Human herpesvirus 6B genome sequence: coding content and comparison with human herpesvirus 6A

Abstract

Human herpesvirus 6 variants A and B (HHV-6A and HHV-6B) are closely related viruses that can be readily distinguished by comparison of restriction endonuclease profiles and nucleotide sequences. The viruses are similar with respect to genomic and genetic organization, and their genomes cross-hybridize extensively, but they differ in biological and epidemiologic features. Differences include infectivity of T-cell lines, patterns of reactivity with monoclonal antibodies, and disease associations. Here we report the complete genome sequence of HHV-6B strain Z29 [HHV-6B(Z29)], describe its genetic content, and present an analysis of the relationships between HHV-6A and HHV-6B. As sequenced, the HHV-6B(Z29) genome is 162,114 bp long and is composed of a 144,528-bp unique segment (U) bracketed by 8,793-bp direct repeats (DR). The genomic sequence allows prediction of a total of 119 unique open reading frames (ORFs), 9 of which are present only in HHV-6B. Splicing is predicted in 11 genes, resulting in the 119 ORFs composing 97 unique genes. The overall nucleotide sequence identity between HHV-6A and HHV-6B is 90%. The most divergent regions are DR and the right end of U, spanning ORFs U86 to U100. These regions have 85 and 72% nucleotide sequence identity, respectively. The amino acid sequences of 13 of the 17 ORFs at the right end of U differ by more than 10%, with the notable exception of U94, the adeno-associated virus type 2 rep homolog, which differs by only 2.4%. This region also includes putative cis-acting sequences that are likely to be involved in transcriptional regulation of the major immediate-early locus. The catalog of variant-specific genetic differences resulting from our comparison of the genome sequences adds support to previous data indicating that HHV-6A and HHV-6B are distinct herpesvirus species.

FIG. 1

(A) HHV-6B genomic and genetic architecture. The upper portion shows the positions and arrangements of the major repeat elements R0, R1, R2A, R2B, and R3, the origin of replication (oriLyt), and the structure of the DR termini. ORF sizes, orientation, and location are indicated. HHV-6B unique ORFs are labeled B1 through B9. The orientation and order of the seven conserved herpesvirus core gene blocks are indicated, as is the block (U2 to U19) found only in betaherpesviruses. 101K, antigenic virion protein 101K; PA, polymerase processivity factor; RR, large ribonucleotide reductase; Teg, large tegument protein; Pol, DNA polymerase; gB, glycoprotein B; MDBP, major DNA binding protein; HP, helicase/primase complex; gH, glycoprotein H; MCP, major capsid protein; Exo, alkaline exonuclease; OBP, origin binding protein; IE1, major immediate-early gene; Rep, parvovirus rep homolog. (B) Nucleotide sequence comparison between HHV-6A and HHV-6B genomes. Genomes were aligned in segments by using GAP with gap and length weights of 50 and 3, respectively, except for the region spanning residues 124000 through 144500 (dashed line), for which weights of 25 and 1, respectively, were used to maximize the alignment. After concatenation of the aligned segments, identities between the aligned sequences were plotted by using PLOTSIMILARITY with a window of 1,000 residues. The horizontal dashed line represents mean identity of 88% across the whole alignment. Several regions with scores less than the mean are labeled; variable intergenic regions are identified by their flanking genes, e.g., U10/U11; the region spanning R2A and R2B is indicated as R2A-R2B. Nucleotide identity (NI) for the indicated regions was determined using GAP for aligned degapped sequences, with gap and length weights of 50 and 3, respectively, except for the segment spanning the right end of U, where weights 250 and 25, respectively, were used in order to omit gaps.

FIG. 1

(A) HHV-6B genomic and genetic architecture. The upper portion shows the positions and arrangements of the major repeat elements R0, R1, R2A, R2B, and R3, the origin of replication (oriLyt), and the structure of the DR termini. ORF sizes, orientation, and location are indicated. HHV-6B unique ORFs are labeled B1 through B9. The orientation and order of the seven conserved herpesvirus core gene blocks are indicated, as is the block (U2 to U19) found only in betaherpesviruses. 101K, antigenic virion protein 101K; PA, polymerase processivity factor; RR, large ribonucleotide reductase; Teg, large tegument protein; Pol, DNA polymerase; gB, glycoprotein B; MDBP, major DNA binding protein; HP, helicase/primase complex; gH, glycoprotein H; MCP, major capsid protein; Exo, alkaline exonuclease; OBP, origin binding protein; IE1, major immediate-early gene; Rep, parvovirus rep homolog. (B) Nucleotide sequence comparison between HHV-6A and HHV-6B genomes. Genomes were aligned in segments by using GAP with gap and length weights of 50 and 3, respectively, except for the region spanning residues 124000 through 144500 (dashed line), for which weights of 25 and 1, respectively, were used to maximize the alignment. After concatenation of the aligned segments, identities between the aligned sequences were plotted by using PLOTSIMILARITY with a window of 1,000 residues. The horizontal dashed line represents mean identity of 88% across the whole alignment. Several regions with scores less than the mean are labeled; variable intergenic regions are identified by their flanking genes, e.g., U10/U11; the region spanning R2A and R2B is indicated as R2A-R2B. Nucleotide identity (NI) for the indicated regions was determined using GAP for aligned degapped sequences, with gap and length weights of 50 and 3, respectively, except for the segment spanning the right end of U, where weights 250 and 25, respectively, were used in order to omit gaps.

