Next-generation sequence analysis of the genome of RFHVMn, the macaque homolog of Kaposi's sarcoma (KS)-associated herpesvirus, from a KS-like tumor of a pig-tailed macaque

Abstract

The complete sequence of retroperitoneal fibromatosis-associated herpesvirus Macaca nemestrina (RFHVMn), the pig-tailed macaque homolog of Kaposi's sarcoma-associated herpesvirus (KSHV), was determined by next-generation sequence analysis of a Kaposi's sarcoma (KS)-like macaque tumor. Colinearity of genes was observed with the KSHV genome, and the core herpesvirus genes had strong sequence homology to the corresponding KSHV genes. RFHVMn lacked homologs of open reading frame 11 (ORF11) and KSHV ORFs K5 and K6, which appear to have been generated by duplication of ORFs K3 and K4 after the divergence of KSHV and RFHV. RFHVMn contained positional homologs of all other unique KSHV genes, although some showed limited sequence similarity. RFHVMn contained a number of candidate microRNA genes. Although there was little sequence similarity with KSHV microRNAs, one candidate contained the same seed sequence as the positional homolog, kshv-miR-K12-10a, suggesting functional overlap. RNA transcript splicing was highly conserved between RFHVMn and KSHV, and strong sequence conservation was noted in specific promoters and putative origins of replication, predicting important functional similarities. Sequence comparisons indicated that RFHVMn and KSHV developed in long-term synchrony with the evolution of their hosts, and both viruses phylogenetically group within the RV1 lineage of Old World primate rhadinoviruses. RFHVMn is the closest homolog of KSHV to be completely sequenced and the first sequenced RV1 rhadinovirus homolog of KSHV from a nonhuman Old World primate. The strong genetic and sequence similarity between RFHVMn and KSHV, coupled with similarities in biology and pathology, demonstrate that RFHVMn infection in macaques offers an important and relevant model for the study of KSHV in humans.

Fig 1

Comparative map of the rhadinovirus and lymphocryptovirus genomes. The positions and transcription directions of the ORFs identified in the RFHVMn genome (KF703446; this study) are compared to the corresponding ORFs in KSHV (NC_009333), RRV (AF210726 and NC_003401), and EBV (NC_007605), as identified in the NCBI accession records (the basic design of this map was patterned after Fig. 1 of reference 22). The numbering of the KSHV, RFHVMn, and RRV ORFs is patterned after the genome structure of the prototype rhadinovirus, HVS, with KSHV–specific ORFs designated K1 to K15 and their homologs in RFHVMn and RRV designated RF1 to RF15 and R1 to R15, respectively. The EBV ORFs are indicated, using the typical EBV nomenclature above the ORF and the corresponding rhadinovirus designation below. The approximate positions of the ORFs are shown with regard to their positions within the KSHV genome (i.e., 1 to 140 kb). The sizes of the ORF markers are approximately consistent with the sizes of the actual encoded proteins. Vertical black lines within an ORF indicate splicing events or internal initiations, while longer–range splices of protein–coding exons are indicated with bars. The ORFs are color coded with regard to their conservation in other herpesvirus genomes, showing core HV genes conserved in most herpesvirus families, genes conserved in beta– and gammaherpesviruses, gammherpesviruses only, gamma–2–herpesviruses, RV1 and RV2 rhadinoviruses, RV1 rhadinoviruses only, KSHV only, and EBV only, as indicated. The RRV nomenclature from AF210726 was used for consistency.

Fig 2

Nucleotide dot plot alignment of the complete RFHVMn and KSHV genomes. The nucleotide sequence of the RFHVMn genome (KF703446) was compared to the sequence of the KSHV genome (NC_009333) using the Java Dot Plot alignment program (JDotter). The positions of specific ORFs and the divergent loci are indicated.

