled receptor homologues (67). Family members include UL33 (Rh56), UL78 (Rh107), and US28 (Rh214, Rh215, Rh216, Rh218, and Rh220). There is no apparent RhCMV homologue to HCMV US27, also a seven-transmembrane family member.

(iv) UL82 family. HCMV UL82 family members comprise the upper and lower matrix proteins pp71 and pp65. RhCMV homologues to these proteins are Rh110 (pp71) and Rh111/Rh112 (pp65). The RhCMV pp71 protein has 39% identity with HCMV pp71. The two pp65 copies of RhCMV have 32% (Rh111) and 35% (Rh112) identity with the HCMV protein and 39% identity between themselves. Immunologically, the pp65 homologue encoded by Rh112 ORF elicits a greater T-cell response than that generated against Rh111 (L. Picker, personal communication).

(v) US01 family. The US01 family of proteins has three members in HCMV and includes the US01, US31, and US32 proteins. RhCMV encodes homologues to all three of these proteins (Rh181, Rh225 and Rh226, respectively) that are at colinear positions within the genome.

(vi) US06 family. The HCMV US06 family of proteins show both structural and functional similarities with one another. The majority of these family members are glycoproteins, although US06 proteins have been shown to function as downregulators of MHC class I on the cell surface. RhCMV encodes five potential US06 family members capable of immunomodulation: US02 (Rh182), US03 (Rh184), US06 (Rh185), US08 (Rh187), and US11 (Rh189). Historically, US02 and US03 were considered US02 family members, but we have chosen to classify the RhCMV proteins as US06 family proteins due to structural and functional features in common with US06.

(vii) US12 family. There are 11 US12 family members within the US region of RhCMV; four of these (Rh194, Rh195, Rh196, and Rh197) are duplications of US14. The remaining seven ORFs include Rh190 (US12), Rh192 (US13), Rh198 (US17), Rh199 (US18), Rh200 (US19), Rh201 (US20), and Rh202 (US21). Family classification is based on structural similarities among the proteins. The function of these proteins has yet to be elucidated.

(viii) US22 family. US22 family members are found throughout the genomes of both RhCMV and HCMV. RhCMV encodes 10 homologues to HCMV ORFs. These include Rh40, Rh42, Rh47, Rh50, Rh60, Rh61, Rh69, Rh203, Rh204, and Rh230. Proteins of the US22 family have two stretches of hydrophobic sequences and predominantly acidic carboxy termini; the RhCMV proteins meet both of these criteria.

Phylogenetic analysis. A phylogenetic analysis of the relationship of RhCMV to other herpesviruses is shown in Fig. 4. Six ORFs found in HSV-1, KSHV, MCMV, RCMV, and ChCMV were examined by bootstrap analysis with the maximum parsimony method. Alignments were performed with ClustalW and analyzed to ensure that sequences were properly aligned. ClustalW data were then used to generate a phylogenetic tree for DNA polymerase, glycoprotein B, helicase, major capsid protein, single-stranded-DNA-binding protein, and uracil N-glycosylase. The HCMV proteins were used as the root for the analysis, and thus the figure shows divergence from these proteins. The tree indicates that the virus most closely related to HCMV is ChCMV, followed by RhCMV, MCMV, RCMV, KSHV, and finally HSV-1.



