Genomic characterization of Japanese macaque rhadinovirus, a novel herpesvirus isolated from a nonhuman primate with a spontaneous inflammatory demyelinating disease

Abstract

Japanese macaque rhadinovirus (JMRV) is a novel gamma-2 herpesvirus that was isolated from a Japanese macaque (JM) with an inflammatory demyelinating encephalomyelitis referred to as Japanese macaque encephalomyelitis, a disease that possesses clinical and histopathological features resembling multiple sclerosis in humans. Genomic DNA sequence analysis reveals that JMRV is a gammaherpesvirus closely related to rhesus macaque rhadinovirus (RRV) and human herpesvirus 8. We describe here the complete nucleotide sequence and structure of the JMRV genome, as well as the sequence of two plaque isolates of this virus. Analysis of the JMRV genome not only demonstrates that this virus shares a number of genes with RRV that may be involved in pathogenesis but also indicates the presence of unique JMRV genes that could potentially contribute to disease development. The knowledge of the genomic sequence of JMRV, and the ability to easily propagate the virus in vitro, make JMRV infection of JM an attractive model for examining the potential role of an infectious viral agent in the development of demyelinating encephalomyelitis disease in vivo.

Fig 1

Restriction digest analysis of JMRV DNA. A 2.5-μg portion of purified viral genomic DNA was digested overnight with HindIII or BamHI and run on a 0.7% agarose gel. The patterns obtained for both digestions correlate with the predicted fragment sizes for the JMRV genomic sequence, as listed in Table 1. L1 and L2 represent DNA ladders run as size standards.

Fig 2

Map of the JMRV genome. All predicted JMRV ORFs are depicted by arrows, and the scale (in base pairs) indicates their approximate genomic location. ORFs are numbered from left to right as described in Table 3, with only selected ORFs labeled for purposes of clarity. JMRV ORFs that are homologous to RRV ORFs are shaded light gray, those homologous to both RRV and HHV-8 ORFs are shaded dark gray, and those unique to JMRV are white. Internal repeat regions are indicated by hashed boxes and are labeled with the corresponding repeat name above their location. Hashed boxes at the ends of the genome depict the location of TRs, and dashed lines at the genome termini indicate the approximate location of the partial TR sequences.

Fig 3

Identification of the TR sequence. (A) Purified viral DNA was digested with HindIII and run on a 1% agarose gel, resulting in the identification of an ∼1.6-kb digestion product corresponding to the terminal repeat (TR) unit of JMRV. This fragment was purified, cloned, and sequenced. (B) The TR unit of JMRV is 1,564 bp in length and possesses defining features of a herpesvirus TR, including the presence of direct repeats (DR1 and DR2) and packaging motifs (pac-1 and pac-2). The TR also possesses three potential ORFs: JM1, JM171, and JM170-TR. JM170-TR is a variant of JM170 that possesses an alternate 49 nt at its 3′ end. The alternate 49-nt sequence of JM170-TR is denoted in the TR sequence by italicized lettering, and the location of the junction of the LUR and partial TR located at the right end of the genome is marked by an asterisk. (C) Diagram depicting the predicted layout of TR units at the termini of the JMRV genome. The size marker indicates the fragment produced by HindIII digestion of viral DNA, in relation to the orientation of the TR units. The exact number of copies of the TR at the ends of the genome is unknown and varies between individual viral genomes.

Fig 4

Phylogenetic tree of selected gammaherpesviruses. The protein sequence of DNA polymerase from JMRV, RRV, HHV-8, HVS, RLCV, MHV-68, and EBV were subjected to bootstrap analysis (1,000 replicates) using the neighbor joining method. Numbers represent the percentage of bootstrap trees that contain the same branch point.

Fig 5

Analysis of JMRV-unique gene expression. RNA was purified from primary rhesus fibroblasts infected with JMRV and subjected to RT-PCR using primers specific for JMRV-unique ORFs. Cells were infected at an MOI of 5, treated with CHX, GCV, or left untreated, and collected at 24, 48, and 72 h, respectively. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) reactions with RT (+RT) serve as controls for the presence of RNA, and GAPDH reactions without RT (−RT) demonstrate absence of DNA in each sample.