Complete sequence and comparative analysis of the genome of herpes B virus (Cercopithecine herpesvirus 1) from a rhesus monkey

Abstract

The complete DNA sequence of herpes B virus (Cercopithecine herpesvirus 1) strain E2490, isolated from a rhesus macaque, was determined. The total genome length is 156,789 bp, with 74.5% G+C composition and overall genome organization characteristic of alphaherpesviruses. The first and last residues of the genome were defined by sequencing the cloned genomic termini. There were six origins of DNA replication in the genome due to tandem duplication of both oriL and oriS regions. Seventy-four genes were identified, and sequence homology to proteins known in herpes simplex viruses (HSVs) was observed in all cases but one. The degree of amino acid identity between B virus and HSV proteins ranged from 26.6% (US5) to 87.7% (US15). Unexpectedly, B virus lacked a homolog of the HSV gamma(1)34.5 gene, which encodes a neurovirulence factor. Absence of this gene was verified in two low-passage clinical isolates derived from a rhesus macaque and a zoonotically infected human. This finding suggests that B virus most likely utilizes mechanisms distinct from those of HSV to sustain efficient replication in neuronal cells. Despite the considerable differences in G+C content of the macaque and B virus genes (51% and 74.2%, respectively), codons used by B virus are optimal for the tRNA population of macaque cells. Complete sequence of the B virus genome will certainly facilitate identification of the genetic basis and possible molecular mechanisms of enhanced B virus neurovirulence in humans, which results in an 80% mortality rate following zoonotic infection.

FIG. 1.

Molecular cloning of B virus genome. The first letter in the clone designations indicates the cloning site: B, BamHI; K, KpnI; P, PstI, S, SalI; X, XhoI. The number in the clone designations reflects the order of clone isolation. Double-sided arrows indicate PCR fragments used for verification of clone junctions. The map scale is in kilobases.

FIG. 2.

Identification of B virus genomic termini. (A) Southern blot of SphI-digested genomic DNA hybridized with digoxigenin-labeled plasmids containing 1.6-kb (lane 1) and 1.8-kb (lane 2) terminal fragments. An autoradiograph of the membranes is shown. The positions of DNA size markers are shown on the left. Arrows indicate terminal small (T_S), terminal large (T_L), and junction (J) fragments. (B) Cloned genomic fragments containing B virus termini. The structural organization of the B virus genome is shown, with the U_L and U_S regions represented by solid lines and the TR_L, IR_L, TR_S, and IR_S regions represented by open boxes. Terminal a sequences and the oppositely oriented internal a′ sequence are indicated. Below the genome diagram, a schematic alignment of the isolated terminal fragments is shown. Arrows denote the locations of a sequence copies and their orientations in the genome. The numbers in parentheses indicate the number of clones sequenced. (C) Alignments of the a sequences of B virus (BV), HSV-1, and HSV-2. Arrows indicate genomic ends. The conserved motifs of pac1 and pac2 signals are shown in bold.

FIG. 3.

Comparison of B virus and HSV-1 origins of replication. The DNA sequences of the oriL and oriS core elements are shown. BV/L1, B virus oriL1; BV/L2, B virus oriL2; BV/S, B virus oriS1 and oriS2; HSV1/L, HSV-1 oriL; HSV1/S, HSV-1 oriS. The OBP-binding sites (box I, box II, and box III) are boxed. Residue substitutions in all B virus origins relative to HSV-1 oriL are shown in bold. Residue substitutions in B virus oriL2 relative to oriL1 are underlined.