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  		FIG. 4. Phylogenetic analysis. Phylogenetic trees for alpha-, beta-, and gammaherpesviruses. Six proteins were selected for phylogenetic analysis with the ClustalX program. Proteins were aligned by ClustalW. The alignments were adjusted to remove any unnecessary gaps, and then tree data was calculated. Trees were drawn with the TreeView program. The measure of divergence is presented as a scale at the bottom of each tree. Proteins used for the analysis were DNA polymerase, glycoprotein B (gB), helicase, major capsid protein, single-stranded-DNA-binding protein, and uracil N-glycosylase.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The RhCMV genome has now been completely sequenced. The RhCMV strain 68-1 genome is 221,459 bp in length and potentially encodes over 200 proteins. Like the other sequenced betaherpesviruses, RhCMV contains ORFs that are homologous to genes from both the unique long (UL) and unique short (US) regions, similar to other viruses within the herpesvirus family. However, unlike human and chimpanzee CMVs, RhCMV does not have repeats between the putative UL and US regions. Restriction analysis of the RhCMV genome further indicated that the genome does not isomerize, which is not uncommon within the betaherpesvirus family. Consistent with this finding, probes specific for each terminus failed to detect the altered terminal fragments that would be predicted by isomerization on Southern blots of RhCMV genomic DNA (13). Moreover, the closely related simian CMV strain Colburn was likewise found by restriction analysis not to isomerize (45). Thus, based on the organization of the RhCMV genome, the genome is most similar to the tupaia herpesvirus, which was recently sequenced and is the prototypic member of the F group of herpesvirus genomes (5).

The complete DNA sequence of RhCMV was compared to all of the published herpesviruses genomes within GenBank. From this analysis, we found that RhCMV and the other herpesvirus genomes are very similar at the genetic and nucleotide levels (34). For HCMV, the G+C content is about 57%, whereas for RhCMV the G+C content is around 49%; however, slight differences are seen at the termini, possibly due to the lack of repeats. Even so, homologous proteins are found within terminal regions, suggesting that host determinants may account for these differences at the nucleotide level.

The sequence analysis revealed that RhCMV encodes homologues to four regions of HCMV that are transcribed at immediate-early times: the major immediate-early transcripts (IE-1 and IE-2), which have been described previously (7), UL36 to UL38, US3, and TRS1. In HCMV infection, these proteins are expressed early during infection and are involved in the regulation of gene expression throughout the virus's life cycle. MCMV does not contain a US3 homologue; however, the US3 locus is dispensable for growth in tissue culture (42). In addition, MCMV does not appear to encode the second transcript of the UL37 gene, which may not be functional. RhCMV encodes both transcripts of the UL37 gene. RhCMV UL37 therefore may function like its HCMV counterpart (1, 14). The TRS1 homologue in RhCMV shows significant homology to the HCMV protein (35% identity). TRS1 in HCMV has been shown to be expressed at immediate-early times and to act as a transactivator of several early promoters (46, 66).

At least two additional RhCMV gene homologues may have regulatory functions. The UL121 homologue Rh155 is located colinearly with the HCMV protein and has homology to the ICP0 protein of HSV-1. ICP0 has been implicated in viral switching between latent and lytic infection and has been shown to transactivate other herpesvirus promoters (20). However, the Rh155 protein does not contain a zinc finger motif and may not function in the same manner as ICP0. Rh155 does contain a highly charged carboxy terminus, which is a feature of some trans-acting transcriptional activators. Rh97 encodes a homologue of the UL69 transactivator of HCMV and is also highly conserved at 59% identity. Taken together, regulatory proteins and transactivators are highly conserved between HCMV and RhCMV.

Examination of the enzyme-coding capacity of RhCMV indicates that the virus encodes the same enzymes as HCMV and, like HCMV, lacks an identifiable thymidine kinase gene. The RhCMV DNA polymerase has been reported previously and has a sequence identical to the one described here (70). Present within the RhCMV genome is a homologue of the UL97 gene of HCMV. The UL97 product has protein kinase activity and has been shown to phosphorylate the nucleoside analog ganciclovir (47). Previous studies have shown that RhCMV is sensitive to ganciclovir treatment (70). Homologues to the HCMV DNA helicase-primase and the DNA repair enzymes dUTPase and DNase were present in RhCMV. The RhCMV enzymes are highly conserved with the HCMV proteins (Table 2). [Unique to the betaherpesviruses are homologues to the UL77 protein.] UL77 is a pyruvoyl decarboxylase homologue that can be found in HCMV virions. Pyruvoyl decarboxylase enzymes are involved in the synthesis of cellular polyamines, which is increased during HCMV infection in vitro (35). When polyamines are decreased, so are the number of HCMV particles released from infected cells (26).

