The R1 gene of RRV is located at a position equivalent to that of the K1 gene of KSHV and the STP gene of HVS. Although R1 shows no homology to STP, it contains sequence motifs and an organization of structural features that are very similar to K1. The R1 and K1 proteins exhibit approximately 27% amino acid identity in their extracellular domains, and both extracellular domains resemble those of the immunoglobulin receptor superfamily. R1 and K1 differ significantly in the length of their cytoplasmic domains, but both contain YXXL motifs that resemble SH2-binding sequences. R1 has five YXXL elements near the C terminus of its cytoplasmic tail. The third and fourth YXXL motifs of R1 show the conserved pattern for a putative ITAM: (D/E)X0–3
). The fourth and fifth YXXL motifs could also possibly function as an ITAM, since the YXXL motifs are separated by 8 amino acids and are preceded by negatively charged amino acids. ITAMs are present in B-cell, T-cell and Fc immunoreceptors and interact with both Src and Syk family protein tyrosine kinases (3
). In addition to the YXXL motifs, there are several other tyrosine residues that could serve as potential phosphorylation sites. These include two YXXP motifs, one YXXA motif, and one YXXV motif. Such motifs have also been shown to interact with SH2 domains present in a variety of tyrosine kinases and other signaling molecules (3
). Hence, R1 has rich potential for affecting cellular signaling through sequence motifs present within its cytoplasmic domain.
The finding of R1 localization predominantly in the cytoplasm was surprising. Much of the staining was in punctate form near the perimeter of the cytoplasm, suggestive of possible endosomal localization. YXXH, where H is a hydrophobic amino acid, can also serve as a signal for internalization of plasma membrane proteins (30
). The cytoplasmic domain of R1 also contains a dileucine repeat at residues 318 and 319 (Fig. ), which can also serve as a signal for endocytosis (30
). Further work is needed to delineate the effects of specific sequence motifs in the cytoplasmic domain of R1 on endocytosis and signaling.
We have sequenced four independent R1 genes. The four rhadinovirus isolates from which the sequences were derived were obtained from different sources. Three of the macaques (Mm26-95, Mm309-95, and Mf23-97) were born and raised at the New England Regional Primate Research Center, and Mn19545 was obtained from the Oregon Regional Primate Research Center. The R1 genes from the two M. mulatta
animals were the most divergent. This suggests that R1 may exhibit polymorphic divergence independent of the species of origin or that one or more of the viruses was derived from cross-species transmission. Although there is some divergence among the R1 sequences, the percent divergence is lower than was observed with the K1 gene of KSHV (16
). Comparison of the primary amino acid sequences of K1 genes isolated from two body cavity-based lymphoma cell lines, BCBL-1 and BC-1, and a Kaposi’s sarcoma biopsy specimen revealed many amino acid substitutions around the transmembrane region of K1, but the carboxy-terminal sequences were the most conserved among the K1 isolates (16
). Despite the divergence seen among individual K1 and R1 sequences, the carboxy-terminal ITAMs in both the R1 and K1 genes are well conserved, suggesting that these motifs may play an important role in the function of these proteins.
STP of HVS and K1 of KSHV both transform rodent fibroblasts and contribute to oncogenic transformation of T lymphocytes from common marmosets (10
). The STP gene of HVS C488 interacts with cellular Ras (12
). K1 has recently been shown to interact with the Syk family of protein kinases (17
). We have now demonstrated that R1 can also transform rodent fibroblasts and induce tumors in nude mice. In addition, an HVSΔSTP/R1 recombinant was capable of immortalizing primary lymphocytes. Demonstration of a role for R1 in the natural history of primary RRV infection, persistence, or disease is dependent on further animal model development.