Fig 3

Comparison of the KSHV ORF K1 and the RFHVMn ORF RF1 protein sequences. The sequences of KSHV ORF K1 (YP_001129350) and the RFHVMn ORF RF1 (AGY30683) positional homolog were aligned. Identical residues are highlighted in black. The regions conserved with the immunoglobulin (Ig) superfamily and the immunoglobulin receptor tyrosine–based activation motif (ITAM) are indicated, and the variable regions (VR1 and VR2) identified in the KSHV subtypes (45) are shown. The residues predicted to form a hydrophobic transmembrane (TM) domain and the positions of two repeat domains present in the RF1 sequence are indicated. Potential N-linked glycosylation sites are underlined, and sites conserved between K1 and RF1 are indicated with asterisks. Dashed lines represent gaps in the sequence, while solid lines represent continuations of the left and right brackets.

Fig 4

Amino acid alignments of the KSHV ORFs K4, K4.1, K4.2, and vBCL2 with the corresponding RFHVMn homologs. The amino acid alignments of ORFs K4 (YP_001129362) and RF4 (AGY30694) (A), K4.1 (YP_001129363) and RF4.1 (AGY30695), (B), K4.2 (YP_001129364) and RF4.2 (AGY30696) (C), and KvBCL2 (YP_001129368) and RFvBCL2 (AGY30698) (D) are shown. Putative hydrophobic TM domains and N-terminal signal sequences with predicted cleavage sites are underlined, and cysteine residues are highlighted. BLAST analysis revealed that the K4/RF4 and K4.1/RF4.1 ORFs are conserved members of the family of interleukin 8-like CC cytokines. The K4.2 and RF4.2 ORFs both contained putative transmembrane domains (two in K4.2 and one in RF4.2). *, identical residue; :, conservative substitution; ., semiconservative substitution.

Fig 5

Comparison of the KSHV ORF K7 and RFHV ORF RF7 protein sequences. The sequences of KSHV ORF K7 (YP_001129367) and the RFHVMn ORF RF7 (AGY30697) positional homolog were aligned. Since the K7 sequence was previously shown to have sequence similarity to human survivin, the sequence of survivin/baculoviral IAP repeat containing protein 5 isoform 2 (NP_001012270) from aa 7 to aa 94 was aligned to the RF7 and K7 sequences. Amino acid residues conserved among all three sequences are highlighted in black, those conserved between RF7 and K7 are highlighted in green, those conserved between RF7 and survivin are highlighted in red, and those conserved between K7 and survivin are highlighted in blue. Putative N-terminal hydrophobic transmembrane domains are underlined.

Fig 6

Phylogenetic analysis of the vIRF homologs. The encoded amino acid sequences of the vIRF homologs of KSHV (K9 [YP_001129411], K10 [YP_001129414], K10.5 [YP_001129413], and K11 [YP_001129412]), RFHVMn (RF9-11 [AGY30740-43; this study]), and RRV (R9.1 [AAF60036], R9.2 [AAF60037], R9.3 [AAF60038], R9.4 [AAF60039], R9.5 [AAF60040], R9.6 [AAF60041], R9.7 [AAF60042], and R9.8 [AAF60043]) were aligned using MUSCLE, and phylogeny was determined by protein maximum likelihood. The human (NP_002154) and rhesus macaque (NP_001252887) cellular interferon regulatory factor 8 sequences were used as an outgroup. The lineage divisions of the RV1 and RV2 rhadinovirus proteins and the cellular proteins are indicated with dashed lines, and the presence of spliced transcripts is noted. The numbers of substitutions per site are indicated.