Another group of proteins that are highly conserved between HCMV and RhCMV are the structural proteins. As expected, RhCMV encodes homologues to the major capsid protein, the minor capsid protein, and the capsid assembly protein. In addition to the capsid proteins, there are homologues to most of the known tegument proteins, including UL32 (pp150), UL48 (large tegument protein), UL82 (pp71), UL83 (pp65), and UL99 (pp28). RhCMV does not encode a copy of the UL65 protein, which is also absent in MCMV. Interestingly, RhCMV encodes an additional copy of the UL83 ORF (pp65). Current data are lacking on whether both proteins are present in virions, but immunological data indicate that there is a T-cell response to both proteins, suggesting that each is expressed (L. Picker, personal communication). The identity between the two RhCMV copies is 39%, while Rh111 has 32% identity and Rh112 has 35% identity to HCMV UL83. Such a low identity score suggests that either a gene duplication event occurred during the evolution of the virus or that these two genes are distinct and have separate functions.

The sequenced betaherpesviruses all contain gene families, although this feature is not common to all herpesviruses. Sequence analysis determined that RhCMV encodes gene homologues to all the HCMV families. Within the HCMV genome, nine gene families have been described; we have described eight for RhCMV. In HCMV, two families that are similar in structure, US2 and US6, have both been shown to downregulate MHC class I expression on the cell surface. We have chosen to classify US02-like family members as US06 family members due to structural similarities.

Immunomodulation is responsible for a large part of a virus's survival and propagation within a host. Herpesviruses encode numerous immunomodulatory proteins that are thought to be involved in immune evasion. The genomic analysis of RhCMV demonstrates that the virus encodes numerous immunomodulators. On average, the identity of these proteins with their HCMV counterparts is 21%. This may reflect host adaptation and not imply a lack of function. A recent publication has shown that RhCMV encodes and expresses an IL-10 homologue (48). The function of the RhCMV protein has yet to be elucidated, but it may function as a cytokine synthesis inhibitory factor. Like HCMV, RhCMV encodes a TNF receptor homologue. A TNF receptor homologue may be used as a mechanism to prevent the cells' progression towards apoptosis. However, there is insufficient evidence to determine if the RhCMV TNF receptor homologues are expressed and if the protein binds cytokines.

Viral infection may induce apoptosis within the cells they infect. In addition to the TNF receptor, HCMV encodes the apoptosis inhibitors vICA (UL36) and vMIA (UL37). The product of the vICA ORF inhibits apoptosis by binding to the prodomain of caspase-8, preventing activation (62). RhCMV has two potential ORFs that show homology to the UL36 gene. However, it is not known if these genes are spliced to yield one message or if they function independently of one another. HCMV's vMIA protein acts by inhibiting Fas-mediated apoptosis at a point downstream of caspase-8 activation and Bid cleavage but upstream of cytochrome c release (27). Rh62 and Rh66 are homologous to vMIA; however, the function of these proteins is not known.

RhCMV potentially encodes spliced transcripts homologous to the Fc{gamma} receptor glycoproteins (4). The Fc{gamma} receptors have recently been identified in HCMV. The HCMV proteins belong to the immunoglobulin gene superfamily and represent a diverse group of Fc{gamma} receptor structures. As mentioned above, RhCMV encodes several homologues to US06 family members. US06 proteins are present in HCMV, in other herpesviruses, and in poxviruses. Many of these have been characterized and shown to downregulate MHC class I on the cell surface. MHC class I presentation of antigen is a significant host defense directed towards the clearance of pathogens. RhCMV also encodes two ORFs that show low sequence homology to the immunoglobulin heavy chain (rh96 and rh98). These RhCMV proteins may prove to be additional herpesvirus immunomodulators or the equivalent of the HCMV UL16 protein.