FIG. 2

(A) DR_L and DR_R in linear and concatemeric genomes. The locations of terminal sequences from this and other works are indicated relative to the pac1-het(TAACCC)_n and (TAACCC)_n-pac2 elements at the left and right termini of DR, respectively. The dashed lines inside DR indicate the relative lengths of the TRS arrays. The black bars below the DRs represent the relative location of the sequences aligned in panel B; sequence designations are as shown below the lines. Sources for the sequences are as follows: B2L_DR and B2R_DR are from this work and were generated from amplimers TL and DRL/U, respectively (Table 1); P3, P10, P15, P4, P9, P1, P7 are DR_L-DR_R junction-spanning clones derived by PCR using 5′gcggatccTAACCCATCCCCCAACGCGC as the rightward primer (RP) and 5′cggaattcTTAGGGTTAATACCCCCCTTT as the leftward primer (LP) (the BamHI and EcoRI sites used in cloning are in lowercase) on template from HHV- 6B(Z29)-infected Molt-3 cells. The locations of primers RP and LP are indicated in panel B; A1L_DR, A1R_DR, and A1 (GenBank accession no. X79798, X79799, and X79800, respectively) are from Gompels and Macaulay (18); B1L_DR, B1R_DR, B1, and B2 (GenBank accession numbers L22337, L23336, and L22335, respectively) are from Thomson et al. (57). (B) Alignment of terminal sequences. Sequences were aligned by using PILEUP with gap creation and extension penalties of 5 and 1, respectively. Sequences A1, A1R_DR, and A1L_DR are from HHV-6A(U1102); the rest are from HHV-6B(Z29). Differences between the variants are shown in lowercase, and differences between HHV-6B sequences are underlined. Nucleotides found only in DR_R-DR_L junction-spanning clones are italicized. Putative terminal nucleotides are in bold. (C) Summary diagram of the DR termini. Designations are as for panel A; N indicates any nucleotide.

FIG. 3

Alignment of HHV-6B and HHV-6A consensus R3 sequences. Individual R3 units of HHV-6A (28 copies) (19) and HHV-6B strains HST (24 copies) (27) and Z29 (26 copies) were aligned. Alignments were done by using PILEUP with gap creation and extension penalties of 5 and 1, respectively. Each consensus sequence represents 80% plurality between the aligned units of each virus. Sequences common to all three viruses are in uppercase. Conserved pockets are underlined, and the binding site for the transcription factor PEA3 is in bold. Symbols: Y, C or T; R, A or G; S, C or G; W, A or T; M, A or C; K, G or T; H, A or C or T; V, A or C or G.

FIG. 4

ORF length versus Ka/Ks ratio. Ka/Ks ratios were calculated by using DIVERGE for 88 aligned HHV-6A and HHV-6B coding sequences. Protein and nucleic acid sequences were aligned by using PILEUP with gap and length weights of 12 and 4, respectively, for proteins and 5 and 1, respectively, for nucleic acids. Ka/Ks ratios for spliced genes were determined for the spliced proteins and are designated by the 5′-proximal exon. ORFs with less than 80% amino acid (aa) identity are labeled.

FIG. 5

Graphical representation of intra- and intervariant alignments in the region encompassing U75 to the 3′ end of U94 [HHV-6B(Z29) coordinates 113977 to 143096). The HHV-6B(Z29) and HHV-6B(HST) DNA segments were aligned by using GAP with gap and length weights of 50 and 3, respectively, and the graphical representations were generated with GAPSHOW. The corresponding region of HHV-6A(U1102) was aligned with that of HHV-6B(Z29) by using GAP with gap and length weights of 25 and 1, respectively. Nucleotide sequence differences are represented by the longest vertical bars; gaps are depicted by the shorter vertical lines. Regions spanning R1 and R3 are indicated. Locations of R1, R2A, R2B, R3, and ORFs are indicated.