Fig 7

Comparison of the divergent D loci of RFHVMn and KSHV. (A) Graphical representation of the structure of the Ori-Lyt-R regions of KSHV (NC_009333) and RFHVMn (KF703446) between ORF69 and ORF71; the presence of encoded proteins and microRNAs is shown. The position of the 18-bp TATA repeat (red box) and C/EBP palindromic motifs (stars) are indicated. The known KSHV microRNA genes (purple) and the predicted RFHVMn candidate microRNAs with bona fide pre-microRNA hairpins (blue) are shown. (B) Alignment of ORF K12 (YP_001129428) and the positional ORF RF12 (AGY30757) homolog. Identical residues and the basic domain of RF12 are highlighted. (C) Alignment of the kshv-miR-K12-10a-3p and rfhvmn-miRc-RF9-3p pre-miRNAs. The predicted hairpin structures are indicated by parentheses. Boldface and underlined letters denote sequences of the mature kshv-miR-K12-10a-3p RNA (74) and the mature rfhvmn-miRc-RF9-3p, as predicted (pred) according to the guidelines of Zeng et al. (75). The conserved seed sequences are highlighted.

Fig 8

Comparison of the long-inverted-repeat regions of Ori-Lyt-R and Ori-Lyt-L of KSHV and RFHVMn. (A and B) Dot plot of the nucleotide sequence alignment of the inverted-repeat regions of Ori-Lyt-L and Ori-Lyt-R for KSHV (A) and RFHVMn (B), with the approximate positions of the C/EBP palindromes (M1/2, M5/6, M7/8, and M9/10), the 18-bp TATATA repeat, and adjacent microRNA genes, illustrated in panel C. (C) Alignment of the nucleotide sequences of KSHV (U75698) containing the Ori-Lyt-L (bp 23223 to 23685) (KSL), the inverted sequence of KSHV containing the Ori-Lyt-R region bp (119245 to 119782) (KSRi), the nucleotide sequences of RFHVMn (KF703446; this study) containing the putative Ori-Lyt-L (bp 21937 to 22462) (RFL), and the inverted sequence of RFHVMn containing the putative Ori-Lyt-R (bp 111254 to 111800) (RFRi). The conserved AT-rich motifs (blue) and C/EBP motifs (red) are indicated (see the text). Other conserved regions (CR1 to CR6) are highlighted in yellow or gray. The kshv-miR-K12-K9 microRNA with the mature microRNA (purple) and stem-loops (green) and the predicted rfhvmn-miRc-RF6 are shown.

Fig 9

Conservation of the bidirectional ORF73 LANA and K14/RF14 promoter structure of KSHV and RFHVMn. (A and B) Diagrammatic rendering (A) and sequence alignment (B) of the promoter region of the KSHV LANA/K14 genes (NC_009333) and the RFHV LANA/RF14 genes, showing the initiating codons and the positions of the conserved splice donor (SD) and splice acceptor (SA) sites generating the latent spliced LANA transcript from the constitutive promoter (LANApc). The Rta-inducible LANApi promoter of KSHV LANA is indicated, with its associated RBP-Jκ binding sites and Rta response element (RRE) located within the intron region spliced out of the LANApc transcript (see the text). The position of a putative LANApi promoter for RFHV LANA is shown. (C) Alignment of the RBP-Jκ binding motifs of KSHV and RFHV indicated in panels A and B. Nucleotide numbers are based on the LANA translational initiation codon.

Fig 10

Conservation of the junctional regions of the K8 and K8.1 genes and their homologs in RFHVMn and EBV. The nucleotide sequences of KSHV (NC_009333), RFHV (this study), and EBV (NC_007605) from the junctional regions of K8/K8.1, RF8/RF8.1, and bZIP/GP42 were aligned, and the translational stop and initiation codons are highlighted. The transcriptional start site of the K8.1 mRNA is indicated, and the polyadenylation signal for EBV bZIP is marked with asterisks (see the text). The conserved AATATTAA motif within the promoters for K8.1, RF8.1, and GP42 is highlighted.

