Analysis of 4.3 kilobases of divergent locus B of macaque retroperitoneal fibromatosis-associated herpesvirus reveals a close similarity in gene sequence and genome organization to Kaposi's sarcoma-associated herpesvirus

Abstract

We previously identified retroperitoneal fibromatosis-associated herpesvirus (RFHV) as a simian homolog of Kaposi's sarcoma-associated herpesvirus (KSHV) in a fibroproliferative malignancy of macaques that has similarities to Kaposi's sarcoma. In this report, we cloned 4.3 kb of divergent locus B (DL-B) flanking the DNA polymerase gene from two variants of RFHV from different species of macaque with a consensus degenerate hybrid oligonucleotide primer approach. Within the DL-B region of RFHV, viral homologs of the cellular interleukin-6, dihydrofolate reductase, and thymidylate synthase genes were identified, along with a homolog of the gammaherpesvirus open reading frame (ORF) 10. In addition, a homolog of the KSHV ORF K3, the modulator of immune recognition-1, was identified. Our data show a close similarity in sequence conservation, gene content, and genomic structure between RFHV and KSHV which strongly supports the grouping of these viral species within the same RV-1 rhadinovirus lineage and the hypothesis that RFHV is the macaque homolog of KSHV.

FIG. 1.

PCR strategy for cloning a region of divergent locus B (DL-B) of RFHVMm. The order and orientation of the genes within the DL-B region of KSHV flanking the DNA polymerase genes are shown (the size of each ORF is indicated in base pairs). The positions of the DNA polymerase-specific PCR primers QIRQB and DRIPA and the PCR product obtained by partial inverse amplification of the ORF 10 flanking region of RFHVMm are indicated. The positions of the DMGLB and RHFGA primer pools used for CODEHOP amplification of a region of the RFHVMm TS gene are also shown. Finally, the 3.6-kb PCR fragment of RFHVMm obtained by long-range PCR amplification between the ORF 10-specific primer F6565 and the TS-specific primer R20432 is indicated.

FIG.2.

CODEHOP PCR amplification of a region of the RFHVMm vTS gene with a thermal gradient. PCR amplification of DNA from the retroperitoneal fibromatosis tumor from MmuYN91 was performed with CODEHOPs derived from two conserved TS motifs, RHFGA and DMGLB. A gradient of annealing temperatures ranging from 55 to 70^oC was used. The expected 284-bp fragment is indicated. Lane temperatures: 1, 70°C; 2, 68.9°C; 3, 67.1°C; 4, 64.3°C; 5, 60.5°C; 6, 57.9°C; 7, 56.1°C; 8, 55.0°C.

FIG. 3.

Relative organization of ≈7.7 kb of the RFHV genome between the glycoprotein B (gB) and vTS genes in comparison to the genomes of other primate rhadinoviruses, including human KSHV, macaque RRV, and South American squirrel monkey HVS. Ateline herpesvirus 3 from the South American spider monkey displays the same organization as HVS, a close relative, except it lacks a vDHFR homolog (2). The relative position and orientation of the ORFs identified within the respective 7,739- and 7,756-bp fragments of the RFHVMm and RFHVMn genomes assembled from overlapping sequences from the previously published partial gB and complete DNA polymerase (DNA Pol) (40) and the 3.6 to 4.1 kb of the DL-B region targeted in the present study are shown. Missing genes are indicated with a dashed line, and noncontiguous regions of the genome are indicated with a double slanted line.

FIG. 4.

Comparison of viral ORF 70 and cellular TS homologs. (A) Amino acid sequence alignment of the partial vTS sequences obtained from RFHVMm and RFHVMn with the comparable regions of other viral and cellular TS homologs. The numbering is derived from the KSHV vTS sequence. (B) Phylogenetic analysis of the alignment in A with the protein maximum-likelihood method. Bootstrap values from 100 replica samplings and the scale for substitutions per site are provided. VZV, human varicella-zoster virus (NP_040136); VZVpm, patas monkey varicella-zoster virus (NP_077428); human TS (YXHUT); mouse TS (YXMSI); yeast TS, Saccharomyces cerevisiae (P06785); insect TS, Anopheles sp. (EAA07160); plant TS, Arabidopsis thaliana (AAA32788). See Table 1 for other accession numbers.

FIG. 4.

Comparison of viral ORF 70 and cellular TS homologs. (A) Amino acid sequence alignment of the partial vTS sequences obtained from RFHVMm and RFHVMn with the comparable regions of other viral and cellular TS homologs. The numbering is derived from the KSHV vTS sequence. (B) Phylogenetic analysis of the alignment in A with the protein maximum-likelihood method. Bootstrap values from 100 replica samplings and the scale for substitutions per site are provided. VZV, human varicella-zoster virus (NP_040136); VZVpm, patas monkey varicella-zoster virus (NP_077428); human TS (YXHUT); mouse TS (YXMSI); yeast TS, Saccharomyces cerevisiae (P06785); insect TS, Anopheles sp. (EAA07160); plant TS, Arabidopsis thaliana (AAA32788). See Table 1 for other accession numbers.

FIG. 5.