The viral cyclooxygenase-2 homologue may also have an immunomodulatory function. Cellular cyclooxygenase-2 is produced during an inflammatory response when immunoglobulin molecules are cross-linked. This triggers a rapid permeabilization of the lipid membrane through the production of arachidonic acid and releases inflammatory mediators into the surrounding area. Many anti-inflammatory drugs such as aspirin are inhibitors of arachidonic acid metabolism. Recently, Zhu et al. demonstrated that cyclooxygenase-2 is induced during HCMV infection and performs an important function supporting viral replication (76). Increased cyclooxygenase-2 activity early in infection promotes accumulation of the viral transcriptional regulatory protein IE-2, which is in turn required for subsequent viral transcription and DNA replication. RhCMV-encoded cyclooxygenase-2 could play an analogous role. RhCMV cyclooxygenase-2 could also possibly function as a transcriptional control molecule or be involved in latency by slowing viral replication. It remains to be established whether the RhCMV homologue is active and, if so, how the gene's expression is regulated.

The seven-transmembrane family member proteins are G protein-coupled receptors that are present on the cell surface and couple to G proteins to activate extracellular ligands (11). Once activated, the G proteins transduce signals from the cell surface to an effector molecule in order to modulate intracellular functions. RhCMV encodes all but one of the G protein-coupled receptors found in HCMV. However, RhCMV does encode five potential copies of the US28 protein, which has been shown to function as a chemokine receptor and to elicit cell migration in vitro. (68). Structurally, the G protein-coupled receptor homologues are very similar to their HCMV counterparts, although identity between the proteins is about 36%. All of the G protein-coupled receptors in HCMV are transcribed. It will be an interesting point of study in RhCMV to determine whether the seven-transmembrane family members are transcribed and whether they function.

Phylogenetic analysis of HCMV and comparison with ChCMV, RhCMV, MCMV, RCMV, KSHV, and HSV-1 for several different conserved genes revealed that the primate cytomegaloviruses are most closely related to HCMV. The analysis also demonstrates that both MCMV and RCMV are very distant from HCMV on the evolutionary tree and therefore may not be the best model with which to study HCMV pathogenesis and host immune response to the virus. Although chimpanzees are most closely related to humans evolutionarily, limited numbers of chimpanzees in the wild make ChCMV infection of chimps an unsuitable study model. With the availability of the genomic sequence, RhCMV will offer significant advantages over the other cytomegalovirus model systems. RhCMV is highly homologous to HCMV, encodes many of the same proteins, and has a similar genomic structure, and there is a relevant animal model with which to study both cytomegalovirus and cytomegalovirus/SIV infections in the natural host.


   ACKNOWLEDGMENTS
 
This work was supported by NIH grants RR15094 (D.G.A. and S.W.W.), AI31249 (D.G.A.), RR00163 (L.I.S. and S.W.W.), and CA75922 (S.W.W.).

We thank Robert P. Searles, Jay A. Nelson, and Heather Meyers for contributions and preliminary analysis of the sequence; Klaus Früh and Peter Barry for helpful discussions during the annotation of the genome; Don Siess for technical assistance; the Wadsworth Center Molecular Genetics Core for sequencing; Andrew Townsend for graphics expertise; and Lori Boshears for proofreading the manuscript.


   FOOTNOTES
 
* Corresponding author. Mailing address for David G. Anders: The David Axelrod Institute, Wadsworth Center for Molecular Genetics, Albany, NY 12201-2002. Phone: (518) 474-8969. Fax: (518) 474-3181. E-mail: anders{at}wadsworth.org. Scott W. Wong, Vaccine and Gene Therapy Institute, OHSU, West Campus, 505 NW 185th Avenue, Beaverton, OR 97006. Phone: (503) 690-5285. Fax: (503) 418-2719. E-mail: wongs{at}ohsu.edu. Back


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 Discussion
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Journal of Virology, June 2003, p. 6620-6636, Vol. 77, No. 12
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.12.6620-6636.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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