Molecular Characterization of the Rhesus Rhadinovirus (RRV) ORF4 Gene and the RRV Complement Control Protein It Encodes

Abstract

The diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamily Gammaherpesvirinae and currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein as RCP) in infected rhesus macaque fibroblasts mapped the ends of the transcripts of both strains. They revealed that H26-95 encodes a full-length, unspliced RCP transcript, while 17577 RCP generates a full-length unspliced mRNA and two alternatively spliced transcripts. Western blotting confirmed that infected cells express RCP, and immune electron microscopy disclosed this protein on the surface of RRV virions. Functional studies of RCP encoded by both RRV strains revealed their ability to suppress complement activation by the classical (antibodymediated) pathway. These data provide the foundation for studies into the biological significance of gammaherpesvirus complement regulatory proteins in a tractable, non-human primate model.

Characterization of the Complement Inhibitory Function of Rhesus Rhadinovirus Complement Control Protein (RCP)

“…These proteins were named RCP (6). Despite predicted high amino acid similarity between most of the proteins encoded by the two RRV strains, their RCP genes differed significantly from each other; RCP from strain 17577 (referred to as RCP-1) consists of 645 amino acids and contains eight CCP domains, whereas RCP from strain H2695 (RCP-H) consists of 395 amino acids and contains four CCP domains (18), consistent with the four CCP domains found in KCP (19). Both RCP-1 and RCP-H have a C-terminal transmembrane spanning region, as does KCP.…”

Disruption of LANA in Rhesus Rhadinovirus Generates a Highly Lytic Recombinant Virus

“…Our group has demonstrated that the transcription program of RRV is similar to that of KSHV (17,25). Several laboratories have used the RRV model to study viral gene products, including the viral interleukin-6 homologue (37), Rta/Orf50, R8/RAP, R8.1 (17,50), R1 (13,14), R15 (46,63), RRV Orf4 (55), and the RRV-encoded microRNAs (74). We previously reported the kinetics of gene expression with regard to immediate-early, early, and late genes during RRV lytic infection and have reported that they are similar to those seen during KSHV reactivation (17,25).…”

Complete Genome Sequence of Pig-Tailed Macaque Rhadinovirus 2 and Its Evolutionary Relationship with Rhesus Macaque Rhadinovirus and Human Herpesvirus 8/Kaposi's Sarcoma-Associated Herpesvirus

“…1; spliced sites indicated with a thin black bar within colored ORFs or as connections between colored ORFs). While alternate splicing has been detected in the KSHV ORF4 KCP (44) and RRV17577 ORF4 CCP (45), no conserved splice donor or acceptor sites were unambiguously identified by sequence analysis within the ORF4 homologs of the MneRV2 or RRV26-95 genomes. Additional spliced transcripts have been detected in noncoding regions of the KSHV K13, ORF72, and ORF73, but lack of sequence similarity within the MneRV2 homologs of these genes precluded the identification of conserved splice sites.…”

“…Previous studies have shown that the ORF4 homolog of RRV is different in the two RRV strains. RRV26-95 encodes a protein (AAF59982) with four CCP N-terminal domains, while RRV17577 encodes a larger protein (NP_570746) with eight CCP domains (45). Analysis of the encoded amino acid sequences revealed that the MneRV2 ORF4 was closely related to the RRV26-95 and MfuRV2 ORF4 sequences with an N-terminal signal peptide, four CCP domains, and a conserved serine/threonine (S/T) domain prior to the first cysteine residue (Fig.…”