Comparison of ORF 10 homologs. (A) Alignment of the gammaherpesvirus ORF 10 homologs and the distantly related ORF 11 homologs of KSHV and RRV. Residues conserved in four of the six ORF 10 homologs are highlighted to show ORF 10 conservation. Residues in the ORF 11 homologs which were conserved in two other ORF 10 or ORF 11 homologs are also highlighted to indicate the similarity between the ORF 10 and ORF 11 homologs. (B) Phylogenetic analysis with the protein maximum-likelihood method. ORF 10 homolog sequences from the Old World primate rhadinoviruses RFHVMn, RFHVMm, KSHV, and RRV, the New World primate rhadinoviruses HVS and AtHV3, the ungulate rhadinoviruses BHV4, AHV1, and EHV2, the murine rhadinovirus MHV68, and the lymphocryptovirus EBV (LF1 gene) were aligned with ClustalW and analyzed with the PROML program. Bootstrap values from 100 replica samplings and the scale for substitutions per site are provided. The ORF 11 homologs of KSHV and RRV were included as an outgroup.

FIG. 6.

Comparison of viral and cellular IL-6 homologs. (A) Amino acid sequence alignment of the RFHVMm and RFHVMn vIL-6 homologs compared to the vIL-6 of KSHV and RRV and the human (NP_000591) and rhesus macaque (L26028) cellular IL-6. Amino acids conserved between the KSHV and RFHV sequences are highlighted; a potential N-linked glycosylation site and a cysteine residue conserved between KSHV and RFHV are indicated with an asterisk and an open circle, respectively. The predicted signal peptide cleavage site for the vIL-6 homologs is shown (arrow) and corresponds to the cleavage site determined for human IL-6. The four major alpha-helical regions (A, B, C, and D) and the minor helical region (E) determined for human IL-6 are indicated (47). The residues in sites II and III of KSHV vIL-6 which interact with the gp130 receptor subunit (see text) are indicated with φ and θ, respectively (7). The residues in site I which interact with the IL-6R subunit are indicated with π (23). The amino acid numbering is relative to the KSHV vIL-6 sequence. (B) Phylogenetic analysis of the alignment in A with the protein maximum-likelihood method. Bootstrap values from 100 replica samplings and the scale for substitutions per site are provided.

FIG. 6.

Comparison of viral and cellular IL-6 homologs. (A) Amino acid sequence alignment of the RFHVMm and RFHVMn vIL-6 homologs compared to the vIL-6 of KSHV and RRV and the human (NP_000591) and rhesus macaque (L26028) cellular IL-6. Amino acids conserved between the KSHV and RFHV sequences are highlighted; a potential N-linked glycosylation site and a cysteine residue conserved between KSHV and RFHV are indicated with an asterisk and an open circle, respectively. The predicted signal peptide cleavage site for the vIL-6 homologs is shown (arrow) and corresponds to the cleavage site determined for human IL-6. The four major alpha-helical regions (A, B, C, and D) and the minor helical region (E) determined for human IL-6 are indicated (47). The residues in sites II and III of KSHV vIL-6 which interact with the gp130 receptor subunit (see text) are indicated with φ and θ, respectively (7). The residues in site I which interact with the IL-6R subunit are indicated with π (23). The amino acid numbering is relative to the KSHV vIL-6 sequence. (B) Phylogenetic analysis of the alignment in A with the protein maximum-likelihood method. Bootstrap values from 100 replica samplings and the scale for substitutions per site are provided.

FIG. 7.

Comparison of viral and cellular DHFR homologs. (A) Amino acid sequence alignment of the vDHFR of KSHV, RFHVMn, RFHVMm, RRV, and HVS and comparison with the human (NP_000782) and mouse (NP_034179) cellular DHFR. Residues conserved between KSHV, RFHVMn, and RFHVMm are highlighted. An asterisk indicates the presence of an insertion or deletion difference with RRV. (B) Phylogenetic analysis of the alignment in A with the protein maximum-likelihood method. Bootstrap values from 100 replica samplings and the scale for substitutions per site are provided.

FIG. 8.

Comparison of ORF RF3 homologs. (A) Amino acid sequence alignment of the MIR homologs of KSHV (K3 and K5), RFHVMn (RF3Mn), RFHVMm (RF3Mm), BHV4 (Bo5 and Bo4), MHV68 (MK3), HVS (ORF 12), and myxoma virus (MV-LAP; AAK00734). The BKS zinc finger domain is indicated, with the positions of the hydrophobic transmembrane domains and the conserved region (CR) in the C-terminal domain shown. Residues identical within the K3, K5, RF3Mn, and RF3Mm sequences are highlighted. A consensus sequence for the BKS zinc finger domain is shown. (B) Phylogenetic analysis of the complete ORFs with the protein maximum-likelihood method with the addition of the 5L protein of yaba-like disease virus (NP_073390). Bootstrap values from 100 replica samplings and the scale for substitutions per site are provided.

FIG. 8.

Comparison of ORF RF3 homologs. (A) Amino acid sequence alignment of the MIR homologs of KSHV (K3 and K5), RFHVMn (RF3Mn), RFHVMm (RF3Mm), BHV4 (Bo5 and Bo4), MHV68 (MK3), HVS (ORF 12), and myxoma virus (MV-LAP; AAK00734). The BKS zinc finger domain is indicated, with the positions of the hydrophobic transmembrane domains and the conserved region (CR) in the C-terminal domain shown. Residues identical within the K3, K5, RF3Mn, and RF3Mm sequences are highlighted. A consensus sequence for the BKS zinc finger domain is shown. (B) Phylogenetic analysis of the complete ORFs with the protein maximum-likelihood method with the addition of the 5L protein of yaba-like disease virus (NP_073390). Bootstrap values from 100 replica samplings and the scale for substitutions per site are provided.