Fig 11

Comparison of the transcripts of KSHV ORF K8.1 and its homolog RFHVMn ORF RF8.1. (A) Gene structure of KSHV ORF K8.1 with the exons color coded and amino acids at the junctions indicated. Exons 2 and 3 are in different reading frames, as indicated, and exon 2B indicates a read-through of a cryptic splice site. Alternate splice variants that have been detected are labeled with red stars (see the text). NCBI accession numbers: K8.1α, AAC63270; K8.1β, AAC63271; K8.1γ, AAB62630. The N-terminal signal peptide (SP) and C-terminal hydrophobic TM are shown. (B) Gene structure of RFHV ORF RF8.1, with regions homologous to the K8.1 exons color coded and labeled. The entire RF8.1 ORF (AGY30733) is in the same reading frame, unlike K8.1.

Fig 12

Comparison of the spliced transcripts of KSHV ORF K8, RFHVMn ORF RF8, and EBV bZIP. (A) Gene structure of KSHV ORF K8, with the exons color coded and amino acids at the junctions indicated. Exons 1 and 2 are in the same reading frame, and exons 2 and 3 are separated by an intron sequence. Transcript K8γ is generated by a read-through of cryptic splice junctions between exons 1A and 1B and between exons 2A and 2B (accession no. AAD25322). Transcript K8α is generated by splicing exons 1A, 2A, and 3 (accession no. AAD25316). Transcript K8β is generated by splicing exons 1A and 2A with read-through of the cryptic splice junction between exons 2A and 2B (accession no. AAD25319). (B) Gene structure of RFHVMn ORF RF8. Exons 1 and 2 are in different reading frames, while exons 2 and 3 are separated by an intron sequence. Possible transcripts corresponding to the K8 transcripts are shown (RF8α; accession no. AGY30732). (C) Gene structure of EBV bZIP, revealing close similarity to the gene structure of RF8. Possible transcripts corresponding to the K8 transcripts are shown, and detected transcripts are indicated with red stars. The bZIP accession no. is NC_007605; for more information regarding bZIPΔ, see reference .

Fig 13

Phylogenetic comparison of KSHV and EBV and their macaque homologs. (A) The amino acid sequences for the homologs of glycoprotein B (gB), ORF64/BPLF1, DNA polymerase (DNA Pol), and ORF37/BGLF5 from KSHV, RFHVMn (this study), RRV, EBV, and rhesus macaque RhLCV were aligned using ClustalW, and a distance tree was generated for each gene using neighbor joining as implemented in Phylip 3.2. The numbers of substitutions per site are indicated, and the ratios of branch lengths between the human and macaque RV1 rhadinoviruses (KSHV and RFHVMn) and human and macaque lymphocryptoviruses (LCV) are shown. (B) Protein maximum-likelihood analysis with a molecular clock of a 1,313-aa concatenated sequence from the DNA polymerase and glycoprotein B of the Old World primate rhadinoviruses, lymphocryptoviruses, and cytomegaloviruses from humans (KSHV, EBV, and hCMV) and rhesus macaques (RFHVMm, RRV, RhLCV, and RhCMV) and New World primate rhadinovirus, lymphocryptovirus, and cytomegalovirus from the squirrel monkey (HVS, SsciLCV, and SsciCMV). Rhadinovirus sequences from the pig-tailed macaque (RFHVMn and MneRV2) were also analyzed. The betaherpesvirus (β), gamma-1-herpesvirus (γ1)/lymphocryptovirus, and gamma-2-herpesvirus (γ2)/rhadinovirus branches are indicated. The tentative evolutionary time scale in millions of years before the present) is positioned on each branch based on the same date for separation of the Old and New World herpesvirus lineages as for separation of the Old and New World primate lineages (76) (open triangles). The separation of the macaque and human herpesvirus lineages is indicated with solid triangles. Protein sequences were obtained from the complete genome sequences of KSHV (NC_009333), RFHVMn (KF703446; this study), RRV (NC_003401), HVS (NC_001350), EBV (NC_007605), RhLCV (AY037858), hCMV (X17403), RhCMV (NC_006150), and SsciCMV (FJ483967) and partial sequences of SsciLCV (AY139024), RFHVMm (AF005479), and MneRV2 (unpublished data).